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Purification and characterization of an organic-solvent-tolerant halophilic α-amylase from the moderately halophilic Nesterenkonia sp. strain F
Abstract
A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively. The amylase was active over a wide range of salt concentrations (0-4 M) with maximum activity at 0.75-1 M NaCl. The α-amylase activity was stimulated by Ca(2+) and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by β-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform, toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries.
... As compared to unmodified α-amylase, SA-αamylase displayed lower stability at all concentrations of β-mercaptoethanol tested. The decrease in α-amylase activity in the presence of β-mercaptoethanol indicated that the disulphide bridges present in the enzyme structure are essential for enzyme activity [59]. The enhanced stability of modified α-amylases in β-mercaptoethanol suggested improved masking of the disulphide bridges, thereby protecting the tertiary structure of the enzyme. ...
... Ca 2+ was found to have a stimulatory effect on the activity of unmodified α-amylase. In some cases, Ca 2+ has been found to inhibit α-amylase [61,62], whereas in some studies, Ca 2+ was found to stimulate α-amylase activity [55,59] which was in agreement with the present findings. The stimulatory effect conferred by Ca 2+ on the enzyme activity is an indication that α-amylase is a metallo-enzyme and has a calcium-binding site [59]. ...
... In some cases, Ca 2+ has been found to inhibit α-amylase [61,62], whereas in some studies, Ca 2+ was found to stimulate α-amylase activity [55,59] which was in agreement with the present findings. The stimulatory effect conferred by Ca 2+ on the enzyme activity is an indication that α-amylase is a metallo-enzyme and has a calcium-binding site [59]. However, except for PA-α-amylase, chemical modification was seen to decrease the enzyme activity of the modified α-amylases in the presence of Ca 2+ in comparison to the unmodified α-amylase. ...
The present study describes the chemical modification of α-amylase using succinic anhydride (SA), phthalic anhydride (PA) and a novel modifier viz. 2-octenyl succinic anhydride (2-OSA). SA-, PA- and 2-OSA-α-amylases displayed a 50%, 91% and 46% increase in stability at pH 9, respectively; as compared to unmodified α-amylase. PA-α-amylase showed a significant increase in Ea and ΔHa#, and a concomitant decrease in ΔSa#. The modified α-amylases exhibited improved thermostability as reflected by significant reductions in Kd and ΔSd#, and increments in t1/2, D-, Ed, ΔHd# and ΔGd# values. The modified α-amylases displayed variable stabilities in the presence of different surfactants, inhibitors, metal ions and organic solvents. Interestingly, the chemical modification was found to confer resistance against inactivation by Hg²⁺ on α-amylase. The conformational changes in modified α-amylases were investigated using intrinsic tryptophan fluorescence, ANS (extrinsic) tryptophan fluorescence, and dynamic fluorescence quenching. Both intrinsic and extrinsic tryptophan fluorescence spectra showed increased fluorescence intensity for the modified α-amylases. Chemical modification was found to induce a certain degree of structural rigidity to α-amylase, as shown by dynamic fluorescence quenching. Analysis of the CD spectra by the K2d method using the DichroWeb online tool indicated evident changes in the α-helix, β-sheet and random coil fractions of the α-amylase secondary structure, following chemical modification using anhydrides. PA-α-amylase exhibited the highest productivity in terms of hydrolysis of starch at 60 °C over a period of 5 h indicating potential in varied biotechnological applications.
... Earlier, a novel halophilic, SDS and surfactant stable, raw starch digesting and two organic solvent tolerant amylases from a moderately halophilic bacterium Nesterenkonia sp. strain F were purified and characterized (Shafiei et al. 2010(Shafiei et al. , 2011(Shafiei et al. , 2012. ...
... Due to the importance of alpha-amylase in the industrial sector and assigning of 15 to 20 % of the world market in saccharification sector to this enzyme (Mojsov 2012), today, many halophilicamylases from various sources have been purified and characterized. In our previous studies, we purified and characterized three halophilic alpha-amylases from Nesterenkonia sp.strain F (Shafiei et al. 2010(Shafiei et al. , 2011(Shafiei et al. , 2012. However, genetic studies and understanding the molecular adaptation to extreme salinity of the halophilic enzymes can reveal the new biotechnological potential and uses of these enzymes in industry. ...
... After affinity chromatography, single protein band with a molecular weight of approximately 52 kDa on the SDS-PAGE gel was observed which indicate successful purification and homogeneity of the purified enzyme. The value of molecular mass of the recombinant alpha-amylase was comparable with that obtained for the organic-solventtolerant halophilic alpha-amylase previously purified from Nesterenkonia sp.F (57 kDa) (Shafiei et al. 2011), but different from those which were obtained for the other purified alpha-amylases of Nesterenkonia sp. F (110 and 100 kDa) (Shafiei et al. 2010(Shafiei et al. , 2012. ...
A novel pH and thermo-tolerate halophilic alpha-amylase from moderately halophilic bacterium, Nesterenkonia sp.strain F was cloned and expressed in Escherichia coli. 16S rRNA sequence of the strain shared 99.46% similarities with closely related type species. Also, the genome sequence shared ANI values below 92% and dDDH values below 52% with the closely related type species. Consequently, it is proposed that strain F represents a novel species. The AmyF gene was 1390 bp long and encodes an alpha-amylase of 463 amino acid residues with pI of 4.62. The deduced AmyF shared very low sequence similarity (< 24%) with functionally characterized recombinant halophilic alpha-amylases. The recombinant alpha-amylase was successfully purified from Ni–NTA columns with a molecular mass of about 52 KDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active over a wide range of temperature (25–75 °C) and pH (4–9) with optimum activity at 45 °C and 7.5, respectively. Also, although it was active over a various concentrations of NaCl and KCl (0–4 M), increasing activity of the enzyme was observed with increasing concentration of these salts. Low concentrations of Ca2+ ion had no activating effect, but high concentrations of the ion (40–200 mM) enhanced activity of AmyF. The enzyme activity was increased by increasing concentrations of Mg2+, Zn2+, Hg2+ and Fe3+. However, it was inhibited only at very high concentrations of these metal ions. Cu2+ did not decrease the amylase activity and the highest activity was observed at 100 mM of the ion. These properties indicate wide potential applications of this recombinant enzyme in starch processing industries. This is the first isolation, cloning and characterization of a gene encoding alpha-amylase from Nesternkonia genus.
... Previous studies on the individual optimization of amylase and protease have established starch and yeast extract as the invariable substrate for the amylase and protease production, respectively (Juwon and Emannuel 2012;Saxena and Singh 2014;Shukla and Singh 2015;Kikani et al. 2020). Production of amylase and protease in halophiles is salt-dependent (Zhang and Zeng 2008;Shafiei et al. 2011;Mostafa et al. 2012;Gohel and Singh 2018;Sharma et al. 2020). Moreover, an increase in the NaCl concentration in the production medium considerably increased the secretion of halophilic extracellular enzymes (Shafiei et al. 2011;Sharma et al. 2020). ...
... Production of amylase and protease in halophiles is salt-dependent (Zhang and Zeng 2008;Shafiei et al. 2011;Mostafa et al. 2012;Gohel and Singh 2018;Sharma et al. 2020). Moreover, an increase in the NaCl concentration in the production medium considerably increased the secretion of halophilic extracellular enzymes (Shafiei et al. 2011;Sharma et al. 2020). Actinomycetes require a prolonged growth period for optimum enzyme production (Gohel and Singh 2018;Rathore et al. 2019;Sheikh et al. 2019, Sharma et al. 2020). ...
... Usually, the crude fermentation products compared to the purified preparations are more stable under the adverse conditions (Kiran and Chandra 2008;Bennett et al. 2009;Sharma et al. 2020). Among the possible reasons of the stability of the crude products, shielding/protecting the native molecules by certain stabilizing agents appears important (Shafiei et al. 2011;Hammami et al. 2018;Sharma et al. 2020). Further, the extracellular enzymes account for a lower content of the soluble proteins compared to their intracellular counterparts, which are rather difficult to purify (Shukla and Singh 2015;Kikani et al. 2020). ...
Amylases and proteases are among the industrially most important enzymes for food processing, animal feed, brewing, starch processing, detergents, healthcare, leather processing, and biofuel production. In this study, we investigated the growth kinetics and statistically optimized the co-production of amylase and protease in a phylogenetically novel haloalkaliphilic actinomycete, Streptomyces lopnurensis KaM5 of seawater. The Plackett-Berman design using Minitab 14.0 software was employed to assess the impact of the nutritional factors, temperature, pH, and incubation time. Further, starch, yeast extract, NaCl concentrations, and incubation time were optimized by Box-Behnken design at their three levels. The Pareto charts, contour, surface plots, and individual factorial analysis expressed the variability and levels for the optimal enzyme production. ANOVA analysis admitted the statistical fitness and significance level among the variables. A two-fold increase in enzyme production was achieved by cost-effective co-production media. The study was further extended to growth kinetics associated with enzyme production. Specific growth rate (μ), maximal cell mass (Xmax), volumetric product formation (Pmax), rate of product formation (Qp), and generation time (g) were computed and analyzed. These parameters significantly improved when compared with the pre-optimized conditions, and the production economics of the enzyme was industrially viable. The initial studies on the characteristics of the enzymes suggested its ability to function under the combination of alkaline pH and high salt concentrations. The co-production of enzymes from extremophiles can be a potentially viable option for large-scale production and applications.
... This enhances their range of applications (Klibanov 2001). As a consequence of their unique properties, halophilic enzymes are of tremendous interest for biotechnological applications in food processing, environmental bioremediation, and biosynthetic processes (Shafiei et al. 2011;Kumar et al. 2016). ...
... Alpha-amylase, an enzyme which cleaves the Oglycosidic bonds in starch, is one of the most commonly studied halophilic enzymes because of its importance in food, fermentation, textile, and paper industries (Kumar et al. 2016). Most studies on halophilic amylases have been reported, understandably, from halophilic bacteria and archaea which are evolutionarily adapted to extreme salt conditions (Amoozegar et al. 2003;Shafiei et al. 2011;Uzyol et al. 2012;Kumar et al. 2016). It might be expected that mesophiles, in general, will not produce halophilic enzymes. ...
... Halophilic enzymes have adapted to high-salt environments by acquiring a relatively large number of negatively charged amino acid residues on their surfaces to prevent precipitation. Consequently, their solubility is often very poor in surroundings with lower salt concentrations (Shafiei et al. 2011(Shafiei et al. , 2012. Further work on amino acid composition of the alpha-amylase of Ulkenia sp. ...
