CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion.

BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis. RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter Para for Cas9 were used, along with the background expression of the HR repair system. CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system.

Degradation of long-chain n-alkanes by a novel thermal-tolerant Rhodococcus strain.

A novel bacterial strain, CH91, was isolated from a high-temperature oil reservoir. Morphological characterization, phylogenetic analyses of 16S rRNA gene sequence and genome relatedness indicated that the strain is a potential new species in the genus Rhodococcus. Strain CH91 could grow in the temperature range of 25-50 °C (optimally at 37 °C) and utilize a broad range of long-chain n-alkanes from hexadecane to hexatriacontane. The utilization of the n-alkanes mixture of strain CH91 revealed that the degradation rate was correlated to the length of the carbon chain. Two novel alkB genes encoding alkane 1-monooxygenase were found in the genome of this strain. The protein sequences of both alkane 1-monooxygenases showed a remarkable phylogenetic distance to other reported AlkB protein sequences. These results would help broaden our knowledge about alkane degradation by Rhodocuccus and its potential ecological role. The ability of the strain in the long-chain alkane degradation and thermal tolerance could also be further exploited for bioremediation of oil contaminations and microbial enhanced oil recovery.

Structure of an Alkaline Pectate Lyase and Rational Engineering with Improved Thermo-Alkaline Stability for Efficient Ramie Degumming.

Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed ß-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca2+-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔTm values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T5015 value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T5015 value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions (4 h, 60 °C, pH 10.0), compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases.

A Review of the Recent Developments in the Bioproduction of Polylactic Acid and Its Precursors Optically Pure Lactic Acids.

Lactic acid (LA) is an important organic acid with broad industrial applications. Considered as an environmentally friendly alternative to petroleum-based plastic with a wide range of applications, polylactic acid has generated a great deal of interest and therefore the demand for optically pure l- or d-lactic acid has increased accordingly. Microbial fermentation is the industrial route for LA production. LA bacteria and certain genetic engineering bacteria are widely used for LA production. Although some fungi, such as Saccharomyces cerevisiae, are not natural LA producers, they have recently received increased attention for LA production because of their acid tolerance. The main challenge for LA bioproduction is the high cost of substrates. The development of LA production from cost-effective biomasses is a potential solution to reduce the cost of LA production. This review examined and discussed recent progress in optically pure l-lactic acid and optically pure d-lactic acid fermentation. The utilization of inexpensive substrates is also focused on. Additionally, for PLA production, a complete biological process by one-step fermentation from renewable resources is also currently being developed by metabolically engineered bacteria. We also summarize the strategies and procedures for metabolically engineering microorganisms producing PLA. In addition, there exists some challenges to efficiently produce PLA, therefore strategies to overcome these challenges through metabolic engineering combined with enzyme engineering are also discussed.

Bio-production of high-purity propionate by engineering L-threonine degradation pathway in Pseudomonas putida.

Propionic acid (PA) is widely used in the food, agricultural, and pharmaceutical industries. Since the petrochemical PA is unsustainable, biological production of PA from renewable substrates is gaining attention. In this study, we engineered the strain Pseudomonas putida KT2440 to transform L-threonine to PA with only CO2 released as by-product. The cell factory was created by chromosomal incorporation of heterologous L-threonine deaminase, permease, and acyl-CoA thioesterase, deletion of branch pathways as well as overproduction of the endogenous branched-chain alpha-keto acid dehydrogenase complex. The final engineered strain could produce 399 mM PA from 400 mM L-threonine in a batch biotransformation process, with a molar yield of 99.8% under the optimized conditions in 48 h. The PA titer further reached to 50.3 g/L (679 mM) with a productivity of 0.6 g/L/h in a fed-batch conversion process. No obvious by-products, such as acetate and succinate, were detected in the broth, which would significantly facilitate downstream purification steps. Thus, this study offers an alternative route for biological production of PA.

Tepidiphilus baoligensis sp. nov., a Novel Bacterium of the Family Hydrogenophilaceae Isolated from an Oil Reservoir.

