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(a) Effect of temperature on protease stability (●) 50°C; (◯) 55°C; (▾) 60°C. (b) Effect of stabilizers on heat inactivation at 50°C. (c) Effect of stabilizers on heat inactivation at 55°C. (d) Effect of stabilizers on heat inactivation at 60°C. For (b), (c), and (d) the original activity before preincubation was taken as 100%. Values are means of three independent determinations.
Source publication
Paecilomyces lilacinus (Thom) Samson LPS 876, a locally isolated fungal strain, was grown on minimal mineral medium containing “hair waste,” a residue from the hair-saving unhairing process, and produced a protease with keratinolytic activity. This enzyme was biochemically characterized. The optimum reaction conditions, determined with a response s...
Citations
... After incubation, cloth pieces were rinsed with tap water and dried. Visual examination of various pieces showed the effect of crude enzyme in the removal of stains [19,20]. ...
Alkaline amylase producers were isolated from the marine environment of Arabian Sea coast. A total of 10 isolates were obtained from the soil and water samples collected from the marine environment. Five isolates namely; BCSS, JBWS, RBWS, BCWS and ALSS showed good growth on alkaline M9 media with 10% NaCl having starch as the primary carbon source. Based on their maximal amylase activity, three microbial cultures (BCSS, JBWS, and RBWS) were selected for the study. The crude enzyme from these purified cultures was extracted and partial characterization was carried out. The partial characterization study involved the effect of metal ions and surfactants and determination of optimal pH and temperature and its stability studies. The amylase activity was optimum for all three cultures at 60 0C whereas, the optimum pH was found to be pH 7, pH 8 and pH 9 for RBWS, JBWS and BCSS. The pH and temperature stability studies showed maximum amylase activity at its optimal conditions for 5h (300 mins) in the order BCSS>RBWS>JBWS. The amylase activity was found to increase with sodium (BCSS), manganese (RBWS) and potassium (JBWS) at 1:1 dilution. The cultures were stable with the surfactants (Tween -20, SDS and Triton-X) at 1:10 dilution of 1% surfactant and enzyme. BCSS was the most stable at high temperature and pH among all the isolates with an optimal amylase activity at pH 9 and temperature of 60 0C. The isolate was identified to be Bacillus nanhaiisediminis NH3. These remarkable properties indicated its great potential in industrial applications.
... The endogenous enzyme present in detergents was inactivated by heating the diluted detergent for 1 hr at 70 C prior to addition of enzyme preparation. The partially purified enzyme was incubated with deactivated detergent solution for 1 hr at 50 C and then the remaining activities were determined separately for enzyme under standard assay conditions (Haddar et al. 2010;Cavello et al. 2012;Bhange et al. 2016). The keratinase activity in the absence of any detergent was taken as control (100%) (Mukherjee et al. 2008). ...
... Response Surface Methodology (RSM) is a compilation of mathematical and statistical techniques that are helpful in the modelling and analysis of problems wherein the response of interest is affected by numerous variables. Thus, the main objective of RSM is the optimisation of that response (Cavello et al. 2012;Kumar and Kushwaha 2012). This optimisation process comprises three main steps: conducting of statistically designed experiments, estimation of coefficients in a mathematical model and forecasting the response and verifying the adequacy of the model. ...
