Detergent-resistant α-amylase derived from Anoxybacillus karvacharensis K1 and its production based on whey.

In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg-1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg-1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg-1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5'-dithio-bis-[2-nitrobenzoic acid (DNTB), ß-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme's activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL-1 and 1580.3 ± 183.7 µmol mg-1 protein min-1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with ß-glucosidase for 24 h.

Thermodynamics of a hyperthermostable carboxylesterase from Anoxybacillus geothermalis D9.

Hyperthermostable enzymes are highly desirable biocatalysts due to their exceptional stability at extreme temperatures. Recently, a hyperthermostable carboxylesterase EstD9 from Anoxybacillus geothermalis D9 was biochemically characterized. The enzyme exhibited remarkable stability at high temperature. In this study, we attempted to probe the conformational adaptability of EstD9 under extreme conditions via in silico approaches. Circular dichroism revealed that EstD9 generated new ß-sheets at 80 °C, making the core of the hydrolase fold more stable. Interestingly, the profiles of molecular dynamics simulation showed the lowest scores of radius of gyration and solvent accessible surface area (SASA) at 80 °C. Three loops were responsible for protecting the catalytic site, which resided at the interface between the large and cap domains. To further investigate the structural adaptation in extreme conditions, the intramolecular interactions of the native structure were investigated. EstD9 revealed 18 hydrogen bond networks, 7 salt bridges, and 9 hydrophobic clusters, which is higher than the previously reported thermostable Est30. Collectively, the analysis indicates that intramolecular interactions and structural dynamics play distinct roles in preserving the overall EstD9 structure at elevated temperatures. This work is relevant to both fundamental and applied research involving protein engineering of industrial thermostable enzymes.

Characterization of a highly thermostable recombinant xylanase from Anoxybacillus ayderensis.

Xylanases are the main enzymes to hydrolyze xylan, the major hemicellulose found in lignocellulose. Xylanases also have a wide range of industrial applications. Therefore, the discovery of new xylanases has the potential to enhance efficiency and sustainability in many industries. Here, we report a xylanase with thermophilic character and superior biochemical properties for industrial use. The new xylanase is discovered in Anoxybacillus ayderensis as an intracellular xylanase (AAyXYN329) and recombinantly produced. While AAyXYN329 shows significant activity over a wide pH and temperature range, optimum activity conditions were determined as pH 6.5 and 65 °C. The half-life of the enzyme was calculated as 72 h at 65 °C. The enzyme did not lose activity between pH 6.0-9.0 at +4 °C for 75 days. Km, kcat and kcat/Km values of AAyXYN329 were calculated as 4.09824 ± 0.2245 µg/µL, 96.75 1/sec, and 23.61/L/g.s -1, respectively. In conclusion, the xylanase of A. ayderensis has an excellent potential to be utilized in many industrial processes.

Identification and Characterization of a Thermostable GH36 α-Galactosidase from Anoxybacillusvitaminiphilus WMF1 and Its Application in Synthesizing Isofloridoside by Reverse Hydrolysis.

An α-galactosidase-producing strain named Anoxybacillus vitaminiphilus WMF1, which catalyzed the reverse hydrolysis of d-galactose and glycerol to produce isofloridoside, was isolated from soil. The α-galactosidase (galV) gene was cloned and expressed in Escherichia coli. The galV was classified into the GH36 family with a molecular mass of 80 kDa. The optimum pH and temperature of galV was pH 7.5 and 60 °C, respectively, and it was highly stable at alkaline pH (6.0-9.0) and temperature below 65 °C. The specificity for p-nitrophenyl α-d-galactopyranoside was 70 U/mg, much higher than that for raffinose and stachyose. Among the metals and reagents tested, galV showed tolerance in the presence of various organic solvents. The kinetic parameters of the enzyme towards p-nitrophenyl α-d-galactopyranoside were obtained as Km (0.12 mM), Vmax (1.10 × 10-3 mM s-1), and Kcat/Km (763.92 mM-1 s-1). During the reaction of reverse hydrolysis, the enzyme exhibited high specificity towards the glycosyl donor galactose and acceptors glycerol, ethanol and ethylene glycol. Finally, the isofloridoside was synthesized using galactose as the donor and glycerol as the acceptor with a 26.6% conversion rate of galactose. This study indicated that galV might provide a potential enzyme source in producing isofloridoside because of its high thermal stability and activity.

Whole-Genome Sequence Data Analysis of Anoxybacillus kamchatkensis NASTPD13 Isolated from Hot Spring of Myagdi, Nepal.

