Halothermothrix Orenii Research Articles

Halonatronomonas betaini gen. nov., sp. nov., a haloalkaliphilic isolate from soda lake capable of betaine degradation and proposal of Halarsenatibacteraceae fam. nov. and Halothermotrichaceae fam. nov. within the order Halanaerobiales

A search for the organisms responsible for anaerobic betaine degradation in soda lakes resulted in isolation of a novel bacterial strain, designated Z-7014T. The cells were Gram-stain-negative, non-endospore-forming rods. Growth occurred at 8–52 °C (optimum 40–45 °C), pH 7.1–10.1 (optimum pH 8.1–8.8) and 1.0–3.5 M Na+ (optimum 1.8 M), i.e. it can be regarded as a haloalkaliphile. The strain utilized a limited range of substrates, mostly peptonaceous but not amino acids, and was able to degrade betaine. Growth on betaine occurred only in the presence of peptonaceous substances which could not be replaced by vitamins. The G + C content of the genomic DNA of strain Z-7014T was 36.1 mol%. The major cellular fatty acids (>5% of the total) were C16:0 DMA, C18: 0 DMA, C16:1ω8, C16:0, C18:1 DMA, C16:1 DMA, C18:1ω9, and C18:0. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain Z-7014T formed a distinct evolutionary lineage in the order Halanaerobiales with the highest similarity to Halarsenitibacter silvermanii SLAS-1T (83.6%), Halothermothrix orenii H168T (85.6%), and Halocella cellulosilytica DSM 7362T (85.6%). AAI and POCP values between strain Z-7014T and type strains of the order Halanaerobiales were 51.7–57.8%, and 33.8–58.3%, respectively. Based on polyphasic results including phylogenomic data, the novel strain could be distinguished from other genera, which suggests that strain Z-7014T represents a novel species of a new genus, for which the name Halonatronomonas betaini gen. nov., sp. nov. is proposed. The type strain is Z-7014T (=KCTC 25237T = VKM B-3506T). On the basis of phylogenomic data, it is also proposed to evolve two novel families Halarsenitibacteraceae fam. nov. and Halothermotrichaceae fam. nov. within the current order Halanaerobiales.

Glycosyl benzoates as novel substrates for glycosynthases.

The development of a procedure for the one-pot synthesis of glycosyl benzoates directly from unprotected sugars in aqueous media using 2-chloro-1,3-dimethylimidazolium chloride (DMC), thiobenzoic acid, and triethylamine is reported. These glycosyl donors are excellent substrates for wild-type and mutant glycosidases. β-Glucosyl benzoate was hydrolysed by the GH1 β-glucosidase derived from Halothermothrix orenii (HorGH1). Subsequent use of this substrate in thioligase-mediated glycosylation of p-nitrothiophenol demonstrated their superiority as donors compared to their p-nitrophenol counterparts with excellent conversions. Using a series of arene nucleophiles, we also demonstrate good to excellent conversions (up to 94%) of β-glucosyl benzoate to the corresponding p-nitrophenyl- and thioglycosides.

Characterization of a recombinant arginine deiminase from Halothermothrix orenii and its application in citrulline production.

In recent years, arginine deiminase (ADI, EC 3.5.3.6) has attracted much attention as a biocatalyst that produces the functional amino acid l-citrulline from l-arginine and also as an anticancer enzyme. Here, we identified and characterized a putative ADI from the thermophilic bacterium Halothermothrix orenii. The H. orenii ADI (H-ADI) protein was expressed in Escherichia coli BL21(DE3) with a specific activity of 91.8 U/mg protein at 55°C and pH 6.5. The enzyme remained at 74% relative activity after incubation at 45°C for 180 min, only 25% at 50°C. The melting temperature was 56°C. H-ADI is not a metal-requiring enzyme; Ni2+ slightly improved the catalytic activity. The Km and Vmax for l-arginine were 55.5 mM and 156.8 μmol/min/mg protein, respectively. Moreover, three residues (Arg183, Arg237, and His273) were key to the formation of l-citrulline, as analyzed by alanine-scanning mutagenesis. Finally, the enzymatic synthesis of l-citrulline was carried out at 50°C with a conversion ratio reaching 99.03%. Together, these findings show that H-ADI is a promising biocatalyst for the production of l-citrulline.

