Recombinant Expression and Characterization of a Novel Thermo-Alkaline Lipase with Increased Solvent Stability from the Antarctic Thermophilic Bacterium Geobacillus sp. ID17.

Lipases are enzymes that hydrolyze long-chain carboxylic esters, and in the presence of organic solvents, they catalyze organic synthesis reactions. However, the use of solvents in these processes often results in enzyme denaturation, leading to a reduction in enzymatic activity. Consequently, there is significant interest in identifying new lipases that are resistant to denaturing conditions, with extremozymes emerging as promising candidates for this purpose. Lip7, a lipase from Geobacillus sp. ID17, a thermophilic microorganism isolated from Deception Island, Antarctica, was recombinantly expressed in E. coli C41 (DE3) in functional soluble form. Its purification was achieved with 96% purity and 23% yield. Enzymatic characterization revealed Lip7 to be a thermo-alkaline enzyme, reaching a maximum rate of 3350 U mg-1 at 50 °C and pH 11.0, using p-nitrophenyl laurate substrate. Notably, its kinetics displayed a sigmoidal behavior, with a higher kinetic efficiency (kcat/Km) for substrates of 12-carbon atom chain. In terms of thermal stability, Lip7 demonstrates stability up to 60 °C at pH 8.0 and up to 50 °C at pH 11.0. Remarkably, it showed high stability in the presence of organic solvents, and under certain conditions even exhibited enzymatic activation, reaching up to 2.5-fold and 1.35-fold after incubation in 50% v/v ethanol and 70% v/v isopropanol, respectively. Lip7 represents one of the first lipases from the bacterial subfamily I.5 and genus Geobacillus with activity and stability at pH 11.0. Its compatibility with organic solvents makes it a compelling candidate for future research in biocatalysis and various biotechnological applications.

Identification and biochemical characterization of a novel halolysin from Halorubellus sp. PRR65 with a relatively high temperature activity.

Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp154-His193-Ser348. Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The Km, Vmax and Kcat for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg-1 and 1538.09 S-1, respectively, under 60 °C, pH 8.0 and 4.0 M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities.

A functionally uncharacterized type-2 malate/L-lactate dehydrogenase family protein from Thermus thermophilus HB8 catalyzes stereospecific reduction of 2-keto-3-deoxy-D-gluconate.

2-Keto-3-deoxy- D-gluconate (KDG) is an important intermediate found in various sugars, sugar acids and polysaccharide catabolic pathways. Here, we report that a functionally uncharacterized type-2 malate/L-lactate dehydrogenase family protein (TTHB078) from Thermus thermophilus HB8 catalyzes a novel reaction, NAD(P)H-dependent reductase activity on KDG. This enzyme, designated KdgG, utilizes both NADH and NADPH as electron donors, but higher activity was observed with NADH. Analysis of the reaction product revealed that KdgG catalyzes reversible reduction of KDG to form 3-deoxy-D-mannonate. Molecular phylogenetic analysis indicated that KdgG and its homologs distributed in the genus Thermus form a novel clade among type-2 malate/L-lactate dehydrogenase family proteins.

Evaluation of temperature, pH and nutrient conditions in bacterial growth and extracellular hydrolytic activities of two Alicyclobacillus spp. strains.

Extremophile bacteria have developed the metabolic machinery for living in extreme temperatures, pH, and high-salt content. Two novel bacterium strains Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2, were isolated from crater lake El Chichon in Chiapas, Mexico. Phylogenetic tree analysis based on the 16SrRNA gene sequence revealed that the strain Alicyclobacillus sp. PA1 and Alicyclobacillus sp. PA2 were closely related to Alicyclobacillus species (98% identity and 94.73% identity, respectively). Both strains were Gram variable, and colonies were circular, smooth and creamy. Electron microscopy showed than Alicyclobacillus sp. PA1 has a daisy-like form and Alicyclobacillus sp. PA2 is a regular rod. Both strains can use diverse carbohydrates and triglycerides as carbon source and they also can use organic and inorganic nitrogen source. But, the two strains can grow without any carbon or nitrogen sources in the culture medium. Temperature, pH and nutrition condition affect bacterial growth. Maximum growth was produced at 65 °C for Alicyclobacillus sp. PA1 (0.732 DO600) at pH 3 and Alicyclobacillus sp. PA2 (0.725 DO600) at pH 5. Inducible extracellular extremozyme activities were determined for ß-galactosidase (Alicyclobacillus sp. PA1: 88.07 ± 0.252 U/mg, Alicyclobacillus sp. PA2: 51.57 ± 0.308 U/mg), cellulose (Alicyclobacillus sp. PA1: 141.20 ± 0.585 U/mg, Alicyclobacillus sp. PA2: 51.57 ± 0.308 U/mg), lipase (Alicyclobacillus sp. PA1: 138.25 ± 0.600 U/mg, Alicyclobacillus sp. PA2: 175.75 ± 1.387 U/mg), xylanase (Alicyclobacillus sp. PA1: 174.72 ± 1.746 U/mg, Alicyclobacillus sp. PA2: 172.69 ± 0.855U/mg), and protease (Alicyclobacillus sp. PA1: 15.12 ± 0.121 U/mg, Alicyclobacillus sp. PA2: 15.33 ± 0.284 U/mg). These results provide new insights on extreme enzymatic production on Alicyclobacillus species.

