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Characterization of alkaline thermostable keratinolytic protease from thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity
Abstract
A thermostable alkaline protease produced from Bacillus sp. JB 99 exhibited significant keratinolytic and dehairing activity. The enzyme was purified by ammonium sulphate precipitation followed by CM-cellulose and Sephadex G-100 chromatography and resulted in 13.6 fold purification with 23.8% of recovery. The specific activity of purified enzyme was 2989.6 U mg−l. Purified protease had a molecular weight of 29 kDa and appeared as a single band. Gelatin zymogram analysis also revealed a clear hydrolytic zone, which corresponded to the band obtained with SDS-PAGE. The optimum pH and temperature for the keratinolytic activity was pH 11.0 and 70 °C respectively and half life of protease was 70 °C for 4 h. N-terminal amino acid sequence of purified enzyme exhibited extensive homology with other thermostable alkaline proteases and inhibition by PMSF indicated serine type of protease. The Km and Vmax of protease for keratin substrate were 3.8 ± 0.5 mg ml−1 and 15.1 ± 1.6 μm min−1 mg−1 and casein were 3.3 ± 0.4 mg ml−l and 15.6 ± 0.9 μm min−1 mg−1 respectively. The enzyme efficiently dehaired buffalo and goat hide without damaging the collagen layer, which makes it a potential candidate for application in leather industry to avoid pollution problem associated with the use of chemicals in the industry. The enzyme also degraded chicken feathers in presence of reducing agent which can help poultry industry in management of keratin-rich waste and obtaining value added products.
... Additionally, after a 3 h incubation, the enzyme retained more than 60% of its activity between 50-60 °C (Fig. 4d), whilst enzyme activity was almost completely inactivated at 70 °C. Similarly, thermostable-alkaline keratinases from different microbes have been reported previously, exhibiting activities in the 8-12 pH range, and stable at 55-70 °C [24,28,30]. Therefore, keratinase_SW-X could be classified as a thermostablealkaline keratinase. ...
... Ag + , Cu 2+ , Co 2+ , and Fe 3+ decreased keratinase_SW-X activity with the remaining activity of 11-46%. In agreement with our observations, several other studies reported that the keratinase activity of B. halodurans JB 99 [30], B. pumilus Content courtesy of Springer Nature, terms of use apply. Rights reserved. ...
... Prakash et al. [28] reported that the K m and V max of keratinase from B. halodurans PPKS-2 were 6.5 mg/mL and 22.09 μmol/ min/mg, respectively when using keratin as a substrate. The K m and V max of purified keratinase from B. halodurans JB 99 towards keratin, were 3.8 mg/mL and 15.12 ± 1.6 μmol/ min/mg, respectively [30]. ...
The production, purification, and characterization of thermostable-alkaline keratinase from Bacillus halodurans SW-X (keratinase_SW-X), and its applicability towards the production of white chicken feather hydrolysate and bioplastic starch/keratin production were investigated. A maximal keratinase activity of 16.05 ± 0.25 U/mL was achieved by cultivation of B. halodurans SW-X in 5 L stirred tank bioreactor at 47 °C, 200 rpm, and 2 vvm for 48 h. Keratinase_SW-X was purified as a monomeric protein with the estimated MW of 25 kDa, 14.4-fold, and 20% recovery yield. The protein was classified as thermostable-alkaline keratinase due to its optimal pH (pH 10) and temperature (70 °C), pH stability (pH 9.0–11), thermostability (> 3 h at 50–60 °C), and it showed the highest specificity towards chicken feather keratin with Km of 0.45 mg/mL, and Vmax of 3.51 μmol/min/mg. Crude keratinase_SW-X biodegraded white chicken feathers into two types of low (10–20 kDa) and high (≥ 30 kDa) chicken feather (keratin) hydrolysates, with total recovery yields of 30%. The bioplastic starch/keratin was synthesized using cassava starch (70%, w/w), glycerol (30%, w/w), and keratin hydrolysate (0–0.30%, w/w). The optimal concentration of keratin hydrolysate was 0.10% (w/w), resulting in improved strength and elongation at break, with a good appearance of the bioplastic starch/keratin. Our study suggests that B. halodurans SW-X is a potential strain for thermostable-alkaline keratinase production. Moreover, from environmental perspectives, keratinases_SW-X is a promising candidate enzyme that valorizes low cost chicken feather waste to a high value added product, indicating a novel approach for feather waste treatment and utilization.
Graphic Abstract
... retaining more than 80% of its original activity after incubation at 4°C for 24 h (Fig. S1b). The optimal pH of protease_SE5 was quite similar to previous report of alkaline protease from Bacillus halodurans JB99, which showed optimal pH at 11.0 [28]. An alkaline protease from Bacillus pumilus CBS was active and stable in a pH range between 8.0-10.6, ...
... This result indicated protease_SE5 belongs to serine protease. Also, serine protease from Bacillus halodurans JB99 was strongly inhibited by PMSF [28]. The activity of protease_SE5 was stimulated by 10 mM Ca 2+ . ...
... The negative effects of Fe 3+ and Zn 2+ on thermostable proteases from Bacillus spp. have been reported [28,29]. The heavy metal ions relate to the denaturation of proteins by binding a variety of organic ligands leading to enzyme denaturation [33]. ...
... The research showed high keratinolytic and unhairing activity. The protease effectively removed hair from goat and buffalo hide and did not damage the collagen layer and the color of the hide did not change significantly [20]. Another study with protease from B. halodurans PPKS-2 also showed the possibility of using enzyme in unhairing. ...
... There are studies with different size proteases that can be effectively used in leather processing. For example, during one of the studies, a 39.5 kDa size protease was produced, that can be used in dehairing; in different research, B. halodurans protease was purified with 29 kDa [20,34]. The molecular size distribution in the protease mixture may affect protein hydrolysis. ...
Enzymes are biological catalysts that exist in all living organisms. Proteases are one of the most important enzymes in the industry; microbial proteases are widely used in the food, textile, detergent, and leather industries. Traditionally, alkaline proteases are used in the leather industry for bating, however, due to environmental issues, there are many investigations for the application of proteolytic enzymes in other operations such as soaking, unhairing, and derma opening up. This study aimed to produce a proteolytic enzyme from B. halodurans BCRC 910501 and apply it to the soaking of salted hide. After cultivation, the crude enzyme was used for further analysis and leather processing. The results showed an enzyme with higher caseinolytic activity compared to keratinolytic activity. A soaking study found that enzyme improves rehydration, non-collagenous protein, and salt removal. Enzymatic soaking affected collagenous proteins more than the control, but did not significantly impact wet-blue leather properties. Using this enzyme in leather processing might be suitable to reduce the duration of soaking and the number of process steps.
... Various methods of feather degradation have been studied with results of complete degradation varying from 24 h to days. These methods include inoculation of bacterial cultures into whole chicken feathers [74,75] while others have used heat and chemical treatment in addition to bacterial degradation [76,77]. Other studies, like herein, have used enzymatic extracts from bacteria for feather degradation. ...
... However, an addition of 2 % (w/v) sodium azide and 5 mM calcium chloride was used to enhance thermostability of the protease, therefore, could have contributed to the rapid feather degradation as the enzyme's stability in the presence of heat (50 • C) was maintained. Similarly, feather degradation by crude protease extract from Bacillus halodurans JB 99 [77] supplemented with 0,1 % β-mercaptoethanol enhanced the rate of feather degradation, resulting in up to 85 % degradation after 24 h, leaving the basal shaft (rachis) intact. In comparison to their experiment where β-mercaptoethanol was not used, feather degradation with β-mercaptoethanol supplementation yielded better results [78]. ...
Research on proteases and secondary metabolites from endophytes is an area that requires attention from researchers. In this study, proteases from Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17 endophytes were characterised, and their potential biotechnological applications were investigated. Optimum protease production was achieved when isolates were grown in media containing (g/L): glucose 10g, casein 5g, yeast extract 5g, KH2PO4 2g, Na2CO3 10g at pH 9. The crude protease extracts were active in alkaline environments, thus referred to as alkaline proteases with optimal pH of 10. Additionally, Bacillus sp. strain MHSD 16 and Bacillus sp. strain MHSD17 proteases were active at high temperatures, with optimum enzyme activity at 50 °C. Thermostability profiles of these proteases showed that the enzymes were highly stable between (40–60 °C), maintaining over 85 % stability after 120 min incubation at 60 °C. Furthermore, the enzymes were stable and compatible with various household and laundry detergents. In the presence of commercial laundry detergent, OMO® 68 % and 72 % activity was retained for Bacillus sp. strain MHSD16 and Bacillus sp. strain MHSD17, respectively, while 67 % and 68 % activity were retained in the presence of Sunlight®. The potential application for use in detergents was investigated through the removal of blood stains with the crude alkaline extracts displaying efficient stain removal abilities. Feather degradation was also investigated and Bacillus sp. MHSD17 exhibited feather keratin degrading properties more effectively than Bacillus sp. MHSD16.
... Previously reported that pH 7.0 is the optimum pH for purified keratinases from some Bacillus strains. Generally, the keratinolytic enzymes are optimally active at the range of pH 7-9.5 [48][49][50][51][52][53][54]. Recently, Srivastava et al. immobilized keratinase on chitosan and chitosan grafted-β-cyclodextrin beads, and it was found that the pH optima for both free and immobilized forms were 11.0 [55]. ...
... This improved performance at higher temperatures may be due to the nanoparticle coating, which keeps the enzyme aggregates away from thermal denaturation. For free enzyme, this activity was quite similar to temperature optima (30-80 °C) for keratinases from B. strains [50][51][52][53][54]56]. Concerning the immobilized enzyme, our results are in line with those of Srivastava et al., who reported the higher keratinolytic activity for immobilized form [56]. ...
Due to the increasing development of the poultry industry and increasing environmental pollution, especially keratin waste, efficient keratin hydrolyzing biocatalyst has become an attractive goal for researchers. In the current research, a Bacillus keratinase has been immobilized based on the cross-linked enzyme aggregates (CLEAs) method on the functionalized Fe3O4 nanoparticles. Immobilization efficiency and the relative activity of the immobilized enzyme were 87 and 84%, respectively. Results exhibited that the immobilized keratinase showed 7.96 folds more Vmax value than free enzyme in the presence of keratin substrate. Thermostability results showed that the mCLEAs-keratinase improved the enzyme stabilities by about 3.5 and 5.8 folds compared to free enzyme after 3 h incubation at 70 and 80 °C, respectively. The substrate specificity results showed that the activity of keratinase 2S1 toward keratin was five times more than the casein substrate. The activity of the immobilized enzyme in the presence of acetonitrile increased about 7.3 times compared to the free keratinase. The results showed that 43% of the enzyme activity was maintained after ten cycles of reusing. The hydrolysis degree of keratin wastes by immobilized enzyme was three folds more than free enzyme after 2 h of incubation at 42 °C. These results indicated that the free and immobilized keratinases had high potential activities for recycling keratin waste.
Graphic Abstract
... The type of bacteria that is widely used for protease production comes from the genus Bacillus 7 . Several studies of protease gene cloning have been reported from Bacillus pumilus, Bacillus halodurans JB99, and Bacillus licheniformis 2709 8,9,10 . Several species of Bacillus sp. are also known to have the ability for producing alkalothermophilic protease. ...
... The highest activity of the protease enzyme was obtained at pH 9 of 13.66 U / mL, which proved that the protease enzyme produced by the recombinant B. subtilis DB104 worked best at alkaline pH (Fig 5B). Protease enzymes that are able to work in the range of neutral to alkaline pH (7)(8)(9)(10)(11)(12) are grouped into alkaline proteases 26,27 . The protease enzyme produced by B. subtilis DB104 recombinant has good activity at pH 7-9, so it belongs to the alkalthermophilic protease group. ...