Simple sequence repeat insertion induced stability and potential 'gain of function' in the proteins of extremophilic bacteria.

Here, we analysed the genomic evolution in extremophilic bacteria using long simple sequence repeats (SSRs). Frequencies of occurrence, relative abundance (RA) and relative density (RD) of long SSRs were analysed in the genomes of extremophilic bacteria. Thermus aquaticus had the most RA and RD of long SSRs in its coding sequences (110.6 and 1408.3), followed by Rhodoferax antarcticus (77.0 and 1187.4). A positive correlation was observed between G + C content and the RA-RD of long SSRs. Geobacillus kaustophilus, Geobacillus thermoleovorans, Halothermothrix orenii, R. antarcticus, and T. aquaticus preferred trinucleotide repeats within their genomes, whereas others preferred a higher number of tetranucleotide repeats. Gene enrichment showed the presence of these long SSRs in metabolic enzyme encoding genes related to stress tolerance. To analyse the functional implications of SSR insertions, three-dimensional protein structure modelling of SSR containing diguanylate cyclase (DGC) gene encoding protein was carried out. Removal of SSR sequence led to an inappropriate folding and instability of the modelled protein structure.

L-Fucose Synthesis Using a Halo- and Thermophilic l-Fucose Isomerase from Polyextremophilic Halothermothrix orenii

l-Fucose isomerase (l-FucI)-mediated isomerization is a promising biotechnological approach for synthesizing various rare sugars of industrial significance, including l-fucose. Extremozymes that can retain their functional conformation under extreme conditions, such as high temperature and salinity, offer favorable applications in bioprocesses that accompany harsh conditions. To date, only one thermophilic l-FucI has been characterized for l-fucose synthesis. Here, we report l-FucI from Halothermothrix orenii (HoFucI) which exhibits both halophilic and thermophilic properties. When evaluated under various biochemical conditions, HoFucI exhibited optimal activities at 50–60 °C and pH 7 with 0.5–1 M NaCl in the presence of 1 mM Mn2+ as a cofactor. The results obtained here show a unique feature of HoFucI as a polyextremozyme, which facilitates the biotechnological production of l-fucose using this enzyme.

In Silico Analysis of Fungal and Chloride-Dependent α-Amylases within the Family GH13 with Identification of Possible Secondary Surface-Binding Sites.

This study brings a detailed bioinformatics analysis of fungal and chloride-dependent α-amylases from the family GH13. Overall, 268 α-amylase sequences were retrieved from subfamilies GH13_1 (39 sequences), GH13_5 (35 sequences), GH13_15 (28 sequences), GH13_24 (23 sequences), GH13_32 (140 sequences) and GH13_42 (3 sequences). Eight conserved sequence regions (CSRs) characteristic for the family GH13 were identified in all sequences and respective sequence logos were analysed in an effort to identify unique sequence features of each subfamily. The main emphasis was given on the subfamily GH13_32 since it contains both fungal α-amylases and their bacterial chloride-activated counterparts. In addition to in silico analysis focused on eventual ability to bind the chloride anion, the property typical mainly for animal α-amylases from subfamilies GH13_15 and GH13_24, attention has been paid also to the potential presence of the so-called secondary surface-binding sites (SBSs) identified in complexed crystal structures of some particular α-amylases from the studied subfamilies. As template enzymes with already experimentally determined SBSs, the α-amylases from Aspergillus niger (GH13_1), Bacillus halmapalus, Bacillus paralicheniformis and Halothermothrix orenii (all from GH13_5) and Homo sapiens (saliva; GH13_24) were used. Evolutionary relationships between GH13 fungal and chloride-dependent α-amylases were demonstrated by two evolutionary trees—one based on the alignment of the segment of sequences spanning almost the entire catalytic TIM-barrel domain and the other one based on the alignment of eight extracted CSRs. Although both trees demonstrated similar results in terms of a closer evolutionary relatedness of subfamilies GH13_1 with GH13_42 including in a wider sense also the subfamily GH13_5 as well as for subfamilies GH13_32, GH13_15 and GH13_24, some subtle differences in clustering of particular α-amylases may nevertheless be observed.