To augment the keratinolytic ability of Bacillus halotolerans L2EN1 isolated from the poultry farm (Nahan, District Sirmour) of Himachal Pradesh, different cultural conditions were optimised using One Variable at a Time (OVAT) approach accompanied by Response Surface Methodology (RSM). Optimisation (OVAT) results revealed that after 3rd day of incubation, maximum enzyme activity was attained at 45 °C, pH 11.0 with 12.5% of inoculum size in the presence of Mn2+ and EDTA in the production medium. Sucrose (1.5%) and yeast extract (2.0%) were observed to be best carbon and nitrogen sources, respectively. A significant increase of 73.19 per cent in the keratinase activity was observed using Central Composite Design (CCD) of RSM. The SDS-PAGE results revealed that crude keratinase is a heterotetramer made up of four polypeptide chains with molecular weights of 17, 37, 40 and 60 kDa. Partial purification by 90 − 100 percent ammonium sulphate gave maximum keratinase production of 22.66 U/mL with purification of 1.68 and yield of 11.47 per cent. The enzyme showed compatibility with different commercial detergents and retained its activity in the order: Reshma (97.77%) > Speed (93.44%) > Tide (79.93%) >Ariel (70.18%) > Surf excel (67.98%) at 50 °C after 1 h of incubation. Wash performance analysis demonstrated that washing with tap water at 18, 28, 35 and 45 °C for 30, 45 and 60 min removed some amount of blood stains from the cotton cloth pieces. However, replacement of detergent’s enzyme (Reshma) with crude keratinase achieved complete blood stain removal under same conditions, suggesting its suitability as a potential cleaning additive in detergents for the removal of blood (proteinaceous) stains for long washing cycles (1 h).
... A biodetergent formulated with alkaline keratinase (from Paenibacillus woosongensis TKB2) can remove blood and egg yolk stains efficiently (Paul et al., 2014). Keratinolytic enzymes produced by Paecilomyces lilacinus (Cavello et al., 2012), Gibberella intermedia (Zhang et al., 2016) and B. pumilus (Gong et al., 2015) have also shown potential in detergent applications. Additionally, keratinolytic enzymes may find use in detergents for cleaning drains and clogged pipes caused for example by hair (Gupta et al., 2013a). ...
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.
... However, in the 2nd and 4th samplings, it was detected in both the sites. In the context of the preservation and conservation of the geological heritage, the literature relates the deleterious action of P. lilacinum to its enzymatic production (Nuhoglu et al. 2006;Mitchell and Gu 2010;Cavello et al. 2012). ...
The present study aims to explore the diversity patterns of cultivable filamentous fungi and establish the ecological interactions that occur within the mycological community on soapstone in two distinct places: Architectural Complex of Caraça and Congonhas town, in Minas Gerais state, Brazil. A total of 200 isolates of filamentous fungi were detected and 72 distinct taxa were identified using molecular tools. The rock-inhabiting fungal communities were comprised of cosmopolitan taxa, which displayed high diversity and richness, but low dominance indexes at two of the sampling sites over the course of 12 months. The detection of organic acids secreted by the isolated filamentous fungal species revealed the potential for deterioration of the model stone by these fungi. Our results suggest that the isolated fungal communities of the two Brazilian localities have the ability to adapt to environmental adversities over the four seasons of the year, predicting adaptive population dynamics in the stone colonization. We conclude that this study on the ecological dynamics of the rock-inhabiting fungal communities can help to understand the competitive interactions between the environmental fungi. In addition, these fungi may contribute to the preservation of the Brazilian historical heritage that is vulnerable to environmental conditions.
... Human hair is periodically shaved away because it grows constantly in the growth process of mammals. 2,3 The hair has a composite structure, and it is mainly divided into three regions. The outermost region is the cuticle layer that is a thick sheath of several cell-like scales. ...
In this study, hollow microtubes were prepared via stepwise thermal treatment of human hair waste in the middle temperature range from 200 to 450 °C. The effect of the treatment temperature on the thermal decomposition of the hair was examined in terms of the dimension and wall thickness of the microtubes. The thermal decomposition behavior of the hair was analyzed via differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The structure and morphology of the hollow microtubes were examined using scanning electrochemical microscopy (SEM) and elemental analysis (EA). The thermal and morphological observations reveal that the thermal decomposition of the hair started from the core medulla section with the melting of the α-helical structure in the cortex at about 250 °C. As the heat treatment temperature further increased to 450 °C, the degradation of the cortical structure expanded to the outside, and the wall thickness of the hollow microtube decreased. Finally, the hollow microtube structure was completed, and the scaled structure in the cuticle layer became compact and smooth. Therefore, the hollow microtube structure might be a result of the melting and degradation of the cortex and medulla. The adsorption capacity of the hollow microtubes was also evaluated using n-hexane and water.