Proteases are potential enzymes that utilized in various industrial fields, and the demand of these enzymes is increasing. Bacillus halodurans CM1 is Indonesia indigenous bacterium which is detected to be able to produce alkalotermophilic protease enzyme. In this study, we subcloned the protease gene consist of Open Reading Frame of protease gene and its promoter from Bacillus halodurans CM1 in Bacillus subtilis DB104 via conjugation, and analyzed the expression of the recombinant protease. The protease gene is 1 417 bp length including the open reading frame and the promoter, and obtained by PCR and cloned into pGEM T easy. After confirmed by sequencing, the gene was subcloned into vector pBBRE194, then the recombinant plasmid was transformed into E. coli S17-1. This E.coli was then conjugated to Bacillus subtilis DB104. The target recombinant B. subtilis DB104 has been obtained confirmed by plasmid verification and erythromycin resistance. The recombinant protease produced showed the highest enzyme activity at 50oC and pH 9 (with pH range 5-9) which with protease activity 13.66 U/mL.
... The difference in dehairing results might be due to the duration of incubation time, pH and temperature. This study conducted the dehairing process for 6 h at 60°C and uncontrolled pH, while others used alkaline pH at higher temperature with prolonging incubation time [8,32,33]. It has been reported that the hair is much more easily removed from the hides after 24 h incubation when incubated at 37°C than at 25°C. ...
... Enzymatic dehairing potential from microorganisms is influenced by the presence of either keratinolytic or collagenolytic activity [35]. Collagenolytic activity is needed for the removal of dye in the treated hides, but it will affect the collagen layer of the hides [33]. ...
The growing industrial demand for stable proteases has driven this study to purify and partially characterize a protease from a bacterial strain isolated from undersea fumaroles. Phylogenetic analysis of the 400bp conserved area of the 16S rRNA gene indicated that the isolate was related to Geobacillus thermoleovorans strain SGAir 0734. Purification by ammonium sulfate precipitation and anion exchange chromatography produced 118-fold purity. The optimum activity (646 U mL⁻¹) of the pure enzyme was observed at 60 °C, pH 7 and with 5M sodium chloride addition. The enzyme retained 73% of its activity when in 50% n-hexane, while the activity was only 40% of the control when in 50% ethyl acetate. The enzyme was tested for its leather dehairing capability. It only caused a slight dehairing and produced soft leather. The enzyme may be applied in skin rejuvenation, wrinkle-smoothing, and high-quality suede production industries.
... Various keratinous materials such as chicken feather, feather, wool, meal and bovine hair have been employed as inducers of keratinases [14] . In the present study, we have purified the keratinase and it was similar to purification made from Bacillus sp [15] . The current investigation was successfully explored purification of Keratinase enzyme by the presence of distinct band on the SDS-PAGE which showed approximately 40 kD and results contemporaneous similar with previous reports were displayed by Keratinase from B. licheniformis FK14 (35 kDa), Streptomyces Pactum DSM 40530 and Doratomyces microsporus (30 -33 kDa) [16] . ...
... The enzyme was stable at the range of 6 -8 [8,13] Bacillus sp., FK 46 at 37 °C [20] , B. licheniformis PWD-1 at 50°C [21] . But in contrast to this alkaline keratinases with optimal pH 11.0 were also observed from Bacillus halodurans [15] . They are effectively active approximately pH10 with a molecular range between 10 and 30 kDa and isoelectric point around pH 9.0. ...
For the first time, the enzyme was purified and characterized by examined exploitation of Keratinase industrially in hydrolysis, the keratin substrate by using the Bti to produce the mosquitocidal toxins. Thus, the earlier report showing some Bacillus strains produced keratinolytic enzyme[8]. However, there is no report existing as on date on keratinolytic action of Bti to degrade keratin from feathers as functional unit. Most microbial keratinases are inducible enzymes[13]. Various keratinous materials such as chicken feather, feather, wool, meal and bovine hair have been employed as inducers of keratinases[14]. In the present study, we have purified the keratinase and it was similar to purification made from Bacillus sp[15]. The current investigation was successfully explored purification of Keratinase enzyme by the presence of distinct band on the SDS-PAGE which showed approximately 40 kD and results contemporaneous similar with previous reports were displayed by Keratinase from B. licheniformis FK14 (35 kDa), Streptomyces Pactum DSM 40530 and Doratomyces microsporus (30 – 33 kDa)[16]. However, keratinase with wide molecular mass range (18, 40 and 130 kDa were reported from Streptomyces albidoflavus, Streptomyces thermoviolaceus and Fervidobacterium pennavorans respectively[17] and Chrysosporium keratinophilum (69 kDa).
The appropriate pH of Bti Keratinase was found at neutral pH which was similar to earlier report by[19] for Bacillus sp. This is in difference from other alkaline keratinases. The enzyme was stable at the range of 6 - 8[8,13] Bacillus sp., FK 46 at 37°C[20], B. licheniformis PWD-1 at 50°C[21]. But in contrast to this alkaline keratinases with optimal pH 11.0 were also observed from Bacillus halodurans[15]. They are effectively active approximately pH10 with a molecular range between 10 and 30 kDa and isoelectric point around pH 9.0. This type of protease is produced by the variety of Bacillus species like B. halodurans, B. subtilis and B. licheniformis[22].
The concentration PMSF and EDTA can inhibit purified Keratinase activity even at 1mM, which reveals the serine type protease inhibitor. Similar examination was accounted for proteases exhibiting keratinolytic activity from Streptomyces and Bacillus species[22]. The strong inhibitory effect by EDTA indicated the importance of metal ions for activity/stability[23]. Present results indicated the magnitude of MgCl2 in feather degradation which was acknowledged by the results demonstrated that Amazonian bacterium (Bacillus sp. P45) enhanced the feather degradation by the presence of MgCl2 in feather media[23]. Similarly, protease production by a haloalkaliphilic bacterium was shown to be slightly stimulated by MgCl2, but inhibited MgCl2 [24]. The majority of Keratinase enzymes produced from bacteria are discharged in extracellular. Enzymatic degradation of chicken feather waste required the combined effect of reducing agents, because of single agent alone cannot be feasible to improve the enzymatic activity. The admiring feather concentration for the Keratinase was decided to be 4 mg/ml. Earlier report showed that the high concentration of feather waste was the reason for substrate inhibition or suppression of fabrication[20], on the other hand, high substrate concentration might enhance the medium viscosity, which possibly results in oxygen limitation for bacterial growth[25].
... Sundararajan et al. 54 stated that alkaline protease from Bacillus cereus VITSN04 has potential dehairing activity. Shrinivas and Naik 55 ...
... Sundararajan et al. 54 stated that alkaline protease from Bacillus cereus VITSN04 has potential dehairing activity. Shrinivas and Naik 55 ...
... Previous studies have also revealed that highest activity of alkaline proteases against the casein compared to other substrates [53] . At 70 °C and pH 11.0, the kinetic parameters of a protease from Bacillus halodurans was determined using casein concentrations ranging between 0.5 and 7.5 mg/ml [50] ; the K m and V max of this purified protease was found to be 3.3 mg/ml and 15 U/mg protein, respectively. The results for commercial papain "Paya" reported by Elsson et al . ...
Papain is a globular cysteine-protease family consisting of a single polypeptide chain with three disulfide bridges and a sulfhydryl group necessary for proteolytic activity. Its application is extensive to the fields of medicine and food. The aim of this study is to extract, purify and characterize papain enzymes from the leaves of Carica papaya. Crude extracts containing the enzymes were extracted from the leaves of papaya in Mountain Top University, Nigeria, and purified in a 3-step purification using 70% NH4(SO4)2, DEAE-Cellulose column chromatography and Sephadex G-25 column chromatography to achieve a purification fold of 1.6424. The effects of temperature, pH, substrate concentration and Mg²⁺ concentrations on the activity of the enzymes were determined. From the author's extensive search, this is the first paper in the open literature to report the presence of five enzymes with different measures of binding affinity and catalytic efficiencies between the cysteine-proteases and the casein-substrate. The purified enzymes from Carica papaya are optimally active from 50°C - 59°C and pH 4.5 – 6.6. Its activity is generally enhanced by the addition of Mg²⁺. The Km and Vmax values for the enzyme in the various pools ranged from 1.47mg/ml - 8.70mg/ml and 0.42µmol/ml/min - 0.4167µmol/ml/min. In this study, papain E displayed optimum activity at pH 5.5 and 50°C in the presence of Mg²⁺ with the best catalytic efficiency (Kcat/Km 59.776 - pmL/mg.min) and binding affinity (Km - 0.83 mg/mL) when compared to the isolated enzymes. Values of the parameters assessed from this study could be put to use by the industry in the production of papain and also for household and medical use in storing the product.
... There are a few possible ways to adopt enzymes for the unhairing process. The first one is when the enzymes are applied for a pure enzymatic process, in such a case, the unhairing effect is achieved, owing to the enzyme used (Shrinivas and Naik, 2011;More et al., 2017). The next way is to use enzyme and oxidising agents. ...
The aim of the paper is to obtain new byproducts based on surfactants (gemini – polymethylene-α, ω-bis (N, N-dialkyl-N-deoxy-d-glucitolammonium iodides or bolaform – demecarium bromide) and protein hydrolysates (keratin and collagen) with micro and macro nutrients for applications in agriculture. A method was developed to include micro and macronutrients in keratin and collagen hydrolysates, in order to obtain new byproducts-bioemulsions (stable because of surfactants), with final goal of application as a new class of root fertilizers for cereals (e.g., corn). The newly obtained byproducts (bioemulsions based on surfactants) were characterized by: dynamic light scattering measurements, contact angle, optical microscopy and microbiological tests against fungal attack of Fusarium spp. and Botrytis cinerea. Better results were obtained for gemini surfactant based on sugar – polymethylene-α, ω-bis (N, N-dialkyl-N-deoxy-d-glucitolammonium iodides) due to the properties such as: biodegradability, nontoxicity and adherence to surfaces. The new fertilizer created in this research – bioemulsions based on surfactants, can support the general structure of the grains as well as the chlorophyll content, increasing the growth yield. The fertilizer is indicated for any type of crops and soils, with recommended use as additional fertilizer for plants (cereals) in the vegetation and growth phases, with a maximum need for nutrients.
... There are a few possible ways to adopt enzymes for the unhairing process. The first one is when the enzymes are applied for a pure enzymatic process, in such a case, the unhairing effect is achieved, owing to the enzyme used (Shrinivas and Naik, 2011;More et al., 2017). The next way is to use enzyme and oxidising agents. ...
Various methods of neutralisation of hide unhaired with sodium silicate and sodium sulphide have been investigated seeking to reduce or refuse ammonium sulphate conventionally applied for the neutralisation. Due to the high release of ammonia into the environment, alternative materials are being sought that do not impair the properties of the hide being processed and that a high-quality chromed semi-finished product of leather can be obtained. In the research, different methods of hide neutralisation and their influence on subsequent hide treatment processes qualitative indexes of the obtained leather were analysed and described. It has been found that after unhairing with sodium silicate and sodium sulphide hide, it is appropriate to neutralise and bate the hide by adding 1 % ammonium sulphate, 0.8 % lactic acid and 0.15 % proteolytic enzyme preparation OROPON ON2. To obtain the best quality of chromed semi-finished product of leather, it is advisable to pickle the hide using not more than 1 % sulfuric acid. In this way, neutralised-bated, pickled and chromed leather’s quality meets the quality requirements for chromed semi-finished product of leather.
... Iqbal et al. (2015) also reported that a thermostable alkaline serine protease from G. stearothermophilus B1172 was stable at 90 °C and pH 9. The alkaline protease from B. cereus strain S8 showed optimum temperature and pH of 70 ºC and 10, respectively (Lakshmi et al. 2018). Besides, Shrinivas and Naik (2011) reported thermostable alkaline protease from Bacillus sp. JB 99 showed an optimal temperature at 70 ºC and pH op timum at 11. Thermostable alkaline protease in this study which is stable at 70°C and pH 9.6 could be applied in the detergent industry. ...