Probing the dynamics between the substrate and the product towards glucose tolerance of Halothermothrix orenii β-glucosidase

Most β-Glucosidase (B8CYA8) are prone to inhibition by glucose. Experimentally observed specific activity of B8CYA8 on 20 mM, 50 mM, and 100 mM p-nitrophenyl-β-D-glucopyranoside (pNPGlc) substrate concentrations show surprise dependence on the presence of 0–3 M glucose at 335 K. We found that at high substrate concentration, the enzyme shows stimulation in specific activity with glucose and the glucose inhibition curve shifts toward the right with the increase in the substrate concentration. We employed atomistic molecular dynamics simulations of β-Glucosidase from Halothermothrix orenii at different glucose and pNPGlc concentrations to provide microscopic explanations to the experimentally observed non-monotonic glucose concentration dependence of the enzyme activity. Our results show that accumulation of substrate (pNPGlc) near the B8CYA8 catalytic site residues E166 and E354 and in the active site tunnel increases up to 0.5 M glucose when the specific activity is the highest. The number of pNPGlc in the tunnel decreases drastically when glucose concentration is more than 0.5 M, and hence the specific activity decreases. Potential of mean force (PMF) calculations showed that the most favorable interaction between pNPGlc and β-Glucosidase exists at 0.5 M glucose while at deficient and high glucose concentrations, the binding energy between the substrate and β-Glucosidase is very low. These studies provide the molecular basis towards understanding inhibition and stimulation of β-Glucosidase activity in the presence of glucose and may enable the optimum use of enzymes for the efficient conversion of high biomass loading saccharification reactions. Communicated by Ramaswamy H. Sarma

In Silico Study and Optimization of Bacillus megaterium alpha-Amylases Production Obtained from Honey Sources.

This study aimed to screen alpha-amylase producing microorganisms from honey as a low water activity medium, a suitable source for selecting stable and cost-beneficial bacterial enzyme production systems. Plackett-Burman method was used to select twelve effective factors including pH, inoculum size, temperature, time, corn starch, KH2PO4, peptone, MgSO4, CaCl2, NaCl, glycerin, and yeast extract concentrations on bacterial alpha-amylases production yield. The Box-Behnken method was utilized to optimize the level of selected significant factors. The stability of bacterial alpha-amylases was also determined in low pH and high-temperature conditions. In addition, in silico study was used to create the alpha-amylase structure and study the stability in high-temperature and low water available condition. Among all isolated and characterized microorganisms, Bacillus megaterium produced the highest amount of alpha-amylases. The in silico data showed the enzyme 3D structure similarity to alpha-amylase from Halothermothrix orenii and highly negative charge amino acids on its surface caused the enzyme activity and stability in low water conditions. Based on Box-Behnken results, the temperature 35 °C, pH 6 and starch 40g/l were determined as the optimum level of significant factors to achieve the highest alpha-amylases unit (101.44 U/ml). This bacterial alpha-amylases enzyme showed stability at pH 5 and a range of temperatures from 40 to 60 °C that indicates this enzyme may possess the potential for using in industrial processes.

Elucidating the regulation of glucose tolerance in a β-glucosidase from Halothermothrix orenii by active site pocket engineering and computational analysis

β-Glucosidase catalyzes the hydrolysis of β-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of β-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with β-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity on p-nitrophenyl D-glucopyranoside stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the β-glucosidase at lower concentrations of glucose that, however, does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of β-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

Engineering novel S-glycosidase activity into extremo-adapted β-glucosidase by rational design.

The breakdown of sulphur glycosidic bonds in thioglycosides can produce isothiocyanate, a chemoprotective agent linked to the prevention of cancers; however, only a handful of enzymes have been identified that are k0nown to catalyse this reaction. Structural studies of the myrosinase enzyme, which is capable of hydrolysing the thioglycosidic bond, have identified residues that may play important roles in sulphur bond specific activity. Using rational design, two extremo-adapted β-glycosidases from the species Thermus nonproteolyticus (TnoGH1) and Halothermothrix orenii (HorGH1) were engineered towards thioglycoside substrates. Twelve variants, six for TnoGH1and six for HorGH1, were assayed for activity. Remarkable enhancement of the specificity (kcat/KM) of TnoGH1 and HorGH1 towards β-thioglycoside was observed in the single mutants TnoGH1-V287R (2500M-1s-1) and HorGH1-M229R (13,260M-1s-1) which showed a 3-fold increase with no loss in turnover rate when compared with the wild-type enzymes. Thus, the role of arginine is key to induce β-thioglycosidase activity. Thorough kinetic investigation of the different mutants has shed light on the mechanism of β-glycosidases when acting on the native substrate.Key Points •Key residues were identified in the active site of Brevicoryne brassicae myrosinase. •Rationally designed mutations were introduced into two extremo-adapted β-glycosidases. •β-glycosidases mutants exhibited improved activity against thioglycosidic bonds. •The mutation to arginine in the active site yielded the best variant.

