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Alkaliphiles are considered more suitable chassis than traditional neutrophiles due to their excellent resistance to microbial contamination. Alkaliphilic Bacillus sp. N16-5, an industrially interesting strain with great potential for the production of lactic acid and alkaline polysaccharide hydrolases, can only be engineered genetically by the laborious and time-consuming homologous recombination. In this study, we reported the successful development of a CRISPR/Cas9-based genome editing system with high efficiency for single-gene deletion, large gene fragment deletion and exogenous DNA chromosomal insertion. Moreover, based on a catalytically dead variant of Cas9 (dCas9), we also developed a CRISPRi system to efficiently regulate gene expression. Finally, this efficient genome editing system was successfully applied to engineer the xylose metabolic pathway for the efficient bioproduction of D-lactic acid. Compared with the wild-type Bacillus sp. N16-5, the final engineered strain with XylR deletion and AraE overexpression achieved 34.3% and 27.7% increases in xylose consumption and D-lactic acid production respectively. To our knowledge, this is the first report on the development and application of CRISPR/Cas9-based genome editing system in alkaliphilic Bacillus, and this study will significantly facilitate functional genomic studies and genome manipulation in alkaliphilic Bacillus, laying a foundation for the development of more robust microbial chassis., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Thermo-alkaline xylanases are widely applied in paper pulping industry. In this study, a novel thermostable and alkaline tolerant GH10 xylanase (Xyn30Y5) gene from alkaliphilic Bacillus sp. 30Y5 was cloned and the surface-layer homology (SLH) domains truncated enzyme (Xyn30Y5-SLH) was expressed in Escherichia coli. The purified Xyn30Y5-SLH was most active at 70 °C and pH 7.0 and showed the highest specific activity of 349.4 U mg -1 . It retained more than 90% activity between pH 6.0 to 9.5 and was stable at pH 6.0-10.0. To improve the activity, 47 mutants were designed based on eight rational strategies and 21 mutants showed higher activity. By combinatorial mutagenesis, the best mutant 3B demonstrated specific activity of 1016.8 U mg -1 with a doubled catalytic efficiency (k cat /K m ) and RA60 1/2h value, accompanied by optimal pH shift to 8.0. The molecular dynamics simulation analysis indicated that the increase of flexibility of α5 helix and loop7 located near to the catalytic residues is likely responsible for its activity improvement. And the decrease of flexibility of the most unstable regions is vital for the thermostablity improvement. This work provided not only a novel thermostable and alkaline tolerant xylanase with industrial application potential but also an effective mutagenesis strategy for xylanase activity improvement., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pullulanase with high catalytic efficiency has attracted great attention from both the academic and industrial communities for its wide application in cold starch hydrolysis. A novel pullulanase gene pul703 was cloned from a mesophilic bacteria Bacillus pseudofirmus 703. Pul703 was characterized to be a type I pullulanase with maximal activity at 45°C and good low-temperature stability, more than 70% of activity was detected after incubation at 25-35°C for 72h. Pul703 obtained the maximal activity around pH 7.0-8.0, and was highly active and stable over a wide pH range of 5.5-9.5, more than 80% of activity was retained after 12h incubation in these pHs. Pul703 was EDTA-resistant and detergent-tolerant, with a relative activity of 100, 99, and 114.8% at the presence of 10mM EDTA, 10% of Triton X-100 and Tween 20, respectively. Pul703 can efficiently hydrolyze pullulan with a specific activity of 270U/mg, which was higher than all reported type I pullulanases. In addition, Pul703 can act synergistically with α-amylase BLA to efficiently hydrolyze amylopectin. These results suggested that Pul703 was a good candidate for cold starch hydrolysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bacillus sp. N16-5 is an alkaliphile with a great ability to utilize mannan. Its mannan utilization gene cluster has been identified in a previous study. The ManR protein encoded by the cluster was predicted to be a LacI family regulator, and the transcription level of the mannan utilization gene cluster was upregulated after the manR gene was deleted, indicating that ManR is the repressor of this cluster. The transcription of the related genes was downregulated when manH, encoding the extracellular substrate-binding domain of the manno-oligosaccharide transporter, was deleted. Furthermore, isothermal titration calorimetry revealed that mannotetraose and mannopentose are ligands of ManR. These results all corroborate the hypothesis that the mannan utilization gene cluster is repressed by the transcription regulator ManR, and that the repression is removed when it binds to manno-oligosaccharides, which are generated by mannan degradation and transported into the cell by a specific transporter., (© FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Debranching of (1, 6)-α-linkages in starch is of great significance as it is widely used in different industries. In recent years, identifying a single potential enzyme that could debranch (1, 6)-α-linkages and cleave (1, 4)-α-linkages brought great interest as amylopectin possessed both (1, 4)-α- and (1, 6)-α-linkages. In the present study, a 64\,kDa exo-α-l, 4-glucosidase from Bacillus pseudofirmus 703, annotated as Amy112, was cloned and characterized. Biochemical analysis indicated that heterologous Amy112 expressed in Escherichia coli exhibited a high activity against amylopectin, with the optimal temperature and pH of 40°C and pH 7.0, respectively. Addition of K + ions improved the amy112 activity by 12%, but Li + , Ca 2+ and Mg 2+ ions showed no significant effect. Amy112 sequence homology revealed that it belonged to glycoside hydrolase family 13, showing 65% identity with α-glucosidase GSJ from Geobacillus sp. HTA-462. This is the first report indicating that Amy112 from B. pseudofirmus 703 belongs to GH13 enzyme family, having both exo-α-1, 4-glucosidase and oligo-l, 6-glucosidase activities. However, no transglycosylation activity was detected in LC-MS analysis. Amy112 would be of great significance of being utilized to debranch starch in different industries in a cost effective manner., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The chitosanase from Bacillus sp. TS (CsnTS) is an enzyme belonging to the glycoside hydrolase family 8. The sequence of CsnTS shares 98 % identity with the chitosanase from Bacillus sp. K17. Crystallography analysis and site-direct mutagenesis of the chitosanase from Bacillus sp. K17 identified the important residues involved in the catalytic interaction and substrate binding. However, despite progress in understanding the catalytic mechanism of the chitosanase from the family GH8, the functional roles of some residues that are highly conserved throughout this family have not been fully elucidated. This study focused on one of these residues, i.e., the aspartic acid residue at position 318. We found that apart from asparagine, mutation of Asp318 resulted in significant loss of enzyme activity. In-depth investigations showed that mutation of this residue not only impaired enzymatic activity but also affected substrate binding. Taken together, our results showed that Asp318 plays an important role in CsnTS activity.

