Search

Cellulomonas sp. strain PS-H5 was isolated from Sekinchan Beach in Selangor, Malaysia, using an ex situ cultivation method. The present work reports a high-quality draft annotated genome sequence of this strain and suggests its potential glycoside hydrolase enzymes for cellulose, hemicellulose, and starch degradations.

Anoxybacillus sp. SK 3-4 is a Gram-positive, rod-shaped bacterium and a member of family Bacillaceae. We had previously reported that the strain is an aluminum resistant thermophilic bacterium. This is the first report to provide a detailed analysis of the global transcriptional response of Anoxybacillus when the cells were exposed to 600 mg L-1 of aluminum. The transcriptome was sequenced using Illumina MiSeq sequencer. Total of 708 genes were differentially expressed (fold change >2.00) with 316 genes were up-regulated while 347 genes were down-regulated, in comparing to control with no aluminum added in the culture. Based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the majority of genes encoding for cell metabolism such as glycolysis, sulfur metabolism, cysteine and methionine metabolism were up-regulated; while most of the gene associated with tricarboxylic acid cycle (TCA cycle) and valine, leucine and isoleucine metabolism were down-regulated. In addition, a significant number of the genes encoding ABC transporters, metal ions transporters, and some stress response proteins were also differentially expressed following aluminum exposure. The findings provide further insight and help us to understand on the resistance of Anoxybacillus sp. SK 3-4 toward aluminium.

A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2&Delta, CBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2&Delta, CBM had an identical optimum temperature at 70 &deg, C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2&Delta, CBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s&minus, 1, but this was reduced by 40% for XynRA2&Delta, CBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.

Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0-6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60 °C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26-72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362-370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme.

Xylanases (EC 3.2.1.8) are essential enzymes due to their applications in various industries such as textile, animal feed, paper and pulp, and biofuel industries. Halo-thermophilic Rhodothermaceae bacterium RA was previously isolated from a hot spring in Malaysia. Genomic analysis revealed that this bacterium is likely to be a new genus of the family Rhodothermaceae. In this study, a xylanase gene (1140 bp) that encoded 379 amino acids from the bacterium was cloned and expressed in Escherichia coli BL21(DE3). Based on InterProScan, this enzyme XynRA1 contained a GH10 domain and a signal peptide sequence. XynRA1 shared low similarity with the currently known xylanases (the closest is 57.2–65.4% to Gemmatimonadetes spp.). The purified XynRA1 achieved maximum activity at pH 8 and 60 °C. The protein molecular weight was 43.1 kDa XynRA1 exhibited an activity half-life (t1/2) of 1 h at 60 °C and remained stable at 50 °C throughout the experiment. However, it was NaCl intolerant, and various types of salt reduced the activity. This enzyme effectively hydrolyzed xylan (beechwood, oat spelt, and Palmaria palmata) and xylodextrin (xylotriose, xylotetraose, xylopentaose, and xylohexaose) to produce predominantly xylobiose. This xylanase is the first functionally characterized enzyme from the bacterium, and this work broadens the knowledge of GH10 xylanases.

Rhodothermaceae bacterium RA is a halo-thermophile isolated from a saline hot spring. Previously, the genome of this bacterium was sequenced using a HiSeq 2500 platform culminating in 91 contigs. In this report, we report on the resequencing of its complete genome using a PacBio RSII platform. The genome has a GC content of 68.3%, is 4,653,222 bp in size, and encodes 3711 genes. We are interested in understanding the carbohydrate metabolic pathway, in particular the lignocellulosic biomass degradation pathway. Strain RA harbors 57 glycosyl hydrolase (GH) genes that are affiliated with 30 families. The bacterium consists of cellulose-acting (GH 3, 5, 9, and 44) and hemicellulose-acting enzymes (GH 3, 10, and 43). A crude cell-free extract of the bacterium exhibited endoglucanase, xylanase, β-glucosidase, and β-xylosidase activities. The complete genome information coupled with biochemical assays confirms that strain RA is able to degrade cellulose and xylan. Therefore, strain RA is another excellent member of family Rhodothermaceae as a repository of novel and thermostable cellulolytic and hemicellulolytic enzymes. Electronic supplementary material The online version of this article (10.1007/s13205-018-1391-z) contains supplementary material, which is available to authorized users.