The effect of ionic liquid on the structure of active site pocket and catalytic activity of a β-glucosidase from Halothermothrix orenii

Abstract Understanding the inhibition of cellulase activity in the presence of ionic liquids (IL) is extremely important towards engineering IL tolerant enzymes for biomass saccharification and subsequent biofuel production. Here we report the all-atom molecular dynamics simulations of a highly active and thermostable GH1 β-glucosidase (EC 3.2.1.21) B8CYA8 from Halothermothrix orenii in the presence of the IL, 1-ethyl-3-methylimidazolium chloride ([EMIM][Cl]). In the presence of IL, we observed a lack of destabilization of the overall protein structure but instead a localized increase in fluctuations of active site pocket residues corresponding to the loop region, gatekeeper region, aglycone binding site, and glycone binding site. The role of specific residues was confirmed by the Principal Component Analysis (PCA) and Chemical Shift Perturbation (CSP) analyses. Since the enzymatic saccharification requires the proper arrangement of catalytic side chains, residue fluctuation within the active site tunnel affects the reaction rate. Indeed, while B8CYA8 exhibits very high IL tolerance, high concentrations of IL inhibit the enzyme, our analyses show that in high concentrations of IL, the IL accumulates at the active site pocket entrance and near the active site catalytic residues to restrict substrate accessibility to the catalytic site and decreases enzyme activity. Unlike the reaction product glucose, the IL does not change the overall tunnel diameter to constrict substrate access. To understand how IL viscosity may affect enzyme behavior, we determined the self-diffusion constant and show that the presence of IL has a significant effect on substrate access to the catalytic site due to the low diffusivity of the substrate in IL. These results highlight the role played by the residues in active site pocket and the IL properties and may guide in the design of better protein and IL pairs for efficient catalysis.

Probing the Effect of Glucose on the Activity and Stability of β-Glucosidase: An All-Atom Molecular Dynamics Simulation Investigation

β-Glucosidase (EC 3.2.1.21) plays an essential role in the removal of glycosyl residues from disaccharide cellobiose to produce glucose during the hydrolysis of lignocellulosic biomass. Although there exist a few β-glucosidase that are tolerant to large concentrations of glucose, these enzymes are typically prone to glucose inhibition. Understanding the basis of this inhibition is important for the production of cheaper biofuels from lignocellulose. In this study, all-atom molecular dynamics simulation at different temperatures and glucose concentrations was used to understand the molecular basis of glucose inhibition of GH1 β-glucosidase (B8CYA8) from Halothermothrix orenii. Our results show that glucose induces a broadening of the active site tunnel through residues lining the tunnel and facilitates the accumulation of glucose. In particular, we observed that glucose accumulates at the tunnel entrance and near the catalytic sites to block substrate accessibility and inhibit enzyme activity. The reduction of enzyme activity was also confirmed experimentally through specific activity measurements in the presence of 0–2.5 M glucose. We also show that the increase in glucose concentrations leads to a decrease in the number of water molecules inside the tunnel to affect substrate hydrolysis. Overall, the results help in understanding the role of residues along the active site tunnel for the engineering of glucose-tolerant β-glucosidase.

Recyclable Thermoresponsive Polymer-β-Glucosidase Conjugate with Intact Hydrolysis Activity.

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose and is a rate-limiting enzyme in the conversion of lignocellulosic biomass to sugars toward biofuels. Since the cost of enzyme is a major contributor to biofuel economics, we report the bioconjugation of a temperature-responsive polymer with the highly active thermophilic β-glucosidase (B8CYA8) from Halothermothrix orenii toward improving enzyme recyclability. The bioconjugate, with a lower critical solution temperature (LCST) of 33 °C withstands high temperatures up to 70 °C. Though the secondary structure of the enzyme in the conjugate is slightly distorted with a higher percentage of β-sheet like structure, the stability and specific activity of B8CYA8 in the conjugate remains unaltered up to 30 °C and retains more than 70% specific activity of the unmodified enzyme at 70 °C. The conjugate can be reused for β-glucosidic bond cleavage of cellobiose for at least four cycles without any significant loss in specific activity.