Thermostable proteases that optimally withstand the high‐temperature conditions of thermophilic bacteria could be produced and purified, which would be highly beneficial for use in industry. Geobacillus sp. is a thermophilic bacterium that can be found in various environmental conditions. The goal of this study was to isolate and characterize thermostable serine protease that had been produced by thermophilic Geobacillus sp. strain DS3. The proteolytic index was measured in a solid medium. The expression of protease was optimized by Geobacillus sp. DS3 at 50 °C for 18 h. Targeted protease was purified using ammonium sulfate (40‐60%) and DEAE Sephadex A‐25 resin. Using SDS‐PAGE, the molecular weight of the enzyme was predicted to be around 32 kDa. Purified thermostable protease was highly activated at 70 °C, pH 9.6 stable for 1 h, and inhibited by PMSF. Therefore, this enzyme is classified as a thermostable alkaline serine protease. Its kinetic study revealed specific activity of 0.41 U/mg (Vmax) and 0.25 mg/mL (KM). Overall, a thermostable alkaline serine protease from Geobacillus sp. DS3 showed high activity at high temperatures and alkaline pH, which is vital for application in industries such as leather processing and detergent formulation.
... For example, the temperature and pH in each step of conventional leather processing are different, and the pH varies from 2.6 to 13.0 and the temperature changes from 25 to 50 °C (Wu et al. 2014;Lei et al. 2020). Similarly, in the reported researches on enzymatic dehairing, pH and temperature generally vary from 7.0 to 10.0 and 25-45 °C, respectively, and the enzyme dosage is added based on the proteolytic activity of protease under various pH and temperature conditions [60][61][62][63]. As is well known, proteases from microbes are important industrial enzymes and can be classified by pH conditions for optimal activity (acid, neutral or alkaline proteases) [64]. ...
Collagen is the most abundant fibrous structural protein, and therefore, the quantitative evaluation of the effect of protease on collagen has a profound influence on enzyme application. In this research, unlabeled native bovine hide powder was utilized to detect collagen hydrolytic activity of the protease. The optimum conditions of the determination method were as follows: 30 mg/mL substrate concentration, 30 min reaction time, and 2–9 U/mL enzyme concentration. Then, several typical industrial protease preparations were chosen to measure collagenolytic activities at different temperatures and pH values, whose change trends were quite distinct from those of proteolytic activity assay method based on casein or dye-labeled hide powder substrate. Especially, in the pH 5–7, casein hydrolytic activities of these proteases showed sharper peaks with relative activity from 6% to 100%, whereas, their collagen hydrolytic activities based on native hide powder exhibited 30–100% with broader peaks. And collagen hydrolytic activities resulted from using dye-labeled substrate reached a lower optimum pH value than that of other methods. Besides, the results of these measurements displayed a moderate degree of reproducibility. This method is more reasonable than the protease assay method using casein or labeled hide powder as the substrate in many fields.
... Although keratinolytic proteases have been studied since 90s, keratinases from thermophilic or thermotolerant Bacillus is not common, being the list of this type of keratinolytic microorganisms quite short. B. licheniformis K-508 [25], B. subtilis RM-01 [26], Brevibacillus thermoruber T1E [27], and B. halodurans JB99 [28] are representatives of this group of microorganisms. Our work has the aim to enlarge the list of these moderate extremophiles studying the production and characterization of the proteolytic enzymes produce by novel strains of the thermotolerant keratinolytic bacteria: Bacillus cytotoxicus. ...
Background:
Argentina's geothermal areas are niches of a rich microbial diversity. In 2020, species of Bacillus cytotoxicus were isolated for the first time from these types of pristine natural areas. Bacillus cytotoxicus strains demonstrated the capability to grow and degrade chicken feathers with the concomitant production of proteases with keratinolytic activity, enzymes that have multitude of industrial applications. The aim of this research was to study the production of the proteolytic enzymes and its characterization. Also, feather protein hydrolysates produced during fermentation were characterized.
Results:
Among the thermotolerant strains isolated from the Domuyo geothermal area (Neuquén province, Argentina), Bacillus cytotoxicus LT-1 and Oll-15 were selected and put through submerged cultures using feather wastes as sole carbon, nitrogen, and energy source in order to obtain proteolytic enzymes and protein hydrolysates. Complete degradation of feathers was achieved after 48 h. Zymograms demonstrated the presence of several proteolytic enzymes with an estimated molecular weight between 50 and > 120 kDa. Optimum pH and temperatures of Bacillus cytotoxicus LT-1 crude extract were 7.0 and 40 °C, meanwhile for Oll-15 were 7.0 and 50 °C. Crude extracts were inhibited by EDTA and 1,10 phenanthroline indicating the presence of metalloproteases. Feather protein hydrolysates showed an interesting antioxidant potential measured through radical-scavenging and Fe3+-reducing activities.
Conclusion:
This work represents an initial approach on the study of the biotechnological potential of proteases produced by Bacillus cytotoxicus. The results demonstrated the importance of continuous search for new biocatalysts with new characteristics and enzymes to be able to cope with the demands in the market.
... There are a few possible ways to adopt the enzymes for the unhairing process. The first one is when the enzymes are applied for a pure enzymatic process, in such a case, the unhairing effect is achieved, owing to the enzyme used [14][15][16]. Unfortunately, so far, such an unhairing method has been applied merely on laboratory scale [13]. ...
Recently, increasing attention has been paid to the application of enzymes in a wide variety of leather production processes. The aim of the present study was to investigate the action of enzymatic pickling on derma’s collagen and the influence of this action on subsequent processes and properties of chromed and finished leather. The application of active in acidic medium proteolytic enzymes in the pickling process led to an additional impact on derma structure: collagen was more strongly affected and the porosity of the pelt dermis was reduced, but the hide became more thermally stable. The enzymatically pickled pelt bonded more chromium and reached higher shrinkage temperature while chroming; dyes penetrated deeper; such leather bonded more fatliquors. On the other hand, the action of enzymes worsened the physical–mechanical properties of the leather, as the experimental leather was weaker than the conventional one. The first was characterised by weaker grain layer and had significantly higher relative elongation. Therefore, as some properties improve and others worsen during such a process, the application of every enzyme should be carefully investigated and optimized to produce a leather with defined properties.
... The enzyme produced from Bacillus subtilis [12], Aspergillus tamarii [62], Streptomyces avermectinus NRRL B-8165 [63], Bacillus sp. JB 99 [64], and Bacillus halodurans JB 99 [65] are useful for dehairing. ...
Proteases are important industrial biocatalysts that constitute the largest group of enzymes acting as proteinases, peptidases,
and amidases with a broad range of industrial applications. In this review, particular attention has been given to comprehensively scrutinize the proteases. After the succinct introduction, classifcation of proteases as exopeptidases (amino and
carboxy proteases) and endopeptidases (serine, aspartic, cysteine, and metalloproteases), sources of alkaline, acidic and
neutral protease like animal, plant and microbial sources along with the multi-industrial applications have been dissertated.
Now a day’s, mostly proteases, which are present in the market, are produced from microbial sources because of the fast
production rate and the limited requirement of cultivation. In addition to this, a critique on the applications of proteases in
food, detergent, leather, pharmaceutical, cosmetics, silk degumming, silver recovery, chemical industry, and wastewater
treatment industries is also concisely addressed. Finally, protein engineering and immobilization strategies to improve the
catalytic properties of protease are thoroughly vetted. The quest for novel sources of protease enzyme has been encouraged
to fulfll their ever-increasing demands for industrial exploitation.
... Therefore, dehairing, using a microbial keratinase, is considered an easy alternative [40]. There are many reports of dehairing of goat/bovine skin, employing purified/semi-purified keratinases [41][42][43]. In the present study, the crude protease with keratinolytic activity from Laceyella sacchari strain YNDH completely dehaired goat hides without affecting the skin quality. ...
Background:
Due to a multitude of industrial applications of keratinolytic proteases, their demands are increasing. The present investigation studied the production and monitoring of the most possible multi-functional applications of YNDH thermoalkaline keratin-degrading enzyme.
Results:
This work is considered the first that reported YNDH strain closely related to Laceyella sacchari strain; YNDH is a producer of protease/keratinase enzyme and able to degrade natural keratin such as feathers, wool, human hairs, and nails. Experimental design Plackett-Burman (PBD) was applied to evaluate culture conditions affecting the production of thermoalkaline protease/keratinase. Afterwards, Box-Behnken design (BBD) was applied to find out the optimum level of significant variables namely, NH4Cl, yeast extract, and NaNO3 with a predicted activity of 1324.7 U/ml. Accordingly, the following medium composition and parameters were calculated to be optimum (%w/v): NH4Cl, 0.08; feather, 1; yeast extract, 0.04; MgSO4.7H2O, 0.02; NaNO3, 0.016; KH2PO4, 0.01; K2HPO4, 0.01; pH, 8; inoculum size; 5%, cultivation temperature (Temp.) 45 °C and incubation time 48 h. The studied enzyme can degrade keratin-azure, remove proteinaceous materials, and is able to remove hairs from goat hides. These interesting characteristics make this enzyme a good candidate in many applications especially in detergent (Det.), in leather industries, and in pharmaceuticals particularly in nail treatment.
Conclusion:
The promising properties of the newly keratin-degrading protease enzyme from Laceyella sacchari strain YNDH would underpin its efficient exploitation in several industries to cope with the demands of worldwide enzyme markets.
... Many reports have demonstrated the effect of reducing agents such as cysteine, β-mercaptoethanol (β-ME), dithiothreitol (DTT), sodium sulfide, and thioglycolate on promoting the degradation of native keratins. For example, Shrinivas et al. (2011) reported that enzymatic degradation of chicken feather required the combined effect of reducing agent (β-ME) and keratinolytic activity of Bacillus sp. JB 99. ...
Microbial thermostable alkaline keratinases play an important role in enzymatic degradation of poultry waste, especially chicken feather waste which can be converted to high value products such as keratin hydrolysate, bioplastic, and hydrogels. However, the biotechnological application of keratinase requires not only good characteristics and high specificity of enzyme, but the high titer of enzyme activity is also necessary. Therefore, this study emphasizes on the optimization of thermostable alkaline keratinase production by Bacillus halodurans SW-X using a statistical approach. The Plackett-Burman (PB) design was applied to screen the significant factors affecting keratinase production and found that chicken feather was the only significant factor (P≤0.15). The optimal conditions for thermostable alkaline keratinase production were further investigated using response surface methodology (RSM) approach via a central composite design (CCD). The optimal medium compositions and cultivation conditions were chicken feather of 7.38 g/L, initial pH of 9.67, 47.0°C, 36-h of cultivation time, and shaking speed of 250 rpm. Under the optimized conditions, an overall 20-folds increase in activities of protease (13,000 U/mL) and keratinase (7.40 U/mL) were achieved. Moreover, the production of keratinase was also implemented in 5.0-L stirred tank bioreactor with a working volume of 2.5-L of optimized medium and cultivation conditions with aeration rate of 2.0 vvm. The maximal protease and keratinase activities of 15,001±103 and 15.9±0.16 U/mL were obtained at 48-h of cultivation time. In addition, this crude enzyme was used for keratin hydrolysate production from chicken feather. The result revealed that the promising soluble protein concentration of 3.04±0.01 mg/mL or 304 mg/g chicken feather was obtained at 72-h of reaction time. This study suggests the promising biotechnological valorization of chicken feather into high value product of keratin hydrolysate by an effective keratinase.
... I40 [43], Bacillus subtilis (B. subtilis) RM-01 [44], Brevibacillus thermoruber T1E [45], B. halodurans JB99 [46], and Thermoactinomyces sp. YT06 [47] are representatives of keratinolytic bacteria that can be consider moderately thermophilic aerobic bacteria (50-60 • C). ...