Properties and Applications of Extremozymes from Deep-Sea Extremophilic Microorganisms: A Mini Review.

The deep sea, which is defined as sea water below a depth of 1000 m, is one of the largest biomes on the Earth, and is recognised as an extreme environment due to its range of challenging physical parameters, such as pressure, salinity, temperature, chemicals and metals (such as hydrogen sulphide, copper and arsenic). For surviving in such extreme conditions, deep-sea extremophilic microorganisms employ a variety of adaptive strategies, such as the production of extremozymes, which exhibit outstanding thermal or cold adaptability, salt tolerance and/or pressure tolerance. Owing to their great stability, deep-sea extremozymes have numerous potential applications in a wide range of industries, such as the agricultural, food, chemical, pharmaceutical and biotechnological sectors. This enormous economic potential combined with recent advances in sampling and molecular and omics technologies has led to the emergence of research regarding deep-sea extremozymes and their primary applications in recent decades. In the present review, we introduced recent advances in research regarding deep-sea extremophiles and the enzymes they produce and discussed their potential industrial applications, with special emphasis on thermophilic, psychrophilic, halophilic and piezophilic enzymes.

Haloferax sulfurifontis GUMFAZ2 producing xylanase-free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa-India.

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.

Cloning, expression, and characterization of thermophilic L-asparaginase from Thermococcus kodakarensis KOD1.

The present study demonstrates cloning, expression, and characterization of hyperthermostable L-asparaginase from Thermococcus kodakarensis KOD1 in Escherichia coli BLR(DE3). The recombinant 6× His-tagged protein L-asparaginase from T. kodakarensis (TkAsn), was purified to homogeneity by heat treatment followed by affinity chromatography using a nickel-nitrilotriacetic acid (Ni-NTA) column. The molecular mass of the purified enzyme was found to be approximately 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzymatic properties, such as optimum temperature and pH, were 90 °C and 8.0, respectively. Its appearent Km , Vmax , and Kcat values were 2.6 mM, 1121 µmol min(-1) mg(-1) , and 694 S(-1) , respectively. The enzyme displayed high thermal stability at optimum temperature with an insignificant loss in enzymatic activity, retaining almost 90% of its activity over a time period of 32 h. The relative activity of the enzyme was significantly inhibited by the supplementation of Cu(2+) and Ni(2+) ions, while moderately inhibited by other ions. In contrast, Mg(2+) ions enhanced the relative activity compared to the control. The acrylamide contents in baked dough were reduced to sixty percent after treatment with recombinant TkAsn as compared to the untreated control. Results of the present study revealed that the enzyme was highly active at broader range of temperatures and pH, which reflect the potential of recombinant TkAsn in the food processing industry. In addition, the high thermal stability of the enzyme may facilitates its handling, storage, and transportation.

Apoptosis and cell cycle arrest of leukemic cells by a robust and stable L-asparaginase from Pseudomonas sp. PCH199.

Biomolecules obtained from microorganisms living in extreme environments possess properties that have pharmacokinetic advantages. Enzyme assay revealed recombinant L-ASNase, an extremozyme from Pseudomonas sp. PCH199 is to be highly stable with 90 % activity (200 h) at 37 °C. The stability of the enzyme in human serum (50 % activity maintained in 63 h) reveals high therapeutic potential with less dosage. The enzyme exhibited cytotoxicity to K562 blood cancer cell lines with IC50 of 0.37 U/mL without affecting the IEC-6 normal epithelial cell line. Due to the depletion of L-asparagine, K562 cells experience nutritional stress that results in the abruption of metabolic processes and eventually leads to apoptosis. Comparative studies on MCF-7 cells also revealed the same fate. Due to nutritional stress induced by L-ASNase treatment, mitochondrial membrane potential was lost, and reactive oxygen species were increased to 48 % (K562) and 21 % (MCF-7) as indicated by flow cytometric analysis. DAPI staining with prominent nuclear morphological changes visualized under the fluorescent microscope confirmed apoptosis in both cancer cells. Treatment increases pro-apoptotic Bax protein, and eventually, the cell cycle is arrested at the G2/M phase in both cell lines. Therefore, the current study paves the way for PCH199 L-ASNase to be considered a potential chemotherapeutic agent for treating acute lymphoblastic leukemia.