To improve the extracellular production of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K m value (11 mg/mL) of 32a-ManAR2 was higher than that (1.46 mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k cat/K m value of 602.7 ml/mg s. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25 °C), lower IPTG concentration (0.01-0.05 mM), and Triton X-100 supplement (0.1 %) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10 L fermentor. The productivity of the total soluble mannanase reached 9284.64 U/mL with the extracellular rate of 74 % at 46 h of fermentation, which was the highest productive level of alkaline β-mannanase in recombinant E. coli to date.

Background: Bacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron-sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene., Results: Bacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP(+)-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300% compared with the wild-type strain., Conclusions: IspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.

High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase's properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca(2+) were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center.

Background: Polylactic acid (PLA) is one important chemical building block that is well known as a biodegradable and a biocompatible plastic. The traditional lactate fermentation processes need CaCO3 as neutralizer to maintain the desired pH, which results in an amount of insoluble CaSO4 waste during the purification process. To overcome such environmental issue, alkaliphilic organisms have the great potential to be used as an organic acid producer under NaOH-neutralizing agent based fermentation. Additionally, high optical purity property in D-lactic acid is now attracting more attention from both scientific and industrial communities because it can improve mechanical properties of PLA by blending L- or D-polymer together. However, the use of low-price nitrogen source for D-lactate fermentation by alkaliphilic organisms combined with NaOH-neutralizing agent based process has not been studied. Therefore, our goal was the demonstrations of newly simplify high-optical-purity D-lactate production by using low-priced peanut meal combined with non-sterile NaOH-neutralizing agent based fermentation., Results: In this study, we developed a process for high-optical-purity D-lactate production using an engineered alkaliphilic Bacillus strain. First, the native L-lactate dehydrogenase gene (ldh) was knocked out, and the D-lactate dehydrogenase gene from Lactobacillus delbrueckii was introduced to construct a D-lactate producer. The key gene responsible for exopolysaccharide biosynthesis (epsD) was subsequently disrupted to increase the yield and simplify the downstream process. Finally, a fed-batch fermentation under non-sterile conditions was conducted using low-priced peanut meal as a nitrogen source and NaOH as a green neutralizer. The D-lactate titer reached 143.99 g/l, with a yield of 96.09 %, an overall productivity of 1.674 g/l/h including with the highest productivity at 16 h of 3.04 g/l/h, which was even higher than that of a sterile fermentation. Moreover, high optical purities (approximately 99.85 %) of D-lactate were obtained under both conditions., Conclusions: Given the use of a cheap nitrogen source and a non-sterile green fermentation process, this study provides a more valuable and favorable fermentation process for future polymer-grade D-lactate production.

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.

Thermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to chemical-based ramie degumming processes. In particular, the alkaline pectate lyase from Bacillus sp. strain N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca(2+). However, BspPelA displays poor thermostability and is inactive after incubation at 50°C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone PCR and screening of >12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that single E124I, T178A, and S271G substitutions were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that the formation of a hydrophobic cluster and new H-bond networks was the key factor contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant, EAET, exhibited a 140-fold increase in the t50 (time at which the enzyme loses 50% of its initial activity) value at 50°C, accompanied by an 84.3% decrease in activity compared with that of wild-type BspPelA, while the most advantageous combined mutant, EA, exhibited a 24-fold increase in the t50 value at 50°C, with a 23.3% increase in activity. Ramie degumming with the EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggest that the EA mutant, exhibiting remarkable improvements in thermostability and activity, has the potential for applications in ramie degumming in the textile industry., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

To understand the molecular basis of higher pH catalytic adaptation of family 11 xylanases, we compared the structures of alkaline, neutral, and acidic active xylanases and analyzed mutants of xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5. It was revealed that alkaline active xylanases have increased charged residue content, an increased ratio of negatively to positively charged residues, and decreased Ser, Thr, and Tyr residue content relative to non-alkaline active counterparts. Between strands β6 and β7, alkaline xylanases substitute an α-helix for a coil or turn found in their non-alkaline counterparts. Compared with non-alkaline xylanases, alkaline active enzymes have an inserted stretch of seven amino acids rich in charged residues, which may be beneficial for xylanase function in alkaline conditions. Positively charged residues on the molecular surface and ionic bonds may play important roles in higher pH catalytic adaptation of family 11 xylanases. By structure comparison, sequence alignment and mutational analysis, six amino acids (Glu16, Trp18, Asn44, Leu46, Arg48, and Ser187, numbering based on Xyn11A-LC) adjacent to the acid/base catalyst were found to be responsible for xylanase function in higher pH conditions. Our results will contribute to understanding the molecular mechanisms of higher pH catalytic adaptation in family 11 xylanases and engineering xylanases to suit industrial applications.