Anoxybacillus kamchatkensis NASTPD13 isolated from Paudwar hot spring of Myagdi, Nepal, upon morphological and biochemical analysis revealed to be Gram-positive, straight or slightly curved, rod-shaped, spore-forming, catalase, and oxidase-positive facultative anaerobes. It grows over a wide range of pH (5.0-11) and temperature (37-75°C), which showed growth in different reduced carbon sources such as starch raffinose, glucose, fructose, inositol, trehalose, sorbitol, mellobiose, and mannitol in aerobic conditions. Furthermore, the partial sequence obtained upon sequencing showed 99% sequence similarity in 16S rRNA gene sequence with A. kamchatkensis JW/VK-KG4 and was suggested to be Anoxybacillus kamchatkensis. Moreover, whole-genome analysis of NASTPD13 revealed 2,866,796 bp genome with a G+C content of 41.6%. Analysis of the genome revealed the presence of 102 RNA genes, which includes sequences coding for 19 rRNA and 79 tRNA genes. While the 16S rRNA gene sequence of strain NASTPD13 showed high similarity (>99%) to those of A. kamchatkensis JW/VK-KG4, RAST analysis of NASTPD13 genome suggested that A. kamchatkensis G10 is actually the closest neighbor in terms of sequence similarity. The genome annotation by RAST revealed various genes encoding glycoside hydrolases supporting that it can utilize several reduced carbon sources as observed and these genes could be important for carbohydrate-related industries. Xylanase pathway, particularly the genomic region encoding key enzymes for xylan depolymerization and xylose metabolism, further confirmed the presence of the complete gene in xylan metabolism. In addition, the complete xylose utilization gene locus analysis of NASTPD13 genome revealed all including D-xylose transport ATP-binding protein XylG and XylF, the xylose isomerase encoding gene XylA, and the gene XylB coding for a xylulokinase supported the fact that the isolate contains a complete set of genes related to xylan degradation, pentose transport, and metabolism. The results of the present study suggest that the isolated A. kamchatkensis NASTPD13 containing xylanase-producing genes could be useful in lignocellulosic biomass-utilizing industries where pentose polymers could also be utilized along with the hexose polymers.

Resistance, removal, and bioaccumulation of Ni (II) and Co (II) and their impacts on antioxidant enzymes of Anoxybacillus mongoliensis.

In this study, it was hypothesis that A. mongoliensis could be used as bioindicator for Ni (II) and Co (II). Thus, Ni (II) and Co (II) resistance, removal, bioaccumulation, and the impacts of them on antioxidant enzyme systems of thermophilic Anoxybacillus mongoliensis were investigated in details. The bioaccumulation of Ni (II) and Co (II) on the cell membrane of thermophilic A. mongoliensis, variations on surface macrostructure and functionality by FT-IR and SEM, and determination of antioxidant enzyme activities were also tested. The highest bioaccumulation values of Co (II) and Ni (II) were detected as 102.0 mg metal/g of dry bacteria at 10 mg/L for the 12th h and 90.4 mg metal/g of dry bacteria for the 24th h, respectively, and the highest Ni (II) and Co (II) cell membrane bioaccumulation capacities of A. mongoliensis were determined as 268.5 and 274.9 mg metal/g wet membrane, respectively at the 24th h. In addition, increasing on SOD and CAT activities were observed on depend of concentration of Ni (II) and Co (II) with respect to control. The antioxidant enzyme activity results also indicated that A. mongoliensis might be used as a bioindicator for Ni (II) and Co (II) pollution in environmental water specimens.

Characterization of a thermostable, allosteric L-asparaginase from Anoxybacillus flavithermus.

L-asparaginase is an important enzyme with diverse applications in food industry and therapeutics. However, the enzyme currently employed in the treatment of leukaemias, comes with undesired L-glutaminase activity. A gene encoding 38 kDa L-asparaginase form Anoxybacillus flavithermus was cloned and expressed in Escherichia coli as a soluble and active enzyme. Heat treatment and Ni-affinity column chromatography provided highly purified enzyme possessing a specific activity of 165 units mg-1. The enzyme exhibited allosteric behaviour with a Hill coefficient of 1.60 and K0.5 of 25 mM with L-asparagine as specific substrate. No detectable activity was observed in the presence of D-asparagine, l-glutamine and d-glutamine. Purified AfASNase showed optimum activity at 60 °C and pH 7.0. The enzyme had ability to withstand up to 6 M urea and showed complete inactivation when treated with 1 M guanidine hydrochloride. Protein-Ligand docking and molecular dynamic simulations indicated that the regulatory site is formed by T262-T263-C265-G269-Thr294 and is located on a domain different from the one carrying the well-established active site. AfASNase is reported as first thermostable L-asparaginase with allosteric regulation. Hitherto, AfASNase presents the first characterization of recombinant L-asparaginase from the genus Anoxybacillus.

