Design of thermostable rhamnogalacturonan lyase mutants from Bacillus licheniformis by combination of targeted single point mutations

Rhamnogalacturonan I lyases (RGI lyases) (EC 4.2.2.-) catalyze cleavage of α-1,4 bonds between rhamnose and galacturonic acid in the backbone of pectins by β-elimination. In the present study, targeted improvement of the thermostability of a PL family 11 RGI lyase from Bacillus licheniformis (DSM 13/ATCC14580) was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. These were selected by using a consensus approach together with assessing protein stability changes (PoPMuSiC) and B-factor iterative test (B-FIT). The second-generation mutants involved combinations of two to seven individually favorable single mutations. Thermal stability was examined as half-life at 60 °C and by recording of thermal transitions by circular dichroism. Surprisingly, the biggest increment in thermal stability was achieved by producing the wild-type RGI lyase in Bacillus subtilis as opposed to in Pichia pastoris; this effect is suggested to be a negative result of glycosylation of the P. pastoris expressed enzyme. A ~ twofold improvement in thermal stability at 60 °C, accompanied by less significant increases in T _m of the enzyme mutants, were obtained due to additive stabilizing effects of single amino acid mutations (E434L, G55V, and G326E) compared to the wild type. The crystal structure of the B. licheniformis wild-type RGI lyase was also determined; the structural analysis corroborated that especially mutation of charged amino acids to hydrophobic ones in surface-exposed loops produced favorable thermal stability effects.     

Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method

Good protein thermostability is very important for the protein application. In this report, we propose a strategy which contained a prediction method to select residues related to protein thermal stability, but not related to protein function, and an experiment method to screen the mutants with enhanced thermostability. The prediction strategy was based on the calculated site evolutionary entropy and unfolding free energy difference between the mutant and wild-type (WT) methyl parathion hydrolase enzyme from Ochrobactrum sp. M231 [Ochr-methyl parathion hydrolase (MPH)]. As a result, seven amino acid sites within Ochr-MPH were selected and used to construct seven saturation mutagenesis libraries. The results of screening these libraries indicated that six sites could result in mutated enzymes exhibiting better thermal stability than the WT enzyme. A stepwise evolutionary approach was designed to combine these selected mutants and a mutant with four point mutations (S274Q/T183E/K197L/S192M) was selected. The T _m and T ₅₀ of the mutant enzyme were 11.7 and 10.2 °C higher, respectively, than that of the WT enzyme. The success of this design methodology for Ochr-MPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering.     

The Effects of One Amino Acid Substitutions at the C-Terminal Region of Thermostable L2 Lipase by Computational and Experimental Approach

The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris–HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris–HCl pH 8, while for L2 lipase it was at 70 °C in Glycine–NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T _m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.     

Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods

Thermostability has been considered as a requirement in the starch processing industry to maintain high catalytic activity of pullulanase under high temperatures. Four data driven rational design methods (B-FITTER, proline theory, PoPMuSiC-2.1, and sequence consensus approach) were adopted to identify the key residue potential links with thermostability, and 39 residues of Bacillus acidopullulyticus pullulanase were chosen as mutagenesis targets. Single mutagenesis followed by combined mutagenesis resulted in the best mutant E518I-S662R-Q706P, which exhibited an 11-fold half-life improvement at 60 °C and a 9.5 °C increase in T_m. The optimum temperature of the mutant increased from 60 to 65 °C. Fluorescence spectroscopy results demonstrated that the tertiary structure of the mutant enzyme was more compact than that of the wild-type (WT) enzyme. Structural change analysis revealed that the increase in thermostability was most probably caused by a combination of lower stability free-energy and higher hydrophobicity of E518I, more hydrogen bonds of S662R, and higher rigidity of Q706P compared with the WT. The findings demonstrated the effectiveness of combined data-driven rational design approaches in engineering an industrial enzyme to improve thermostability.     