The alkaliphilic halotolerant bacterium Bacillus sp. N16-5 is often exposed to salt stress in its natural habitats. In this study, we used one-colour microarrays to investigate adaptive responses of Bacillus sp. N16-5 transcriptome to long-term growth at different salinity levels (0%, 2%, 8%, and 15% NaCl) and to a sudden salt increase from 0% to 8% NaCl. The common strategies used by bacteria to survive and grow at high salt conditions, such as K+ uptake, Na+ efflux, and the accumulation of organic compatible solutes (glycine betaine and ectoine), were observed in Bacillus sp. N16-5. The genes of SigB regulon involved in general stress responses and chaperone-encoding genes were also induced by high salt concentration. Moreover, the genes regulating swarming ability and the composition of the cytoplasmic membrane and cell wall were also differentially expressed. The genes involved in iron uptake were down-regulated, whereas the iron homeostasis regulator Fur was up-regulated, suggesting that Fur may play a role in the salt adaption of Bacillus sp. N16-5. In summary, we present a comprehensive gene expression profiling of alkaliphilic Bacillus sp. N16-5 cells exposed to high salt stress, which would help elucidate the mechanisms underlying alkaliphilic Bacillus spp. survival in and adaptation to salt stress.

The chitosanase gene from a Bacillus sp. strain isolated from soil in East China was cloned and expressed in Escherichia coli. The gene had 1224 nucleotides and encoded a mature protein of 407 amino acid residues. The optimum pH and temperature of the purified recombinant chitosanase were 5.0 and 60 °C, respectively, and the enzyme was stable below 40 °C. The K m, V max, and specific activity of the enzyme were 1.19 mg mL(-1), 674.71 μmol min(-1) at 50 °C, and 555.3 U mg(-1), respectively. Mn(2+) was an activator of the recombinant chitosanase, while Co(2+) was an inhibitor. Hg(2+) and Cu(2+) inhibited the enzyme at 1 mM. The highest level of enzyme activity (186 U mL(-1)) was achieved in culture medium using high cell-density cultivation in a 7-L fermenter. The main products of chitosan hydrolyzed by recombinant chitosanase were (GlcN)3-6. The chitosanases was successfully secreted to the culture media through the widely used SecB-dependent type II pathway in E. coli. The high yield of the extracellular overexpression, relevant thermostability, and effective hydrolysis of commercial grade chitosan showed that this recombinant enzyme had a great potential for industrial applications.

A xylanase gene (xyn11A) was cloned from the genomic library of alkalophilic Bacillus sp. SN5. It encoded a polypeptide of 366 amino acids, consisting of a family 11 glycoside hydrolase, a short linker region, and a family 36 carbohydrate-binding module (CBM). The intact xylanase Xyn11A and the CBM-linker-truncated Xyn11A-LC were expressed in Escherichia coli BL21 (DE3). Both purified recombinant proteins exhibited the highest activity at 55 °C. The optimal pH for Xyn11A activity was 7.5, whereas Xyn11A-LC showed a broad pH profile (>80% activity at pH 5.5-8.5) with optimal activity at pH 5.5 and 7.5-8.0. They had high alkali tolerance, retaining over 80% residual activity after preincubation at pH 8.5-11.0 at 37 °C for 1 H. Xyn11A-LC showed better thermal stability, lower affinity, and lower catalytic activity to insoluble xylan than Xyn11A, whereas its specific activity for soluble beechwood xylan (4,511.9 U/mg) was greater than that of Xyn11A (3,136.4 U/mg). These results implied that the CBM of Xyn11A could change the enzymatic properties and play a role in degrading insoluble xylan. Xyn11A-LC is a family 11 alkali-tolerant cellulase-free xylanase with high specific activity, which qualifies it as a potential candidate for industrial applications, especially in the paper industry., (© 2014 International Union of Biochemistry and Molecular Biology, Inc.)

Thermostable alkaline pectate lyases can be potentially used for enzymatically degumming ramie in an environmentally sustainable manner and as an alternative to the currently used chemical-based ramie degumming processes. To assess its potential applications, pectate lyase from Bacillus pumilus (ATCC 7061) was cloned and expressed in Escherichia coli. Evolutionary strategies were applied to generate efficient ramie degumming enzymes. Obtained from site-saturation mutagenesis and random mutagenesis, the best performing mutant enzyme M3 exhibited a 3.4-fold higher specific activity on substrate polygalacturonic acid, compared with the wild-type enzyme. Furthermore, the half-life of inactivation at 50 °C for M3 mutant extended to over 13 h. In contrast, the wild-type enzyme was completely inactivated in less than 10 min under the same conditions. An upward shift in the optimal reaction temperature of M3 mutant, to 75 °C, was observed, which was 10 °C higher than that of the wild-type enzyme. Kinetic parameter data revealed that the catalysis efficiency of M3 mutant was higher than that of the wild-type enzyme. Ramie degumming with M3 mutant was also demonstrated to be more efficient than that with the wild-type enzyme. Collectively, our results suggest that the M3 mutant, with remarkable improvements in thermoactivity and thermostability, has potential applications for ramie degumming in the textile industry.