Extremozymes and compatible solute production potential of halophilic and halotolerant bacteria isolated from crop rhizospheric soils of Southwest Saurashtra Gujarat.

Halophiles are one of the classes of extremophilic microorganisms that can flourish in environments with very high salt concentrations. In this study, fifteen bacterial strains isolated from various crop rhizospheric soils of agricultural fields along the Southwest coastline of Saurashtra, Gujarat, and identified by 16S rRNA gene sequencing as Halomonas pacifica, H. stenophila, H. salifodinae, H. binhaiensis, Oceanobacillus oncorhynchi, and Bacillus paralicheniformis were investigated for their potentiality to produce extremozymes and compatible solute. The isolates showed the production of halophilic protease, cellulase, and chitinase enzymes ranging from 6.90 to 35.38, 0.004-0.042, and 0.097-0.550 U ml-1, respectively. The production of ectoine-compatible solute ranged from 0.01 to 3.17 mg l-1. Furthermore, the investigation of the ectoine-compatible solute production at the molecular level by PCR showed the presence of the ectoine synthase gene responsible for its biosynthesis in the isolates. Besides, it also showed the presence of glycine betaine biosynthetic gene betaine aldehyde dehydrogenase in the isolates. The compatible solute production by these isolates may be linked to their ability to produce extremozymes under saline conditions, which could protect them from salt-induced denaturation, potentially enhancing their stability and activity. This correlation warrants further investigation.

Evaluation of Antibiotic Biodegradation by a Versatile and Highly Active Recombinant Laccase from the Thermoalkaliphilic Bacterium Bacillus sp. FNT.

Laccases are industrially relevant enzymes that have gained great biotechnological importance. To date, most are of fungal and mesophilic origin; however, enzymes from extremophiles possess an even greater potential to withstand industrial conditions. In this study, we evaluate the potential of a recombinant spore-coat laccase from the thermoalkaliphilic bacterium Bacillus sp. FNT (FNTL) to biodegrade antibiotics from the tetracycline, ß-lactams, and fluoroquinolone families. This extremozyme was previously characterized as being thermostable and highly active in a wide range of temperatures (20-90 °C) and very versatile towards several structurally different substrates, including recalcitrant environmental pollutants such as PAHs and synthetic dyes. First, molecular docking analyses were employed for initial ligand affinity screening in the modeled active site of FNTL. Then, the in silico findings were experimentally tested with four highly consumed antibiotics, representatives of each family: tetracycline, oxytetracycline, amoxicillin, and ciprofloxacin. HPLC results indicate that FNTL with help of the natural redox mediator acetosyringone, can efficiently biodegrade 91, 90, and 82% of tetracycline (0.5 mg mL-1) in 24 h at 40, 30, and 20 °C, respectively, with no apparent ecotoxicity of the products on E. coli and B. subtilis. These results complement our previous studies, highlighting the potential of this extremozyme for application in wastewater bioremediation.

Industrial Biotechnology Based on Enzymes From Extreme Environments.

Biocatalysis is crucial for a green, sustainable, biobased economy, and this has driven major advances in biotechnology and biocatalysis over the past 2 decades. There are numerous benefits to biocatalysis, including increased selectivity and specificity, reduced operating costs and lower toxicity, all of which result in lower environmental impact of industrial processes. Most enzymes available commercially are active and stable under a narrow range of conditions, and quickly lose activity at extremes of ion concentration, temperature, pH, pressure, and solvent concentrations. Extremophilic microorganisms thrive under extreme conditions and produce robust enzymes with higher activity and stability under unconventional circumstances. The number of extremophilic enzymes, or extremozymes, currently available are insufficient to meet growing industrial demand. This is in part due to difficulty in cultivation of extremophiles in a laboratory setting. This review will present an overview of extremozymes and their biotechnological applications. Culture-independent and genomic-based methods for study of extremozymes will be presented.