Halophilic bacteria are well known to produce highly salt-tolerant enzymes that have unusual applications in biotechnology. Production of halophilic proteins is generally not expected in mesohaline microorganisms. Ulkenia sp. AH-2, a mesohaline, marine straminipilan thraustochytrid isolated from waters of a mangrove ecosystem, produces halophilic alpha-amylases. Four enzyme fractions, viz.., A, B, C, and D, were obtained upon ammonium sulfate fractionation and gel filtration. These had a broad salinity tolerance ranging from 0 to 4 M NaCl, with an optimum at 3 M NaCl. Pools A, C, and D each resolved as a single band on PAGE and zymogram analysis, and the purified proteins were designated Amy a, Amy c, and Amy h. The major activity resided in “pool B,” consisting of several amylases which could not be further resolved into pure fractions. Together, these had an optimum at 2 M NaCl. All the enzymes were stable to storage for 2 to 24 h at 4 °C in a range of salt concentrations and even showed enhanced activity following such incubations. True to halophilic enzymes, the complex of “pool B” amylases showed improved activity in the presence of a wide range of organic solvents at 20% concentration. These enzymes are of particular interest by virtue of their constitutive nature as well as production under culture conditions that do not require salinity beyond that of seawater.
... thermoleovorans CCB US3 UF5, AmyF from Nesterenkonia sp. Strain F, BmaN2 from B. megaterium NL3, and AT23 from Anoxybacillus gonesis AT23 have relatively low K m values towards soluble starch with the value of 6.1, 5.8, 5.1, and 1.7 mg mL − 1 , respectively [31,42,48,49]. The k cat values of BaqA and BaqAΔC (Table 1) were quite similar to that of B. amyloliquefaciens, which is 450 s − 1 [50]. ...
BaqA is a raw starch degrading α-amylase produced by the marine bacterium Bacillus aquimaris MKSC 6.2, associated with soft corals. This α-amylase belongs to a new subfamily Glycoside Hydrolases (GH) 13_45 which has several unique characteristics, namely, a pair of tryptophan residues Trp201 and Trp202, a distinct LPDIx signature in the Conserved Sequence Region-V (CSR-V), and an elongated C-terminus containing five aromatic residues. The research aims to investigate physicochemical, kinetics, and biochemical properties of BaqA. In this study, the full-length enzyme (BaqA) and a truncated form of BaqA (designated as BaqAΔC), lacking the C-terminal 34 amino acids were constructed and expressed in Escherichia coli ArcticExpress (DE3). BaqA formed inclusion bodies, while BaqAΔC was produced as a soluble protein. Purified and refolded BaqA exhibited a catalytic efficiency (kcat/Km) of 53.1 ± 6.3 mL mg⁻¹ s⁻¹ at 40 °C and pH 7.5, whereas the purified BaqAΔC displayed kcat/Km of 11.4 ± 1.3 mL mg⁻¹ s⁻¹ under the optimum condition of 50 °C and pH 6.5. Moreover, BaqAΔC showed a slight reduction in the binding affinity towards sago granules. Interestingly, BaqAΔC displayed robust stability and halotolerant properties compared to BaqA. BaqAΔC maintained 50 % amylolytic activity for up to 6 h, whereas BaqA lost over 50 % of its activity within 90 min. Furthermore, BaqAΔC showed a remarkable increase in amylolytic activity upon the addition of NaCl, with an optimum concentration of 0.5 M. Even at a high salt concentration (1.5 M NaCl), BaqAΔC retained over 50 % of its residual activity. Taken together, its solubility, amylolytic activity, stability, ability to degrade raw starch, and moderate halotolerance make BaqAΔC a promising candidate for various starch processing industries.
... Shafiei and coworkers also reported that butanol decreased the activity of α-amylase from Nesterenkonia sp. 38 Structural changes in the domain nearby active site A/B were revealed to be the causal effect of inactivation. However, in the presence of ether or ethanol, α-amylase was able to maintain its activity. ...
Enzymatic reactions can be modulated by the incorporation of organic solvents, leading to alterations in enzyme stability, activity, and reaction rates. These solvents create a favorable microenvironment that enables hydrophobic reactions, facilities enzyme-substrate complex formation, and reduces undesirable water-dependent side reactions. However, it is crucial to understand the impact of organic solvents on enzymatic activity, as they can also induce enzyme inactivation. In this study, the enzymatic performance of Aspergillus oryzae α-amylase (Taka-amylase) in various organic solvents both experimentally and computationally was investigated. The results demonstrated that ethanol and ether sustain Taka-amylase activity up to 20% to 25% of the organic solvents, with ether providing twice the stability of ethanol. Molecular dynamics simulations further revealed that Taka-amylase has a more stable structure in ether and ethanol relative to other organic solvents. In addition, the analysis showed that the loop located near the active site in the AB-domain is a vulnerable site for enzyme destabilization when exposed to organic solvents. The ability of Taka-amylase to preserve the secondary loop structure in ether and ethanol contributed to the enzyme’s activity. In addition, the solvent accessibility surface area of Taka-amylase is distributed throughout all enzyme structures, thereby contributing to the instability of Taka-amylase in the presence of most organic solvents.
... Modified method for haloalkaliphilic BMS3 amylase activity assay. Amylase activity was assayed by modifying the method of Shafiei et al (Shafiei et al., 2011). in which, 0.5 mL of suitably diluted crude BMS3 amylase solution was added to 2 mL of 1% (w/v) soluble starch in 20 mM Tris-HCl (pH 8) containing 10% NaCl and incubated at 30 • C for 10 min. ...
... The halophilic alpha-amylase produced by Nesterenkonia sp. strain F retained its catalytic activity in the organic solvents such as chloroform, cyclohexane, benzene, and toluene (Shafiei et al., 2011). The selection of halophiles is also beneficial due to its stability at low water conditions owed to acidic residues on the surface (Madern et al., 2000;Fukuchi et al., 2003). ...
... strain F was purified and characterized. The enzyme was highly active over a wide range of NaCl concentrations with stability in methanol, ethanol, and DMSO [55]. Thermally stable amylases with significant catalysis and stability in organic solvents have been described from the thermophilic and haloalkaliphiic bacteria and actinobacteria [44,[56][57][58]. ...
Accepted for publication on 12 March 2023 in "International Journal of Biological Macromolecules (ISSN: 0141-8130, Elsevier, IF 8.10)"
This review article is about solvent tolerance and the ability to catalyze reactions by enzymes from extremophilic microorganisms. Usually, the enzymes do not or insignificantly function in solvents, most likely due to the interference of the solvents between enzyme and water molecules at the interface. Enzyme catalysis, under the influence of solvents, has many avenues of applications, such as the synthesis of peptides, esters, and other transesterification products. In this review, the enzymes from the extremophilic microorganisms have been described for their structural attributes and prospects of modifying them to catalyze the reactions under non-aqueous conditions. The basic theme is the strategies and mechanisms evolved in these microorganisms and their enzymes to sustain and function under solvent stress.
Keywords
Extremophiles; Non aqueous enzymes; Solvent stability; Immobilization; Adaptations
... A variety of bacteria have been reported for α-amylase production. Bacillus species are the most common bacterial sources, however amylases from Geobacillus, Lactobacillus, Pseudomonas, Corynebacterium, Nesterenkonia species were also reported [5,7,8,9]. ...
A novel amylase producing bacterium FAD4 was isolated from the wastewater of a textile factory located in Soke (Aydın/Turkey). The amylase production ability of gram positive, coccoidal FAD4 strain was confirmed with plate assay. Morphological and 16S rRNA sequence analyses revealed that FAD4 belongs to the Brachybacterium paraconglomeratum species with a sequence similarity of 99.8%. The optimal conditions for amylase production were determined as 72 h at 30 °C with supplementation of 1% starch. Optimum temperature and pH of the amylase were 50 °C and 7.0 respectively. Different starch, carbon and nitrogen sources were investigated for amylase production. A high enzyme production was observed with 1% potato starch and among nitrogen sources peptone was induced the production of amylase. Lactose, galactose, and fructose were also increased the enzyme production as carbon sources.
... Moreover, bacteria tend to secrete amylases outside the cells to perform extracellular digestion of starch into sugars (Luang-In et al. 2019). To date, many bacteria have been isolated and documented as amylaseproducing bacteria, including genera Arthrobacter, Escherichia, Micrococcus, Proteus, Pseudomonas, Serratia and Streptomyces (Kafilzadeh et al. 2012;Shafiei et al. 2011). Many bacteria in the genus Bacillus are also capable of producing a high amount of amylase (Gopinath et al. 2017;Joshi et al. 2021;Praney et al. 2019). ...
Klinfoong R, Thummakasorn C, Ungwiwatkul S, Boontanom P, Chantarasiri A. 2022. Diversity and activity of amylase-producing bacteria isolated from mangrove soil in Thailand. Biodiversitas 23: 5519-5531. Mangrove forests are a potential ecosystem for the isolation of various economic enzymes derived from mangrove-associated bacteria. The knowledge of amylase-producing bacteria isolated from mangrove forests in the Southeast Asian region has been scarce. This study aimed to investigate the isolation, genetic identification, and activity characterization of amylase-producing bacteria from mangrove soils in Thailand. The amylase-producing bacteria isolated from mangrove soils in the present were genetically belong to the genera Bacillus, Desulfurella, Peribacillus, Priestia, and Pseudomonas. Several amylase-producing bacteria such as Bacillus proteolyticus, Desulfurella, Pseudomonas entomophila, and Pseudomonas putida found in this study have hardly ever been reported. The Bacillus paralicheniformis strain DNP0507 was the most active amylolytic bacterium with 2.395 0.133 U/mg of amylase activity. The optimum temperature and pH for amylolytic activity were determined to be 50°C at a pH of 7.0 with a thermal stability range of 20-60°C at a neutral pH of 7.0-8.0. The enzyme activity was significantly enhanced by Cu 2+ , Co 2+ , and Pb 2+ and was inhibited considerably by a chelating agent EDTA. Finally, the most active amylolytic B. paralicheniformis strain DNP0507 could be applied in baking industries, food industries, and starchy waste valorization.
... strain F presented a half-life in the absence of an organic solvent of ca. 54 days but presented a half-life longer than 79 days in the presence of 20% of organic solvents with log P ow ≥ 1.97 [46]. Application of an amylase from the marine bacterium Pseudoalteromonas undina NKMB 0074 enabled the conversion of sugars for bioethanol production from microalgae biomass under saline conditions without a desalinization step, which would be necessary if amylases from terrestrial origin were to be used [47]. ...
Oceans possess tremendous diversity in microbial life. The enzymatic machinery that marine bacteria present is the result of extensive evolution to assist cell survival under the harsh and continuously changing conditions found in the marine environment. Several bacterial cells and enzymes are already used at an industrial scale, but novel biocatalysts are still needed for sustainable industrial applications, with benefits for both public health and the environment. Metagenomic techniques have enabled the discovery of novel biocatalysts, biosynthetic pathways, and microbial identification without their cultivation. However, a key stage for application of novel biocatalysts is the need for rapid evaluation of the feasibility of the bioprocess. Cultivation of not-yet-cultured bacteria is challenging and requires new methodologies to enable growth of the bacteria present in collected environmental samples, but, once a bacterium is isolated, its enzyme activities are easily measured. High-throughput screening techniques have also been used successfully, and innovative in vitro screening platforms to rapidly identify relevant enzymatic activities continue to improve. Small-scale approaches and process integration could improve the study and development of new bioprocesses to produce commercially interesting products. In this work, the latest studies related to (i) the growth of marine bacteria under laboratorial conditions, (ii) screening techniques for bioprospecting, and (iii) bioprocess development using microreactors and miniaturized systems are reviewed and discussed.