Unlabelled: The alkaliphilichemicellulolytic bacterium Bacillus sp. N16-5 was isolated from Lake Wudunao in Inner Mongolia. It has a broad substrate spectrum and exhibits a capacity to utilize complex carbohydrates such as galactomannan, xylan and pectin. Previous transcriptional analysis of differential carbohydrate utilization by Bacillus sp. N16-5 has identified a putative gene cluster related to xylan utilization. It contains a putative xylo-oligosaccharide ATP- binding cassette (ABC) transporter encoded by xynEFG gene cluster., Objective: xynE gene is predicted to encode an extracellular solute-binding protein of the ABC transporter. Here, the physiological roles of xynE on the xylan utilization was investigated by gene deletion., Methods: We obtained the xynE deficient strain N16-5 (ΔxynE) through homologous recombination using a temperature sensitive shuttle vector pNNB194. The effects of xynE on xylan utilization by N16-5 were detected by comparing the growth profiles on xylan of the wild type and mutant strains as well as the variation of reducing sugars concentration in the medium during cultivation. We further verified the phenotype by constructing the complementary strain. Moreover, the substrate specificity of XynE was illustrated by the HPLC analysis results of the xylan medium components, which was supplemented with the growth profiles of the wild type strain and N16-5 (ΔxynE) strain on xylose., Results: Compared with the wild type strain, strain N16-5 (ΔxynE) had a delayed exponential phase, obtained a lower maximum optical intensity OD600 value, and presented the accumulation and depletion of reducing sugars during cultivation. The complementary strain retrieved the phenotype of wild type strain, and grown slightly better than it. HPLC analysis showed that N16-5 ( ΔxynE) strain degraded the xylan substrates more slowly than wild type, xylo- oligosaccharides like xylotetraose, xylotriose and xylobiose began to accumulate after 16 h cultivation. Moreover it still maitained a large number of the degradation product xylobiose in the medium after 60 h. When cultured in xylose medium, strain N16-5 (ΔxynE) performed similar growth profile with the wild type strain., Conclusion: XynE played an important role in rapidly and effectively utilizing xylan in Bacillus sp. N16-5 and specifically related with xylo-oligosaccharide uptake.

Lactic acid is one of the top 30 potential building-block chemicals from biomass, of which the most extensive use is in the polymerization of lactic acid to poly-lactic-acid (PLA). To reduce the cost of PLA, the search for cheap raw materials and low-cost process for lactic acid production is highly desired. In this study, the final titer of produced L-lactic acid reached a concentration of 185 g·L(-1) with a volumetric productivity of 1.93 g·L(-1)·h(-1) by using sugarcane bagasse hydrolysate as the sole carbon source simultaneously with cottonseed meal as cheap nitrogen sources under the open fed-batch fermentation process. Furthermore, a lactic acid yield of 0.99 g per g of total reducing sugars was obtained, which is very close to the theoretical value (1.0 g g(-1)). No D-isomer of lactic acid was detected in the broth, and thereafter resulted in an optical purity of 100%, which exceeds the requirement of lactate polymerization process. To our knowledge, this is the best performance of fermentation on polymer-grade L-lactic acid production totally using lignocellulosic sources. The high levels of optically pure L-lactic acid produced, combined with the ease of handling and low costs associated with the open fermentation strategy, indicated the thermotolerant Bacillus sp. P38 could be an excellent candidate strain with great industrial potential for polymer-grade L-lactic acid production from various cellulosic biomasses.

A Gram-positive, alkaliphilic bacterium, designated strain Zby6(T), was isolated from Zhabuye Lake in Tibet, China. The strain was able to grow at pH 8.0-11.0 (optimum at pH 10.0), in 0-8 % (w/v) NaCl (optimum at 3 %, w/v) and at 10-45 °C (optimum at 37 °C). Cells of the isolate were facultatively anaerobic and spore-forming rods with polar flagellum. The predominant isoprenoid quinone was MK-7, and its cell wall peptidoglycan contained meso-diaminopimelic acid. The major cellular fatty acids were iso-C(15:0), C(16:0) and anteiso-C(15:0). The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine. The genomic DNA G+C content of the isolate was 38.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Zby6(T) was a member of the genus Bacillus and most closely related to Bacillus cellulosilyticus DSM 2522(T) (97.7 % similarity). The DNA-DNA relatedness value between strain Zby6(T) and B. cellulosilyticus DSM 2522(T) was 59.2 ± 1.8 %. Comparative analysis of genotypic and phenotypic features indicated that strain Zby6(T) represents a novel species of the genus Bacillus, for which the name Bacillus alkalicola sp. nov. is proposed; the type strain is Zby6(T) (=CGMCC 1.10368(T) = JCM 17098(T) = NBRC 107743(T)).

The alkaline xylanase Xyn11A-LC from the alkalophilic Bacillus sp. SN5 was expressed in E. coli, purified and crystallized. The crystal structure was determined at a resolution of 1.49 Å. Xyn11A-LC has the β-jelly roll structure typical of family 11 xylanases. To improve its thermostability and thermophilicity, a mutant SB3 was constructed by introducing three arginines on the different sides of the protein surface. SB3 increased the optimum temperature by 5 °C. The wild type and SB3 had the half-lives of 22 and 68 min at 65 °C at pH 8.0 (Tris/HCl buffer), respectively. CD spectroscopy revealed that the melting temperature (T m) of the wild type and SB3 were 55.3 and 66.9 °C, respectively. These results showed that the introduction of arginines enhance the thermophilicity and thermostability of Xyn11A-LC.