Engineering and introduction of <Emphasis Type="Italic">de novo</Emphasis> disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement

The organophosphorus hydrolase (OPH) has been used to degrade organophosphorus chemicals, as one of the most frequently used decontamination methods. Under chemical and thermal denaturing conditions, the enzyme has been shown to unfold. To utilize this enzyme in various applications, the thermal stability is of importance. The engineering of de novo disulphide bridges has been explored as a means to increase the thermal stability of enzymes in the rational method of protein engineering. In this study, Disulphide by Design software, homology modelling and molecular dynamics simulations were used to select appropriate amino acid pairs for the introduction of disulphide bridge to improve protein thermostability. The thermostability of the wild-type and three selected mutant enzymes were evaluated by half-life, ΔG inactivation (ΔGi) and structural studies (fluorescence and far-UV CD analysis). Data analysis showed that half-life of A204C/T234C and T128C/E153C mutants were increased up to 4 and 24 min, respectively; however, for the G74C/A78C mutant, the half-life was decreased up to 9 min. For the T128C/E124C mutant, both thermal stability and Catalytic efficiency (kcat) were also increased. The half-life and ΔGi results were correlated to the obtained information from structural studies by circular dichroism (CD) spectrometry and extrinsic fluorescence experiments; as rigidity increased in A204C/T2234C and T128C/E153C mutants, half-life and ΔGi also increased. For G74C/A78C mutant, these parameters decreased due to its higher flexibility. The results were submitted a strong evidence for the possibility to improve the thermostability of OPH enzyme by introducing a disulphide bridge after bioinformatics design, even though this design would not be always successful.     

Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy

As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t _1/2) inactivation at 65 °C, 18 °C increase in apparent T _m value, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.

     

Enhancement of thermostability of fungal deglycating enzymes by directed evolution

Fructosyl peptide oxidases are valuable for the determination of glycoproteins such as hemoglobin A1c. For practical use in clinical diagnosis, we applied directed evolution to improve the thermostability of these enzymes. After two rounds of random mutagenesis and high-throughput screening, six thermostabilizing amino acid substitutions were identified. Therefore, site-directed and cassette mutageneses were applied to combine these six stabilizing mutations. The simultaneous mutants showed that the stabilizing effect of the amino acid replacement was cumulative. The sextuple mutant enzyme, R94K/G184D/F265L/N272D/H302R/H388Y, had a half-life of thermal inactivation at 50°C that was 79.8-fold longer than that of the parental fructosyl peptide oxidase. The thermostable variants also showed increased tolerance to digestion by a protease. The sextuple mutant enzyme did not lose its activity on incubation with neutral protease, while the wild-type enzyme almost completely lost its activity. Furthermore, three amino acid substitutions were introduced into another fructosyl peptide oxidase with a different substrate specificity. The half-life of inactivation at 50°C was 3.61-fold longer than that of the parent enzyme. These engineered fructosyl peptide oxidases will be useful for industrial application to clinical diagnosis.     

Directed evolution of <Emphasis Type="Italic">Streptomyces lividans</Emphasis> xylanase B toward enhanced thermal and alkaline pH stability

The thermal and alkaline pH stability of Streptomyces lividans xylanase B was improved greatly by random mutagenesis using DNA shuffling. Positive clones with improved thermal stability in an alkaline buffer were screened on a solid agar plate containing RBB-xylan (blue). Three rounds of directed evolution resulted in the best mutant enzyme 3SlxB6 with a significantly improved stability. The recombinant enzyme exhibited significant thermostability at 70°C for 360 min, while the wild-type lost 50% of its activity after only 3 min. In addition, mutant enzyme 3SlxB6 shows increased stability to treatment with pH 9.0 alkaline buffer. The K _m value of 3SlxB6 was estimated to be similar to that of wild-type enzyme; however k _cat was slightly decreased, leading to a slightly reduced value of k _cat/K _m, compared with wild-type enzyme. DNA sequence analysis revealed that eight amino acid residues were changed in 3SlxB6 and substitutions included V3A, T6S, S23A, Q24P, M31L, S33P, G65A, and N93S. The stabilizing effects of each amino acid residue were investigated by incorporating mutations individually into wild-type enzyme. Our results suggest that DNA shuffling is an effective approach for simultaneous improvement of thermal and alkaline pH stability of Streptomyces lividans xylanase B even without structural information.     

Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase

A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4^G-β-d-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.     

Structure-based engineering of alkaline α-amylase from alkaliphilic Alkalimonas amylolytica for improved thermostability

This study aimed to improve the thermostability of alkaline α-amylase from Alkalimonas amylolytica through structure-based rational design and systems engineering of its catalytic domain. Separate engineering strategies were used to increase alkaline α-amylase thermostability: (1) replace histidine residues with leucine to stabilize the least similar region in domain B, (2) change residues (glycine, proline, and glutamine) to stabilize the highly conserved α-helices in domain A, and (3) decrease the free energy of folding predicted by the PoPMuSiC program to stabilize the overall protein structure. A total of 15 single-site mutants were obtained, and four mutants — H209L, Q226V, N302W, and P477V — showed enhanced thermostability. Combinational mutations were subsequently introduced, and the best mutant was triple mutant H209L/Q226V/P477V. Its half-life at 60 °C was 3.8-fold of that of the wild type and displayed a 3.2 °C increase in melting temperature compared with that of the wild type. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50 °C to 55 °C, the optimum pH shifted from 9.5 to 10.0, the stable pH range expanded from 7.0–11.0 to 6.0–12.0, the specific activity increased by 24 %, and the catalytic efficiency (k _cat/K _m) increased from 1.8×10⁴ to 3.5?×?10⁴ l/(g min). Finally, the mechanisms responsible for the increased thermostability were analyzed through comparative analysis of structure models. The structure-based rational design and systems engineering strategies in this study may also improve the thermostability of other industrial enzymes.     

Improved thermostability of a Bacillus subtilis esterase by domain exchange

A moderately thermostable esterase from Geobacillus stearothermophilus (BsteE) and its homolog from Bacillus subtilis (BsubE) show a high structural similarity with more than 95 % homology and 74 % amino acid identity. Interestingly, their thermal stability differs significantly by 30 °C in their melting temperature. In order to identify the positions that are responsible for this difference, most of the flexible amino acids assumed to confer instability were found to be in the cap region. For this reason, a 30 amino acid long cap domain fragment containing ten differing positions derived from BsteE was incorporated into the homologous gene encoding for the more labile BsubE by spliced overlap-extension PCR. The melting temperature of the two wild-type esterases and the mutant was evaluated by circular dichroism spectroscopy, while the kinetic parameters and the stability were determined with a photometric assay. The cap domain mutant maintained its activity, with a catalytic efficiency more similar to BsteE, while it exhibited an increase of the melting temperature by 4 °C compared to BsubE. Additional point mutations based on the differences of the parent enzymes gave a further increase of the thermostability up to 11 °C compared to BsubE; however, a significant reduction in activity was observed.     

Identification of critical residues for the activity and thermostability of Streptomyces sp. SK glucose isomerase

The role of residue 219 in the physicochemical properties of d-glucose isomerase from Streptomyces sp. SK strain (SKGI) was investigated by site-directed mutagenesis and structural studies. Mutants G219A, G219N, and G219F were generated and characterized. Comparative studies of their physicochemical properties with those of the wild-type enzyme highlighted that mutant G219A displayed increased specific activity and thermal stability compared to that of the wild-type enzyme, while for G219N and G219F, these properties were considerably decreased. A double mutant, SKGI F53L/G219A, displayed a higher optimal temperature and a higher catalytic efficiency than both the G219A mutant and the wild-type enzyme and showed a half-life time of about 150 min at 85 °C as compared to 50 min for wild-type SKGI. Crystal structures of SKGI wild-type and G219A enzymes were solved to 1.73 and 2.15 Å, respectively, and showed that the polypeptide chain folds into two structural domains. The larger domain consists of a (β/α)₈ unit, and the smaller domain forms a loop of α helices. Detailed analyses of the three-dimensional structures highlighted minor but important changes in the active site region as compared to that of the wild-type enzyme leading to a displacement of both metal ions, and in particular that in site M2. The structural analyses moreover revealed how the substitution of G219 by an alanine plays a crucial role in improving the thermostability of the mutant enzyme.     