... Currently, the genus includes 24 validly published species (Parte et al. 2020). Nesterenkonia members were reported to be organic-solvent-tolerant (Shafiei et al. 2011;. They possess a large number of carbohydrate-related genes, as well as genes involved in butanol fermentation and monosaccharide/polysaccharide utilization. ...
In the present study, a comparative genome analysis of the novel alkaliphilic actinobacterial Nesterenkonia haasae with other members of the genus Nesterenkonia was performed. The genome size of Nesterenkonia members ranged from 2,188,008 to 3,676,111 bp. N. haasae and Nesterenkonia members of the present study encode the essential glycolysis and pentose phosphate pathway genes. In addition, some Nesterenkonia members encode the crucial genes for Entner-Doudoroff pathways. Some Nesterenkonia members possess the genes responsible for sulfate/thiosulfate transport system permease protein/ ATP-binding protein and conversion of sulfate to sulfite. Nesterenkonia members also encode the genes for assimilatory nitrate reduction, nitrite reductase, and the urea cycle. All Nesterenkonia members have the genes to overcome environmental stress and produce secondary metabolites. The present study helps to understand N. haasae and Nesterenkonia members’ environmental adaptation and niches specificity based on their specific metabolic properties. Further, based on genome analysis, we propose reclassifying Nesterenkonia jeotgali as a later heterotypic synonym of Nesterenkonia sandarakina .
... The ability to withstand organic solvents could be a paramount property of xylanases as several industrial and biotechnological applications are carried out in the presence of solvents. For instance, ethanol-tolerant xylanases are employed in bioethanol production; solvent-tolerant xylanases are used in the bioremediation of solvent-contaminated industrial wastewaters; solvent-and surfactant-tolerant xylanases are used in the deinking of recycled paper; solvent tolerance makes it easier to selectively precipitate, recover, and use enzymes [55][56][57][58], as well as in the bioconversion of lignocellulose [59]. The xylanase under the current study revealed significant stability with hydrophobic (log p > 2.0) and hydrophilic (log p < 2.0) organic solvents. ...
In the present study, an extracellular alkali-thermo-tolerant xylanase from Bacillus paramycoides was produced in the presence of an organic solvent. The enzyme was purified by ammonium sulphate precipitation, gel filtration, and ion exchange chromatography, with an overall recovery of 25.9%. The purified enzyme hada 70 kDa molecular weight (MW) confirmed by SDS-PAGE gel analysis. The maximum enzyme activity was reported at 55 °C and pH 7.0. Xylanase activity and stability were improved in the presence of 30% (v/v) n-dodecane, iso-octane, n-decane, and cyclohexane (7 days). The enzyme activity was improved by Co2+, EDTA, and Triton-X-100 while vigorously repressed by Hg2+ and Cu2+. The purified enzyme showed 1.473 mg/mL Km and 654.017 µg/mL/min Vmax values. The distinctive assets of the isolate verified the potential application in the field of biomass conversion into fuel and other industrial processes. Organic solvent-tolerant xylanases can be used for concurrent saccharification and bioethanol production, the amplification of intoxicating beverages, and the fermenting industry.
... Generally, enzymes are easily denatured and their activities disappear in the presence of organic solvents. Therefore, enzymes that remain stable in the presence of organic solvents might be useful for biotechnological applications in which such solvents are used(Shafiei et al., 2011). Because salt reduces water activity, a feature in common with organic solvent systems, halophilic enzymes are thought to be valuable tools as biocatalysts in other low-water-activity environments, such as in aqueous/organic and nonaqueous media (Marhuenda-Egea &Bonete, 2002). ...
As an alternate to the conventional methods, in the present study, response
surface methodology was employed for the optimization of biodiesel production from
Neochloris aquatica RDS02 using different growth medium viz., BBM, BG11, Modified
CHU10 and COMBO. A box-behnken design was three axials, four factorial points and seven replicates were analyzed to 4 factorial models for optimizing the microalgal growth, lipid, total chlorophyll, and biomass and biodiesel production process. Mathematical equations were formulated by software programming with a box-Behnken model using the design expert(version 7.0.0) software. The present model was analyses 2-D and 3-D response surface graphs.The optimal process using four different algal growth medium obtained for achieving the maximum yield from Neochloris aquatica RDS02 were BBM=75ml/L, BG11=12.5ml/L, Modified CHU 10 =10.5 ml/L and COMBO=10 ml/L respectively. The maximum predicted value of lipid, total chlorophyll, biomass and biodiesel obtained 219.98 mg/L, 327.32 mg/L,14.49 g/L, 5.7 ml/L respectively. The results conclude that the RSM approach eventually helps in the optimization and bulk production of Neochloris aquatica RDS02 which possess various industrial applications.
Keywords: Algal growth medium, Lipid, Total chlorophyll, Biomass and Biodiesel
productivity, Response surface methodology
... strain S-1 (Fukushima et al. 2005), Nesterenkonia sp. strain F (Shafiei et al. 2011), Salimicrobium halophilum strain LY20 and glutamate dehydrogenase from Halobacterium salinarum strain NRC-36014 (Munawar and Engel 2012). Enzyme production varies according to the salt concentrations (10 to 15% NaCl), e.g., Halogeometricum sp. ...
This chapter discusses the importance of nanoparticles for human beings in general, the physical parameters of halophiles, biological application of halophilic compounds, using various microorganisms, and their application synthesis of nanoparticles. Halophilic microorganisms are the most dominant species present in saline waters and with multiple biochemical reactions providing great benefits to pharmaceutical and biological applications. Microorganisms have the capacity to detoxify toxic chemicals and heavy metals that enter the body due to their chemical detoxification technique using reductase enzyme and energy-dependent efflux from the cell by membrane proteins, thus reducing metal ions into metal nanoparticles. Scientists mainly prefer the extracellular method of biological synthesis to avoid the down streaming process during the recovery of nanoparticles, such as sonication for lysis of the cell wall, washing, and centrifugation to purify synthesized nanoparticles. Nanotechnology plays a significant role in diverse life science fields such as pharmaceuticals, agriculture, and industrial purposes.
... This result was obtained through 24 h incubation and by measuring the breakage of the α-1,4-glycosidic link [49], therefore displayed a rapid starch hydrolysis. Interestingly, Vibrio alginolyticus Jme3À20 produced both αand β-amylases, adding a new bacteria genus to the most studied amylolytic bacteria from genera Bacillus, Streptomyces, Micrococcus, Arthrobacter, Escherichia, Pseudomonas, Proteus, and Serratia [50,51]. In a previous report, Kanimozhi et al. [52] successfully recovered four strains of Bacillus sp from mangrove soils, with the ability to produce α-amylases. ...
Mangrove-associated bacteria are of industrial interest due to their diverse and versatile enzyme properties. This study investigates the culturable bacteria from a wide range of habitat in a Bruguiera cylindrica L. mangrove ecosystem in North Sumatra. Screening of extracellular hydrolytic enzymes showed multiple potential traits in amylase, cellulase, chitinase, phosphatase, protease, and urease production by bacterial isolates. Molecular identification based on 16S rDNA region of a potential strain, Vibrio alginolyticus Jme3-20 is then reported as a newly proteolytic agent. The strain also showed a stable growth under salinity (NaCl) stress with considerable phosphate solubilization activities. Protease activity was enhanced by optimizing the 0.5% (w/v) sucrose and soy peptone in the fermentation medium. SDS-PAGE and zymogram analysis showed a 35-kDa MW of protease. Hence, our study revealed important insights into the bacterial diversity and activity in mangrove ecosystems, evidencing the importance of microbial exploration in this ecosystem.
... Amylase vi sinh vật đáp ứng được nhu cầu công nghiệp và có thể gia tăng sự tổng hợp enzyme bằng các kỹ thuật di truyền, phương pháp nuôi cấy liên tục, cảm ứng và tối ưu hóa các điều kiện sinh trưởng [8]. Các chủng vi sinh vật thường được sử dụng để sản xuất amylase thuộc chi Bacillus, Streptomyces, Micrococcus, Pseudomonas, Arthrobacter, Escherichia, Proteus, Aspergillus và Serratia, trong đó các chủng xạ khuẩn thuộc chi Streptomyces ngày càng được quan tâm [5,[9][10][11][12]. ...
Vi sinh vật có vai trò quan trọng trong việc phân hủy các hợp chất hữu cơ trong tự nhiên và là đối tượng cho việc sản xuất enzyme trên thế giới. Trong đó, amylase là một trong ba enzyme được sử dụng nhiều nhất trên thị trường enzyme công nghiệp và việc tìm kiếm các đối tượng vi sinh vật sản xuấtamylase mạnh và có khả năng hoạt động tốt trong các điều kiện cực đoan là cần thiết, đặc biệt xạ khuẩn là đối tượng tiềm năng cho sự sản xuất amylase công nghiệp. Trong nghiên cứu này, 23 chủng xạ khuẩn đã được phân lập từ nhiều mẫu đất khác nhau, trong đó chủng xạ khuẩn RB-XK3 đã được tuyển chọn và định danh thuộc loài Streptomyces canus. Chủng xạ khuẩn này thể hiện khả năng sinh tổng hợp amylase tốt trong môi trường Gause II chứa 1% cơ chất tinh bột và 0,5% NH4NO3 với pH 8,0 tại 37C trong thời gian lên men là 96 giờ. Chủng xạ khuẩn RB-XK3 cho thấy tiềm năng sản xuất amylase trong môi trường kiềm thay vì trung tính như các chủng xạ khuẩn khác và có thể ứng dụng trong nhiều lĩnh vực khác nhau.
... strain F (Shafiei et al., 2011). ...
Lignocellulosic biofuels and chemicals have great potential to reduce our dependence on fossil fuels and mitigate air pollution by cutting down on greenhouse gas emissions. Chemical, thermal, and enzymatic processes are used to release the sugars from the lignocellulosic biomass for conversion to biofuels. These processes often operate at extreme pH conditions, high salt concentrations, and/or high temperature. These harsh treatments add to the cost of the biofuels, as most known biocatalysts do not operate under these conditions. To increase the economic feasibility of biofuel production, microorganisms that thrive in extreme conditions are considered as ideal resources to generate biofuels and value‐added products. Halophilic archaea (haloarchaea) are isolated from hypersaline ecosystems with high salt concentrations approaching saturation (1.5–5 M salt concentration) including environments with extremes in pH and/or temperature. The unique traits of haloarchaea and their enzymes that enable them to sustain catalytic activity in these environments make them attractive resources for use in bioconversion processes that must occur across a wide range of industrial conditions. Biocatalysts (enzymes) derived from haloarchaea occupy a unique niche in organic solvent, salt‐based, and detergent industries. This review focuses on the use of haloarchaea and their enzymes to develop and improve biofuel production. The review also highlights how haloarchaea produce value‐added products, such as antibiotics, carotenoids, and bioplastic precursors, and can do so using feedstocks considered “too salty” for most microbial processes including wastes from the olive‐mill, shell fish, and biodiesel industries.