Alkaline α-amylases are of great interest in desizing processes and detergent industries. Here, an alkaline α-amylase gene amy703 from an alkaliphilic Bacillus pseudofirmus strain was cloned and sequenced. Its encoding product, Amy703, might represent a new clade of α-amylase family, because it shared only 35 % highest identity with all amylases characterized up to date and was not clustered into any subfamilies with amylase activity in glycoside hydrolase family 13. Heterologous expression and characterization of Amy703 showed that it is a metalloenzyme with maximal activity at 40 °C and pH 9.0. Its activity was significantly enhanced by 2- and 2.48-fold at the presence of 10 mM Ca2+ and Mg2+, respectively, while Hg2+ was a strong inhibitor of Amy703. Amy703 has a higher affinity (Km = 3.92 mg/ml) for soluble starch compared to many other alkaline amylases. The computer modeling of its structure indicated that Amy703 contains typical amylase domains and a loop region appearing to bind the substrates. Site-directed mutagenesis suggested that a conserved residue Glu550 was essential for the activity of Amy703, and proposed it working together with other two residues to constitute a catalytic triad (Asp521, Glu550, and Asp615).

In this study, efficient polymer-grade L-lactic acid production was achieved with the strain Bacillus sp. P38 by using cellulosic hydrolysate as the sole carbon source. In fed-batch fermentation, 180 g L(-1)L-lactic acid was obtained with a volumetric productivity of 2.4 g L(-1)h(-1) and a yield of 0.96 g g(-1) total reducing sugars. No D-isomer of lactic acid was detected in the broth. Strain P38 tolerated up to 10 g L(-1) 2-furfural, and lactate production was sharply inhibited only when the 2-furfural concentration was higher than 6 g L(-1). Moreover, strain P38 also tolerated high concentrations (>6 g L(-1)) of other fermentation inhibitors in cellulosic hydrolysate, such as vanillin and acetic acid, although it was slightly sensitive to formic acid. The efficient L-lactic acid production, combined with high inhibitor tolerance and efficient pentose utilization, indicate that Bacillus sp. P38 is a promising producer of polymer-grade L-lactic acid from cellulosic biomass., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, a newly isolated alkaliphile Exiguobacterium sp. strain 8-11-1 was used to produce optically pure L-lactate. With an initial glucose concentration of 80 g/L, a high overall L-lactic acid productivity of 8.15 g/L/h was achieved using NaOH as a neutralizing agent. The fed-batch fermentations were carried out under both sterile and nonsterile conditions. Under the nonsterile condition, 125 g/L L-lactic acid was obtained with a high percent yield and average productivity of 98.33% and 3.79 g/L/h, respectively. These values were consistent with the results from sterile condition. No d-isomers of lactic acid were detected, resulting in an optical purity of 100% in both conditions. The high levels of optically pure L-lactic acid produced by Exiguobacterium sp. 8-11-1, combined with the ease of handling and low costs associated with the open fermentation strategy, provide a novel and potentially important approach for L-lactic acid production in the future., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Jerusalem artichoke is a low-requirement crop, which does not interfere with food chain, and is a promising carbon source for industrial fermentation. Microbial conversion of such a renewable raw material to useful products, such as lactic acid, is an important objective in industrial biotechnology. In this study, high-optical-purity l-lactate was efficiently produced from the hydrolysates of Jerusalem artichoke powder by a thermophilic bacterium, Bacillus coagulans XZL4. High l-lactate production (134gl(-1)) was obtained using 267gl(-1) Jerusalem artichoke powder (total reducing sugars of 140gl(-1)) and 10gl(-1) of corn steep powder in fed-batch fermentation, with an average productivity of 2.5gl(-1)h(-1) and a yield of 0.96gg(-1) reducing sugars. The final product optical purity is 99%, which meets the requirement of lactic acid polymerization. Our study represents a cost-effective and promising method for polymer-grade l-lactate production using a cheap raw bio-resource., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities., Methodology/principal Findings: Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2-C12) and triglycerides (C2-C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220., Conclusion: EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these enzymes showed hugely different thermal stabilities, indicating their diverse thermal adaptations via just changing a few amino acid residues.

The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources.

A xylanase gene (xyn10A) was cloned from Bacillus sp. SN5 and expressed in Escherichia coli. It encoded a 348-residue polypeptide of ~45 kDa. The deduced amino acid sequence had 68 % identity with the endo-1,4-beta-xylanase from Paenibacillus lactis 154 that belonged to family 10 of the glycoside hydrolases. Purified recombinant Xyn10A had maximum activity at 40 °C and pH 7.0, with the specific activity of 105 U/mg and a Km of 0.6 mg/ml for beechwood xylan. Xyn10A retained more than 80 % activity between 25 and 45 °C and 29 % activity at 5 °C. It exhibited the highest activity (134 %) in 0.5 M NaCl and still retained 90 % activity in 2.5 M NaCl. It retained about 87 % activity after incubation in 2 M NaCl for 24 h. The cold-active and halo-tolerant properties of Xyn10A make it promising for application in the food industry, especially in the processing of saline food and sea food.

Highly efficient polymer-grade L-lactic acid production was achieved by an alkaliphilic strain Bacillus sp. WL-S20 using inexpensive peanut meal as nitrogen source and sodium hydroxide as neutralizing agent. In multi-pulse fed-batch fermentation of Bacillus sp. WL-S20, a L-lactic acid concentration of 225 g/l with a yield of 99.3% was obtained. In single-pulse fed-batch fermentation, a concentration of 180 g/l was obtained with a yield of 98.6%. No D-isomers of lactic acid were detected. The production of a high concentration of optically pure L-lactic acid by alkaliphilic Bacillus sp. WL-S20, combined with a low-cost nutrient and environment-friendly NaOH-based process, represent a potentially novel way for L-lactic acid production at an industrial scale., (Copyright © 2012. Published by Elsevier Ltd.)