Recombinant cellobiohydrolase of Myceliophthora thermophila: characterization and applicability in cellulose saccharification.

A codon optimized cellobiohydrolase (CBH) encoding synthetic gene of 1188 bp from a thermophilic mold Myceliophthora thermophila (MtCel6A) was cloned and heterologously expressed in Escherichia coli for the first time. In silico analysis suggested that MtCel6A is a GH6 CBH and belongs to CBHII family, which is structurally similar to Cel6A of Humicola insolens. The recombinant MtCel6A is expressed as active inclusion bodies, and the molecular mass of the purified enzyme is ~ 45 kDa. The rMtCel6A is active in a wide range of pH (4-12) and temperatures (40-100 °C) with optima at pH 10.0 and 60 °C. It exhibits T1/2 of 6.0 and 1.0 h at 60 and 90 °C, respectively. The rMtCel6A is an extremozyme with organic solvent, salt and alkali tolerance. The Km, Vmax, kcat and kcat/Km values of the enzyme are 3.2 mg mL-1, 222.2 µmol mg-1 min-1, 2492 s-1 and 778.7 s-1 mg-1 mL-1, respectively. The product analysis of rMtCel6A confirmed that it is an exoenzyme that acts from the non-reducing end of cellulose. The addition of rMtCel6A to the commercial cellulase mix (Cellic CTec2) led to 1.9-fold increase in saccharification of the pre-treated sugarcane bagasse. The rMtCel6A is a potential CBH that finds utility in industrial processes such as in bioethanol, paper pulp and textile industries.

Cloning, expression, purification and characterization of chitin deacetylase extremozyme from halophilic Bacillus aryabhattai B8W22.

Chitin deacetylase (CDA) (EC 3.5.1.41) is a hydrolytic enzyme that belongs to carbohydrate esterase family 4 as per the CAZY database. The CDA enzyme deacetylates chitin into chitosan. As the marine ecosystem is a rich source of chitin, it would also hold the unexplored extremophiles. In this study, an organism was isolated from 40 m sea sediment under halophilic condition and identified as Bacillus aryabhattai B8W22 by 16S rRNA sequencing. The CDA gene from the isolate was cloned and overexpressed in E. coli Rosetta pLysS and purified using a Ni-NTA affinity chromatography. The enzyme was found active on both ethylene glycol chitin (EGC) and chitooligosaccharides (COS). The enzyme characterization study revealed, maximum enzyme velocity at one hour, optimum pH at 7 with 50 mM Tris-HCl buffer, optimum reaction temperature of 30 ºC in standard assay conditions. The co-factor screening affirmed enhancement in the enzyme activity by 142.43 ± 7.13% and 146.88 ± 4.09% with substrate EGC and COS, respectively, in the presence of 2 mM Mg2+. This activity was decreased with the inclusion of EDTA and acetate in the assay solutions. The enzyme was found to be halotolerant; the relative activity increased to 116.98 ± 3.87% and 118.70 ± 0.98% with EGC and COS as substrates in the presence of 1 M NaCl. The enzyme also demonstrated thermo-stability, retaining 87.27 ± 2.85% and 94.08 ± 0.92% activity with substrate EGC and COS, respectively, upon treatment at 50 ºC for 24 h. The kinetic parameters K m, V max, and K cat were 3.06E-05 µg mL-1, 3.06E + 01 µM mg-1 min-1 and 3.27E + 04 s-1, respectively, with EGC as the substrate and 7.14E-07 µg mL-1, 7.14E + 01 µM mg-1 min-1 and 1.40E + 06 s-1, respectively, with COS as the substrate. The enzyme was found to be following Michaelis-Menten kinetics with both the polymeric and oligomeric substrates. In recent years, enzymatic conversion of chitosan is gaining importance due to its known pattern of deacetylation and reproducibility. Thus, this BaCDA extremozyme could be used for industrial production of chitosan polymer as well as chitosan oligosaccharides for biomedical application. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03073-3.

Discovery of hyperstable carbohydrate-active enzymes through metagenomics of extreme environments.

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate-active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 ß-mannanase/ß-1,3-glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+ -dependent GH109 with a previously unreported ß-N-acetylglucosaminide/ß-glucoside specificity. DATABASES: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

Crystal Structure and Active Site Engineering of a Halophilic γ-Carbonic Anhydrase.