Construction of a highly thermostable 1,3-1,4-β-glucanase by combinational mutagenesis and its potential application in the brewing industry

Objectives

To improve the thermostability and catalytic property of a mesophilic 1,3-1,4-β-glucanase by combinational mutagenesis and to test its effect in congress mashing.

Results

A mutant β-glucanase (rE-BglTO) constructed by combinational mutagenesis showed a 25 °C increase in optimal temperature (to 70 °C) a 19.5 °C rise in T ₅₀ value and a 15.6 °C increase in melting temperature compared to wild-type enzyme. Its half-life values at 60 and 70 °C were 152 and 99 min, which were 370 and 800 % higher than those of wild-type enzyme. Besides, its specific activity and k _cat value were 42,734 U mg^?1 and 189 s^?1 while its stability under acidic conditions was also improved. In flask fermentation, the catalytic activity of rE-BglTO reached 2381 U ml^?1, which was 63 % higher than that of wild-type enzyme. The addition of rE-BglTO in congress mashing decreased the filtration time and viscosity by 21.3 and 9.6 %, respectively.

Conclusions

The mutant β-glucanase showed high catalytic activity and thermostability which indicated that rE-BglTO is a good candidate for application in the brewing industry.

     

Involvement of cysteine 306 and alanine 63 in the thermostability and oligomeric organization of glucose isomerase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. SK

The implication of the original alanine 63 (Ala63) and the unique cysteine 306 (Cys306) residues in the thermostability of the Streptomyces sp. SK glucose isomerase (SKGI) were investigated by site-directed mutagenesis and homology modelling. The Cys306 to Ala mutation within SKGI dramatically affected its thermal stability by decreasing the half-life from 80 to 15 min at 90°C while the Ala63 to Ser replacement shifted this half-life to 65 min. The electrophoretic analysis proves that the residue Cys306 participates in oligomerization of the SKGI. Its stabilizing role is materialized by hydrogen bonds established with arginines at positions 284 and 259, as deduced from the constructed three-dimensional model. We have also shown that the presence of an Ala63 instead of Ser63 seems to be more suitable for enzyme thermostability by maintaining hydrophobic pocket that contributes to the protection of the enzyme active site.     

Statistical Optimization of Culture Variables for Enhancing Agarase Production by <Emphasis Type="Italic">Dendryphiella arenaria</Emphasis> Utilizing <Emphasis Type="Italic">Palisada perforata</Emphasis> (Rhodophyta) and Enzymatic Saccharification of the Macroalgal Biomass

Agarase is a promising biocatalyst for several industrial applications. Agarase production was evaluated by the marine fungus Dendryphiella arenaria utilizing Palisada perforata as a basal substrate in semi-solid state fermentation. Seaweed biomass, glucose, and sucrose were the most significant parameters affecting agarase production, and their levels were further optimized using Box-Behnken design. The maximum agarase activity was 7.69 U/mL. Agarase showed a degree of thermostability with half-life of 99 min at 40 °C, and declining to 44.72 min at 80 °C. Thermodynamics suggested an important process of protein aggregation during thermal inactivation. Additionally, the enzymatic saccharification of the seaweed biomass using crude agarase was optimized with respect to biomass particle size, solid/liquid ratio, and enzyme loadings. The amount of biosugars obtained after optimization was 26.15 ± 1.43 mg/g. To the best of our knowledge, this is the first report on optimization of agarase in D. arenaria.     

Enhancement of the thermal and alkaline pH stability of <Emphasis Type="Italic">Escherichia coli</Emphasis> lysine decarboxylase for efficient cadaverine production

Objective

To enhance the thermal and alkaline pH stability of the lysine decarboxylase from Escherichia coli (CadA) by engineering the decameric interface and explore its potential for industrial applications.