The pectate lyase (Bsp165PelA) from Bacillus sp. N16-5 has great potential in industrial applications because it shows high specific activity under extremely alkaline conditions. Besides, activity measurement of Bsp165PelA does not require addition of calcium, in a way different from the other pectate lyases. Here we report crystal structures of Bsp165PelA in apo-form and in complex with trigalacturonate. The parallel β-helix, active site residues and substrate binding cleft are similar to those in the other pectate lyases from Polysaccharide Lyase family 1. However, some of the highly conserved Ca(2+) binding residues and secondary structures are altered in Bsp165PelA, making it difficult to coordinate with Ca(2+) as in the other pectate lyases. We found Bsp165PelA forms some direct enzyme-substrate interactions instead of using Ca(2+) ions bridging in the extremely alkaline environment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A Gram-positive, alkaliphilic bacterium, designated strain DLS13T, was isolated from Dali Lake in Inner Mongolia Autonomous Region, China. The isolate was able to grow at pH 7.5-11.0 (optimum at pH 9), in 0-8 % (w/v) NaCl (optimum at 2 %, w/v) and at 10-45 °C (optimum at 30 °C). Cells of the isolate were facultatively anaerobic, spore-forming rods with peritrichous flagella. The predominant isoprenoid quinone was MK-7 and its cell wall peptidoglycan contained meso-diaminopimelic acid. The major polar lipids consisted of phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids were anteiso-C15:0, anteiso-C17:0 and iso-C15:0. The genomic DNA G+C content of the isolate was 43.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain DLS13T was a member of the genus Bacillus and most closely related to Bacillus saliphilus DSM 15402T (96.9 % similarity). The DNA-DNA relatedness value between strain DLS13T and B. saliphilus DSM 15402T was 38.7±1.9 %. Comparative analysis of genotypic and phenotypic features indicated that strain DLS13T represents a novel species of the genus Bacillus, for which the name Bacillus daliensis sp. nov. is proposed; the type strain is DLS13T (=CGMCC 1.10369T=JCM 17097T=NBRC 107572T).

Alkaliphilic and halophilic Bacillus sp. BG-CS10 was isolated from Zabuye Salt Lake, Tibet. The gene celB, encoding a halophilic cellulase was identified from the genomic library of BG-CS10. CelB belongs to the cellulase superfamily and DUF291 superfamily, with an unknown function domain and less than 58% identity to other cellulases in GenBank. The purified recombinant protein (molecular weight: 62 kDa) can hydrolyze soluble cellulose substrates containing beta-1,4-linkages, such as carboxylmethyl cellulose and konjac glucomannan, but has no exoglucanase and β-glucosidase activities. Thus, CelB is a cellulase with an endo mode of action and glucomannanase activity. Interestingly, the enzyme activity was increased approximately tenfold with 2.5 M NaCl or 3 M KCl. Furthermore, the optimal temperatures were 55°C with 2.5 M NaCl and 35°C without NaCl, respectively. This indicates that NaCl can improve enzyme thermostability. The K ( m ) and k (cat) values of CelB for CMC with 2.5 M NaCl were 3.18 mg mL(-1) and 26 s(-1), while the K ( m ) and k (cat) values of CelB without NaCl were 6.6 mg mL(-1) and 2.1 s(-1). Thus, this thermo-stable, salt and pH-tolerant cellulase is a promising candidate for industrial applications, and provides a new model to study salt effects on the structure of protein.

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Objective: To isolate and identify an alkaliphilic mannanase producing bacterium, purify and characterize mannanase thereof., Methods: Mannanase-producing alkaliphilic bacterium HMTS15 was isolated by alkaline agar with konjak from water sample of Hamatai Lake in Inner Mongolia, China. The morphological, biochemical and physiological characteristics and 16S rRNA gene were analyzed to identify the taxonomic position of strain HMTS15. Mannanase produced by strain HMTS15 was purified by four steps including (NH4)2SO4 precipitation, cellulose DEAE-sepharose, twice Superdex 200. The enzyme properties including optimal temperature, optimal pH, thermal stability, pH stability, NaCl tolerance, metal ion tolerance, EDTA and SDS tolerance were tested., Results: Strain HMTS15 was Gram-positive rod. Its growth pH ranged from 7.0 to 11.0 and growth temperature ranged from 10 degrees C to 45 degrees C. The G + C content of the DNA was 40 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons indicated that strain HMTS15 was a member of Bacillus. The extracellular mannanase from strain HMTS15 was purified as a single band with molecular weight of about 45 kD on SDS-PAGE. The optimal catalytic activity was showed at 75 degrees C and pH 10. The mananase was stable up to 60 degrees C and retained about 60% residual activity at 65 degrees C for 30 min. The ions Fe2+, Mn2+, Co2+, Zn2+, Ag+, Hg2+ and EDTA inhibited the acitivity of the mannanase., Conclusion: Polyphasic taxonomy revealed that strain HTMS15 was a new member of Bacillus agaradhaerens. The alkaline mannanase produced by strain HMTS15 hold the valuable property in stability at high temperature and broad range of pH.