A Gram-negative, aerobic, motile, non-spore-forming and rod-shaped bacterium, designated strain B18-69 T, was isolated from oil-well production liquid in Baolige oilfield, China. The strain was able to grow at pH 6-9.5 (optimum at pH 7), in 0-4% (w/v) NaCl (optimum at 0.5-1%, w/v) and at 35-60 °C (optimum at 55 °C). Major cellular fatty acids were C16:0, C19:0 cyclo ω8c, C17:0 cyclo and C18:1 ω7c. The predominant respiratory quinone was ubiquinone 8. Major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylcholine (PC). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B18-69 T was most closely related to Tepidiphilus margaritifer DSM 15129 T (98.8% similarity). The draft genome of strain B18-69 T was composed of 2,250,419 bp, and the G+C content was 64.6 mol%. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain B18-69 T and T. margaritifer DSM 15129 T were 90.9% and 68.9%, respectively. Genotypic and phenotypic features indicate that strain B18-69 T represents a novel species of the genus Tepidiphilus, for which the name Tepidiphilus baoligensis sp. nov. is proposed. The type strain is B18-69 T (= CGMCC 1.13573 T = KCTC 62782 T).

Tepidicella baoligensis sp. nov., A Novel Member of Betaproteobacterium Isolated from an Oil Reservoir.

A Gram-negative, non-pigmented, aerobic bacterium, designated strain B18-50T was isolated from oil-well production water in Baolige oilfield, China. The strain was able to grow at pH 6.5-10.5 (optimum at pH 7.5-8.5), in 0-3% (w/v) NaCl (optimum at 0-0.5%, w/v) and at 20-60 °C (optimum at 45 °C). Cells of the isolate were motile with a single polar flagellum and non-spore-forming rods. Organic acids and amino acids were used as carbon and energy sources, but sugars and polyols were not assimilated. The major cellular fatty acids were C16:0, C16:1ω6c/ω7c, and C18:1ω7c. Ubiquinone 8 was the predominant respiratory quinone. The major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The genomic DNA G+C content of the isolate was 62.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B18-50T was most closely related to Tepidicella xavieri DSM 19605T (97.5% similarity). Comparative analysis of genotypic and phenotypic features indicate that strain B18-50T represents a novel species of the genus Tepidicella, for which the name Tepidicella baoligensis sp. nov. is proposed. The type strain is B18-50T (= CGMCC 1.13575T = KCTC 62779T).

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline ß-mannanase from an alkaliphilic Bacillus clausii strain S10.

BACKGROUND: ß-Mannanase catalyzes the cleavage of ß-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable ß-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline ß-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high ß-mannanase activity at alkaline condition. In this study, this ß-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied. RESULTS: A thermo-alkaline ß-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 °C with good stability at pH 7.0-11.5 and below 80 °C. BcManA demonstrated high cleavage capability on polysaccharides containing ß-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg-1 was observed on konjac glucomannan with the Km and kcat value of 0.62 g l-1 and 1238.9 s-1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml-1 with secretory rate of 98.5% was obtained using SPlipA and P43 after 72 h cultivation in 2 × SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml-1 after 72 h cultivation with 6% inoculum size by shake flask fermentation. CONCLUSIONS: The thermo-alkaline ß-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported ß-mannanases and was a promising thermo-alkaline ß-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml-1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline ß-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other ß-mannanases in B. subtilis.

Coralloluteibacterium thermophilus sp. nov., A Gammaproteobacterium Isolated from an Oil Reservoir.

A Gram-negative, yellow-pigmented, aerobic bacterium, designated strain B51-30T, was isolated from oil-well production liquid in Baolige oilfield, China. The strain was able to grow at pH 6-10 (optimum at pH 7.5), in 0-6% (w/v) NaCl (optimum at 1%, w/v) at 15-55 °C (optimum at 45 °C). Cells of the isolate were non-motile and non-spore-forming rods. The major cellular fatty acids were iso-C15:0, iso-C11:0, iso-C11:0 3OH, iso-C17:1 ω9c, and iso-C17:0. Ubiquinone 8 was the predominant respiratory quinone. The major polar lipids consisted of phosphatidylethanolamine and diphosphatidylglycerol. The genomic DNA G+C content of the isolate was 70.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain B51-30T was most closely related to Coralloluteibacterium stylophorae KCTC 52167T (98.7% similarity). The two strains showed DNA-DNA relatedness values of 58.5%. Genotypic and phenotypic features indicate that strain B51-30T represents a novel species of the genus Coralloluteibacterium, for which the name Coralloluteibacterium thermophilus sp. nov. is proposed. The type strain is B51-30T (= CGMCC 1.13574T = KCTC 62780T).

Cloning, expression, and characterization of an alkaline protease, AprV, from Vibrio sp. DA1-1.