Environments previously thought to be uninhabitable offer a tremendous wealth of unexplored microorganisms and enzymes. In this paper, we present the discovery and characterization of a novel γ-carbonic anhydrase (γ-CA) from the polyextreme Red Sea brine pool Discovery Deep (2141 m depth, 44.8°C, 26.2% salt) by single-cell genome sequencing. The extensive analysis of the selected gene helps demonstrate the potential of this culture-independent method. The enzyme was expressed in the bioengineered haloarchaeon Halobacterium sp. NRC-1 and characterized by X-ray crystallography and mutagenesis. The 2.6 Å crystal structure of the protein shows a trimeric arrangement. Within the γ-CA, several possible structural determinants responsible for the enzyme's salt stability could be highlighted. Moreover, the amino acid composition on the protein surface and the intra- and intermolecular interactions within the protein differ significantly from those of its close homologs. To gain further insights into the catalytic residues of the γ-CA enzyme, we created a library of variants around the active site residues and successfully improved the enzyme activity by 17-fold. As several γ-CAs have been reported without measurable activity, this provides further clues as to critical residues. Our study reveals insights into the halophilic γ-CA activity and its unique adaptations. The study of the polyextremophilic carbonic anhydrase provides a basis for outlining insights into strategies for salt adaptation, yielding enzymes with industrially valuable properties, and the underlying mechanisms of protein evolution.

Purification and Characterization of Cellulase from Obligate Halophilic Aspergillus flavus (TISTR 3637) and Its Prospects for Bioethanol Production.

A cellulase from the extreme obligate halophilic fungus, Aspergillus flavus, isolated from a man-made solar saltern in Phetchaburi, Thailand, was purified by ammonium sulfate precipitation and using Sephadex G-100 gel filtration column chromatography. The cellulase was found to be approximately 55 kDa by SDS-PAGE. Using CMC as a substrate, the specific activity of the cellulase was 62.9 units (U) mg-1 with Vmax and Km values of 37.87 mol min-1 mg-1 and 3.02 mg mL-1, respectively. Characterization of the enzyme revealed it to be an extremozyme, having an optimum activity at pH 10, 60 °C, and 200 g L-1 of NaCl. The enzyme activity was not significantly altered by the addition of divalent metal cations at 2 mM and neither did ß-mercaptoethanol, while EDTA was found strongly inhibiting the cellulase. Compared with commercial cellulase, the purified cellulase from A. flavus was more active in the extremity of conditions, especially at pH 10, 60 °C, and 150 g L-1 NaCl, whereas the commercial cellulase had a very low activity.

A polyextremophilic alcohol dehydrogenase from the Atlantis II Deep Red Sea brine pool.

Enzymes originating from hostile environments offer exceptional stability under industrial conditions and are therefore highly in demand. Using single-cell genome data, we identified the alcohol dehydrogenase (ADH) gene, adh/a1a, from the Atlantis II Deep Red Sea brine pool. ADH/A1a is highly active at elevated temperatures and high salt concentrations (optima at 70 °C and 4 m KCl) and withstands organic solvents. The polyextremophilic ADH/A1a exhibits a broad substrate scope including aliphatic and aromatic alcohols and is able to reduce cinnamyl-methyl-ketone and raspberry ketone in the reverse reaction, making it a possible candidate for the production of chiral compounds. Here, we report the affiliation of ADH/A1a to a rare enzyme family of microbial cinnamyl alcohol dehydrogenases and explain unique structural features for halo- and thermoadaptation.

Enzymatic Characteristics of a Highly Thermostable ß-(1-4)-Glucanase from Fervidobacterium islandicum AW-1 (KCTC 4680).

A highly thermostable ß-(1-4)-glucanase (NA23_08975) gene (fig) from Fervidobacterium islandicum AW-1, a native-feather degrading thermophilic eubacterium, was cloned and expressed in Escherichia coli. The recombinant FiG (rFiG) protein showed strong activity toward ß-D-glucan from barley (367.0 IU/mg), galactomannan (174.0 IU/mg), and 4-nitrophenyl-cellobioside (66.1 IU/mg), but relatively weak activity was observed with hydroxyethyl cellulose (5.3 IU/mg), carboxymethyl cellulose (2.4 IU/mg), and xylan from oat spelt (1.4 IU/mg). rFiG exhibited optimal activity at 90°C and pH 5.0. In addition, this enzyme was extremely thermostable, showing a half-life of 113 h at 85°C. These results indicate that rFiG could be used for hydrolysis of cellulosic and hemicellulosic biomass substrates for biofuel production.