Geothermal areas are the niches of a rich microbial diversity that is not only part of the intangible patrimony of a country but also the source of many microbial species with potential biotechnological applications. Particularly, microbial species in geothermal areas in Argentina have been scarcely explored regarding their possible biotechnological uses. The purpose of this work was to explore the proteolytic and keratinolytic enzymatic potential of microorganisms that inhabit in the Domuyo geothermal area in the Neuquén Province. To this end, we did enrichment cultures from two high-temperature natural samples in mineral media only supplemented with whole chicken feathers. After the isolation and the phylogenetic and morphologic characterization of different colonies, we obtained a collection of Bacillus cytotoxicus isolates, a species with no previous report of keratinolytic activity and only reported in rehydrated meals connected with food poisoning outbreaks. Its natural habitat has been unknown up to now. We characterized the proteolytic and keratinolytic capacities of the B. cytotoxicus isolates in different conditions, which proved to be remarkably high compared with those of other similar species. Thus, our work represents the first report of the isolation as well as the keratinolytic capacity characterization of strains of B. cytotixicus obtained from a natural environment.
... Keratinolytic activity was determined by using keratin as substrates (Shrinivas and Naik 2011). After diluting 100 times, to measure the soluble substrates, 250 μL diluted purified KerT was dissolved in 250 μL Gly-NaOH buffer (pH 10.0) containing 1% (w/v) keratin and BSA. ...
To promote enzymatic unhairing for leather production, a new unhairing enzyme is developed. The Keratinase (kerT) gene, which is amplified from B. amyloliquefaciens TCCC11319 by PCR, is expressed in B. subtilis WB600. The recombinant KerT reduces the collagenolytic protease content as well as improving the keratinase content effectively. Therefore, the improved keratinase leads to the obviously unhairing effect, whereas the low collagenolytic protease ensures the integrity of collagen fibers in hide. It represents, the leather grain surface isn’t destroyed thereby the value of finished leather can be maintained. In addition, by analyzing the properties of KerT, tits activity isn’t inhibited with Na⁺, K⁺ and Ca²⁺ which are commonly used in leather production. The freeze-dried fermentation broth can be used directly as unhairing enzyme without addition of traditional sulfide chemicals. By evaluating the properties of unhaired hide, the results show that the collagen degradation ability of this new unhairing enzyme is slightly and it does not cause any adverse effects on the leather quality. Besides, this unhairing enzyme doesn’t further degrade collagen in the time range of 8 h to 24 h, thus it is safely and easy-control in actual production. In conclusion, the enzymatic unhairing method with recombinant KerT has the potential to be more sustainable and efficient alternative than current sulphur-lime method, and it does not require the further purification thereby saving the cost.
... Its N-terminal amino acid sequence showed homology with other thermostable alkaline proteases. It is a serine protease because it was inhibited by PMSF (Shrinivas and Naik 2011). ...
Keratins are insoluble, fibrous, and structural proteins that are present in the epidermis and its appendages and these include feather, hair, wool, nail, hoof, and horns. Keratins adhere epidermal cells to one another and provide protection on the skin. They are structurally stabilized by their tightly packed peptide chains and the existence of several cross-linkages by disulphide bonds, hydrogen bonding, and hydrophobic interactions. Keratin-containing materials are generated abundantly as by-products of agro-industrial processing and constitute nuisance in the environment as a result of their recalcitrance to degradation by regular proteolytic enzymes like pepsin, trypsin, and papain. The traditional physical and chemical techniques for their treatment are expensive, energy consuming, can damage some essential amino acids, and non-environmentally benign. However, degradation by a variety of microorganisms had proven to be a viable alternative means of keratin treatment. A vast variety of bacteria, fungi, and actinomycetes have been recognized as keratin degraders. They degrade keratins mainly with their keratinases, which sometimes act synergistically with other enzymes like disulfide reductases and cysteine dioxygenase for effective degradation of keratins. The microbial keratinases hydrolyze keratins into soluble proteins, peptides, and amino acids. They are utility enzymes with very diverse biotechnological applications. Biodegradation of keratin-rich wastes by microorganisms is therefore an efficient, cheap, and eco-friendly method of waste management and production of products of high biotechnological value. The present review examines the trends in the role of microorganisms for the biotechnological treatment of keratin-rich wastes.
... The digestion of the young dermal tissue was better with the "mixed" enzyme solution, and the tissue specimens treated with our solution gave rise to more fibroblasts that reached 95% confluence more rapidly than the ones obtained with the standard enzyme digestion method. Furthermore, our method included an elevated calcium concentration and a higher temperature than usual because calcium ions are important for the ability of collagenase to bind to collagen fibres and start digestion, and the activity of this enzyme is higher at slightly elevated temperatures (22,23). The only major exception occurred when tissue samples from older donors were investigated. ...
Cultivated fi broblasts have been widely used in a large number of in vitro studies. Although they readily proliferate under cell culture conditions, improvements in methods for their isolation are necessary. Here, we present our modifi ed enzyme digestion method and compare its effi ciency with commonly used techniques. Th ree foreskin samples from young, middle-aged and old donors were used. Th e classical explant, standard enzyme digestion method with collagenase and our improved enzyme digestion method were compared for effi ciency of fi broblast isolation and the time needed to achieve 95% confl uence in a 30-mm Petri dish. Th e explant method was the slowest to achieve fi bro-blast confl uence, especially with the tissues from the older donors (up to 23 days). With the standard enzyme digestion method, the skin tissue was partially digested, but the fi bro-blasts reached confl uence much faster (the younger donor cells needed approximately 7 days to reach confl uence). Our modifi ed "mixed" enzyme digestion method was the fastest (the fi broblasts from the younger donors needed up to 5 days to reach confl uence). For studies requiring the primary isolation and cultivation of dermal fi broblasts, the best method to achieve this goal is the tissue digestion method with the multiple enzyme solution. SAŽETAK Kultivisani fi broblasti se često upotrebaljavaju u brojnim "in vitro" studijama. Iako oni relativno lako proliferišu u uslo-vima ćelijskih kultura, standarne metode primarne izolacija fi broblasta nisu dovoljno efi kasne. U ovom radu mi prikazuje-mo modifi kovanu metodu enzimske digestije tkiva i upoređu-jemo njene rezultate sa standardnim metodama. U eksperimenu su korišćena tri uzorka dobijena nakon cirkumcizije kod mladih, sredovečnih i starih pacijeneta. Upoređivana je efi kasnost primarne izolacije fi broblasta ko-rišćenjem eksplant metode, standardne enzimske metode uz korišćenje kolagenaze i naše modifi kovane metode enzimske digestije tkiva. Upoređivano je vreme neophodno za dostiza-nje 95% konfl uencije fi broblasta u 30mm Petri šoljama. Eksplant metoda je najsporija kada je u pitanju dostiza-nje konfl uencije i to posebno kod starih donora (do 23 dana). Standardna metoda enzimske digestije dovodi do nepotpune disocijacije tkiva humane kože, ali je dostizanje konfl uencije fi broblasta bilo znatno brže nego kod eksplanta (kod mladih donora polovinom 7. dana). Naša, modifi kovana enzimska metoda sa mešavinom enzima je najbrže dovela do konfl u-encije ćelija (kod mladih donora 5. dana). Kada dizajn neke studije zahteva efi kasnu izolaciju der-malnih fi broblasta i visok procenat vijabilnosti ćelija, najbo-lja metoda je digestija tkiva sa mešavinom enzima. Ključne reči: dermalni fi rboblasti, izolacija, kultivacija, enzimska digestija 66
... Most of the previous studies have revealed that highest activity of alkaline proteases showed with casein as compared to other substrates. The Kinetic parameters (K M and V max ) were determined for thermostable protease from Bacillus halodurans JB 99 by [5] at 70°C and pH 11.0 for concentrations ranging between 0.5 and 7.5 mg/ml of casein. The K M and V max of purified protease was found to be 3.3 mg/ml and 15 U/mg protein respectively. ...
Proteases are the hydrolytic enzymes which hydrolyzes peptide bond between proteins with paramount applications in pharmaceutical and industrial sector. Therefore production of proteases with efficient characteristics of biotechnological interest from novel strain is significant. Hence, in this study, an alkaline serine protease produced by Bacillus cereus strain S8 (MTCC NO 11901) was purified and characterized. The alkaline protease was purified by ammonium sulfate precipitation (50%), ion exchange (DEAE-Cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. As a result of this purification, a protein with specific activity of 300U/mg protein was obtained with purification fold 17.04 and recovery percentage of 34.6%. The molecular weight of the purified protease was determined using SDS-PAGE under non-reducing (71 kDa) and reducing conditions (35 kDa and 22 kDa). Zymogram analysis revealed that proteolytic activity was only associated with 22 kDa. These results indicate that existence of the enzyme as dimer in its native state. The molecular weight of the protease (22 kDa) was also determined by gel filtration (Sephadex G-200) chromatography and it was calculated as 21.8 kDa. The optimum activity of the protease was observed at pH 10.0 and temperature 70 °C with great stability towards pH and temperature with casein as a specific substrate. The enzyme was completely inhibited by PMSF and TLCK indicating that it is a serine protease of trypsin type. The enzyme exhibits a great stability towards organic solvents, oxidizing and bleaching agents and it is negatively influenced by Li²⁺ and Co²⁺ metal ions. The purified protein was further characterized by Matrix Assisted Laser Desorption Ionization/Mass Spectroscopy (MALDI/MS) analysis which reveals that total number of amino acids is 208 with isoelectric point 9.52.
... The genus Bacillus are in fact the most frequently enquired group of keratinolytic bacteria, the other Bacillus species other than B. licheniformis that has been reported for keratinolytic activity by previous researchers include Bacillus cereus and Bacillus subtilis (Łaba and Szczekała, 2013), Bacillus megaterium (Forgacs et al., 2001), Bacillus polymyxa (Laba and Rodziewicz, 2010), Bacillus pumilus (Huang et al., 2003), Bacillus halodurans (Prakash et al., 2010;Shrinivas and Naik, 2011) and Bacillus polyfermenticus (Dong et al., 2017). ...
Keratinase enzymes are a special type of protease that has a bio-degradative potential for degrading keratin-containing substrates by the enzymes they produced during bioprocessing. The study was carried out to investigate the effect of microbial degraded feather meal on broiler chickens. The strain was previously isolated from a feather dumping site. The effects of crude and immobilized enzyme-degraded feather meal were investigated on growth performance, haematology and intestinal histology of broiler chickens. Maximum activity of the keratinolytic enzyme was at 45 °C, the maximum bio-degradative potential at 48 h of fermentation, while the pH 7 exhibited the maximum keratinolytic activity. The values obtained for feed conversion ratio for birds on feather meal were statistically similar to the value obtained for those on the control diet. The microbial biodegradation of feather wastes could be a better approach to overcome high feed cost and environmental pollution arising from solid waste disposal.
... pennivorans, isolated from a solfataric mud (35-40 C), showed that keratinolytic activity was reported by Ionata et al. (2008). Among moderately thermophilic aerobic bacteria (50-60 C), the keratinolytic microorganisms reported are B. licheniformis PWD-1 (Williams et al. 1990), Streptomyces thermonitrificans MG104 (Mohamedin 1999), Bacillus subtilis RM-01 (Rai et al. 2009), Brevibacillus thermoruber T1E (Bihari et al. 2010) and Bacillus halodurans JB99 (Shrinivas and Naik 2011). M. ruber H328 is the only one that has been isolated from a hot spring, while all the other aerobic bacteria reported until now have been isolated from samples of soil, waste streams, sugarcane molasses and poultry wastes. ...
Thermophilic aerobic bacteria were isolated from two geothermal areas in Neuquén province using two different enrichment methods and a total of 30 isolates were obtained. From chicken feather enrichment cultures, strains affiliated to Bacillus cytotoxicus and B. licheniformis were isolated and all of them demonstrated the capability to degrade completely chicken feather.