Type I pullulanase from Anoxybacillus sp. SK3-4 (PulASK) is an unusual debranching enzyme that specifically hydrolyzes starch α-1,6 linkages at long branches producing oligosaccharides (≥G8), but is nonreactive against short branches; thus, incapable of producing reducing sugars (G1–G7). We report on the effects of both single and co-immobilization of PulASK on product specificity. PulASK was purified and immobilized through covalent attachment to three epoxides (ReliZyme EP403/M, Immobead IB-150P, and Immobead IB-150A) and an amino-epoxide (ReliZyme HFA403/M) activated supports. Following immobilization, all PulASK derivatives were active on both short and long branches in starch producing reducing sugars (predominantly maltotriose) and oligosaccharides (≥G8), respectively, a feature that is absent in the free enzyme. This study also demonstrated that co-immobilization of PulASK and α-amylase from Anoxybacillus sp. SK3-4 (TASKA) on ReliZyme HFA403/M significantly changed the product specificity compared to the free enzymes alone or individually immobilized enzymes. In conclusion, individual or co-immobilization caused changes in the product specificity, presumably due to changes in the enzyme binding pocket caused by the influence of carrier surface properties (hydrophobic or hydrophilic) and the lengths of the spacer arms.

The B domain of CGTase has been generally accepted as a domain involved in thermostability. However, limited work has been performed in which entire B domain is substituted with the thermostable counterpart. Using overlap extension PCR, we replaced the B domain of a variant of CGTase Bacillus sp. G1 by six other B domains from thermostable CGTases. Likely due to distortion in the substrate-binding cleft adjacent to the active site, variants with the domain replacements from Thermoanaerobacter, Thermococcus, Thermococcus kodakarensis, Anaerobranca gottschalkii and Pyrococcus furiosus completely lost their catalytic function. A mutant designated Cgt_ET1 with a domain replacement from a Bacillus stearopthermophilus ET1 CGTase was the only variant that retained activity after domain exchange. Both the parental enzyme and the mutant Cgt_ET1 had an identical optimum temperature at 60 °C. The activity half-life was 22 min for the parental CGTase, whereas a marked increase to 57 min was observed for the mutant. Further mutagenesis on Cgt_ET1 was performed at residue 188 by replacing a Phe residue with Tyr. The mutant Cgt_ET1_F188Y displayed a decreased activity half-life of 28 min. Both mutants exhibited a better cyclodextrin-forming ability and a faster turnover rate (kcat) than the parental CGTase.

In general, biofuel production involves biomass pretreatment and enzymatic saccharification, followed by the subsequent sugar conversion to biofuel via fermentation. The crucial step in the production of biofuel from biomass is the enzymatic saccharification. Many of the commercial cellulase enzyme cocktails, such as Spezyme® CP (Genencor), Acellerase™ 1000 (Genencor), and Celluclast® 1.5L (Novozymes), are ineffectively to release free glucose from the pretreated biomass without additional β-glucosidase. In this study, for the first time, a β-glucosidase DT-Bgl gene (1359 bp) was identified in the genome of Anoxybacillus sp. DT3-1, and cloned and heterologously expressed in Escherichia coli BL21. Phylogenetic analysis indicated that DT-Bgl belonged to glycosyl hydrolase (GH) family 1. The recombinant DT-Bgl was highly active on cello-oligosaccharides and p-nitrophenyl-β-d-glucopyranoside (pNPG). The DT-Bgl was purified using an Ni–NTA column, with molecular mass of 53 kDa using an SDS-PAGE analysis. It exhibited optimum activity at 70 °C and pH 8.5, and did not require any tested co-factors for activation. The K m and V max values for DT-Bgl were 0.22 mM and 923.7 U/mg, respectively, with pNPG as substrate. The DT-Bgl displayed high glucose tolerance, and retained 93 % activity in the presence of 10 M glucose. Anoxybacillus DT-Bgl is a novel thermostable β-glucosidase with low glucose inhibition, and converts long-chain cellodextrins to cellobiose, and further hydrolyse cellobiose to glucose. Results suggest that DT-Bgl could be useful in the development of a bioprocess for the efficient saccharification of lignocellulosic biomass.