A process for efficient production of an alkaline β-mannanases from Bacillus sp. N16-5 was established by heterologous expression using Pichia pastoris. A high producing strain was generated by removing the native β-mannanases signal peptide and increasing the copy number of the mature β-mannanases gene. High cell density fermentation of this strain in 1-L bioreactor led to a production level of 4164 U/mL after 96 h of induction. Sorbitol co-feeding and temperature-lowering strategies both increased the β-mannanase production levels. Combined usage of these two strategies achieved the most effective result-the enzyme level reached 6336 U/mL within 84 h, which to our best knowledge is the highest production level reported for the expression of extreme β-mannanase thus far. The strategy described in this work can also be adapted to express other important industrial enzymes with extreme properties., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bacillus coagulans 2-6 is an efficient producer of lactic acid. The genome of B. coagulans 2-6 has the smallest genome among the members of the genus Bacillus known to date. The frameshift mutation at the start of the d-lactate dehydrogenase sequence might be responsible for the production of high-optical-purity l-lactic acid.

A truncated alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 (MAN330) was expressed and secreted in Kluyveromyces cicerisporus. The recombinant engineered strain for MAN330 production was stable during 80 generations, and the maximum yield of MAN330 reached 3,795 U/ml in 15 l fermenter. MAN330 exhibited similar pH optima, temperature optima, and substrate specificities to its full-length protein (MAN493). However, stability of MAN330 was about 7% higher than that of MAN493 from pH 9-11. MAN330 had about 10% higher stability than MAN493 from 60°C to 80°C.

Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)(8)-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)(8)-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pK(a) calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline adaptation mechanism.

To determine the impact of carbohydrates on the metabolic pathway in alkaliphiles, proteomes were obtained from cultures containing different carbohydrates and were resolved on two-dimensional gel electrophoresis (2-DE). The proteomes were compared to determine differentially expressed proteins. A novel alkaliphilic bacterium (alkaliphilic Bacillus sp. N16-5 isolated from Wudunur Soda Lake, China) was isolated in media with five different carbon sources (glucose, mannose, galactose, arabinose, and xylose). Comparative proteome analysis identified 61 differentially expressed proteins, which were mainly involved in carbohydrate metabolism, amino acid transport, and metabolism, as well as energy production and conversion. The comparison was based on the draft genome sequence of strain N16-5. The abundance of enzymes involved in central metabolism was significantly changed when exposed to various carbohydrates. Notably, catabolite control protein A (CcpA) was up-regulated under all carbon sources compared with glucose. In addition, pentose exhibited a stronger effect than hexose in CcpA-mediated carbon catabolite repression. These results provided a fundamental understanding of carbohydrate metabolism in alkaliphiles.

Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×10(5) transformants per µg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity.

Lignocellulosic biomass-derived sugars are considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentations of bulk chemicals such as lactic acid. In the present study, corncob molasses containing a high content of xylose, which is one of the lignocellulosic biomasses and a waste by-product from xylitol production, was used for L-lactic acid production via a newly isolated xylose utilizing Bacillus sp. strain XZL9. Bacillus sp. strain XZL9 can utilize the mixture of sugars including xylose, arabinose, and glucose in corncob molasses for L-lactic acid production. High concentration of L-lactic acid (74.7 g l⁻¹) was obtained from corncob molasses (initial total sugars of 91.4 g l⁻¹) in fed-batch fermentation. This study provides an encouraging means of producing L-lactic acid from lignocellulosic resource such as the low-cost corncob molasses., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A thermophilic Bacillus sp. strain 2-6 was used in completely open repeated batch fermentation for producing optically pure L-lactic acid. Up to 107 g l(-1) L-lactic acid of optical purity 99.8% was obtained with NaOH as pH regulator. Unexpectedly, accumulated cells did not necessarily lead to increased L-lactic acid production. Kinetic and viable cells analyses revealed that L-lactic acid production by Bacillus sp. 2-6 was closely related to the growth of viable cells. Accumulated unviable cells had a slight or negative effect on production efficiency. Therefore, the number of repeated batches should be limited to avoid the accumulation of unviable cells. In this study, repeated open operation was shown to be feasible for optically pure L-lactic acid production, and new light shed on the roles of viable and unviable cells in repeated batch fermentation., (Copyright 2010 Elsevier Ltd. All rights reserved.)

An alkaline pectate lyase, Bsp165PelA, was purified to homogeneity from the culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme showed a specific activity as high as 1000 U/mg and had optimum activity at pH 11.5 and 50 degrees . It was composed of a single polypeptide chain with a molecular of 42 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 6.0. It could efficiently depolymerize polygalacturonate and pectin. Characterization of product formation revealed unsaturated digalacturonate and trigalacturonate as the main product. The pectate lyase gene (pelA) contained an open reading frame (ORF) of 1089 bp, encoding a 36-amino acids signal peptide and a mature protein of 326 amino acids with a calculated molecular mass of 35.943 Da. The deduced amino acid sequence from the pelA ORF exhibited significant homology to those of known pectate lyases in polysaccharide lyase family 1. Some conserved active-site amino acids were found in the deduced amino acid sequence of Bsp165PelA. Ca2+ was not required for activity on pectic substrates.