A preliminary research on biotechnological enzymes potential demonstrated that all the isolates displayed at least one of the extracellular hydrolytic enzyme tested. Most of the isolates showed protease, inulinase and/or pectinase activities while cellulase and xylanase activities were less common. In light of these findings, geothermal areas of Argentina may be considered a potential source of thermophilic bacteria able to produce many industrially relevant enzymes.
... Extremophilic bacteria adapted to more than one environmental stressor such as haloalkaliphiles which survive under saline and alkaline conditions, also produce alkaline proteases (Raval et al. 2014). Thermophilic alkaliphiles, thermophilic haloalkaliphiles, psychrophilic, and psycrothrophic bacteria have also been reported to produce valuable alkaline proteases (Kasana 2010, Shrinivas andNaik 2011). ...
In addition to their ecological importance in the acquisition of nitrogen-rich organic compounds, extracellular proteases also have interesting biotechnological applications. Particularly, alkaline proteases represent one of the most important groups of commercial enzymes. First, we introduce the classification and catalytic mechanisms of proteases. Then, this chapter reviews the advances in the bioprospection of alkaline proteases produced by bacteria adapted to selective conditions from different environments of Patagonia (Argentina). Among them, the arid soils of the Patagonian Monte are propitious for the development of alkaliphilic microorganisms. Thus, we focus on the description of the species Bacillus patagoniensis and the biochemical and catalytic properties of its alkaline protease. Then, we discuss investigations about alkaline protease-producing bacteria from the southern Patagonian coast, the prevalence of psychrophilic and psychrotolerant strains, and the response of their extracellular proteases to temperature.
... The use of enzyme based products is currently being explored in many areas of leather making process, with increasing importance in the dehairing process, thus eliminating the use of hazardous sodium sulfide (Thanikaivelann et al., 2004). Due to the increasing demand of enzyme in the leather industry, there arises a need for new proteases (Shrinivas and Naik, 2011). ...
... In fact, the specific activity displayed by SPVP was significantly high compared to those previously reported for other alkaline proteases from Bacillus strains. For example, the protease produced by Bacillus halodurans strain JB 99 showed a specific activity of 2989.6 U/mg [44], the protease from Bacillus sp. strain SM2014 had specific activity of 657.14 U/mg [33], the alkaline protease SAPB from Bacillus pumilus strain CBS showed a specific activity of 25,500 U/mg [23], and the protease produced by Bacillus licheniformis strain UV-9 had a specific activity of 12,102.96 ...
... As the denaturation of the enzymatic protein occurs at elevated temperatures therefore, after certain level of temperature increase (above 90°C), the enzyme activity decreased rapidly. Shrinivas et al. (2011) reported a highly thermostable alkaline xylanase was purified to homogeneity from culture supernatant of Bacillus sp. JB 99, enzyme was optimally active at 70°C. ...
A potential bacterial isolate exhibiting extracellular cellulase-free xylanase activity by Bacillus tequilensis SH0 was
isolated from compost. The isolate demonstrating maximal xylanase activity after optimization using COFAT i.e. basal
salt medium at 96 h, pH 5.5, temperature 45ºC, inoculums size 10%, carbon source-wheat bran (1.25%). The multistep
purification techniques used were ion exchange chromatography and gel exclusion chromatography. The molecular
weight was found in range of 14kDa- 97.4 kDa. The purified xylanase showed optimal activity of 41.30 IU/ml at 90ºC,
pH 6.0 on xylan and also depicted cellulase free nature. The Km and Vmax of partially purified xylanase from B.tequilensis
SH0 were 1.55 mg/ml and 125.0 µ mol/mg/min. Encoding of genes responsible for xylanase production was done using
gradient PCR.
... After 20 min of incubation 2 ml of 10% TCA was add-ed to stop the reaction. The reaction mixture was centrifuged at 12 000 rpm for 10 min and absorbance was measured at 280 nm (Shrinivas & Naik, 2011). One unit of protease activity was defined as the amount of enzyme required to release 1 µg of tyrosine per min under experimental conditions. ...
As part of the contribution to the global efforts in research of thermostable enzymes being of industrial interest , we focus on the isolation of thermophilic bacteria from Tunisian hot springs. Among the collection of 161 strains of thermophilic Bacillus isolated from different samples of thermal water in Tunisia, 20% are capable of growing at 100°C and the rest grow at 70°C or above. Preliminary activity tests on media supplemented with enzyme-substrates confirmed that 35 strains produced amylases, 37 – proteases, 43 – cellulases, 31 – xylanases and 37 – mannanases. The study of the effect of temperature on enzyme activity led to determination of the optimal temperatures of activities that vary between 60 and 100°C. Several enzymes were active at high temperatures (80, 90 and 100°C) and kept their activity even at 110°C. Several isolated strains producing enzymes with high optimal temperatures of activity were described for the first time in this study. Both strains B62 and B120 are producers of amylase, protease, cellulase, xylanase, and mannanase. The sequencing of 16S DNA identified isolated strains as Geobacillus kaustophillus, Aeribacillus pallidus, Geobacillus galactosidasus and Geo-bacillus toebii.
The aim of this study was to isolate and characterize keratinolytic thermophilic aerobic bacilli from Armenian geothermal springs. In total 20 thermophilic aerobic bacilli strains have been isolated using chicken feather enrichment cultures. Among these, four strains affiliated (based on 16S rRNA genes sequences) as Bacillus licheniformis (95–97% similarity) and Bacillus borbori (> 99% similarity) demonstrated the capability to completely degrade chicken feathers at 55 °C. The highest rate of feather hydrolyses in mono-species cultures was observed with 40 g L−1 substrate. Notably, enhanced keratin weight loss (≥ 80%) was observed in dual co-cultures involving B. borbori M14, highlighting superior degradative potential of this strain. Keratinolytic enzyme production was dedected during the late exponential growth phase, reached its maximum activity (0.013 U mL−1) during the stationary phase, suggesting growth-associated enzyme synthesis. High-performance liquid chromatography (HPLC) of the hydrolysis end products revealed that aspartic acid and isoleucine were the predominant amino acids, followed by leucine, phenylalanine, alanine, tyrosine and glutamic acid. These findings confirm that the newly isolated strains are promising sources of keratinolytic proteases, with potential applications in circular bioeconomy based processes.
The aim of this study was to isolate and characterize keratinolytic thermophilic aerobic bacilli from Armenian geothermal springs. In total 20 thermophilic aerobic bacilli strains have been isolated using chicken feather enrichment cultures. Among these, four strains affiliated as Bacillus licheniformis (95–97% similarity) and Bacillus borbori (> 99% similarity) demonstrated the capability to completely degrade chicken feathers at 55°C. The highest rate of feather hydrolyses in mono-species cultures was observed with 40 g L − 1 substrate. Notably, enhanced keratin weight loss (≥ 80%) was observed in dual co-cultures involving B. borbori M14, highlighting superior degradative potential of this strain. Keratinolytic enzyme production was dedected during the late exponential growth phase, reached its maximum activity (0.013 U mL − 1 ) during the stationary phase, suggesting growth-associated enzyme synthesis. High-performance liquid chromatography (HPLC) of the hydrolysis end products revealed that aspartic acid and isoleucine were the predominant amino acids, followed by leucine, phenylalanine, alanine, tyrosine and glutamic acid. These findings confirm that the newly isolated strains are promising sources of keratinolytic proteases, with potential applications in circular bioeconomy based processes.
The poultry industry is one of the significant driving sectors in the food industry. On one hand, the enormous growth of this industry has boosted food safety. Still, on the other side, it also generates massive amounts of waste during various stages of food processing. Feathers, viscera, bones, and dead on arrival are some of the solid wastes which are generated. The poultry industry’s most abundant wastes include feathers with approximately 90% protein content, mainly keratin protein. Enzyme technology has been one of the solutions for converting these wastes into valuable products, for example, amino acids, peptides, and other bioactive compounds having a physiological role. For this bioconversion, a keratinase enzyme is of utmost importance. Different microbes, bacteria, and fungi can degrade the feathers by secreting keratinase enzyme. This chapter gives an overview of poultry waste management through enzyme keratinase, its structure, different sources of the enzyme, production methods, and the role of the keratinase enzyme in bioconverting poultry waste into valuable products.
Purified calcium serine metalloprotease from Stenotrophomonas maltophilia strain SMPB12 exhibits highest enzyme activity at pH 9 and temperature range between 15°C-25°C. Enzyme supplemented with 40 µM Ca-Hap-NP (NP-protease) showed maximum elevated activity of 17.29 µmole/min/ml (1.9-fold of original protease activity). The thermostability of the enzyme was maintained for 1 h at 60°C over an alkaline pH range 7.5-10, as compared to the NP untreated enzyme whose activity was of 8.97 µmole/min/ml. A significant loss of activity with EDTA (1.05 µmole/min/ml, 11.75%), PMSF (0.93 µmole/min/ml, 10.46%) and Hg2+ (3.81 µmole/min/ml, 42.49%) was also observed. Kinetics study of NP-protease showed maximum decreases in Km (28.11%) from 0.28 mM (NP untreated enzyme) to 0.22 mM (NP-protease) along with maximum increase in Vmax (42.88%) from 1.25 µmole/min/ml to 1.79 µmole/min/ml at varying temperatures. The enhanced activity of NP-protease was able to efficiently degrade recalcitrant solid wastes like feather to produce value-added products like amino acids and helps in declogging recalcitrant solid wastes. The nano-enabled protease may be utilized in a smaller amount for degrading in bulk recalcitrant solid proteinaceous waste at 15°C temperature as declogging agents providing an eco-friendly efficient process.
Abstract
In the present study, the production of keratinase from Stenotrophomonas maltophilia strain Kb2 was optimised. Plackett-Burman Design (PBD) followed by Central Composite Design (CCD) were used for the determination of significant variables for keratinase synthesis, and the concentration of three variables—feather meal (FM), starch, and MgSO4—were optimised for higher keratinase production. Further an interaction of the selected variables, Partial Least Square Regression (PSLR) analysis was performed. Optimised culture medium led to maximum keratinase activity of 70.8 ± 3.9 U/ml after 24 h of growth, which was 4.91 fold higher than the keratinase obtained from un-optimised media. The keratinase yield was very close to the real projected value. The statistical and multivariate analysis assisted in maximising the keratinase production. Further, crude keratinase was studied for its utilization as a dehairing agent. The results of the present study clearly showed enhanced production of keratinase by employing PBD and CCD followed by the PSLR approach.
The investigation was aimed at isolating and characterizing the culturable
feather degrading bacteria from soils of the poultry waste dump site along
Enugu - Port Harcourt express way by Gariki, Awkunanaw, Enugu. The bacteria that were isolated were tested for their capability to grow on feather meal
agar (FMA). The proteolytic bacteria were tested for feather degradation and
were further identified according to their morphological and biochemical characteristics.
The investigation was aimed at isolating and characterizing the culturable feather degrading bacteria from soils of the poultry waste dump site along Enugu - Port Harcourt express way by Gariki, Awkunanaw, Enugu. The bacteria that were isolated were tested for their capability to grow on feather meal agar (FMA). The proteolytic bacteria were tested for feather degradation and were further identified according to their morphological and biochemical characteristics. The isolate were gram positive, rod shaped and spore-former and were able to utilize glucose, sucrose and lactose. They were also catalase and oxidase positive. They showed typical characteristics of Bacillus sp., thus were from the Bacillus genus. This Bacillus strain is therefore a promising strain for the management of chicken feather waste through biotechnological processes.
Algeria harbor over 240 hot springs, increasing in number as we approach the Northeast side of the country. These ecosystems are a privileged field of investigation to look for new thermophilic strains which produce biomolecules including enzymes.
Our work focuses on the study of enzymatic activities of bacterial strains isolated from thermal aquifers. In total, 79 aerobic and anaerobic strains were isolated and characterized. Phylogenetic identification of these strains by molecular approach (sequencing of the 16S rRNA gene) allowed us to classify them as members of these genera: Albidovulum, Hydrogenophilus, Meiothermus, Thermoanaerobacter, Thermovibrio, Thermotoga, Thermanaerothrix, Tepidimonas, Anoxybacillus, Geobacillus, Gordonia, Sphingopyxis and, Caldicoprobacter.