A bacterial strain, designated RA, was isolated from water sample of a hot spring on Langkawi Island of Malaysia using marine agar. Strain RA is an aerophilic and thermophilic microorganism that grows optimally at 50-60°C and is capable of growing in marine broth containing 1-10% (w/v) NaCl. 16S rRNA gene sequence analysis demonstrated that this strain is most closely related (90% sequence identity) to Rhodothermaceae, which currently comprises of six genera: Rhodothermus (two species), Salinibacter (three species), Salisaeta (one species), Rubricoccus (one species), Rubrivirga (one species), and Longimonas (one species). Notably, analysis of average nucleotide identity (ANI) values indicated that strain RA may represent the first member of a novel genus of Rhodothermaceae. The draft genome of strain RA is 4,616,094 bp with 3630 protein-coding gene sequences. Its GC content is 68.3%, which is higher than that of most other genomes of Rhodothermaceae. Strain RA has genes for sulfate permease and arylsulfatase to withstand the high sulfur and sulfate contents of the hot spring. Putative genes encoding proteins involved in adaptation to osmotic stress were identified which encode proteins namely Na(+)/H(+) antiporters, a sodium/solute symporter, a sodium/glutamate symporter, trehalose synthase, malto-oligosyltrehalose synthase, choline-sulfatase, potassium uptake proteins (TrkA and TrkH), osmotically inducible protein C, and the K(+) channel histidine kinase KdpD. Furthermore, genome description of strain RA and comparative genome studies in relation to other related genera provide an overview of the uniqueness of this bacterium.

A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo and GtamyII), Bacillus aquimaris (BaqA) and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca2+ ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca2+ ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.

Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as “parent CGTase” herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.

Trehalose has similar chemical formula as maltose. In terms of price, trehalose is much more expensivethan maltose. A pink-pigmented bacterium identified as Meiothermus sp. SK3-2 was found to be able to convert maltose to trehalose. Based on fatty acids analysis, the Meiothermus sp. SK3-2 may be a new strain that produce trehalose synthase (TreS). Meiothermus sp. SK3-2 achieved a better biomass growth in MM medium containing maltose. Besides that, TreS activity yield was also higher in MM medium, approximately 3.5, 1.8 and 0.3 fold than that in PY medium, thermophilic Bacillus medium and Castenholz medium, respectively. The optimum working temperature and pH for Meiothermus sp. SK3-2 TreS was 65°C and pH 6.0, respectively. Ammonium chloride at 10 mM increased the activity significantly, while calcium chloride at 5 mM decreased the activity by about 80% and the activity wasfully retarded by 10 mM CaCl2. It was found that the product specificity of this TreS was influenced by factors like temperature, pH and buffer system used. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,890 bp which encoded a 963 amino acid protein. In conclusion, Meiothermus sp. SK3-2 TreS could serve as an alternative source to trehalose production.

Low reaction yields and the high cost of obtaining a single type of pure CD make γ-CD costly. Using rational design and with the aid of 3D modeling structures, recombinant CGTase from Bacillus sp. G1 was molecularly engineered with the aim of producing a higher percentage of γ-CD. A single mutation at subsite −3, denoted H43T, was found to increase γ-CD production from 10% to approximately 39% using tapioca starch. This novel increment was probably the result of reduced steric hindrance to the formation of γ-CD because of the shortened side chain together with the shortened loop at positions 86–89, at substrate-binding subsite −3. A mutation (Tyr188 → Trp) and a deletion at loop 139–144 showed little effect on product specificity; however, mutagenesis at these sites affected cyclization, coupling and hydrolysis activities as well as the kinetic properties of the mutant CGTase. Based on rational design, three further mutations of the mutant H43T (denoted H43T/Δ(139–144)/S134T/A137V/L138D/V139I, H43T/S85G and H43T/Y87F) were constructed and produced γ-CD with yields of 20%, 20% and 39%, respectively. The mutant H43T/Δ(139–144)/S134T/A137V/L138D/V139I had very low cyclization and coupling activities, however their hydrolysis activity was retained. Double mutation (H43T/S85G) caused the enzyme to exhibit higher starch hydrolysis activity, approximately 26 times higher than the native CGTase G1. Although the mutants H43T and H43T/Y87F could produce the same percentage (39%) of γ-CD, the latter was more efficient as the total amount of CD produced was higher based on the Vmax and kcat values.