The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View.

Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including α-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, α-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM) and the surface-binding site (SBS) of α-amylase could facilitate the substrate binding to the enzyme's active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera α-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various α-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in α-amylase to increase its catalytic activity, especially towards the insoluble starch.

Exploiting non-conserved residues to improve activity and stability of Halothermothrix orenii β-glucosidase.

β-glucosidase (EC 3.2.1.21; BG) cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules. In an effort towards designing better BGs, we focused on the role of non-conserved residues across an otherwise homologous BG active site tunnel and designed mutants across the aglycone-binding site (V169C) and the gatekeeper residues (I246A) of the active site tunnel. We expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase (BG) in Halothermothrix orenii. The overexpressed and purified wild-type (B8CYA8) has a high specific activity of 345μmol/min/mg on pNPGlc and a half-life of 1.13h when assayed with pNPGlc at pH 7.1 and 70°C. The specific activities of V169C and I246A were 1.7 and 1.2 times higher than that of wild-type (WT) enzyme with the model substrate pNPGlc, while the activity on the natural substrate cellobiose was slightly higher to the WT. The two mutants were kinetically stable with 4.4- to 11-fold longer half-life compared to the WT enzyme. When the two mutations were combined to generate the V169C/I246A mutant, the specific activity increased to nearly twofold higher than WT on both substrates and the half-life increased fivefold. The two single mutants also show enhanced saccharification of insoluble natural biomass on supplementation of Trichoderma viride cellulase cocktail. These enhanced properties suggest the need for a closer look at the active site tunnel of these enzymes, especially across residues that are not conserved towards improving catalytic efficiencies.

Engineering a thermostable Halothermothrix orenii β-glucosidase for improved galacto-oligosaccharide synthesis.

Lactose is produced in large amounts as a by-product from the dairy industry. This inexpensive disaccharide can be converted to more useful value-added products such as galacto-oligosaccharides (GOSs) by transgalactosylation reactions with retaining β-galactosidases (BGALs) being normally used for this purpose. Hydrolysis is always competing with the transglycosylation reaction, and hence, the yields of GOSs can be too low for industrial use. We have reported that a β-glucosidase from Halothermothrix orenii (HoBGLA) shows promising characteristics for lactose conversion and GOS synthesis. Here, we engineered HoBGLA to investigate the possibility to further improve lactose conversion and GOS production. Five variants that targeted the glycone (−1) and aglycone (+1) subsites (N222F, N294T, F417S, F417Y, and Y296F) were designed and expressed. All variants show significantly impaired catalytic activity with cellobiose and lactose as substrates. Particularly, F417S is hydrolytically crippled with cellobiose as substrate with a 1000-fold decrease in apparent kcat, but to a lesser extent affected when catalyzing hydrolysis of lactose (47-fold lower kcat). This large selective effect on cellobiose hydrolysis is manifested as a change in substrate selectivity from cellobiose to lactose. The least affected variant is F417Y, which retains the capacity to hydrolyze both cellobiose and lactose with the same relative substrate selectivity as the wild type, but with ~10-fold lower turnover numbers. Thin-layer chromatography results show that this effect is accompanied by synthesis of a particular GOS product in higher yields by Y296F and F417S compared with the other variants, whereas the variant F417Y produces a higher yield of total GOSs.

Biochemical and structural characterization of a thermostable β-glucosidase from Halothermothrix orenii for galacto-oligosaccharide synthesis.

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial β-glucosidases (BGL) with significant β-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galacto-oligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific β-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5–7.5), with maximal β-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65–70 °C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from HoBGLA transgalactosylation are β-d-Galp-(1→6)-d-Lac (6GALA) and β-d-Galp-(1→3)-d-Lac (3GALA), indicating that d-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-d-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type HoBGLA and shown to be favorably accommodated.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-6015-x) contains supplementary material, which is available to authorized users.