Three new hydrolytic enzymes: keratinase (KERCA), protease (SAPCG), and xylanase (XYN35) produced by strains belonging to the genus Caldicoprobacter were purified and characterized. These enzymes may be used respectively in the treatment of leather, in detergents, and in the bleaching of paper pulp, respectively. The results of the characterization of these enzymes are very promising.
Key words: Hot spring, Thermostable enzymes, Keratinase, Protease, Xylanase
The poultry industry is one of the major revenue generating industries around the world. This industry generates a lot of wastes including huge amounts of chicken feathers, that are recalcitrant, thus, difficult to dispose of and have a negative environmental impact. The main methods of disposal of feathers are either incineration or dumping them with other waste materials that hinder their biodegradability by soil microorganisms. These methods are energy-intensive and also lead to the loss of valuable protein keratin that forms the major part of chicken feathers. Recently, biodegradation/metabolism of chicken feathers by microorganisms has been considered an excellent alternative for the disposal of keratin waste. Keratin degradation leads to the formation of free amino acids and soluble proteins that are a precursor of many valuable metabolites and value-added products. Thus, the present review deliberated on the importance of chicken feathers and also other keratin waste materials to be considered as an important bio-resource and their complete valorization into high-value chemicals and development of cost-effective technologies to convert poultry wastes into useful products.
Synthetic chemical usage enhanced day by day at both industrial level and medical fields, together causing vulnerability in the ecological pyramids and leads to loss of ecological equilibrium around the globe. In addition, the enormous growth of industries, affecting the health of both environment and community health. For the betterment of life to achieve in the community, step has taken to develop the different processes for eco-friendly products. Instead of chemical usage, enzymatic processes are the alternatives to attain the healthy environment. Take a glance on the Alkaline proteases derived from fungi having various industrial applications and eco-friendly in nature, includes textile, detergent, leather, feed, waste, water recycling, bioremediation, tannery effluent, effluent treatment and medical applications. The present discussion focused on alkaline proteases applications and properties. Type of production with various influencing parameters.
Interpreting the results and depicting the multifunctional properties used to promote fungal alkaline proteases became versatile in the fields of eco-friendly and medicinal applications, which has gained prominent usage of stable properties.
Keratin is an insoluble and protein-rich epidermal material found in e.g. feather, wool, hair. It is produced in substantial amounts as co-product from poultry processing plants and pig slaughterhouses. Keratin is packed by disulfide bonds and hydrogen bonds. Based on the secondary structure, keratin can be classified into α-keratin and β-keratin. Keratinases (EC 3.4.-.- peptide hydrolases) have major potential to degrade keratin for sustainable recycling of the protein and amino acids. Currently, the known keratinolytic enzymes belong to at least 14 different protease families: S1, S8, S9, S10, S16, M3, M4, M14, M16, M28, M32, M36, M38, M55 (MEROPS database). The various keratinolytic enzymes act via endo-attack (proteases in families S1, S8, S16, M4, M16, M36), exo-attack (proteases in families S9, S10, M14, M28, M38, M55) or by action only on oligopeptides (proteases in families M3, M32), respectively. Other enzymes, particularly disulfide reductases, also play a key role in keratin degradation as they catalyze the breakage of disulfide bonds for better keratinase catalysis. This review aims to contribute an overview of keratin biomass as an enzyme substrate and a systematic analysis of currently sequenced keratinolytic enzymes and their classification and reaction mechanisms. We also summarize and discuss keratinase assays, available keratinase structures and finally examine the available data on uses of keratinases in practical biorefinery protein upcycling applications.
The leather industry earns special attention because of its strong potential for foreign exchange earnings and employment generation prospects. This industry has developed enormously over the past decades; since, leather has become a material of choice in the world of fashion. However, this industry, like many others, is facing stringent environmental regulations worldwide, due to vast usage of toxic chemicals and generation of hazardous waste. Leather manufacturing involves conversion of raw skins and hides into leather through a series of mechanical and chemical operations. Processes like pre-tanning and tanning are known to contribute ~ 80–90% of the total pollution load in tanneries. In order to mitigate the hazards caused by toxic chemicals, enzymes have been identified as a practical alternative for use during processing and as well as for waste management. Even though the use of enzymes in the leather industry dates long back mainly because of their activity on proteins and fat, the complete replacement of chemicals by enzymes has yet to be realized. Earlier, enzymes were derived from animal excreta, and later on from the pancreas of cattle. However, currently, the enzymes are almost entirely produced by microbial fermentation. In light of this, the current review presents a holistic view on the effective utilization of enzymes in leather making, mainly during soaking, dehairing, bating and degreasing processes in order to minimize waste generation, and also in the recovery of valuable and saleable by-products. Globally, ~ 7 million tons of salted bovine hides are used every year for leather making. By using enzymes in bio-preparation, around 8 million gigajoules of energy saving and 0.7 million tons of CO2 savings is estimated to be achieved due to lower processing times and associated energy use. Nevertheless, the search for enzymes for their ultimate application in the leather industry as an eco-friendly alternative continues, since, this process is far safer and more pleasant than the traditional method. The progress made in this field during the past two decades are highlighted and will provide further insight on the scope for utilization of enzymes in this industry. In order to achieve sustainability, clean environment and prevent health hazards, the leather industry ought to adopt the use of eco-friendly alternatives which might primarily depend on research, development and implementation of the potential enzyme technology.
The environment is under constant threat due to the huge accumulation of the keratinaceous wastes produced by poultry processing industries and poultry farms. Millions of tons of feather wastes are produced each year as a by-product that causes severe environmental pollution. Their degradation is tough due to the presence of insoluble, highly stiff, recalcitrant ‘keratin’ polypeptide. Keratin are resistant to degradation by common proteases and chemical catalysts due to high mechanical stability and cross-linked disulphide bonds present in their structure. Microbial keratinases has emerged as a powerful bio-catalyst that can potentially degrade keratin and transform the keratinaceous wastes into value-added products. Keratinase production is receiving worldwide attention for its application in sustainable development and cleaner production. The enzymatic hydrolysis of keratin have the potential for higher productivity, less energy consumption, less effluent generation and waste management. Keratinases add value to poultry and industrial wastes which brings economic feasibility and environmental sustainability. The present review prioritized the various aspects of keratinolytic proteases in bringing sustainable development to the economy of a nation. It also focuses on the recent advancement in the production of microbial keratinases with emphasis on the biochemical characteristics related to enzyme activity and stability, development of novel recombinant and mutant microbial strains along with different optimization strategies leading to expansive production and yield of keratinases. Furthermore, different properties of microbial keratinases which renders them as a green and sustainable material for industrial applications in waste management, textile, leather and detergents with advantages over conventional treatments have also been discussed.
A propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655 U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29 kDa. The optimal temperature and pH of the protease were 45 °C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg²⁺, Zn²⁺, K⁺, Na⁺ and Tween-80 could improve its activity. The calculated Km and Vmax values of the protease towards α-casein were 12.74 mg/mL and 28.57 µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20.
Proteases are hydrolytic enzymes capable of degrading proteins into small peptides and amino acids. They account for nearly 60% of the total industrial enzyme market. Proteases are extensively exploited commercially, in food, pharmaceutical, leather and detergent industry. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. This review summarizes a fraction of the enormous reports available on various aspects of alkaline proteases. Diverse sources for isolation of alkaline protease producing microorganisms are reported. The various nutritional and environmental parameters affecting the production of alkaline proteases in submerged and solid state fermentation are described. The enzymatic and physicochemical properties of alkaline proteases from several microorganisms are discussed which can help to identify enzymes with high activity and stability over extreme pH and temperature, so that they can be developed for industrial applications.
Over a hundred of halophilic/halotolerant microorganisms were screened for alkaline protease production. The bacterium showing the highest enzyme production was characterized and identified as Bacillus halodurans US193 on the basis of 16S rRNA gene analysis. It was alkalophilic, thermophilic and halotolerant since it grew optimally at pH 9.7 and 50 °C with tolerance of up to 125 g NaCl l−1. The alkaline protease was purified 4.9 times with about 40186.1 U/mg as specific activity. It exhibited optimal activity at pH 10, 70 °C and 0.25 M NaCl with perfect stability at wide ranges of pH (6–12), temperatures (30–60 °C) and NaCl concentrations (0–2 M). The serine alkaline protease maintained high stability in the presence of Cu2+, Mg2+, Ba2+ and Ca2+ ions, various organic solvents [50% (v/v)] and ionic and non ionic detergent additives. In addition, it was more compatible with various commercialized detergents than other reported detergent proteases, and was very efficient in blood stain removal. These findings let B. halodurans US193 alkaline protease be an ideal candidate for many industrial processes at harsh conditions, especially as a bio-additive in detergent industry.
A local strain of Bacillus cereus LAU 08 was isolated from a chicken-feather disposal site in Ogbomoso, Southwest Nigeria. Production of keratinase was induced using three sources of keratin, namely: hooves, horn, and feather, at a growth temperature of 37 °C. In each case, high titers were recorded for both keratinolytic and proteolytic activities. Maximum enzyme activities were obtained within 24–96 h of cultivation, depending on the keratin substrate used and the activity under study. While maximum keratinolytic activities of 67.9, 63.1, and 51.7 U ml−1 were obtained using enzymes induced by hooves, horn, and feather, respectively, proteolytic activities of 11 860, 820, and 126 U ml−1 were obtained using enzymes induced by feather, hooves, and horn, respectively. The optimal conditions for the keratinolytic activity were determined to be pH 7.0 and temperature of 50 °C; however, the enzyme displayed more than 50% activities within the broad range of pH 7–9 and temperature of 40–70 °C. In addition, the isolate was able to completely degrade a whole chicken feather within a period of 7 days at room temperature (30 ± 2 °C). B. cereus LAU 08 is therefore a promising strain for the management of chicken feather waste through biotechnological processes.
The present investigation describes microbial production of an alkaline protease and its use in dehairing of buffalo hide.
Bacillus cereus produced extracellular protease when grown on a medium containing starch, wheat bran and soya flour (SWS). The ammonium sulphate
precipitated (ASP) enzyme was applied for dehairing of buffalo hide. Microscopic observation of longitudinal section of buffalo
hide revealed that the epidermis was completely removed and hair was uprooted leaving empty follicles in the hide. The ASP
enzyme was stable for one month at ambient temperature between 25–35°C. Enzymatic dehairing may be a promising shift towards
an environment-friendly leather processing method.
Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and
DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences
revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans.
Three keratinolytic Bacillus spp. isolated from the Brazilian Amazon basin were characterized. The strains P6, P7 and P11 were identified based on morphological
and biochemical characteristics and 16S rDNA sequences. P6, P7 and P11 sequences shared more than 99% similarity with B. subtilis, B. amyloliquefaciens and B. velesensis. The keratinases produced by these bacteria were active on azokeratin and degradation of feather barbules was observed. The
enzymes were inhibited by the serine protease inhibitor PMSF, and showed maximum activity at pH 9.0. Proteins like albumin,
casein and gelatin were hydrolysed by these keratinases. Depilatory studies on bovine pelts revealed that all three strains
were efficient in promoting de-hairing. Microscopic analysis showed that the epidermis was completely removed and the absence
of hair in follicles was observed.
A novel feather-degrading microorganism was isolated from poultry waste, producing a high keratinolytic activity when cultured on broth containing native feather. Complete feather degradation was achieved during cultivation. The bacterium presents potential use for biotechnological processes involving keratin hydrolysis. Chryseobacterium sp. strain kr6 was identified based on morphological and biochemical tests and 16S rRNA sequencing. The bacterium presented optimum growth at pH 8.0 and 30 degrees C; under these conditions, maximum feather-degrading activity was also achieved. Maximum keratinase production was reached at 25 degrees C, while concentration of soluble protein was similar at both 25 and 30 degrees C. Reduction of disulfide bridges was also observed, increasing with cultivation time. The keratinase of strain kr6 was active on azokeratin and azocasein as substrates, and presented optimum pH and temperature of 7.5 and 55 degrees C, respectively. The keratinase activity was inhibited by 1,10-phenanthroline, EDTA, Hg(2+), and Cu(2+) and stimulated by Ca(2+).