Parameter optimization for thermostable lipase production and performance evaluation as prospective detergent additive.

Lipase based formulations has been a rising interest to laundry detergent industry for their eco-friendly property over phosphate-based counterparts and compatibility with chemical detergents ingredients. A thermo-stable Anoxybacillus sp. ARS-1 isolated from Taptapani Hotspring, India was characterized for optimum lipase production employing statistical model central composite design (CCD) under four independent variables (temperature, pH, % moisture and bio-surfactant) by solid substrate fermentation (SSF) using mustard cake. The output was utilized to find the effect of parameters and their interaction employing response surface methodology (RSM). A quadratic regression with R2 = 0.955 established the model to be statically best fitting and a predicted highest lipase production of 29.4 IU/g at an optimum temperature of 57.5 °C, pH 8.31, moisture 50% and 1.2 mg of bio-surfactant. Experimental production of 30.3 IU/g lipase at above conditions validated the fitness of model. Anoxybacillus sp. ARS-1 produced lipase was found to resist almost all chemical detergents as well as common laundry detergent, proving it to be a prospective additive for incorporation.

Study of an Antarctic thermophilic consortium and its influence on the electrochemical behavior of aluminum alloy 7075-T6.

Common alloys used for the manufacture of aircrafts are subject to different forms of environmental deterioration. A major one is corrosion, and there is a strong body of evidence suggesting that environmental microorganisms initiate and accelerate it. The development of an appropriate strategy to reduce this process depends on the knowledge concerning the factors involved in corrosion. In this work, a biofilm forming bacterial consortium was extracted in situ from the corrosion products formed in an aircraft exposed to Antarctic media. Two thermophilic bacteria, an Anoxybacillus and a Staphylococcus strain, were successfully isolated from this consortium. Two extracellular enzymes previously speculated to participate in corrosion, catalase and peroxidase, were detected in the extracellular fraction of the consortium. Additionally, we assessed the individual contribution of those thermophilic microorganisms on the corrosion process of 7075-T6 aluminum alloy, which is widely used in aeronautical industry, through electrochemical methods and surface analysis techniques.

Improvement of the Thermostability and Activity of Pullulanase from Anoxybacillus sp. WB42.

Pullulanase is a commonly used starch-debranching enzyme with broad application in food, chemical and pharmaceutical industries. Since the starch-debranching process requires a high temperature, a thermostable pullulanase is desirable. In this study, based on the strategy of surficial residue replacement and disulfide bond introduction, a mutant pullulanase (PulAC) derived from the pullulanase (PulA) of Anoxybacillus sp. WB42 with higher thermostability and activity was isolated. The surficial residue Lys419 from the wild-type PulA was replaced by arginine, and two disulfide bonds were introduced between Thr245 and Ala326 and Trp651 and Val707. The specific activity and kcat/Km value of the PulAC reached 98.20 U/mg and 12.22 mL/mg/s respectively, 1.5 times greater than that of wild-type PulA. The optimum temperature of the mutant PulAC was 65 °C. The PulAC retained more than 85% activity after incubation at 65 °C for 30 min, which is much higher than the activity maintained by wild-type PulA. Due to its high optimum temperature, thermostability, and specific activity, the mutant PulAC reported here could play an important role in improving hydrolytic efficiency in the starch-debranching process.

Structural and functional consequences of replacement of His403 with Arg near the catalytic site of Anoxybacillus flavithermus cyclomaltodextrinase.

The hydrolytic activity of a thermophilic cyclomaltodextrinase (CMD) from Anoxybacillus flavithermus ZNU-NGA and a representative single mutant were investigated against soluble substrates including α-, ß- and γ-cyclomaltodestrines (CDs). Based on the occurrence of arginine (Arg) at position 403 in some homologue proteins, His403 in Wild-type (WT) CMD was replaced with Arg (H403R variant) with site-directed mutagenesis procedures. According to bioinformatics data, Arg403 in mutant protein is located near Glu357 as one of the catalytic residues in a manner that they are able to create a medium-range attractive electrostatistic interaction. Structural studies by Far UV-CD showed that this mutation is accompanied by conversion of a small fraction of α-helix to ß-form structure. Fluorescence data reveals that, the hydrophobic regions at the surface of protein, as the binding sites for ANS (8-Anilinonaphthalene-1-sulfonic acid) increase in mutant protein, demonstrating relative inflation of H403R variant compared with WT protein. However, the polarity of microenvironment around chromophores did not change upon mutation. Activity measurement in different ranges of pH and temperatures showed that the optimum values of pH and temperature in mutant enzyme is the same as WT enzyme, however; the activity at optimum points increased in H403R variant. It was also revealed that the H403R variant had slightly improved catalytic efficiency for γ-CD. The same value of activation parameters for both protein variants indicates that mutation does not alter the mechanism of catalysis during enzyme-substrate formation.

Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive.

A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.

Influence of Calcium Ions on the Thermal Characteristics of α-amylase from Thermophilic Anoxybacillus sp. GXS-BL.

BACKGROUND: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotechnology. OBJECTIVE: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. METHODS: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. RESULTS: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α- helical contents increasing, and the transition from α-helices to ß-sheet structures, which was obviously different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. CONCLUSION: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

Identification and analysis of binding residues in the CBM68 of pullulanase PulA from Anoxybacillus sp. LM18-11.

Carbohydrate binding module (CBM) as a kind of non-catalytic domain has significant effects on the substrate binding and catalytic properties of glycoside hydrolases. CBM68 of an Anoxybacillus sp. pullulanase (PulA) was identified as a new type of CBM in our previous studies. Then, four key substrate binding amino acid residues (Y14, V91, G92, and R96) were obtained by alanine substitutions in this work. Through kinetic analysis of the mutants, V91A and G92A showed significant reduction both in Km values and kcat values against pullulan. To further identify the changes of substrate affinities of V91A and G92A, devitalized mutants V91A-D413N and G92A-D413N were under measuring by surface plasmon resonance (SPR). Compared with that of PulA-D413N, the substrate affinities of V91A-D413N and G92A-D413N were improved by 1.6-fold and 2.2-fold, respectively. However, as to the product (maltotriose) binding force tested by the isothermal titration calorimetry (ITC), G92A showed higher binding force than that of V91A and PulA by 4.2-fold and 6.2-fold, respectively. That may cause G92A showing significantly lower catalytic efficiency than V91A and PulA. Moreover, four different kinds of amino acids (leucine, serine, glutamic acid and arginine) substitutions for V91 and G92 showed various changes both on the kinetic parameters and enzymatic properties, which demonstrated that V91 and G92 were the critical binding residues in the CBM68. The results of this study made contributed to the rational design for improving the catalytic efficiency of PulA.

A novel ß-xylosidase from Anoxybacillus sp. 3M towards an improved agro-industrial residues saccharification.

An intracellular ß-xylosidase (AbXyl), from the thermoalkaline Anoxybacillus sp. 3M, was purified and characterized. The homodimeric enzyme (140 kDa) was optimally active at 65 °C and pH 5.5, exhibited half life of 10 h at 60 °C, 78 and 88% residual activity after 24 h, at pH 4.5 and 8.0, respectively. Fe2+, Cu2+, Al3+, Ag+ and Hg2+ inhibited the enzyme; the activity was moderately stimulated by SDS and not influenced by ß-mercaptoethanol. In the presence of p-nitrophenyl-ß-d-xylopyranoside, AbXyl exhibited Km of 0.19 mM, Kcat of 453.29 s-1, Kcat Km-1 of 2322 s-1 mM and was moderately influenced by xylose (Ki 21.25 mM). The enzyme hydrolyzed xylo-oligomers into xylose and catalyzed transxylosilation reactions also in presence of alcohols as acceptors, producing xylo-oligosaccharides and alkyl-xylosides. Finally AbXyl was applied towards a statistically optimized process of brewery's spent grain bioconversion, highlighting the important role of this biocatalyst in reaching high yields of fermentable sugars.

Inhibition of α-amylase Activity by Zn2+: Insights from Spectroscopy and Molecular Dynamics Simulations.

BACKGROUND: Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed. OBJECTIVE: In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively). METHODS: Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package. RESULTS: According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and ß-sheet, ß-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme. CONCLUSION: This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

Structural Insight into a Novel Formyltransferase and Evolution to a Nonribosomal Peptide Synthetase Tailoring Domain.

Nonribosomal peptide synthetases (NRPSs) increase the chemical diversity of their products by acquiring tailoring domains. Linear gramicidin synthetase starts with a tailoring formylation (F) domain, which likely originated from a sugar formyltransferase (FT) gene. Here, we present studies on an Anoxybacillus kamchatkensis sugar FT representative of the prehorizontal gene transfer FT. Gene cluster analysis reveals that this FT acts on a UDP-sugar in a novel pathway for synthesis of a 7-formamido derivative of CMP-pseudaminic acid. We recapitulate the pathway up to and including the formylation step in vitro, experimentally demonstrating the role of the FT. We also present X-ray crystal structures of the FT alone and with ligands, which unveil contrasts with other structurally characterized sugar FTs and show close structural similarity with the F domain. The structures reveal insights into the adaptations that were needed to co-opt and evolve a sugar FT into a functional and useful NRPS domain.