... Keratinases have been looked as promising candidates in the detergent industry because many of alkaline keratinases remain stable at wash temperature and are tolerant to surfactants too (Rai et al. 2009;Cavello et al. 2012;Rajput et al. 2010) which make them ideal candidates for detergent formulations. Keratinases enzymes were produced and purified from varied microbes viz. ...
The world’s increasing population and shortage of food and feed is creating an urgently for us to look for new protein sources from waste products like keratinous waste. Poor management of these wastes has made them one of the major recalcitrant pollutants in nature. Microbial keratinases offers an economic and eco-friendly alternative for degrading and recycling keratinous waste into valuable byproducts. Diverse groups of microorganisms viz., bacteria, fungi and actinomycetes have the ability to degrade recalcitrant keratin by producing keratinase enzyme. Microbial keratinases exhibits great diversity in its biochemical properties with respect to activity and stability in various pH and temperature ranges as well as in the range of recalcitrant proteins it degrades like those present in feathers, hairs, nails, hooves etc. Owing to diverse properties and multifarious biotechnological implications, keratinases can be considered as promising biocatalysts for preparation of animal nutrients, protein supplements, leather processing, fiber modification, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical, cosmetic and biomedical industries, and waste management. This review article presents an overview of keratin structure and composition, mechanism of microbial keratinolysis, diversity of keratinolytic microorganisms, and their potential applications in various fields.
... However, our method allows calculation of the standard deviation for each individual data point. Most RSM models determine the standard deviation for the whole model only from data of the central data point of the model, while the other data points are only measured once [8,10,22]. The standard deviations are naturally higher at the borders of an enzyme's activity and illustrate a strong impact on the activity within a slight change of conditions. ...
Background
Glycoside hydrolases are important for various industrial and scientific applications. Determination of their temperature as well as pH optima and range is crucial to evaluate whether an enzyme is suitable for application in a biotechnological process. These basic characteristics of enzymes are generally determined by two separate measurements. However, these lead to a two-dimensional assessment of the pH range at one temperature (and vice versa) and do not allow prediction of the relative enzymatic performance at any pH/temperature combination of interest. In this work, we demonstrate a new method that is based on experimental data and visualizes the relationship among pH, temperature, and activity at a glance in a three-dimensional contour plot.
Results
In this study, we present a method to determine the relative activity of an enzyme at 96 different combinations of pH and temperature in parallel. For this purpose, we used a gradient PCR cycler and a citrate–phosphate-based buffer system in microtiter plates. The approach was successfully tested with various substrates and diverse assays for glycoside hydrolases. Furthermore, its applicability was demonstrated for single enzymes using the endoglucanase Cel8A from Clostridium thermocellum as well as the commercially available complex enzyme mixture Celluclast®. Thereby, we developed a fast and adaptable method to determine simultaneously both pH and temperature ranges of enzymes over a wide range of conditions, an easy transformation of the experimental data into a contour plot for visualization, and the necessary controls. With our method, the suitability of an enzyme or enzyme mixture for any chosen combination of temperature and pH can easily be assessed at a glance.
Conclusions
We propose a method that offers significant advantages over commonly used methods to determine the pH and temperature ranges of enzymes. The overall relationship among pH, temperature, and activity is visualized. Our method could be applied to evaluate exactly what conditions have to be met for optimal utilization of an enzyme or enzyme mixture for both lab-scale and industrial processes. Adaptation to other enzymes, including proteases, should be possible and the method may also lead to a platform for additional applications, such as inactivation kinetics analysis.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-017-0923-9) contains supplementary material, which is available to authorized users.
... Protease activity was quantitatively measured using azo-casein assay according to Cavello et al. [10], with modifications. Reaction mixture containing 20 L of enzyme extract diluted in 0.1 M Tris-HCl buffer (pH: 8) (buffer T) and 50 L of 1% (w/v) azo-casein solution in buffer T was incubated for 60 min at 37 ∘ C in thermocycler machine. ...
... Activation energy ( a ) and 10 for azo-casein hydrolysis of the proteolytic extracts from selected Aspergillus strains.10 determined using the average temperature range. ...