A novel alkaline protease (named AprV) gene from Vibrio sp. DA1-1 was cloned and expressed in Escherichia coli BL21 (DE3) pLysS. The sequence analysis showed the highest homology of 68% with the characterized protease from Alkalimonas collagenimarina AC40T. The recombinant AprV was purified with the molecular weight of 28 kDa. The optimum temperature and pH were determined to be 55 °C and 10.0, respectively. The enzyme activity was slightly enhanced by Ca2+, Mg2+, Zn2+, Ba2+, and, however, was highly inhibited by Sn2+ and EDTA. The AprV was stable in the presence of some surfactants and oxidizing agents, such as 1% Tween 20-80, 1% JFC-2, and 5% JFC-2. Casein was found to be the ideal substrate with specific activity of 1139 U/mg. Moreover, we found that AprV (10,000 U), together with commercial detergent, could completely remove the blood on the cotton. Furthermore, AprV also demonstrated dehairing activity on goat and bull skin. These results indicated that the alkaline protease AprV might be a potential candidate for applications in the detergent and leather industries.

Decalin-Containing Tetramic Acids and 4-Hydroxy-2-pyridones with Antimicrobial and Cytotoxic Activity from the Fungus Coniochaeta cephalothecoides Collected in Tibetan Plateau (Medog).

New tetramic acid derivatives, (±)-conipyridoins A-D (1-4), conipyridoins E (5) and F (6), and new 4-hydroxy-2-pyridone alkaloids (±)-didymellamide E (7), (+)-didymellamide B (8), (+)-N-hydroxyapiosporamide (9), and didymellamides F-H (10-12) were isolated and identified from the solid culture of the fungus Coniochaeta cephalothecoides. Chiral resolution of 1, 2, 3, 4, and 7 gave five pairs of enantiomers: 1a/1b, 2a/2b, 3a/3b, 4a/4b, and 7a/7b, respectively. Stereochemistry of 1a and 1b, and 2a and 2b was established and confirmed by the single-crystal X-ray diffraction and electronic circular dichroism (ECD) methods. Absolute configuration in 3a, 3b, 4a, 4b, 7a, and 7b was assigned by ECD calculations. Compounds 1-6 possess an unprecedented chemical skeleton featuring a decalin ring and a tetramic acid moiety. Compound 11 significantly inhibited the growth of Candida albicans and Aspergillus fumigatus with minimum inhibitory concentration (MIC) of 3.13 and 1.56 µM, respectively, and was further confirmed to be a new chitin synthesis inhibitor. Compound 5 exhibited the strongest activity against the growth of both Staphylococcus aureus and MRSA with MIC value of 0.97 µM. In the light of a co-occurrence of 3-acyl tetramic acids and biogenetically related pyridine alkaloids, the biosynthetic pathway for 1-12 was postulated.

Cloning, evaluation, and high-level expression of a thermo-alkaline pectate lyase from alkaliphilic Bacillus clausii with potential in ramie degumming.

Alkaline pectate lyases (Pels) have potential application in bioscouring of the textile industry. In this study, a thermo-alkaline Pel (BacPelA) gene from an alkaliphilic Bacillus clausii strain was cloned and overexpressed in Escherichia coli. The mature BacPelA exhibited maximum activity at pH 10.5 and 70 °C and showed high cleavage capability on methylated pectins. BacPelA showed the highest specific activity of 936.2 U mg-1 on ≥85% methylated pectin and 675.5 U mg-1 on standard substrate polygalacturonic acid (PGA) upon evaluation of the absorbance at 235 nm (A235). The K m and k cat values for PGA were 0.54 g l-1 and 346.5 s-1, respectively. Moreover, the 3,5-dinitrosalicylic acid (DNS) assay, which detects the released reducing oligogalacturonic acids, was confirmed to be inaccurate and unsuitable for endo-acting pectinase activity assay because of the difference in the reducibility by DNS reagent between the standard galacturonic acid and the catalytic oligomer products. Significant ramie fiber weight loss was observed following treatment with BacPelA (24.8%) and combined enzyme-chemical method (30.9%), which indicated that the degumming efficiency of BacPelA was the highest of all alkaline and thermostable Pels reported to date. The total activity of the recombinant mature BacPelA reached 8378.2 U ml-1 (A235) by high-cell-density cultivation in fed-batch fermentation with productivity of 239.4 U ml-1 h-1 using E. coli as host, which represents the highest Pel yield reported to date. Therefore, BacPelA, with promising properties for bioscouring, shows potential applications for ramie degumming in the textile industry.

Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.

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.

Efficient fermentative production of polymer-grade D-lactate by an engineered alkaliphilic Bacillus sp. strain under non-sterile conditions.

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.

Microbial community dynamics in Baolige oilfield during MEOR treatment, revealed by Illumina MiSeq sequencing.