The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.

The protein 3D structure for CGTase G1 was determined by homology modeling and a good structure was generated after three rounds of energy minimization process. The ERRAT and Verify3D scores for the predicted structure were determined as 98.07 and 90.991%, respectively. The presences of ionic interactions inside the CGTase G1 structure were compared with five CGTases crystal-structures. Mesophilic CGTases have lesser numbers of ionic pairs (average of 72.3 pairs) than of the thermophilic CGTases (average of 78.6 pairs). Most of the interactions in CGTases were involved in the form of networking. The average number for networking ionic pairs in thermostable and mesophilic CGTases is 69.3 and 62.7, respectively. Most of the ionic interactions in CGTases were found in Domain A and the most complex ionic networking was located in this catalytic domain as well. These charged-residues generate interlinking networking that covers a huge area that surrounds the active site groove. A few numbers of secondary structures strands were interlinked by the ionic interactions and this creates a natural protection for the catalytic TIM-barrel structure (Domain A) against heat. Most of the residues involved are consensus, however, slight variations were found. The triad Asp181-Arg185-Asp175 might plays an important factor in the networking which causes the half life of CGTase G1 to be slightly higher compared to other CGTases originally produced by mesophilic strains.

A novel mutant enzyme namely H43T CGTase can produce up to 39% γ-cyclodextrin (γ-CD) compared to the native enzyme which produces only 10% γ-CD. The effect of the reaction conditions on γ-CD production was studied using this mutant CGTase. The effects of substrate–buffer combination, starch pretreatment and concentration, pH, additives and finally the use of a debranching enzyme improved the γ-CD ratio further. The tapioca–acetate pair gave the highest conversion (16% conversion) among four types of starch and four buffer system combinations. Gelatinized starch was preferred compared to raw tapioca starch in producing a high percentage of γ-CD and conversion rate. Higher pH especially pH 8–9 led to a higher proportion of γ-CD, and was relatively more apparent when the concentration of starch was increased. Forty-six percent γ-CD was produced using 2.5% gelatinized tapioca starch at pH 8. Pullulanase enzyme was found to be useful in reducing the viscosity of tapioca starch paste thus increasing the efficiency of utilization of starch by CGTase by at least 20- to 30-fold. Up to 48% γ-CD can be produced when 4% pullulanase-pretreated tapioca starch was reacted with the CGTase mutant. It was also found that the supplementation of the reaction mixture with glucose, toluene, or cyclododecanone improved the γ-CD yield by 42.2, 46.4, 43.4, and 43.4%, respectively. All the parameters involved have been shown to affect the product specificity of the mutant H43T CGTase transglycosylation mechanism.

Anoxybacillus gonensis type strain G2(T) (=NCIMB 13,933(T) =NCCB 100040(T)) has been isolated from the Gönen hot springs in Turkey. This strain produces a number of well-studied, biotechnologically important enzymes, including xylose isomerase, carboxylesterase, and fructose-1,6-bisphosphate aldolase. In addition, this strain is an excellent candidate for the bioremediation of areas with heavy metal pollution. Here, we present a high-quality, annotated, complete genome of A. gonensis G2(T). Furthermore, this report provides insights into several novel enzymes of strain G2(T) and their potential industrial applications.