... Microbial amylase successfully used in medicinal research [11,12]. The most significant bacterial amylases are Bacillus, Streptomyces, Micrococcus, Escherichia, Proteus and Serratia [13,14]. ...
Objective: This study was aimed to isolate potent amylase producing Streptomyces from the marine source.Methods: Soil samples were collected from less explored mangrove regions of Muthupet, Tamilnadu. Isolation of Streptomyces was performed by serial dilution plate technique using starch casein agar (SCA) (pH 7.2 and temp 28 °C). Morphological and biochemical characteristics were studied using Bergey’s manual of systematic bacteriology. Preliminary screening and quantification of amylase activities were analysed in selected Streptomyces isolates by starch agar plate and dinitrosalicylic acid (DNS) method respectively.Results: Totally 65 isolates were separated from the marine soil. Among them, 23 strains showed different morphological features. These strains were subjected to amylase activity. Eight Streptomyces isolates (S1-S8) exhibited positive for amylase activity. The zone of clearance was exhibited in the range of diameters between 4-20 mm. Fermentation was prompted with inorganic salt starch agar, international Streptomyces project (ISP-4) media at 28 °C and incubated in an orbital shaker at 250 rpm for 96 h (pH 7.5). The quantitative estimation of amylase activity was exhibited selected eight isolates in the range between 2.4±0.002-5.9±0.005 (U/ml). The Streptomyces species S4, S5 and S6 exhibited strong amylase activity in both qualitative and quantitative level.Conclusion: This work motivating the amylase producing Streptomyces are originated in mangroves and it proved Streptomyces sp. S6 has a more efficient source of amylase production.
... Microbial amylase successfully used in medicinal research [11,12]. The most significant bacterial amylases are Bacillus, Streptomyces, Micrococcus, Escherichia, Proteus and Serratia [13,14]. ...
Objective: This study was aimed to isolate potent amylase producing Streptomyces from the marine source. Methods: Soil samples were collected from less explored mangrove regions of Muthupet, Tamilnadu. Isolation of Streptomyces was performed by serial dilution plate technique using starch casein agar (SCA) (pH 7.2 and temp 28 °C). Morphological and biochemical characteristics were studied using Bergey's manual of systematic bacteriology. Preliminary screening and quantification of amylase activities were analysed in selected Streptomyces isolates by starch agar plate and dinitrosalicylic acid (DNS) method respectively. Results: Totally 65 isolates were separated from the marine soil. Among them, 23 strains showed different morphological features. These strains were subjected to amylase activity. Eight Streptomyces isolates (S1-S8) exhibited positive for amylase activity. The zone of clearance was exhibited in the range of diameters between 4-20 mm. Fermentation was prompted with inorganic salt starch agar, international Streptomyces project (ISP-4) media at 28 °C and incubated in an orbital shaker at 250 rpm for 96 h (pH 7.5). The quantitative estimation of amylase activity was exhibited selected eight isolates in the range between 2.4±0.002-5.9±0.005 (U/ml). The Streptomyces species S4, S5 and S6 exhibited strong amylase activity in both qualitative and quantitative level. Conclusion: This work motivating the amylase producing Streptomyces are originated in mangroves and it proved Streptomyces sp. S6 has a more efficient source of amylase production.
... Some reports of hydrolases from the genus Nesterenkonia have described proteases, amylases and xylanase (Bakhtiar et al. 2003;Kui et al. 2010;Shafiei et al. 2011;Yang et al. 2008), and two patents have been developed to use these proteases and α-amylases as detergent additives (Hatti-Kaul et al. 2006;Jones and Kolkman 2014). In addition, we describe that the strain HJ01 has a large battery of extracellular enzymes, as it was the strain with the highest production of exoenzymes capable of producing glycolytic hydrolases not previously reported (Table S1). ...
Halophilic bacterias from saline soil from former Lake Texcoco were isolated, identified based on 16 rRNA and tested to produce glucolytic, nucleolytic, proteolytic and lipolytic exoenzymes. The Bacillus, Virgibacillus, Kocuria, Salinicoccus, Gracilibacillus, Halobacillus, Tenuibacillus and Nesterekonia genera where identified. Lipase/eserases and proteases from Nesterenkonia sp. and Nesterenkonia aethiopica showed halotolerant characteristics and were selected to synthesize the oleochemical n-butyl oleate and antioxidant peptides from muscle protein of common carp (Cyprinus carpio), respectively. In organic media (2,2,4-Trimethylpentane), the lipase/esterases from Nesterenkonia sp. (0.6 U/mL) and N. aethiopica (1.2 U/mL) achieved a 62.7% and 53.2% of n-butyl oleate conversion, respectively. The protein hydrolysis from muscle of common carp (C. carpio) showed a degree of hydrolysis of 4.5 ± 0.2% and 2.8 ± 0.1% when proteases from Nesterenkonia sp. and N. aethiopica were used, respectively. Three peptidic fractions ranging molecular masses between 254 and 1002 Da [M + H] show antioxidant scavenging activity, and the principal fraction with a peptide of 547.3 Da [M + H] showed an inhibition of 37.7 ± 1.8% and 16.3 ± 0.6%, when 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were used, respectively. These findings showed that the enzymatic battery of the halophilic bacteria from former lake Texcoco can be used in hydrolysis and synthesis of molecules with applications in different fields as food technology or bioenergy.
... strain F, which produces enzymes with catalytic activity even in the organic solvents such as chloroform, benzene, cyclohexane, and toluene. 22 Due to acidic residues on the surface of halophilic alpha-amylase, this enzyme is stable at low water conditions. [23][24][25] Other types of bacteria can remain alive in low temperatures, such as marine bacteria, and produce coldactive enzymes. ...
Alpha-amylase reputes for starch modification by breaking of 1-4 glycosidic bands and is
widely applied in different industrial sectors. Microorganisms express unique alpha-amylases
with thermostable and halotolerant characteristics dependent on the microorganism’s intrinsic
features. Likewise, genetic engineering methods are applied to produce enzymes with higher
stability in contrast to wild types. As there are widespread application of α-amylase in industry,
optimization methods like RSM are used to improve the production of the enzyme ex vivo.
This study aimed to review the latest researches on the production improvement and stability
of α-amylase.
... strain F, which produces enzymes with catalytic activity even in the organic solvents such as chloroform, benzene, cyclohexane, and toluene. 22 Due to acidic residues on the surface of halophilic alpha-amylase, this enzyme is stable at low water conditions. [23][24][25] Other types of bacteria can remain alive in low temperatures, such as marine bacteria, and produce coldactive enzymes. ...
Alpha-amylase reputes for starch modification by breaking of 1-4 glycosidic bands and is widely applied in different industrial sectors. Microorganisms express unique alpha-amylases with thermostable and halotolerant characteristics dependent on the microorganism’s intrinsic features. Likewise, genetic engineering methods are applied to produce enzymes with higher stability in contrast to wild types. As there are widespread application of α-amylase in industry, optimization methods like RSM are used to improve the production of the enzyme ex vivo. This study aimed to review the latest researches on the production improvement and stability of α-amylase.
... Moreover, a-amylase from halophilic Nesterenkonia sp. strain F was fully active in the presence of benzene, chloroform, toluene, cyclohexane, and 1decanol, and stable when exposed to SDS and surfactants (Shafiei et al., 2010(Shafiei et al., , 2011(Shafiei et al., , 2012. Finally, a-amylase from the halophilic Marinobacter sp. ...
As the primary source of a wide range of industrial products, the study of petroleum-derived compounds is of pivotal importance. However, the process of oil extraction and refinement is among the most environmentally hazardous practices, impacting almost all levels of the ecological chain. So far, the most appropriate strategy to overcome such an issue is through bioremediation, which revolves around the employment of different microorganisms to degrade hazardous compounds, generating less environmental impact and lower monetary costs. In this sense, a myriad of organisms and enzymes are considered possible candidates for the bioremediation process. Amidst the potential candidates is α-amylase, an evolutionary conserved starch-degrading enzyme. Notably, α-amylase was not only seen to degrade n-alkanes, a subclass of alkanes considered the most abundant petroleum-derived compounds but also low-density polyethylene, a dangerous pollutant produced from petroleum. Thus, due to its high conservation in both eukaryotic and prokaryotic lineages, in addition to the capability to degrade different types of hazardous compounds, the study of α-amylase becomes a rising interest. Nevertheless, there are no studies that review all biotechnological applications of α-amylase for bioremediation. In this work, we critically review the potential biotechnological applications of α-amylase, focusing on the biodegradation of petroleum-derived compounds. Evolutionary aspects are discussed, as well for all structural information and all features that could impact on the employment of this protein in the biotechnological industry, such as pH, temperature, and medium conditions. New perspectives and critical assessments are conducted regarding the application of α-amylase in the bioremediation of n-alkanes.
... halophilus showed highest amylase activity when cultured in 2 M NaCl where as when it was grown at lower salt concentrations, the production of amylase was hindered. Shafiei et al., (2011) also reported that increasing salt concentration enhanced amylase production in halotolerant bacterial isolates. Optimized salt concentration for Halomonas meridiana was reported to be 5% (w/v) NaCl and for Bacillus sp. ...
Mangrove region provides a unique ecosystem that shelters diverse life-forms. Owing to
the cyclic fluctuations in salinity of the soil due to tidal movements, such soil harbors
several halotolerant microorganisms. In this study, we aimed at screening of halotolerant
bacterial isolates from three distinct zones (Pirkhali, Jharkhali and Neti Dhopani) of
Sundarban, West Bengal, India. After proper screening, 12 halotolerant isolates were
obtained and subjected to detailed morphological, biochemical and physiological
characterization. The heavy metal tolerance and antibiotic sensitivity of these isolates
were also determined. The isolates were found to produce extra-cellular amylase and
protease which could be further co-related with salinity. Increase in salinity, escalated
amylase production in the strains where as protease production was inhibited. Finally,
16S rRNA gene sequencing and phylogenetic analysis indicated the presence of two new isolates
viz., Falsibacillus pallidus strain CW7 and Virgibacillus marismortui strain 123, that have not
been reported earlier from Sundarban soil having extra-cellular amylase producing potential.
Extremophiles pose challenges to microbiologists and researchers by inhabiting the environments with extreme conditions such as alkalinity, acidity, dryness, salinity, pressure and high temperature. The research on the extremophilic microbial communities such as the thermophiles, halophiles, psychrophiles and piezophiles has increased exponentially in past couple of decades as of their ability to produce an extensive range of enzymes with stability and catalytic activity under harsh conditions. These features of extremophilic enzymes make them an amazing alternative for applications in biotechnological, textile, detergent, pharmaceutical and food industries. Adding more, these enzymes serve as cornerstone for environmentally conscious and industrial sustainability. Further, advancements in omics approaches provide new insights into discovering the diversity of unculturable microbes. The multi-omics approaches provide a means to explore the extremophilic diversity, find out novel genes, enzymes and essential products with industrial relevance. Thus, the study of the extremophiles and their enzymes with potential applications is emerging in the recent years. The present review focuses on the biodiversity of extremozymes producing microbes, omics approaches for exploring these enzymes and major applications in diverse industries.