Results

The mutant T88S was designed for improved structural stability by computational analysis. The optimal pH and temperature of T88S were 7.0 and 55 °C (5.5 and 50 °C for wild-type). T88S showed higher thermostability with a 2.9-fold increase in the half-life at 70 °C (from 11 to 32 min) and increased melting temperature (from 76 to 78 °C). Additionally, the specific activity and pH stability (residual activity after 10 h incubation) of T88S at pH 8.0 were increased to 164 U/mg and 78% (58 U/mg and 57% for wild-type). The productivity of cadaverine with T88S (284 g l-lysine L^?1 and 5 g DCW L^?1) was 40 g L^?1 h^?1, in contrast to 28 g L^?1 h^?1 with wild-type.

Conclusion

The mutant T88S showed high thermostability, pH stability, and activity at alkaline pH, indicating that this mutant is a promising biocatalyst for industrial production of cadaverine.

     

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.     

An intermolecular disulfide bond is required for thermostability and thermoactivity of β-glycosidase from Thermococcus kodakarensis KOD1

Scientists are interested in understanding the molecular origin of protein thermostability and thermoactivity for possible biotechnological applications. The enzymes from extremophilic organisms have been of particular interest in the last two decades. β-glycosidase, Tkβgly is a hyperthermophilic enzyme from Thermococcus kodakarensis KOD1. Tkβgly contains two conserved cysteine residues, C88 and C376. The protein tertiary structure obtained through homology modeling suggests that the C88 residue is located on the surface whereas C376 is inside the protein. To study the role of these cysteine residues, we substituted C88 and C376 with serine residues through site-directed mutagenesis. The wild-type and C376S protein existed in dimeric form and C88S in monomeric form, in an SDS-PAGE gel under non-reducing conditions. Optimal temperature experiments revealed that the wild-type was active at 100 °C whereas the C88S mutant exhibited optimal activity at 70 °C. The half-life of the enzyme at 70 °C was drastically reduced from 266 h to less than 1 h. Although C88 was not present in the active site region, the k _cat/K _m of C88S was reduced by 2-fold. Based on the structural model and biochemical properties, we propose that C88 is crucial in maintaining the thermostability and thermoactivity of the Tkβgly enzyme.     

Stabilization of Escherichia coli uridine phosphorylase by evolution and immobilization

Mutation and immobilization techniques were applied to uridine phosphorylase (UP) from Escherichia coli in order to enhance its thermal stability and hence productivity in a biocatalytic reaction. UP was evolved by iterative saturation mutagenesis. Compared to the wild type enzyme, which had a temperature optimum of 40 °C and a half-life of 9.89 h at 60 °C, the selected mutant had a temperature optimum of 60 °C and a half-life of 17.3 h at 60 °C. Self-immobilization of the native UP as a Spherezyme showed a 3.3 fold increase in thermostability while immobilized mutant enzyme showed a 4.4 fold increase in thermostability when compared to native UP. Combining UP with the purine nucleoside phosphorylase from Bacillus halodurans allows for synthesis of 5-methyluridine (a pharmaceutical intermediate) from guanosine and thymine in a one-pot transglycosylation reaction. Replacing the wild type UP with the mutant allowed for an increase in reaction temperature to 65 °C and increased the reaction productivity from 10 to 31 g l⁻¹ h⁻¹.     

An alkali-tolerant xylanase produced by the newly isolated alkaliphilic <Emphasis Type="Italic">Bacillus pumilus</Emphasis> from paper mill effluent

An alkaline active xylanase, XynBYG, was purified from an alkaliphilic Bacillus pumilus BYG, which was newly isolated from paper mill effluent. It had an optimum pH of 8.0–9.0, and showed good stability after incubated at pH 9.0 for 120 min. The optimum temperature for the activity was 50°C, and the enzyme retained below 55% of its original activity for 30 min at 55°C. The gene coding for XynBYG consists of 687 bp and encodes 229 amino acids. Similarity analysis indicated that XynBYG belong to family 11 glycosyl hydrolases. Site-directed mutagenesis was performed to replace five sites (Tyr/Ser) to Arg/Glu and the results demonstrated that the optimum temperature of the mutant Y7 (S39R-T146E) increased 5°C and the half-life of inactivation (T1/2) at 60 and 65°C was 1 h and 25 min, respectively. Thus, it provides a potential xylanase that can meet the harsh conditions in the industrial applications.