An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol−1 (44.30 kJ mol−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k
cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K
m and k
cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×103 min−1, respectively.
Chicken feather hydrolysate was produced by proteolysis with a keratinolytic bacterium. Feather hydrolysate was produced by using the whole culture on 60gfeathers/l (WCH), or alternatively, using only the culture supernatant of cultivation on 10gfeathers/l (CSH). The amino acid composition of the resulting hydrolysates was determined, indicating deficiency in methionine, lysine and histidine. CSH showed higher amounts of sulfur-containing amino acids than WCH. In vitro digestibility with pepsin plus pancreatin was evaluated. WCH had lower digestibility than soy protein, but higher than feather meal and milled raw feathers. CSH had similar digestibility than casein and soy protein. Predicted nutritional parameters for WCH, CSH and feather meal were calculated. WCH showed higher predicted values of protein efficiency ratio (PER) and biological value (BV) than CSH, which presented higher protein digestibility-corrected amino acid scoring (PDCAAS). These bacterial feather hydrolysates showed potential for utilization as ingredients in animal feed.
Advances in microbial enzyme technology, keratinolytic proteases in this case, offer considerable opportunities for a low-energy consuming technology for bioconversion of poultry feathers from a potent pollutant to a nutritionally upgraded protein-rich feedstuff for livestock. A compendium of recent information on microbial keratinolysis in nature and infection (dermatophytoses) has been provided as underscoring feasible harnessing of the biotechnology for nutritional improvement of feathers, and as an alternative to conventional hydrothermal processing. Supporting evidence of a nutritional (amino acid) upgrading sequel to diverse microbial treatments of feathers, and positive results obtained from growth studies in rats and chicks have been presented. The paper concludes with suggestions for avenues of application of biotechnology for nutritional improvement of feather (and other keratins) as feedstuffs for livestock
A keratin-degrading bacterium strain (K-508) was isolated from partially degraded feathers and characterized. This isolate exhibited a high chicken feather-degrading activity when cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 C. On the basis of its phenotypic characteristics (quickly moving, Gram-positive rods), the results of metabolic tests and rDNA sequence analysis, it was identified as Bacillus licheniformis. Its fermentation broth showed activity on N-Bz-l-Phe-l-Val-l-Arg-p-nitroanilide, N-Suc-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide, N-CBZ-Gly-Gly-l-Leu-p-nitroanilide and N-CBZ-l-Ala-l-Ala-l-Leu-p-nitroanilide as chromogenic protease substrates at near neutral pH. Both trypsin-like and chymotrypsin-like proteases were constitutively secreted by this strain.
Keratin-rich wastes in the form of feathers, hair, nails, and horn are highly available as byproducts of agroindustrial processing.
The increased needs for energy conserving and recycling, summed with the huge increase in poultry industry, have strongly
stimulated the search for alternatives for the management of recalcitrant keratinous wastes. Keratinases, which are produced
by several bacteria that have been often isolated from soils and poultry wastes, show potential use in biotechnological processes
involving keratin hydrolysis. Although these isolates are mostly restricted to the genera Streptomyces and Bacillus, the diversity of keratinolytic bacteria is significantly greater. Bacterial keratinases are mostly serine proteases, although
increased information about keratinolytic metalloproteases, particularly from Gram-negative bacteria, became available. These
enzymes are useful in processes related with the bioconversion of keratin waste into feed and fertilizers. Other promising
applications have been associated with keratinolytic enzymes, including enzymatic dehairing for leather and cosmetic industry,
detergent uses, and development of biopolymers from keratin fibers. The use of keratinases to enhance drug delivery in some
tissues and hydrolysis of prion proteins arise as novel outstanding applications for these enzymes.
The Bacillus sp. no. AH-101 alkaline protease showed higher hydrolysing activity against insoluble fibrous natural proteins such as elastin and keratin in comparison with subtilisins and Proteinase K. The optimum pH of the enzyme toward elastin and keratin was pH 10.5 and pH 11.0–12.0 respectively. The specific activity toward elastin and keratin was 10 600 units/mg protein and 3970 units/mg protein, respectively. The enzymatic activity was not inhibited by p-chloromercuribenzoic acid and iodoacetic acid. Carbobenzoxy-glycyl-glycyl-L-phenylalanyl chloromethyl ketone completely inhibited the caseinolytic activity, but 36% elastolytic activity remained. No inhibitory effect on caseinolytic and elastolytic activity was shown by tosyl-L-phenylalanyl-chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, carbobenzoxy-L-phenylalanyl chloromethyl ketone, and elastatinal. The amino acid composition and amino terminal sequence of the enzyme were determined. The no. AH-101 alkaline protease was compared with subtilisin BPN', subtilisin Carlsberg, no. 221, and Ya-B alkaline proteases. Extensive sequence homology existed among these enzymes.
Thermus sp. Rt41A produced a single extracellular proteinase, as determined by fast protein liquid chromatography and isoelectric focusing. Proteinase activity was expressed from very early in the log phase, and halted when the growth substrate was exhausted. There was no continued proteinase production in the stationary phase. Proteinase production was not stimulated by O2 limitation, not repressed by amino acid growth substrates, and its production could not be correlated to the type or oxidation state of the carbon and energy source or the growth rate on different carbon and energy sources. Growth on certain substrates, e.g. glutamate and glucose, resulted in production of high levels of proteinase, whereas others, such as acetate, resulted in low proteinase levels. Acetate repressed proteinase production in cultures growing on L-glutamate. In continuous culture on L-glutamate, acetate or pyruvate, proteinase production was highest at higher growth (dilution) rates. The kinetics of proteinase production in continuous culture on L-glutamate can be interpreted as evidence for the constitutive nature of proteinase expression byThermus sp. Rt41A. The data obtained show that the control of proteinase production is different to that postulated forThermus sp. Ok6.A1.
Two alkaline protease producing alkaliphilic bacterial strains, designated as AL-20 and AL-89, were isolated from a naturally occurring alkaline habitat. The two strains were identified as Nesternkonia sp. and Bacillus pseudofirmus, respectively. Both strains grew and produced alkaline protease using feather as the sole source of carbon and nitrogen. Addition of 0.5% glucose to the feather medium increased protease production by B. pseudofirmus AL-89 and suppressed enzyme production by Nesternkonia sp. AL-20. The enzymes from both organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, hydrophobic interaction, and gel filtration chromatography. The molecular weight, determined using SDS–PAGE, was 23 kDa for protease AL-20 and 24 kDa for protease AL-89. Protease AL-20 was active in a broad pH range displaying over 90% of its maximum activity between pH 7.5 and 11.5 with a peak at pH 10. The enzyme is unique in that unlike all other microbial serine proteases known so far, it did not require Ca2+ for activity and thermal stability. Its optimum temperature for activity was at 70 °C and was stable after 1 h incubation at 65 °C both in the presence and absence of Ca2+. These properties make protease AL-20 an ideal candidate for detergent application. Protease AL-89 on the other hand require Ca2+ for activity and stability at temperature values above 50 °C. Its optimum activity was at 60 and 70 °C in the absence and presence of Ca2+, respectively. It displayed a pH optimum of 11 and retained about 70% or more of its original activity between pH 6.5 and 11. B. pseudofirmus AL-89, and the protease it produce offers an interesting potential for the enzymatic and/or microbiological hydrolysis of feather to be used as animal feed supplement.
Feather-degrading bacteria were isolated from poultry waste. Among those isolates, three strains identified as Bacillus subtilis, Bacillus pumilis and Bacillus cereus degraded feathers effectively and produced 142, 96 and 109 units of keratinolytic activities, respectively. The production of keratinolytic protease by B. pumilis and B. cereus was inducible with feathers, but B. subtilis produced the enzyme constitutively in the presence of various proteins such as casein, feather and BSA. The optimal conditions for the enzyme production by B. subtilis were 40 °C and pH 5–9, for B. pumilis 40 °C and pH 5–6 and for B. cereus 30 °C and pH 7.0. The maximum keratinolytic activities of B. subtilis and B. pumilis were 161 and 149 units/ml after 84 and 72 h of cultivation, respectively. With B. cereus, the maximum enzyme activity was 117 units/ml after 60 h of cultivation. The strains showed maximum enzyme activities in the late logarithmic growth phase or the beginning of stationary phase, and the production of soluble protein showed the same tendency as that of keratinolytic protease.
A novel protease produced by Bacillus cereus grown on wool as carbon and nitrogen source was purified. B. cereus protease is a neutral metalloprotease with a molecular mass of 45.6 kDa. The optimum activity was at 45 °C and pH 7.0. The substrate specificity was assessed using oxidized insulin B-chain and synthetic peptide substrates. The cleavage of the insulin B-chain was determined to be Asn3, Leu6, His10-Leu11, Ala14, Glu21, after 12 h incubation. Among the peptide substrates, the enzyme did not exhibit activity towards ester substrates; with p-nitroanilide, the kinetic data indicate that aliphatic and aromatic amino acids were the preferred residues at the P1 position. For furylacryloyl peptides substrates, which are typical substrates for thermolysin, the enzyme exhibited high hydrolytic activity with a Km values of 0.858 and 2.363 mM for N-(3-[2-Furyl]acryloyl)-Ala-Phe amide and N-(3-[2-Furyl]acryloyl)-Gly-Leu amide, respectively. The purified protease hydrolysed proteins substrates such as azocasein, azocoll, keratin azure and wool.
Proteolytic enzyme isolated from Aspergillus tamarii prepared by solid state fermentation was studied for dehairing of goat skins in the tannery. The stability of the enzyme for dehairing was optimized at different environmental parameters. Unhairing of goat skins could be obtained at pH 9–11 and temperatures 30–37°C with enzyme concentration of 1% w/v and incubation periods of 18–24 h. The physical properties of the experimental leathers in comparison with the control sets gave better results with respect to tensile strength and elongation at break.
The Amazonian bacterium Bacillus sp. P7 efficiently degraded feather keratin during submerged cultivations, producing extracellular keratinolytic enzymes. Keratinase produced during growth on feather meal broth was partially purified by ammonium sulphate precipitation, gel filtration, and ion-exchange chromatography, resulting in a purification factor of 29.8-fold and a yield of 27%. Zymography revealed two proteolytic bands, mainly inhibited by phenylmethylsulfonyl fluoride (PMSF). Partially purified keratinase had optimal activity at 55 °C and pH 9.0, was stimulated by Ca2+ and Mg2+, and was inhibited by Hg2+, Cu2+ and Zn2+. Organic solvents 2-mercaptoethanol and Triton X-100 slightly affected the enzyme activity, whereas SDS stimulated it. PMSF and ethylenediaminetetraacetic acid (EDTA) inhibited proteolytic activity, which suggests its serine-protease feature, with the requirement of metal ions for maximum activity and/or stability. Alkaline keratinase might be employed in detergent formulations, in leather processing, and in other processes involving protein hydrolysis. The maintenance of enzyme activity in the presence of reducing agent (2-mercaptoethanol) makes this partially purified keratinase interesting for application in the breakdown of recalcitrant keratin wastes.
Bacillus sp. JB-99 was studied for the production of extra-cellular thermostable alkaline cellulase-free xylanase in solid-state fermentation. The xylanase tit was highest on rice bran, among a few easily available lignocellulosic solid substrates tested. Maximum production of xylanase (3644 U/g DBB) was observed bran moistened with mineral salt solution (MSS3) at a substrate-to-moisturizing agent ratio of 1:2.0 (w/v) at 50 °C for 72 h. A 10% (v/w) inoculum was most suitable to attain a high production of xylanase. Yeast extract, beef extract and xylan enhanced enzyme production, while glucose, lactose and fructose strongly repressed the production process.