Extremophiles are micro-organisms that thrive in extreme environments and have evolved unique adaptations that lead to the development of exceptional enzymes. These organisms can be either simple (prokaryotic) or complex (eukaryotic) and include microalgae, fungi, lichens, protists, animals, and plants. They manage to survive in harsh conditions like high temperatures, extreme pH levels (acidic or alkaline), high salinity, and intense pressure. These extremophiles are particularly valuable because they produce enzymes, exopolysaccharides, and various types of compounds, all of which find applications in diverse industries such as food, detergents, pharmaceuticals, leather production, textiles, paper and pulp, bioremediation, and agriculture. The metabolic products of extremophiles possess remarkable qualities, including highly efficient catalytic abilities and stability, tolerance to extreme salinity and alkalinity, resilience to low water activity, resistance to oxidants and bleaching agents, and extended shelf life. The progress in molecular tools such as comparative genomics, proteomics, metagenomics, and transcriptomics has significantly enhanced our ability to uncover the complete metabolic potential of extremophiles. This section will present a comprehensive overview of the different types of extremophiles and the extreme environments they inhabit. It will also delve into the latest advancements in industrial processes and products that rely on enzymes derived from extremophiles. Moreover, the chapter will address the ethical considerations and sustainable practices related to the practice of bioprospecting, which involves the search for valuable biological resources in extreme environments.
Recent geological, hydrochemical, and mineralogical studies performed on hypersaline salt flats have given insights into similar geo-morphologic features on Mars. These salt-encrusted depressions are widely spread across the Earth, where they are characterized by high salt concentrations, intense UV radiation, high evaporation, and low precipitation. Their surfaces are completely dry in summer; intermittent flooding occurs in winter turning them into transitory hypersaline lakes. Thanks to new approaches such as culture-dependent, culture-independent, and metagenomic-based methods, it is important to study microbial life under polyextreme conditions and understand what lives in these dynamic ecosystems and how they function. Regarding these particular features, new halophilic microorganisms have been isolated from some salt flats and identified as excellent producers of primary and secondary metabolites and granules such as halocins, enzymes, carotenoids, polyhydroxyalkanoates, and exopolysaccharides. Additionally, halophilic microorganisms are implemented in heavy metal bioremediation and hypersaline wastewater treatment. As a result, there is a growing interest in the distribution of halophilic microorganisms around the world that can be looked upon as good models to develop sustainable biotechnological processes for all fields. This review provides insights into diversity, ecology, metabolism, and genomics of halophiles in hypersaline salt flats worldwide as well as their potential uses in biotechnology.
Thraustochytrids are saltwater heterotrophic protists, whose main ecological role is organic matter decomposition. These protists have gained notoriety in biotechnology, mainly for their ability to accumulate lipids with high commercial value such as squalene, carotenoids and polyunsaturated fatty acids, especially docosahexaenoic acid. Research on thraustochytrids has been almost exclusively focused on culture optimization for lipid obtention. Considering their ecological role, an enormous potential in bioremediation may not have been fully explored. Thraustochytrids can tolerate, metabolize, adsorb and degrade several environmental pollutants, even very recalcitrant ones like tar balls or aromatic compounds. Additionally, there is evidence of their ability to metabolize a wide array of substrates. Several degrading enzymatic activities with industrial applications have also been described. This review aims to systematize published data on thraustochytrids regarding bioremediation, highlighting their potential in waste treatment and revalorization as they represent a unique opportunity within a biorefinery approach.
A massive amount of waste is generated globally from agriculture sector annually that offers potential feedstock for biorefineries. Undoubtedly α-amylase has become the backbone of starch-based industries. It is a crucial amylolytic enzyme possessing versatile applications. An expensive synthetic substrate that is non-eco-friendly and toxic is not sustainable enough for large scale enzyme production. The agricultural residues should be employed as they are the low-budget production medium with high yield and are eco-friendly. Thus, current study deals with the valorization of agricultural by-products for α-amylase production. Two divergent rice-milled by-products (de-oiled rice bran and rice husk) were investigated to ascertain the best economical medium for α-amylase production. The study deals with production, partial purification and kinetics analysis of α-amylase from rice milled by-products by two bacteria (Staphylococcus aureus MTCC 3160 and Bacillus subtilis MB6). Out of all combinations, the best production (161.45 ± 2.60 U/mL) was obtained in DORB_S. aureus. The Km and Vmax values of DORB_S. aureus were 1.468 mg/mL and 34.722 mg/mL/min respectively. The study provides a roadmap for significant consumption of agricultural by-products. The study highly recommends researchers to explore some more agricultural residues as an eco-friendly and inexpensive medium to synthesize various bio-products under green technology.
Enzymes have a significant role in food processing. Extremozymes are also enzymes obtained from extremophilic microorganisms, which survive at extreme temperature, pH, organic solvents, and salt concentration. Extremophilic enzymes possess higher activity, higher rate of catalysis, better resistance to proteases, and stability when compared to normal enzymes. Extremozymes can be grouped as carbohydrases (e.g., amylases, cellulases, xylanases, pectinases, etc.), proteases, lipases, isomerases, esterases, and dehydrogenases based on mechanism of action and are of great potential in food and agriculture, and chemical, biomedical, and bioprocessing industries. Among all, carbohydrases are extensively used in food processing and demands in food industry are rapidly rising. However, the share of extremophilic carbohydrases in commercial use is still small as the production of extremozymes at large scale is challenging, and their application in the food industry is not fully realized yet.
This chapter aims at reviewing carbohydrases of extremophilic nature, outlining production methods as well as potential applications in food manufacture. Challenges in large-scale production and application to food processing and economic aspects of extremophilic carbohydrases are also to be addressed.
A novel gene encoding an acidic and SDS tolerant halophilic lipase from moderately halophilic bacterium, Nesterenkonia sp. strain F (lipF) was cloned in Escherichia coli and the recombinant lipase (LipF) was characterized. The gene had 819 bp and encoded a protein of 272 amino acids residues (pI=7.0). No sequence similarity between LipF and other halophilic lipolytic enzymes was observed. LipF was grouped into the lipase family XIII because it contained a highly conserved motif GISMG. In contrast to halophilic proteins, equal amount of acidic and basic residues were observed in LipF. Also, a small amount of acidic residues was demonstrated on the surface of the LipF structure. After purification of the recombinant protein, it showed 28.5 kDa on SDS-PAGE. The enzyme was active over a pH range of 3.5 to 8.0 with optimum activity at pH 5.0. The optimum temperature was 55 °C and it was active at 25 to 85 °C. Although LipF was active at 0-4 M of NaCl and KCl, increasing activity was observed with increasing concentration of the salts and maximum activity was at 4 M. LipF was stimulated by Ca2+, Cu2+ and Al3+ but inhibited with Ni2+, PMSF and EDTA. While 50% of the activity was remained in the presence of 0.1% SDS, 0.5% Tween 20 and Triton X-100, 1% SDS increased the activity to 120%. These characteristics indicate wide potential applications of the halophilic lipase in industries. This is the first cloning and characterization of an acidic halophilic lipase from a moderately halophilic bacterium.
Amylases have been widely applied for starch saccharification in the food, paper, pharmaceutical, and textile industries. In the present study, α-amylase DL-3-4−1 from B. amyloliquefaciens was successfully expressed in B. subtilis. The enzymatic activity exhibited high stability in a pH range from 4.0 to 11.0 and moderate thermostability. Furthermore, excellent stability was observed when incubated with metal ions, detergents, inhibitors, and organic solvents. Resistance against ethanol, urea, and guanidine−HCl was also observed. More importantly, a strategy to enhance the extracellular expression of α-amylase DL-3-4−1 was developed using 173 Sec-type signal peptides (SPs) as a library based on a fast and sequence-independent method. Fourteen SPs were identified with significantly increased yields of DL-3-4−1 via a high-throughput assay. Specifically, the enzymatic activity of the α-amylase-producing strain containing the signal peptide SP5D10 (YomL) was 3.74-fold higher than that of the strain with the wild type signal peptide (87.30 U/OD). In brief, the maximum specific activity of an α-amylase-producing strain containing the YomL signal peptide was 326.45 U/OD. This is the first report of an α-amylase that exhibits ethanol, urea, and guanidine−HCl tolerance. Hence, this α-amylase represents a promising candidate for future use in industry.
Microorganisms from extremophilic origins have attracted the scientific community in recent years as they are holding many secrets, stable macromolecules, and molecular evolution of life. Halophiles are important extremophiles that grows in the salt concentration up to 33% (wt/vol). Biosurfactants or bioemulsifiers are amphiphilic in nature with hydrophilic and hydrophobic moieties. There are different types of biosurfactants such as peptides, fatty acids, phospholipids, glycolipids, antibiotics, and polymers. This review discusses about the biosurfactants obtained from marine and solar salterns microbes such as bacteria and actinomycetes with their bioactivities. There are many applications of halophilic biosurfactants, which includes agriculture, bioprocessing, pharmaceuticals, food and cosmoceuticals, intensified oil recovery, and remediation. The biosurfactants derivatives such as glycolipids, lipopeptides, glycoprotein, surfactins, fengycins, rhamnolipid, iturin, and polymers characterized from bacteria, fungi, and actinomycetes, such as Haloferax sp., Kokuria marina, Halomonas sp., Myroides sp., Marinobacter sp., Brevibacterium sp., Bacillus sp., Oceanobacillus sp., Pontibacter korlensis, Nesterenkonia sp., Penicillium sp., Nocardioides sp., Aspergillus sp., and Streptomyces sp., and their health and remediation applications are discussed in this review.
A novel pH and thermo-tolerate halophilic alpha-amylase from moderately halophilic bacterium, Nesterenkonia sp.strain F was cloned and expressed in Escherichia coli . 16S rRNA sequence of the strain shared 99.46 % similarities with closely related type species. Also, the genome sequence shared ANI values below 92 % and dDDH values below 52 % with the closely related type species. Consequently, it is proposed that strain F represents a novel species. The Amy F gene was 1390 bp long and encodes an alpha-amylase of 463 amino acid residues with pI of 4.62. The deduced AmyF shared very low sequence similarity (<24%) with functionally characterized recombinant halophilic alpha- amylases. The recombinant alpha-amylase was successfully purified from Ni-NTA columns with a molecular mass of about 52 KDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was active over a wide range of temperature (25–75 °C) and pH (4–9) with optimum activity at 45 °C and 7.5, respectively. Also, although it was active over a various concentrations of NaCl and KCl (0–4 M), increasing activity of the enzyme was observed with increasing concentration of these salts. Low concentrations of Ca ²⁺ ion had no activating effect, but high concentrations of the ion (40 - 200 mM) enhanced activity of AmyF. The enzyme activity was increased by increasing concentrations of Mg ²⁺ , Zn ²⁺ , Hg ²⁺ and Fe ³⁺ . However, it was inhibited only at very high concentrations of these metal ions. Cu ²⁺ did not decrease the amylase activity and the highest activity was observed at 100 mM of the ion. These properties indicate wide potential applications of this recombinant enzyme in starch processing industries. This is the first isolation, cloning and characterization of a gene encoding alpha-amylase from Nesternkonia genus.