A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semi-quantitative and quantitative evaluation of proteolytic activity was carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics and kinetics features such as: optimum pH and temperature values, thermal stability, activation energy (Ea), quotient energy (Q10), Km and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 showed to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively
... They hydrolyze the fixed proteins on the cloth surface, and can remove stains including keratinous soiled on collars, cuffs, and blood stains on medical aprons (Gupta and Ramnani, 2006). In this respect, keratinases from Bacillus pumilus, Bacillus thuringiensis, Brevibacillus, Paecilomyces lilacinus, Paenibacillus woosongensis TKB2 and Bacillus taquilensis hsTKB2 give better efficiency towards detergent additives (Rajput et al., 2010;Sivakumar et al., 2013;Rai and Mukherjee, 2011;Cavello et al., 2012;Paul et al., 2014a;Paul et al., 2014b). ...
Currently research is centered towards replacement of costly toxic chemicals with the bio-products. This review explores application of bacterial keratinolytic protease to the leather and detergent industries. Keratinolytic proteases, are super proteolytic enzymes, that possesses the ability to degrade the insoluble keratinious waste. They display a huge divergence in physico-chemical properties which makes them superior for leather and detergent industries. In the leather processing industry, enzyme-based products are currently being explored in pre-tanning and tanning processes causing almost 80% reduction in the total pollution like noxious gases, such as hydrogen sulfide, as well as solid wastes, such as lime and chrome sludge. The keratinolytic protease as detergent additives makes it more exciting for green biotechnology. The major-bottleneck in protease research is the improvement of enzyme efficiency and protease stability at the time of marketing. Integrated bio-approach will not only solve the disposal of proper biomass and costly effluent treatment, but also more importantly create a goal-win-goal state for profit, people and climate the three builders of sustainability.
... The amount of hair recovered from a bovine hide after this process is about 3% (dry weight based). Therefore, it was estimated that a tannery, processing 25 ton of salted hides per day, produces about 2.5 ton of wet hair (70% moisture) [1,2]. Nowadays, after the processing of bovine hides, hair wastes are disposed to landfills carrying potent polluting implications. ...
... The biotransformation of hair waste could give rise to a partially degraded organic material with high nitrogen content due to extent of the proteolysis when microorganisms act. This new biomaterial could have different potential uses, that is, as a highly digestible protein source for animal feeding, as a raw material in the fertilizing or chemical industry, and as substrate for the production of enzymes such as proteases and keratinases [2,4,5]. ...
... P. lilacinum LPS #876 was previously reported as a keratinolytic fungus capable of producing keratinases when hair waste was used as substrate under submerged fermentation. Keratinases of P. lilacinum could be used for several purposes as it was stated early [2,5,12]. This report describes experiments that suggest that P. lilacinum LPS #876 is able to produce a cell-free supernatant that can be considered as a multifunctional crude extract with keratinolytic, antifungal, and plant growthpromoting activities. ...
Purpureocillium lilacinum (Thom) Samsom is one of the most studied fungi in the control of plant parasitic nematodes. However, there is not specific information on its ability to inhibit some pathogenic bacteria, fungi, or yeast. This work reports the production of several antifungal hydrolytic enzymes by a strain of P. lilacinum when it is grown in a medium containing hair waste. The growth of several plant-pathogenic fungi, Alternaria alternata, Aspergillus niger, and Fusarium culmorum, was considerably affected by the presence of P. lilacinum’s supernatant. Besides antifungal activity, P. lilacinum demonstrates the capability to produce indoleacetic acid and ammonia during time cultivation on hair waste medium. Plant growth-promoting activity by cell-free supernatant was evidenced through the increase of the percentage of tomato seed germination from 71 to 85% after 48 hours. A 21-day plant growth assay using tomato plants indicates that crude supernatant promotes the growth of the plants similar to a reference fertilizer (
p
>
0.05
). These results suggest that both strain and the supernatant may have potential to be considered as a potent biocontrol agent with multiple plant growth-promoting properties. To our knowledge, this is the first report on the antifungal, IAA production and tomato growth enhancing compounds produced by P. lilacinum LPSC #876.