This study was carried out to understand microbial diversity and function in the microbial enhanced oil recovery (MEOR) process and to assess the impact of MEOR treatment on the microbial community in an oil reservoir. The Illumina MiSeq-based method was used to investigate the structure and dynamics of the microbial community in a MEOR-treated block of the Baolige oilfield, China. The results showed that microbial diversity was high and that 23 phyla occurred in the analyzed samples. Proteobacteria, Firmicutes, Bacteroidetes, Thermotogae, and Euryarchaeota were present in relatively high abundance in all analyzed samples. Injection of bacteria and nutrients resulted in interesting changes in the composition of the microbial community. During MEOR treatment, the community was dominated by the known hydrocarbon-utilizing genera Pseudomonas and Acinetobacter. After the treatment, the two genera decreased in abundance over time while Methanobacteriaceae, as well as known syntrophic genera such as Syntrophomonas, Pelotomaculum, Desulfotomaculum, and Thermacetogenium gradually increased. The change in dominant microbial populations indicated the presence of a succession of microbial communities over time, and the hydrocarbon degradation and syntrophic oxidation of acetate and propionate to methane in the MEOR-treated oilfield. This work contributes to a better understanding of microbial processes in oil reservoirs and helps to optimize MEOR technology.

Evaluation and directed evolution for thermostability improvement of a GH 13 thermostable α-glucosidase from Thermus thermophilus TC11.

BACKGROUND: Thermal stable α-glucosidases with transglycosylation activity could be applied to the industrial production of oligosaccharides as well as conjugation of sugars to biologically useful materials. Therefore, α-glucosidases isolated from thermophiles have gained attention over the past decade. In this study, the characterization of a highly thermostable α-glucosidase and its thermostability improved mutant from newly isolated strain Thermus thermophilus TC11 were investigated. RESULTS: The recombinant α-glucosidase (TtAG) from Thermus thermophilus TC11 was expressed in Escherichia coli BL21 (DE3) and purified. The purified enzyme had a molecular mass of 184 kDa and consisted of 59-kDa subunits; it showed hydrolytic activity for pNP-α-D-glucopyranoside (pNPG), sucrose, trehalose, panose, and isomaltooligosaccharides and very low activity for maltose. The highest specific activity of 288.96 U/mg was observed for pNPG at 90 °C and pH 5.0; Pb(2+) provided a 20 % activity increase. TtAG was stable at 70 °C for more than 7 h and had a half-life of 195 min at 80 °C and 130 min at 90 °C. Transglycosylation activity was also observed with sucrose and trehalose as substrates. TtAG showed differences on substrate specificity, transglycosylation, multimerization, effects of metal ions and optimal pH from other reported Thermus α-glucosidases. One single-substitution TtAG mutant Q10Y with improved thermostability was also obtained from random mutagenesis library. The site-saturation mutagenesis and structural modelling analysis indicated that Q10Y substitution stabilized TtAG structure via additional hydrogen bonding and hydrophobic interactions. CONCLUSION: Our findings indicate that TtAG is a highly thermostable and more acidic α-glucosidase distinct from other reported Thermus α-glucosidases. And this work also provides new insights into the catalytic and thermal tolerance mechanisms of α-glucosidases, which may guide molecular engineering of α-glucosidase and other thermostable enzymes for industrial application.

Directed Evolution and Structural Analysis of Alkaline Pectate Lyase from the Alkaliphilic Bacterium Bacillus sp. Strain N16-5 To Improve Its Thermostability for Efficient Ramie Degumming.

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.

Improving the thermoactivity and thermostability of pectate lyase from Bacillus pumilus for ramie degumming.

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.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Haloarchaeon Halogranum amylolyticum.

Haloarchaea is an important group of polyhydroxyalkanoate (PHA)-accumulating organisms. However, few promising haloarchaeal species for economical and efficient PHA production have been reported. Here, we first discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a high 3-hydroxyvalerate (3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy (TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance ((1)H NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20.1 mol%, which is the highest 3HV fraction thus far reported, as for PHBV produced by the wild-type strains grown on unrelated carbon courses. Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the PHBV production and cell dry weight were increased by approximately eight and four times, respectively, as compared with those of the batch process in shaking flasks. The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and high-efficiency thermal asymmetric interlaced (hiTAIL) PCR methods. This finding suggested that H. amylolyticum shows promising potential in the low-cost biotechnological production of PHBV after further process optimization.

Cloning, expression, and characterization of a novel alkali-tolerant xylanase from alkaliphilic Bacillus sp. SN5.

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.