A raw starch digesting α-amylase from Nile tilapia (Oreochromis niloticus) intestine was identified. The α-amylase, AMY-T, had an estimated molecular weight of 60 kDa and purified to near homogeneity. AMY-T showed an apparent KM 4.78mg/mL and Vmax 0.44mg/mL/min) towards soluble starch. It was highly stable for 24 h in the pH range 3.0-10.0, and to solvents like methanol, isopropanol, butanol, dimethylformamide, DMSO and ethyl-ether. AMY-T was able to digest different carbohydrates, mainly showing endo-activity. Importantly, AMY-T was catalytically efficient and adsorbing towards raw potato starch at temperature documented for other raw starch digesting α-amylases. Thin layer and anion exchange chromatography characterization showed that the end products of raw starch hydrolysis were glucose, maltose and maltodextrins, with degree of polymerisation ranging 1-8. Scanning electron microscopy analysis of the AMY-T treated starch granules documented both granular exo- and endo-attack by AMY-T. These catalytic capabilities suggest high potential for AMY-T for industrial use.
Extracellular α‐amylase and protease were coproduced from halotolerant Virgibacillus dokdonensis VITP14 with banana peels (2% w/v) as substrate. The pH optima for α‐amylase and protease were 6.5 and 7.0 respectively. The temperature optima of α‐amylase and protease were 30°C and 50°C respectively. Both the enzymes were active in the presence of various metal ions (1 mM of Ni2+, Ca2+, Ba2+, Sr2+ and Mg2+), detergents (Tween 20, Tween 80, Triton X‐100) and other additives (2‐mercaptoethanol and urea). Both the enzymes followed Michaelis‐Menten type enzyme kinetics with Vmax of 121.40 μmol min−1 ml−1 and 4.17 μmol min−1 ml−1 and Km of 0.59 mg ml−1 and 0.28 mg ml−1 for amylase and protease respectively. Amylase showed higher activation energy for inactivation (75.55 kJ mol−1 compared to 59.70 kJ mol−1 for protease) and higher thermal stability (reflected by longer half‐life 53.23 min compared to 0.11 min for protease) at 60°C. The coexistence of amylase and protease could be attributed to the difference in the optimum temperatures of activity and thermal stability of the two enzymes. This article is protected by copyright. All rights reserved
Marine habitats harbor many extremophilic microorganisms. Actinomycetes are highly significant due to their abilities to produce secondary metabolites and industrial important enzymes. However majority of the actinomycetes so far are investigated from soil and compost and only limited attention is paid to these microorganisms from different saline habitats. Recent studies have indicated that halophilic actinomycetes are potential candidates for many commercially useful enzymes, suitable in detergent and sugar processing industries and bioremediation. In this report, the study based on the production optimization of an amylase is described. Various physical and chemical parameters affecting the amylase production in submerged fermentation are investigated. The actinobacterial strain Nocardiopsis dassonvillei strain KaS11 was isolated from the sea water of Kachhighadi, near Dwarka Coast. The effect of pH, temperature, incubation time, substrate and NaCl concentrations were assessed and optimized by one variable at a time (OVAT) approach. Three folds increase in the enzyme secretion was attained. The halophilic nature of the enzyme was established as the significant enzyme secretion occurred at 1-2 M of NaCl concentrations. Based on the optimization findings, a complete production media was designed for the amylase production.
Keywords: Amylases, Halophilic actinomycetes, OVAT and Optimization
It is the first time when thermo-tolerant, heavy metal resistance amylase producing strain Bacillus subtilis isolated from soil sample. Amylase was purified 3.8-fold with a specific activity of 11,305.0 U mg ⁻¹ . The molecular weight of purified amylase was 67 kDa as revealed by SDS-PAGE and activity gel analysis. The amylase was active in broad pH and temperature range of 4.0-11.0 and 35-110°C, respectively, with maxima at pH 7.0 and 85°C temperature. The amylase has Km and Vmax value of 2.181 mg ml ⁻¹ and 909.09 µg ml ⁻¹ min ⁻¹ , respectively when starch used as substrate. The amylase was not only stable but also its activity enhanced in the presence of n-dodecane, iso-octane, n-decane, xylene, toluene, n-butanol, acetone, and cyclohexane, after prolonged incubation (7 days). Amylase activity and stability was inhibited in the presence of Hg ²⁺ , benzene, sodium perborate. The unique property of solvent tolerance and heavy metal resistance proves the potential candidature of this isolate not only for starch liquefaction for food application but also for bioremediation strategies involved in environmental cleanup.
The microbial pathway of butanol biosynthesis is a unique route for the production of a biomass-derived advanced biofuel with high potential to be utilized in place of the fossil fuels. In the present study, Nesterenkonia sp. strain F was applied as a beneficial partner for Clostridium acetobutylicum in “starch-to-butanol process”. The capability of Nesterenkonia sp. strain F in secreting organic solvent-tolerant amylase was utilized for upgrading the yield of solvent, i.e., acetone, butanol, and ethanol (ABE), production under aerobic conditions. Monitoring the amylolytic activity and glucose concentration throughout the co-culture revealed higher amylase activity and glucose concentration in comparison with the single culture. After optimizing the culture conditions with response surface methodology (RSM), 10.6 g/L butanol was produced from untreated potato starch (UPS) with a high yield of 0.23 g/g ABE. To assess the performance of the co-culture at larger scale, the fermentation was conducted in a 5 L fermenter continually aerated with a rate of 0.05 vvm and 150 rpm. This led to production of 9.7 g/L butanol, 5.0 g/L acetone, and 0.3 g/L ethanol with a yield and productivity of 0.20 g/g and 0.21 g/L.h, respectively. Furthermore, ABE production from tannin-containing sorghum grain was improved by about 7-fold.
Considering the ever increasing demand for enzymes with improved properties for industrial application, an α-amylase P109 derived from marine metagenomic library of Arabian Sea sediments was tested for its applicability as feed supplement and desizing agent. The physic-chemical properties of the enzyme were analyzed. Its isoelectric point was 6.9, while its purity was confirmed by two dimensional gel electrophoresis also. The ionic detergents tested were more inhibitory on activity of P109 than the non-ionic detergents. Furthermore, oxidizing and reducing agents like hydrogen peroxide, sodium hypochlorite and β-mercaptoethanol did not have a profound inhibitory effect on P109 activity. Apart from stability in the presence of oxidizing and reducing agents, the enzyme could tolerate the presence of solvents. It was also proved that P109 could efficiently desize cloth, and at concentrations ranging from 6.25 to 100 μg/mL did not exhibit cytotoxicity in L929 murine fibroblast cells indicating that it had properties suitable for industrial applications, particularly in textile and food industries.
Amylolytic actinobacteria were isolated from the rumen fluid of ruminants. Eighteen isolates actinobacteria were obtained and the generation of actinobacteria mutants using chemical agent Sodium Azide. The actinobacteria wild type and mutants were then screened for amylase activity using starch agar plate assay. Ten isolates showed the ability to produce amylase enzymes. Submerged fermentation (SmF) was used for microbial production of amylase. It was found that maximum amylase activity (2.53 U/mL) was produced by actinobacteria isolate R-9 M, and 2.43 U/mL was produced by isolate R-2 WT using pollard as a substrate in Submerged fermentation. This preliminary study could provide base information for the discovery of novel actinobacteria from natural resources such as rumen fluid for the production of amylase, which will be used for multipurpose.
The phylum Actinobacteria is a taxonomic group with bioprospecting potential in biotechnology for the discovery of new natural compounds with antimicrobial and / or industrial activity. By means of genome mining, gene clusters associated with secondary metabolites can be explored in these microorganisms and thus facilitate the discovery of new products of pharmaceutical importance. Likewise, through comparative genomics, phylogenetic affiliation can be evaluated with the genomic architecture. The main aim of this study was to evaluate six sequenced Actinobacteria in order to identify gene clusters related to the synthesis of natural products and the ability to tolerate extreme environments through genome mining and comparative genomics. To find genes responsible for the synthesis of natural products, genome assemblies were analyzed using BUSCO v1.22 and QUAST 4.3 software, then the 16S rDNA segments were extracted with RNAmmer 1.2 to identify microorganisms using BLASTn. All genomes were annotated with RAST and the prediction of natural products was made by using AntiSMASH v3.0, BAGEL3 and NaPDoS. Synteny maps of genomes were built by using MAUVE and the phylogenetic tree with 16S rDNA sequences extracted from genomes. These softwares were used to assess the synteny of gene clusters related to cellular stress responses in the sequenced strains. Our results suggest that the three Streptomyces strains from soils of NNP Los Nevados have more predicted natural antimicrobial products than the three halotolerant species isolated from the Zipaquirá salt mine. The main natural products predicted with high % identity were class II and III lantipeptides, tetronomycin, nystatin, avermectin, pristinamycin and actinomycin. All of the microorganisms had bioremediation abilities. We suggest possible horizontal gene transfer (HGT) conferring the ability to survive in extreme environments and there was clear synteny between four extremophiles.
The moderately halophilic heterotrophic aerobic bacteria form a diverse group of microorganisms. The property of halophilism is widespread within the bacterial domain. Bacterial halophiles are abundant in environments such as salt lakes, saline soils, and salted food products. Most species keep their intracellular ionic concentrations at low levels while synthesizing or accumulating organic solutes to provide osmotic equilibrium of the cytoplasm with the surrounding medium. Complex mechanisms of adjustment of the intracellular environments and the properties of the cytoplasmic membrane enable rapid adaptation to changes in the salt concentration of the environment. Approaches to the study of genetic processes have recently been developed for several moderate halophiles, opening the way toward an understanding of haloadaptation at the molecular level. The new information obtained is also expected to contribute to the development of novel biotechnological uses for these organisms.
A metalloprotease secreted by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 when the culture reached the stationary growth phase. This enzyme was purified to homogeneity by acetone precipi-tation and subsequent Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The apparent molecular mass of the protease was 31 kDa by SDS-PAGE, whereas it was estimated as approximately 29 kDa by Sephacryl S-200 gel filtration. The purified protease had a specific activity of 116.8 lmol of tyrosine/ min per mg protein on casein. The optimum temperature and salinity of the enzyme were at 55°C and 0–0.5 M NaCl, although at salinities up to 4 M NaCl activity still remained. The protease was stable and had a broad pH profile (5.0–10.0) with an optimum of 8.5 for casein hydrolysis. The enzyme was strongly inhibited by phe-nylmethyl sulfonylfluoride (PMSF), Pefabloc SC, chy-mostatin and also EDTA, indicating that it belongs to the class of serine metalloproteases. The protease in solutions containing water-soluble organic solvents or alcohols was more stable than that in the absence of organic solvents. These characteristics make it an ideal choice for appli-cations in industrial processes containing organic sol-vents and/or salts.