Purification and characterization of thermostable superoxide dismutase from Anoxybacillus gonensis KA 55 MTCC 12684.

A thermostable superoxide dismutase from thermophilic bacterium Anoxybacillus gonensis KA 55 MTCC 12684 which was isolated from Manikaran hotspring of Himachal Pradesh was purified to apparent homogeneity by fractional ammonium sulphate precipitation and anion exchange chromatography. A purification factor of 33.1-fold was achieved, with the purified enzyme exhibiting specific activity of 5758.4 U/mg protein. The purified superoxide dismutase was optimally active at pH 9.0 and displayed stability over a broad pH range of 7.0-10.0 and was stable up to 70 °C. SOD was localized in polyacrylamide gel by activity staining, based on the reduction of nitroblue tetrazolium (NBT) by superoxide ion. The molecular weight of superoxide dismutase was calculated as 31 kDa by SDS-PAGE. The Km and Vmax values of purified enzyme were found to be 1.002 mM and 14,285.71 U/mg of protein respectively. Tests of inhibitors indicated that the enzyme activity was inhibited by hydrogen peroxide and potassium cyanide but not by sodium azide showing that purified SOD was Cu/ZnSod.

Introduction of novel thermostable α-amylases from genus Anoxybacillus and proposing to group the Bacillaceae related α-amylases under five individual GH13 subfamilies.

Among the thermophilic Bacillaceae family members, α-amylase production of 15 bacilli from genus Anoxybacillus was investigated, some of which are biotechnologically important. These Anoxybacillus α-amylase genes displayed ≥ 91.0% sequence similarities to Anoxybacillus enzymes (ASKA, ADTA and GSX-BL), but relatively lower similarities to Geobacillus (≤ 69.4% to GTA, Gt-amyII), and Bacillus aquimaris (≤ 61.3% to BaqA) amylases, all formerly proposed only in a Glycoside Hydrolase 13 (GH13) subfamily. The phylogenetic analyses of 63 bacilli-originated protein sequences among 93 α-amylases revealed the overall relationships within Bacillaceae amylolytic enzymes. All bacilli α-amylases formed 5 clades different from 15 predefined GH13 subfamilies. Their phylogenetic findings, taxonomic relationships, temperature requirements, and comparisonal structural analyses (including their CSR-I-VII regions, 12 sugar- and 4 calcium-binding sites, presence or absence of the complete catalytic machinery, and their currently unassigned status in a valid GH13 subfamiliy) revealed that these five GH13 α-amylase clades related to familly share some common characteristics, but also display differentiative features from each other and the preclassified ones. Based on these findings, we proposed to divide Bacillaceae related GH13 subfamilies into 5 individual groups: the novel a2 subfamily clustered around α-amylase B2M1-A (Anoxybacillus sp.), the a1, a3 and a4 subfamilies (including the representatives E184aa-A (Anoxybacillus sp.), ATA (Anoxybacillus tepidamans), and BaqA,) all of which were composed from the division of the previously grouped single subfamily around α-amylase BaqA, and the undefinite subfamily formerly defined as xy including Bacillus megaterium NL3.

The N-Terminal Domain of the Pullulanase from Anoxybacillus sp. WB42 Modulates Enzyme Specificity and Thermostability.

Anoxybacillus sp. WB42 pullulanase (PulWB42) is a novel thermophilic amylopullulanase that was assigned to the glycoside hydrolase family 13 subfamily 14 (GH13_14) type I pullulanases in the carbohydrate-active enzymes database. Its N-terminal domain (Met1-Phe101) was identified as the carbohydrate-binding module 68 (CBM68) by homology modeling. The N-domain-deleted PulWB42 exhibited an equivalent Michaelis constant (Km ) for pullulan and significant decreases in pullulytic activity, amylose selectivity, and thermostability relative to PulWB42 having a high α-amylase-to-pullulanase activity ratio. Furthermore, the replacement of Ala90 or Arg93 significantly changed the substrate specificity and catalytic efficiency of PulWB42, whereas Q87A, L173D, and H5A/R6A/T7A showed improvements in thermostability and changes in catalytic kinetics. Therefore, the N domain of PulWB42 is not essential for catalysis, but it does modulate enzyme catalysis, especially with respect to substrate specificity. The modulation was achieved mainly by the Leu86-Arg93 segment adjacent to the CBM48 domain and the catalytic A domain in the modeled structure of PulWB42.