The α-amylases from Anoxybacillus species (ASKA and ADTA), Bacillus aquimaris (BaqA) and Geobacillus thermoleovorans (GTA, Pizzo and GtamyII) were proposed as a novel group of the α-amylase family GH13. An ASKA yielding a high percentage of maltose upon its reaction on starch was chosen as a model to study the residues responsible for the biochemical properties. Four residues from conserved sequence regions (CSRs) were thus selected, and the mutants F113V (CSR-I), Y187F and L189I (CSR-II) and A161D (CSR-V) were characterised. Few changes in the optimum reaction temperature and pH were observed for all mutants. Whereas the Y187F (t1/2 43 h) and L189I (t1/2 36 h) mutants had a lower thermostability at 65°C than the native ASKA (t1/2 48 h), the mutants F113V and A161D exhibited an improved t1/2 of 51 h and 53 h, respectively. Among the mutants, only the A161D had a specific activity, k(cat) and k(cat)/K(m) higher (1.23-, 1.17- and 2.88-times, respectively) than the values determined for the ASKA. The replacement of the Ala-161 in the CSR-V with an aspartic acid also caused a significant reduction in the ratio of maltose formed. This finding suggests the Ala-161 may contribute to the high maltose production of the ASKA.

Species of Anoxybacillus are widespread in geothermal springs, manure, and milk-processing plants. The genus is composed of 22 species and two subspecies, but the relationship between its lifestyle and genome is little understood. In this study, two high-quality draft genomes were generated from Anoxybacillus spp. SK3-4 and DT3-1, isolated from Malaysian hot springs. De novo assembly and annotation were performed, followed by comparative genome analysis with the complete genome of Anoxybacillus flavithermus WK1 and two additional draft genomes, of A. flavithermus TNO-09.006 and A. kamchatkensis G10. The genomes of Anoxybacillus spp. are among the smaller of the family Bacillaceae. Despite having smaller genomes, their essential genes related to lifestyle adaptations at elevated temperature, extreme pH, and protection against ultraviolet are complete. Due to the presence of various competence proteins, Anoxybacillus spp. SK3-4 and DT3-1 are able to take up foreign DNA fragments, and some of these transferred genes are important for the survival of the cells. The analysis of intact putative prophage genomes shows that they are highly diversified. Based on the genome analysis using SEED, many of the annotated sequences are involved in carbohydrate metabolism. The presence of glycosyl hydrolases among the Anoxybacillus spp. was compared, and the potential applications of these unexplored enzymes are suggested here. This is the first study that compares Anoxybacillus genomes from the aspect of lifestyle adaptations, the capacity for horizontal gene transfer, and carbohydrate metabolism.

An amylopullulanase of the thermophilic Anoxybacillus sp. SK3-4 (ApuASK) was purified to homogeneity and characterized. Though amylopullulanases larger than 200 kDa are rare, the molecular mass of purified ApuASK appears to be approximately 225 kDa, on both SDS-PAGE analyses and native-PAGE analyses. ApuASK was stable between pH 6.0 and pH 8.0 and exhibited optimal activity at pH 7.5. The optimal temperature for ApuASK enzyme activity was 60 °C, and it retained 54% of its total activity for 240 min at 65 °C. ApuASK reacts with pullulan, starch, glycogen, and dextrin, yielding glucose, maltose, and maltotriose. Interestingly, most of the previously described amylopullulanases are unable to produce glucose and maltose from these substrates. Thus, ApuASK is a novel, high molecular-mass amylopullulanase able to produce glucose, maltose, and maltotriose from pullulan and starch. Based on whole genome sequencing data, ApuASK appeared to be the largest protein present in Anoxybacillus sp. SK3-4. The α-amylase catalytic domain present in all of the amylase superfamily members is present in ApuASK, located between the cyclodextrin (CD)-pullulan-degrading N-terminus and the α-amylase catalytic C-terminus (amyC) domains. In addition, the existence of a S-layer homology (SLH) domain indicates that ApuASK might function as a cell-anchoring enzyme and be important for carbohydrate utilization in a streaming hot spring.

Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD–Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6–10. At 60°C in the absence of Ca2+, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca2+ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.