Halophilic microorganisms are found as normal inhabitants of highly saline environments and thus are considered extremophiles. They are mainly represented, but not exclusively, by the halobacteria (extremely halophilic aerobic Archaea), the moderate halophiles (Bacteria and some methanogens) and several eukaryotic algae. These extremophilic microorganisms are already used for some biotechnological processes, for example halobacteria are used for the production of bacteriorhodopsin, and the alga Dunaliella is used in the commercial production of β-carotene. Several other present or potential applications of halophiles are reviewed, including the production of polymers (polyhydroxyalcanoates and polysaccharides), enzymes, and compatible solutes, and the use of these extremophiles in enhanced oil recovery, cancer detection, drug screening and the biodegradation of residues and toxic compounds.
A thermostable alkaline α-amylase producing Bacillus sp. ANT-6 was isolated from soil samples. Enzyme synthesis occurred at temperatures between 25 and 45 °C with an optimum of 37 °C. There was a slight variation in amylase synthesis within the pH range 7 and 11 with an optimum pH of 9. The optimum temperatures for amylase production and growth were the same. Analyses of the enzyme by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a single band, which show amylolytic activity, detected in starch gel. The relative molecular mass of the partial purified enzyme was estimated to be 94 500 Da. The enzyme showed optimum activity at pH 10.5 and 80 °C. The partial purified enzyme was highly active in the alkaline range of pH (9.5–13), and it was completely active up to 100 °C retaining 85.5% initial activity at pH 10.5. Enzyme activity was enhanced in the presence of 5 mM CaCl2 (110%) and 3 mM PMSF (103%), and inhibition with 5 mM by Zn, Na, Na-sulphide, EDTA (10 mM), Urea (8 M) and SDS (0.1%) was obtained 36.9, 21.5, 22.2, 4.90, 86% and 10.27, respectively. The enzyme was stable (55%) at high alkaline pH for 24 h. So our result showed that the enzyme was both alkaline, thermostable, thermopile and chelator resistant. The ANT-6 amylase enzyme may be suitable in liquefaction of starch, in detergent and textile industries and in other industrial applications.
This review covers the progress made in research on microbial a-amylase, a highly demanded industrial enzyme in various sectors such as food, pharmaceuticals, textiles, de-tergents, etc. Amylases are of ubiquitous occurrence and hold the maximum market share of enzyme sales. The article surveys the a-amylase family and the major characteristics, microbial sources, production aspects, downstream processing, salient biochemical proper-ties, industrial applications, enzyme engineering and some recent research developments.
Extracellular amylase production by the moderate halophile Halomonas meridiana was optimized and the enzyme was characterized biochemically. The highest amylase production was achieved by growing H. meridiana cultures in media with 5% salts and starch, in the absence of glucose until the end of the exponential phase. The amylase exhibited maximal activity at pH 7.0, being relatively stable in alkaline conditions. Optimal temperature and salinity for activity were 37°C and 10% NaCl, respectively. Moreover, activity at salinity as high as 30% salts was detected. Maltose and maltotriose were the main end products of starch hydrolysis, indicating an α-amylase activity.
Amylases are one of the most important and oldest industrial enzymes. These comprise hydrolases, which hydrolyse starch molecules to fine diverse products as dextrins, and progressively smaller polymers composed of glucose units. Large arrays of amylases are involved in the complete breakdown of starch. However, α-amylases which are the most in demand hydrolyse α-1,4 glycosidic bond in the interior of the molecule. α-Amylase holds the maximum market share of enzyme sales with its major application in the starch industry as well as its well-known usage in bakery. With the advent of new frontiers in biotechnology, the spectrum of α-amylase application has also expanded to medicinal and analytical chemistry as well as in automatic dishwashing detergents, textile desizing and the pulp and paper industry. Amylases are of ubiquitous occurrence, produced by plants, animals and microorganisms. However, microbial sources are the most preferred one for large scale production. Today a large number of microbial α-amylases are marketed with applications in different industrial sectors. This review focuses on the microbial amylases and their application with a biotechnological perspective.
A haloalkaliphilic archaebacterium, Natronococcus sp. strain Ah-36, produced extracellularly a maltotriose-forming amylase. The amylase was purified to homogeneity by ethanol precipitation, hydroxylapatite chromatography, hydrophobic chromatography, and gel filtration. The molecular weight of the enzyme was estimated to be 74,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amylase exhibited maximal activity at pH 8.7 and 55 degrees C in the presence of 2.5 M NaCl. The activity was irreversibly lost at low ionic strength. KCl, RbCl, and CsCl could partially substitute for NaCl at higher concentrations. The amylase was stable in the range of pH 6.0 to 8.6 and up to 50 degrees C in the presence of 2.5 M NaCl. Stabilization of the enzyme by soluble starch was observed in all cases. The enzyme activity was inhibited by the addition of 1 mM ZnCl2 or 1 mM N-bromosuccinimide. The amylase hydrolyzed soluble starch, amylose, amylopectin, and, more slowly, glycogen to produce maltotriose with small amounts of maltose and glucose of an alpha-configuration. Malto-oligosaccharides ranging from maltotetraose to maltoheptaose were also hydrolyzed; however, maltotriose and maltose were not hydrolyzed even with a prolonged reaction time. Transferase activity was detected by using maltotetraose or maltopentaose as a substrate. The amylase hydrolyzed gamma-cyclodextrin. alpha-Cyclodextrin and beta-cyclodextrin, however, were not hydrolyzed, although these compounds acted as competitive inhibitors to the amylase activity. Amino acid analysis showed that the amylase was characteristically enriched in glutamic acid or glutamine and in glycine.
This review makes a comprehensive survey of microbial amylases, i.e. alpha-amylase, beta-amylase and glucoamylase. Amylases are among the most important enzymes and are of great significance in present-day biotechnology. Although they can be derived from several sources, such as plants, animals and micro-organisms, the enzymes from microbial sources generally meet industrial demands. Microbial amylases could be potentially useful in the pharmaceutical and fine-chemical industries if enzymes with suitable properties could be prepared. With the advent of new frontiers in biotechnology, the spectrum of amylase application has widened in many other fields, such as clinical, medicinal and analytical chemistries, as well as their widespread application in starch saccharification and in the textile, food, brewing and distilling industries. In this review, after a brief description of the sources of amylases, we discuss the molecular biology of amylases, describing structures, cloning, sequences, and protoplast fusion and mutagenesis. This is followed by sections on their production and finally the properties of various amylases.
The formation of a disulfide bond between adjacent cysteine residues is accompanied by the formation of a tight turn of the
protein backbone. In nearly 90% of the structures analyzed a type VIII turn was found. The peptide bond between the two cysteines
is in a distorted trans conformation, the omega torsion angle ranges from 159 to –133°, with an average value of 171°. The constrained nature of
the vicinal disulfide turn and the pronounced difference observed between the oxidized and reduced states, suggests that vicinal
disulfides may be employed as a ‘redox‐activated’ conformational switch.
In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.
Halobacterium halobium amylase had optimal activity at pH 6.4 to 6.6 in sodium β-glycerophosphate buffer containing 0.05% NaCl at 55 C; Ca²⁺ was not required. End products from amylose were maltose, maltotriose, and glucose. The amylase, which was devoid of transglucosylase activity, had a multichain attack mechanism.
There are numerous advantages of employing enzymes as catalysts in organic solvents or aqueous solutions containing organic solvents instead of water. A few natural enzymes which are stable in the presence of organic solvents have been discovered. However, almost all natural enzymes are easily denatured and inactivated in the presence of organic solvents. Therefore, several physical and chemical methods, such as immobilization, modification, and entrapment, for stabilizing enzymes in the presence of organic solvents were developed. Protein engineering using site directed mutagenesis and directed evolution are useful for clarifying why organic solvent-stable enzymes are stable in the presence of organic solvents and for developing organic solvent-stable mutant enzymes.
An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe3+, Cu2+, Zn2+ and Al3+ strongly inhibited the enzyme, whereas Ca2+ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. Km value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.
In this study, we have reported novel α-amylase enzyme from less extensively studied marine Streptomyces sp. D1. Enzyme production was determined by using media containing 2% sucrose, 0.35% peptone and 0.15% of malt extract. Optimum temperature for enzyme production and activity was found to be 45 °C and enzyme retained almost 50% of its activity at 85 °C. Enzyme activity was also retained in presence of commercially available detergent and oxidizing agents. The partially purified enzyme from strain D1 exhibited specific activity of 113.64 U/mg protein that corresponds to 2.8-fold purification. SDS-PAGE and zymogram activity staining showed a single band equal to molecular mass of 66 kDa. The reported enzyme may have wide spread application for detergent and pharmaceutical industry.
An organic solvent-stable lipase (LST-03 lipase) secreted into the culture broth of the organic solvent-tolerant Pseudomonas aeruginosa LST-03 was purified by ion-exchange and hydrophobic interaction chromatography in the presence of 2-propanol. The purified enzyme was homogeneous as determined by SDS-PAGE. The molecular mass of the lipase was estimated to be 27.1 kDa by SDS-PAGE and 36 kDa by gel filtration. The optimum pH and temperature were 6.0 and 37°C. LST-03 lipase was stable at pH 5–8 and below 40°C. Its hydrolytic activity was highest against tricaproin (C6), methyl octanoate (C8), and coconut oil respectively among the triacylglycerols, fatty acid methyl esters, and natural oils investigated. The enzyme cleaved not only the 1,3-positioned ester bonds, but also the 2-positioned ester bond of triolein. It exhibited high levels of activity in the presence of n-decane, n-octane, DMSO, and DMF as well as in the absence of an organic solvent. In addition, LST-03 lipase was stabler in the presence of n-decane, ethyleneglycol, DMSO, n-octane, n-heptane, isooctane, and cyclohexane than in the absence of an organic solvent.
Pseudomonas aeruginosa LST-03 grew in a liquid medium in the presence of organic solvents of which the log P value was equal to or greater than 2.4. Its growth in the presence of organic solvent was followed quantitatively by measuring the dry cell mass. Production of the lipolytic enzyme of P. aeruginosa LST-03 cultivated in the presence of cyclohexane was also investigated. Although this strain can grow in the presence of cyclohexane under conditions in which the supply of oxygen is limited, aeration is required for production of lipolytic enzyme. The lipolytic activity of the supernatant of the culture in the presence of cyclohexane was stabler than that in the absence of organic solvent.
The halophilic bacterial strain Chromohalobacter sp. TVSP 101 was shown to produce extracellular, halotolerant, alkali-stable and moderately thermophilic α-amylase activity. The culture conditions for higher amylase production were optimized with respect to NaCl, pH, temperature and substrates. Maximum amylase production was achieved in a medium containing 20% NaCl or 15% KCl at pH 9.0 and 37 °C in the presence of 0.5% rice flour and tryptone. Addition of 50 mM CaCl2 to the medium increased amylase production by 29%. Two kinds of amylase activity, designated amylase I and amylase II, were purified from culture filtrates to homogeneity with molecular masses of 72 and 62 kDa, respectively. Both enzymes had maximal activity at pH 9.0 and 65 °C in the presence of 0–20% (w/v) NaCl but amylase I was much more stable in the absence of NaCl than amylase II. The enzymes efficiently hydrolyzed carbohydrates to yield maltotetraose, maltotriose, maltose, and glucose as the end products.