Objective: To isolate and characterize alkaliphilic bacteria with protease activity., Methods: Protease-producing alkaliphiles were isolated by skim milk agar method. The morphological, biochemical and physiological characteristics, 16S rRNA gene sequence and DNA-DNA hybridization were determined to identify the taxonomic position of strain ZL223. Meanwhile, the enzyme characterization of the protease including optimal temperature and pH range, stability and oxidation resistance were tested by using casein hydrolysis test., Results: A new strain named ZL223 was isolated from Bange Lake in Tibet, China. Cells were Gram-positive, spore-forming, motile rods. Growth pH range was from 7.0 to 10.0, optimum at pH 9.0. Growth temperature range was from 15 degrees C to 45 degrees C, optimum at 37 degrees C. The G + C content of DNA was 40.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Bacillus and most closely related to Bacillus pseudofirmus OF4 with 98.6% sequence similarity. DNA-DNA hybridization analysis confirmed the close relationship of strain ZL223 with Bacillus pseudofirmu OF4 (86% relatedness). Strain ZL223 produced extracellular alkaline protease and its maximal enzyme activity was observed at 40 degrees C and pH 12.0., Conclusion: Base on polyphasic taxonomy, strain ZL223 was a new member of Bacillus pseudofirmus. It produced alkaline protease which was worth further research.

An open reading frame of 1100 bp in the partially sequenced genome sequence of alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative alanine racemase gene (dadX(OF4)), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadX(OF4) was purified to homogeneity by His(6)-tag affinity column, gel filtration and ion-exchange chromatography. The amino acid sequence has highest identity with the known alanine racemase from Oceanobacillus iheyensis HTE831 (48%). The protein was a dimeric, endogenous PLP-dependent enzyme, which was demonstrated by absorption spectra and enzyme activity with or without PLP. The racemization temperature optimum was 40 degrees C and the optimal pH was 10.5. The kinetic parameters K(m) and V(max) at 40 degrees C of alanine racemase, determined by HPLC analysis, were 41.79 mM, 10,500 units/mg for L-alanine and 14.91 mM, 3708 units/mg for D-alanine, respectively.

Alanine racemase (DadX(OF4)), a dimeric endogenous PLP-dependent alkaline enzyme from alkaliphilic Bacillus pseudofirmus OF4, was expressed in Escherichia coli and purified with a His(6) tag in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 291 K using a solution containing 1.4 M sodium/potassium phosphate pH 8.2. The protein crystallized in space group P2(1)2(1)2(1), with two protein molecules in the asymmetric unit.

Background: The demand for lactic acid has been increasing considerably because of its use as a monomer for the synthesis of polylactic acid (PLA), which is a promising and environment-friendly alternative to plastics derived from petrochemicals. Optically pure L-lactic acid is essential for polymerization of PLA. The high fermentation cost of L-lactic acid is another limitation for PLA polymers to compete with conventional plastics., Methodology/principal Findings: A Bacillus sp. strain 2-6 for production of L-lactic acid was isolated at 55 degrees C from soil samples. Its thermophilic characteristic made it a good lactic acid producer because optically pure L-lactic acid could be produced by this strain under open condition without sterilization. In 5-liter batch fermentation of Bacillus sp. 2-6, 118.0 g/liter of L-lactic acid with an optical purity of 99.4% was obtained from 121.3 g/liter of glucose. The yield was 97.3% and the average productivity was 4.37 g/liter/h. The maximum L-lactic acid concentration of 182.0 g/liter was obtained from 30-liter fed-batch fermentation with an average productivity of 3.03 g/liter/h and product optical purity of 99.4%., Conclusions/significance: With the newly isolated Bacillus sp. strain 2-6, high concentration of optically pure L-lactic acid could be produced efficiently in open fermentation without sterilization, which would lead to a new cost-effective method for polymer-grade L-lactic acid production from renewable resources.

A Gram-positive, halophilic bacterium was isolated from a sediment sample from Ai-Ding salt lake in China. The isolate, designated strain 17-5(T), grew at salinities of 8-33 % (w/v) NaCl (optimally at 12 %, w/v). The genomic DNA G+C content of strain 17-5(T) was 48.1 mol%. The predominant isoprenoid quinone was MK-7(H(2)) and the cell-wall peptidoglycan contained meso-diaminopimelic acid. The major polar lipids were diphosphatidylglycerol and an unidentified glycolipid. The major cellular fatty acids were anteiso-C(15 : 0), anteiso-C(17 : 0), iso-C(16 : 0) and C(16 : 0). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 17-5(T) was a member of the genus Bacillus, being most closely related to Bacillus qingdaonensis JCM 14087(T) (96.0 % sequence similarity) and Bacillus salarius DSM 16461(T) (95.6 %). The levels of 16S rRNA gene sequence similarity with respect to other Bacillus species were less than 91.7 %. Comparative analysis of the 16S rRNA gene sequence data, chemotaxonomy and phenotypic features of the novel isolate and related species of Bacillus indicated that strain 17-5(T) represents a novel species within the genus Bacillus, for which the name Bacillus aidingensis sp. nov. is proposed. The type strain is 17-5(T) (=CGMCC 1.3227(T)=DSM 18341(T)).

An alkaline beta-mannanase was purified to homogeneity from a culture broth of alkaliphilic Bacillus sp. N16-5. The enzyme had optimum activity at pH 9.5 and 70 degrees C. It was composed of a single polypeptide chain with a molecular weight of 55 kDa deduced from SDS-PAGE, and its isoelectric point was around pH 4.3. The enzyme efficiently hydrolyzed galactomannan and glucomannan, producing a series of oligosaccharides and monosaccharides. The beta-mannanase gene (manA) contained an open reading frame (ORF) of 1,479 bp, encoding a 32-amino acids signal peptide, and a mature protein of 461 amino acids, with a calculated molecular mass of 50,743 Da. Strain N16-5 ManA, deduced from the manA ORF, exhibited relatively high amino acid similarity to the members of the glycosyl hydrolase family 5. The eight conserved active-site amino acids in family 5 glycosyl hydrolase were found in the deduced amino acid sequence of strain N16-5 ManA.