Improvement of thermal stability of a mutagenised α-amylase by manipulation of the calcium-binding site

Site-directed mutagenesis of an α-amylase isolated from Bacillus megaterium WHO has been performed to evaluate the roles of the calcium binding site residues in enzyme thermostability. The strategy used was to replace residues in the hypothetical calcium binding loops of B. megaterium WHO α-amylase (BMW-amylase) by equivalent positions at Halothermothrix orenii α-amylase (AmyA) as a thermophilic amylase by QuikChange site directed mutagenesis. Asn-75, Ser-76, and His-77 were mutated in the second calcium binding site which resulted in an increase in thermostability. All mutants retained their hydrolytic activity although their kcat parameter decreased in compare to the wild type and in the presence of calcium ions. In S76P and H77E, the Km for starch decreases while overall activity (kcat/Km) was increased. In the presence of calcium, conversion of His-77 to Glu resulted in a 4-fold enhancement in enzyme half life and a 9°C upward shift in T50, which was observed in compare to the wild type. Further analysis suggested the H77E mutant as the most stable which increased the affinity of the enzyme for calcium ion and its optimum temperature was 5°C higher than the wild type.

Tracing the evolution of the α-amylase subfamily GH13_36 covering the amylolytic enzymes intermediate between oligo-1,6-glucosidases and neopullulanases

Among the glycoside hydrolases (GHs) classified within the Carbohydrate-Active enZymes (CAZy) server, the α-amylase family GH13 belongs to the largest GH families. It has been divided into the official 36 subfamilies by the CAZy curators. Originally the subfamilies of oligo-1,6-glucosidase and neopullulanase were defined using the sequence of the fifth conserved sequence region (CSR) as a selection marker. It is localized outside the catalytic α-amylase (β/α)8-barrel in the domain B, that is, in a longer loop connecting the strand β3 with the helix α3 of the barrel. It is sequentially positioned 26–28 residues in front of the invariant aspartic acid residue in the β4-strand acting as the GH13 catalytic nucleophile. The CSR V is characteristic as QpDln and MpKln for the former and latter subfamilies, respectively. A group of intermediate sequences possessing the CSR V as a mix of the two above-mentioned subfamilies, that is, MpDln, was also proposed previously. The present bioinformatics analysis was done in an effort to reveal as many as possible GH13 members of this intermediary group, currently classified as the subfamily GH13_36, and to discuss their evolutionary relationships to known GH13 specificities as well as with regard to their taxonomic origin. Using the BLAST tool with the sequence of the α-amylase from Halothermothrix orenii AmyA exhibiting the intermediary features, 152 GH13 enzymes, and hypothetical proteins were retrieved covering defined specificities (GH13 subfamilies 4, 16, 17, 18, 20, 21, 23, 29, 30, 31, 34, and 35) and intermediary enzymes and proteins (GH13_36). In both evolutionary trees—based on the alignment of CSRs and complete sequences—most of the ‘intermediary’ proteins (i.e., those with MPDLN signature) were positioned in several closely related clusters forming, however, a single GH13_36 large part of the trees. A few novel GH13 subfamilies were proposed as well as the specificity implications were discussed based on the presented in silico analysis. The results may also be helpful in assigning any GH13-like amino acid sequence the subfamily GH13_36 affiliation without additional biochemical characterization.

A highly thermoactive and salt-tolerant α-amylase isolated from a pilot-plant biogas reactor

Aiming at the isolation of novel enzymes from previously uncultured thermophilic microorganisms, a metagenome library was constructed from DNA isolated from a pilot-plant biogas reactor operating at 55 °C. The library was screened for starch-degrading enzymes, and one active clone was found. An open reading frame of 1,461 bp encoding an α-amylase from an uncultured organism was identified. The amy13A gene was cloned in Escherichia coli, resulting in high-level expression of the recombinant amylase. The novel enzyme Amy13A showed the highest sequence identity (75 %) to α-amylases from Petrotoga mobilis and Halothermothrix orenii. Amy13A is highly thermoactive, exhibiting optimal activity at 80 °C, and it is also highly salt-tolerant, being active in 25 % (w/v) NaCl. Amy13A is one of the few enzymes that tolerate high concentrations of salt and elevated temperatures, making it a potential candidate for starch processing under extreme conditions.