The use of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages compared to traditional aqueous reaction systems. Despite the advantages, native enzymes almost universally exhibit low activities and/or stabilities in the presence of organic solvents. This inactivation of the enzymes by organic solvents results in significant limitation of the enzymatic reaction process. Numerous attempts have been made to improve enzyme activity and stability in the presence of organic solvents using methods based on protein engineering and chemical or physical modification. Most enzymes used in these studies did not originally exhibit high activity and stability in the presence of organic solvents because they were not screened as organic solvent-tolerant enzymes. Recently, various attempts have been made to screen enzymes that naturally possess organic solvent-tolerance form various microorganisms including organic solvent-tolerant bacteria, thermophiles, halophiles and mesophiles. These organic solvent-tolerant enzymes are expected to have potential for applications in industrial chemical processes.
A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60-80 degrees C and displayed optimum activity at 70 degrees C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37 degrees C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.
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.
Halotolerant or halophilic microorganisms, able to live in saline environments, offer a multitude of actual or potential applications in various fields of biotechnology. The technical applications of bacteriorhodopsin comprise holography, spatial light modulators, optical computing, and optical memories. Compatible solutes are useful as stabilizers of biomolecules and whole cells, salt antagonists, or stress-protective agents. Biopolymers, such as biosurfactants and exopolysaccharides, are of interest for microbially enhanced oil recovery. Other useful biosubstances are enzymes, such as new isomerases and hydrolases, that are active and stable at high salt contents. Halotolerant microorganisms play an essential role in food biotechnology for the production of fermented food and food supplements. The degradation or transformation of a range of organic pollutants and the production of alternative energy are other fields of applications of these groups of extremophiles.
The aims of this study were to purify and characterize an extracellular alpha-amylase from the salt-tolerant bacterium Bacillus dipsosauri.
An extracellular alpha-amylase from B. dipsosauri strain DD1 was studied using the synthetic substrate 2-chloro-4-nitrophenyl-alpha-D-maltotrioside. Formation of the enzyme was induced by starch, repressed by D-glucose and highest after growth in medium containing 1.0 mol l-1 KCl. The alpha-amylase activity increased with KCl concentration, showed a pH optimum of 6.5, was stable up to 60 degrees C and was stimulated by 1.0 mol l-1 Na2SO4. The enzyme was purified from spent culture medium to apparent homogeneity by precipitation with ethanol, ion-exchange chromatography on DEAE-cellulose, centrifugal membrane filtration and gel-filtration chromatography on BioGel P-100. The purified enzyme had a denatured molecular mass of about 80 kDa but behaved on non-denaturing polyacrylamide gels as if it had a mass of about 30 kDa. The enzyme was partially inhibited by glucose-containing oligosaccharides of increasing length and strongly inhibited by the divalent cations Cd2+ and Zn2+.
The extracellular alpha-amylase from B. dipsosauri strain DD1 was purified to homogeneity and found to exhibit an unusually high degree of salt tolerance.
The alpha-amylase from B. dipsosauri differs from previously described enzymes and may be useful for the processing of starches under high-salt conditions.
The use of halophilic extremozymes in organic media has been limited by the lack of enzymological studies in these media. To explore the behaviour of these extremozymes in organic media, different approaches have been adopted, including the dispersal of the lyophilised enzyme or the use of reverse micelles. The use of reverse micelles in maintaining high activities of halophilic extremozymes under unfavourable conditions could open new fields of application such as the use of these enzymes as biocatalysts in organic media.
The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic alpha-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C. The results also implicated several prototropic groups in the catalytic reaction.
A halophilic archaeon, Haloarcula sp. strain S-1, produced extracellular organic solvent-tolerant alpha-amylase. Molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This amylase exhibited maximal activity at 50 degrees C in buffer containing 4.3 M NaCl, pH 7.0. Moreover, the enzyme was active and stable in various organic solvents (benzene, toluene, and chloroform, etc.). Activity was not detected at low ionic strengths, but it was detected in the presence of chloroform at low salt concentrations. On the other hand, no activity was detected in the presence of ethyl alcohol and acetone.
An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 degrees C and 9.0, respectively. The purified lipase was stable at 45 degrees C and pH 6-9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca2+ and Mg2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography.
Intracellular and extracellular proteins from halophilic archaea face very saline conditions and must be able to maintain stability and functionality at nearly saturated salt concentrations. Haloarchaeal proteins contain specific adaptations to prevent aggregation and loss of activity in such conditions, but these adaptations usually result in a lack of stability in the absence of salt. Here, we present the characterisation of a secreted alpha-amylase (AmyH) from the halophilic archaeon Haloarcula hispanica. AmyH was shown to be very halophilic but, unusually for a halophilic protein, it retained activity in the absence of salt. Intrinsic fluorescence measurements and activity assays showed that AmyH was very stable in high-salt buffer and even maintained stability upon the addition of urea. Urea-induced denaturation was only achieved in the absence of NaCl, demonstrating clearly that the stability of the protein was salt-dependent. Sequencing of the amyH gene showed an amino acid composition typical of halophilic proteins and, moreover, the presence of a signal peptide containing diagnostic features characteristic of export via the Twin-arginine translocase (Tat). Analysis of the export of AmyH showed that it was translocated post-translationally, most likely in a folded and active conformation, confirming that AmyH is a substrate of the Tat pathway.
A solvent tolerant strain of Pseudomonas aeruginosa (PseA) was isolated from soil samples by cyclohexane enrichment in medium. The strain was able to sustain and grow in a wide range of organic solvents. The adaptation of P. aeruginosa cell towards solvents was seen at membrane level in transmission electron micrographs. It also secreted a novel protease, which exhibited remarkable solvent stability and retained most of the activity at least up to 10 days in the presence of hydrophobic organic solvents (log P > or = 2.0) at 25% (v/v) concentrations. The protease was able to withstand as high as 75% concentration of solvents at least up to 48 h. P. aeruginosa strain and its protease, both seem promising for solvent bioremediation, wastewater treatment and carrying out biotransformation in non-aqueous medium.
A moderate halophile, Micrococcus halobius ATCC 21727, produced an extracellular dextrinogenic amylase when cultivated in media containing 1 to 3 M NaCl. The amylase was purified from the culture filtrate to an electrophoretically homogenous state by glycogen-complex formation, diethylaminoethyl-cellulose chromatography, and Bio-Gel P-200 gel filtration. The enzyme had maximal activity at pH 6 to 7 in 0.25 M NaCl or 0.75 M KCl at 50 to 55 degrees C. The activity was lost by dialysis against distilled water. Molecular weight was estimated to be 89,000 by sodium dodecyl sulfate-gel electrophoresis. The action pattern on amylose, soluble starch, and glycogen showed that the products were maltose, maltotriose, and maltotetraose, with lesser amount of glucose.
A highly thermostable alkaline amylase producing Bacillus sp. PN5 was isolated from soil, which yielded 65.23 U mL(-1) of amylase in medium containing (%) 0.6 starch, 0.5 peptone and 0.3 yeast extract at 60 degrees C, pH 7.0 after 60 h of incubation. Maximum amylase activity was at pH 10.0 and 90 degrees C. The enzyme retained 80% activity after 1 h at pH 10.0. It exhibited 65% activity at 105 degrees C and had 100% stability in the temperature range between 80 and 100 degrees C for 1 h. In addition, there was 86.36% stability after 1-h incubation with sodium dodecylsulphate. These properties indicated possible use of this amylase in starch saccharification and detergent formulation.
A moderately halophilic alkalitolerant Bacillus sp. Strain TSCVKK, with an ability to produce extracellular halophilic, alkalitolerant, surfactant, and detergent-stable alpha-amylase was isolated from soil samples obtained from a salt-manufacturing industry in Chennai. The culture conditions for higher amylase production were optimized with respect to NaCl, substrate, pH, and temperature. Maximum amylase production of 592 mU/ml was achieved in the medium at 48 h with 10% NaCl, 1% dextrin, 0.4% yeast extract, 0.2% tryptone, and 0.2% CaCl(2) at pH 8.0 at 30 degrees C. The enzyme activity in the culture supernatant was highest with 10% NaCl at pH 7.5 and 55 degrees C. The amylase that was partially purified by acetone precipitation was highly stable in various surfactants and detergents. Glucose, maltose, and maltooligosaccharides were the main end products of starch hydrolysis indicating that it is an alpha-amylase.
Three isoamylases of Rhyzopertha dominica (termed RdA70, RdA79, and RdA90 according to their relative mobility in gel electrophoresis) were isolated by ammonium sulfate fractionation and hydrophobic interaction chromatography. RdA70 and RdA79 showed an optimal pH of 7.0, whereas for RdA90 the optimal pH was 6.5. The three isoamylases remained stable at 50 degrees C for 1 h, but at 60 degrees C, all lost 50% of their activity in 20 min and were completely inactivated in 1 h. RdA70 and RdA79 were inhibited by albumin extracts from wheat samples varying widely in amylase inhibitory activity; however, RdA90 was highly resistant to inhibition. beta-Mercaptoethanol up to 30 mM increased the activity of the three isoamylases by 2.5-fold. The action pattern of the three isoamylases was typical of endoamylases; however, differences were observed on the hydrolytic efficiency rates measured as V(max)/K(m) ratio on starch, amylopectin, and amylose. The hydrolyzing action of RdA90 on starch and amylopectin (V(max)/K(m)=90.4+/-2.3 and 78.9+/-6.6, respectively) was less efficient than that on amylose (V(max)/K(m)=214+/-23.2). RdA79 efficiently hydrolyzed both amylopectin and amylose (V(max)/K(m)=260.6+/-12.9 and 326.5+/-9.4, respectively). RdA70 hydrolyzed starch and amylose at similar rates (V(max)/K(m)=202.9+/-5.5 and 215.9+/-6.2, respectively), but amylopectin was a poor substrate (V(max)/K(m)=124.2+/-7.4). The overall results suggest that RdA70 and RdA79 appear to belong to a group of saccharifying isoamylases that breaks down long fragments of oligosaccharide chains produced by the hydrolytic action of RdA90. The simultaneous action of the three isoamylases on starch, aside from the high resistance of RdA90 to wheat amylase inhibitors, might allow R. dominica to feed and reproduce successfully on the wheat kernel.
Isolation and partial characterization of three isoamy-lases of Rhyzopertha dominica F
- Cinco-Moroyoqui Fj, Diaz-Malvaez Fi, Alanis-Villa A, Barron-Hoyos Jm, Cardenas-Lopez Jl
- Cortez-Rocha
- Mo
- Wong
- Fj
Cinco-Moroyoqui FJ, Diaz-Malvaez FI, Alanis-Villa A, Barron-Hoyos JM, Cardenas-Lopez JL, Cortez-Rocha MO, Wong-Corral FJ (2008) Isolation and partial characterization of three isoamy-lases of Rhyzopertha dominica F. (Coleoptera: Bostrichidae).