A feather-degrading bacterium was isolated from poultry decomposition feathers in China. The strain, named L1, showed significant feather-degrading activity because it grew and reproduced quickly on basal medium containing 10 g/L of native feather as the source of energy, carbon, and nitrogen. According to the phenotypic characteristics and 16S rRNA profile, the isolate belongs to Stenotrophomonas maltophilia. Keratinase activity of the isolate was determined during cultivation on raw feathers at different temperatures and initial pH. Maximum growth and feather-degrading activity of the bacterium were observed at 40 degrees C and initial pH ranging from 7.5 to 8.0. The crude enzyme was purified by ammonium sulphate precipitation, Sephadex G-100 chromatographic and ceramic hydroxyapatite (CHT) chromatographic. Its molecular mass estimated as 35.2 kDa in SDS-PAGE. The enzyme had an optimum activity at the pH was 7.8 and the temperature was 40 degrees C. The keratinase was wholly inhibited by a serine protease inhibitor, PMSF. Its activity was activated or inhibited by different metal ions. The keratinase activity of enzyme from strain L1 functioned on different keratins, such as feather, hair, wool, horn, and so on.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
A new technique is described for the electrophoretic analysis of plasminogen activators in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized plasminogen and gelatin. The method depends upon the fact that the zymogen and gelatin, when incorporated into the polyacrylamide matrix at the time of casting, are retained during subsequent electrophoresis of enzyme samples, and serve as satisfactory sequential, in situ substrates for the localization of plasminogen activator bands by negative staining. The nonionic detergent, Triton X-100, is used to remove sodium dodecyl sulfate and restore enzyme activity. The method can be used to detect as little as 1 mU of urokinase and effectively distinguishes between melanoma- and urokinase-type plasminogen activators. Plasminogen-independent proteases are detected by omission of plasminogen from the gel.
A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity with different synthetic substrates. The keratinolytic activity of the purified proteinase was examined by incubation with the insoluble substrates keratin azure, feather meal, and native and autoclaved chicken feather downs. The S. pactum proteinase was significantly more active than the various commercially available proteinases. After incubation with the purified proteinase, a rapid disintegration of whole feathers was observed. But even after several days of incubation with repeated addition of enzymes, less than 10% of the native keratin substrate was solubilized. In the presence of dithiothreitol, degradation was more than 70%.
Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications. They perform both degradative and synthetic functions. Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms. Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site. Proteases play a critical role in many physiological and pathophysiological processes. Based on their classification, four different types of catalytic mechanisms are operative. Proteases find extensive applications in the food and dairy industries. Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies. There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents. Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application. Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of the enzyme. Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology.
Thermoalkaliphilic Bacillus sp. JB-99 was grown in a 250 ml Erlenmeyer flask containing 50 ml medium containing (g/l) Pigeon pea waste 10; NaNO3, 5.0; K2HPO4, 5.0; MgSO4 x 2H2O, 0.2 and Na2CO3, 10.0. Incubations were carried out at 50 degrees C on a rotary incubator shaker for 15 h. A high level of extra cellular thermostable protease activity was observed after 24 h incubation. The optimum temperature and pH for activity were 70 degrees C and 11, respectively, so this enzyme showed stable activity at high temperature and under alkaline conditions.
An investigation was carried out on the purification and characterization of an alkaline protease from Bacillus pumilus MK6-5.
An alkalophilic Bacillus pumilus MK6-5 was grown in a laboratory fermenter containing 1% reverse osmosis concentrated cheese whey powder, 0.25% corn steep liquor, 1% glucose, 0.5% tryptone, 1% sodium citrate, 0.02% MgSO4.7H2O and 0.65% Na2CO3 at 35 degrees C and pH 9.6, agitation at 250 rev min(-1) and aeration of 1 vvm for 60 h. When the enzyme was purified using ammonium sulphate precipitation, ion exchange and gel filtration chromatographies, a 26.2% recovery of enzyme with 36.6-fold purification was recorded. The purified protease was found to be homogenous by SDS-PAGE with molecular mass estimate of 28 kDa. The enzyme was optimally active at pH 11.5 and temperature of 55-60 degrees C. The Km and kcat values observed with synthetic substrates at 37 degrees C and pH 8.0 were 1.1 mmol l(-1) and 624 s(-1) for Glu-Gly-Ala-Phe-pNA and 3.7 mmol l(-1) and 826 s(-1) for Glu-Ala-Ala-Ala-pNA, respectively. The kinetic data revealed that small aliphatic and aromatic residues were the preferred residues at the P1 position. Inhibition profile exhibited by PMSF suggested the B. pumilus protease to be an alkaline serine protease.
Bacillus pumilus MK6-5 produced a calcium-dependent, thermostable alkaline serine protease.
The thermostable alkaline protease from Bacillus pumilus MK6-5 will be extremely useful in ultrafiltration membrane cleaning due to its ability to work in broad pH and temperature ranges, and tolerance to detergents, unlike the mesophilic proteases which face these limitations.
Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms, and are essential for cell growth and differentiation. The extracellular proteases are of commercial value and find multiple applications in various industrial sectors. Although there are many microbial sources available for producing proteases, only a few are recognized as commercial producers. A good number of bacterial alkaline proteases are commercially available, such as subtilisin Carlsberg, subtilisin BPN' and Savinase, with their major application as detergent enzymes. However, mutations have led to newer protease preparations with improved catalytic efficiency and better stability towards temperature, oxidizing agents and changing wash conditions. Many newer preparations, such as Durazym, Maxapem and Purafect, have been produced, using techniques of site-directed mutagenesis and/or random mutagenesis. Directed evolution has also paved the way to a great variety of subtilisin variants with better specificities and stability. Molecular imprinting through conditional lyophilization is coming up to match molecular approaches in protein engineering. There are many possibilities for modifying biocatalysts through molecular approaches. However, the search for microbial sources of novel alkaline proteases in natural diversity through the "metagenome" approach is targeting a hitherto undiscovered wealth of molecular diversity. This fascinating development will allow the biotechnological exploitation of uncultured microorganisms, which by far outnumber the species accessible by cultivation, regardless of the habitat. In this review, we discuss the types and sources of proteases, protease yield-improvement methods, the use of new methods for developing novel proteases and applications of alkaline proteases in industrial sectors, with an overview on the use of alkaline proteases in the detergent industry.
Global environmental regulations are changing the leather-processing industry. Pre-tanning and tanning processes contribute 80-90% of the total pollution in the industry and generate noxious gases, such as hydrogen sulfide, as well as solid wastes, such as lime and chrome sludge. The use of enzyme-based products is currently being explored for many areas of leather making. Furthermore, enzymes are gaining increasing importance in the de-hairing process, eliminating the need for sodium sulfide. This review discusses emerging novel biotechnological methods used in leather processing. One significant achievement is the development of a bioprocess-based de-hairing and fiber-opening methodology to reduce toxic waste.
To characterize a new feather-degrading bacterium.
The strain kr10 producing a high keratinolytic activity when cultured on native feather broth was identified as Microbacterium sp., based on phenotypical characteristics and 16S rDNA sequence. The bacterium presented optimum growth and feather-degrading activity at pH 7.0 and 30 degrees C. Complete feather degradation was achieved during cultivation. The keratinase was partially purified by gel filtration chromatography. It was optimally active at pH 7.0 and 55 degrees C. The enzyme was inhibited by 1,10-phenanthroline, EDTA, p-chloromercuribenzoic acid, 2-mercaptoethanol and metal ions like Hg(2+), Cu(2+) and Zn(2+).
A new Microbacterium sp. strain was characterized presenting high feather-degrading activity, which appears to be associated to a metalloprotease-type keratinase. This micro-organism has enormous potential for use in biotechnological processes involving keratin hydrolysis.
A keratin-degrading bacterium was isolated from soil containing deer fur. An axenic culture of the keratin-degrading bacterium was obtained in liquid culture using a keratin enrichment technique. The isolated bacterium was gram negative and catalase- and oxidase-positive. Transmission electron microscopic observations showed that the bacterium was rod-shaped, 1.0-1.3 microm long and 0.7 microm in diameter. Phylogenetic analysis of 16S rDNA revealed that the new isolate has only 90.6% homology with Stenotrophomonas nitritireducens. Hence, this new bacterium was designated as Stenotrophomonas sp. D-1. The optimum temperature was determined to be 20 degrees C for maximum growth and keratinolytic enzyme production. Amino acid data, obtained after treating keratin powder with the supernatant culture, suggest that the major free amino acids resulting from keratin degradation are phenylalanine, tyrosine and valine. In addition, native chicken feather was degraded completely at 20 degrees C in 2.5 d by this bacterium.
Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide "keratin" recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. These enzymes are largely produced in the presence of keratinous substrates in the form of hair, feather, wool, nail, horn etc. during their degradation. The complex mechanism of keratinolysis involves cooperative action of sulfitolytic and proteolytic systems. Keratinases are robust enzymes with a wide temperature and pH activity range and are largely serine or metallo proteases. Sequence homologies of keratinases indicate their relatedness to subtilisin family of serine proteases. They stand out among proteases since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feed and fertilizers. Their application can also be extended to detergent and leather industries where they serve as specialty enzymes. Besides, they also find application in wool and silk cleaning; in the leather industry, better dehairing potential of these enzymes has led to the development of greener hair-saving dehairing technology and personal care products. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.
We isolated the feather-degrading Bacillus
pseudofirmus FA30-01 from the soil sample of poultry farm. The isolate completely degraded feather pieces after liquid culture at 30°C (pH 10.5) for 3 days. Strain FA30-01 is a Gram-positive, spore-forming, rod-shaped bacterium and was identified with B. pseudofirmus based on 16S rDNA analysis. The keratinase enzyme produced by strain FA30-01 was refined using ammonium sulfate precipitation, negative-ion DEAE Toyopearl exchange chromatography, and hydroxyapatite chromatography. The refinement level was 14.5-fold. The molecular weight of this enzyme was 27.5 kDa and it had an isoelectric point of 5.9. The enzyme exhibited activity at pH 5.1–11.5 and 30–80°C with azokeratin as a substrate, although the optimum pH and temperature for keratinase activity were pH 8.8–10.3 and 60°C, respectively. This enzyme is one of the serine-type proteases. Subtilisin ALP I and this enzyme had 90% homology in the N-terminal amino acid sequence. Since this enzyme differed from ALP I in molecular weight, heat resistance and isoelectric point, they are suggested to be different enzymes.
Characterization of a new keratin-degrading bacterium isolated from deer fur Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent Characterization of a novel Stenotrophomonas isolate with high keratinase activity and purification of the enzyme
- S Yamamura
- Y Morita
- Q Hasan
- S R Rao
- Y Murakami
- K Yokoyama
- E Tamiya
- V P Zambare
- Nilegaonkar
- P P Kanekar
Yamamura, S., Morita, Y., Hasan, Q., Rao, S.R., Murakami, Y., Yokoyama, K., Tamiya, E., 2002. Characterization of a new keratin-degrading bacterium isolated from deer fur. Journal of Bioscience and Bioengineering 93, 595e600. Zambare, V.P., Nilegaonkar, Kanekar, P.P., 2007. Production of an alkaline protease by Bacillus cereus MCM B-326 and its application as a dehairing agent. World Journal of Microbiology and Biotechnology 23, 1569e1574. Zhang, J.C., Zhang, Q., Dong, K.W., Chen, L., Wang, J., Zhang, X.Q., Zhou, M.H., 2009. Characterization of a novel Stenotrophomonas isolate with high keratinase activity and purification of the enzyme. Journal of Industrial Microbiology and Biotechnology 36, 181e188. D. Shrinivas, G.R. Naik / International Biodeterioration & Biodegradation 65 (2011) 29e35