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High-level expression of a novel thermostable and mannose-tolerant ?-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli.

ABSTRACT: BACKGROUND: Mannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. ?-Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant ?-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fields as above. RESULTS: A ?-mannosidase gene (Tth man5) of 1,827 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 608 amino acid residues, and was over-expressed in Escherichia coli BL21 (DE3). The results of phylogenetic analysis, amino acid alignment and biochemical properties indicate that the Tth Man5 is a novel ?-mannosidase of glycoside hydrolase family 5. The optimal activity of the Tth Man5 ?-mannosidase was obtained at pH 5.5 and 85°C and was stable over a pH range of 5.0 to 8.5 and exhibited 2 h half-life at 90°C. The kinetic parameters K(m) and V(max) values for p-nitrophenyl-?-D-mannopyranoside and 1,4-?-D-mannan were 4.36±0.5 mM and 227.27±1.59 ?mol min?¹ mg?¹, 58.34±1.75 mg mL?¹ and 285.71±10.86 ?mol min?¹ mg?¹, respectively. The k(cat)/K(m) values for p-nitrophenyl-?-D-mannopyranoside and 1,4-?-D-mannan were 441.35±0.04 mM?¹ s?¹ and 41.47±1.58 s?¹ mg?¹ mL, respectively. It displayed high tolerance to mannose, with a K(i) value of approximately 900 mM. CONCLUSIONS: This work provides a novel and useful ?-mannosidase with high mannose tolerance, thermostability and catalytic efficiency, and these characteristics constitute a powerful tool for improving the enzymatic conversion of mannan through synergetic action with other mannan-degrading enzymes.

SUBMITTER: Shi H

PROVIDER: S-EPMC3852774 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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High-level expression of a novel thermostable and mannose-tolerant β-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli.

Shi Hao H Huang Yingjuan Y Zhang Yu Y Li Wenqian W Li Xun X Wang Fei F

BMC biotechnology 20131008

<h4>Background</h4>Mannan is one of the primary polysaccharides in hemicellulose and is widely distributed in plants. β-Mannosidase is an important constituent of the mannan-degrading enzyme system and it plays an important role in many industrial applications, such as food, feed and pulp/paper industries as well as the production of second generation bio-fuel. Therefore, the mannose-tolerant β-mannosidase with high catalytic efficiency for bioconversion of mannan has a great potential in the fi ...[more]

PMID: 24099409

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Project description:?-Xylosidase is an important constituent of the hemicellulase system and it plays an important role in hydrolyzing xylooligosaccharides to xylose. Xylose, a useful monose, has been utilized in a wide range of applications such as food, light, chemical as well as energy industry. Therefore, the xylose-tolerant ?-xylosidase with high specific activity for bioconversion of xylooligosaccharides has a great potential in the fields as above.A ?-xylosidase gene (Tth xynB3) of 2,322 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 774 amino acid residues, and was expressed in Escherichia coli BL21 (DE3). The phylogenetic trees of ?-xylosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the Tth xynB3 ?-xylosidase was a novel ?-xylosidase of GH3. The optimal activity of the Tth xynB3 ?-xylosidase was obtained at pH 6.0 and 95°C and was stable over a pH range of 5.0-7.5 and exhibited 2 h half-life at 85°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-?-D-xylopyranoside and p-nitrophenyl-?-L-arabinofuranoside were 0.27 mM and 223.3 U/mg, 0.21 mM and 75 U/mg, respectively. The kcat/Km values for p-nitrophenyl-?-D-xylopyranoside and p-nitrophenyl-?-L-arabinofuranoside were 1,173.4 mM-1 s-1 and 505.9 mM-1 s-1, respectively. It displayed high tolerance to xylose, with Ki value approximately 1000 mM. It was stimulated by xylose at higher concentration up to 500 mM, above which the enzyme activity of Tth xynB3 ?-xylosidase was gradually decreased. However, it still remained approximately 50% of its original activity even if the concentration of xylose was as high as 1000 mM. It was also discovered that the Tth xynB3 ?-xylosidase exhibited a high hydrolytic activity on xylooligosaccharides. When 5% substrate was incubated with 0.3 U Tth xynB3 ?-xylosidase in 200 ?L reaction system for 3 h, almost all the substrate was biodegraded into xylose.The article provides a useful and novel ?-xylosidase displaying extraordinary and desirable properties: high xylose tolerance and catalytic activity at temperatures above 75°C, thermally stable and excellent hydrolytic activity on xylooligosaccharides.

Project description:BackgroundMannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis.ResultsThe mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose.ConclusionOur study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.

Project description:BackgroundStreptomyces mobaraenesis transglutaminase (smTG) is widely used to generate protein crosslinking or attachment of small molecules. However, the low thermostability is a main obstacle for smTG application. In addition, it is still hard to achieve the secretory expression of active smTG in E. coli, which benefits the enzyme evolution. In this study, a combined strategy was conducted to improve the thermostability and secretory expression of active smTG in E. coli.ResultsFirst, the thermostable S. mobaraenesis transglutaminase variant S2P-S23V-Y24N-S199A-K294L (TGm1) was intracellularly expressed in pro-enzyme form in E. coli. Fusing the pro-region of Streptomyces hygroscopicus transglutaminase (proH) and TrxA achieved a 9.78 U/mL of intracellular smTG activity, 1.37-fold higher than the TGm1 fused with its native pro-region. After in vitro activation by dispase, the TGm1 with proH yielded FRAPD-TGm1, exhibiting 0.95 ℃ and 94.25% increases in melting temperature and half-life at 60 ℃ compared to FRAP-TGm1 derived from the expression using its native pro-region, respectively. Second, the TGm1 with proH was co-expressed with transglutaminase activating protease and chaperones (DnaK, DnaJ, and GrpE) in E. coli, achieving 9.51 U/mL of intracellular FRAPD-TGm1 without in vitro activation. Third, the pelB signal peptide was used to mediate the secretory expression of active TGm in E. coli, yielding 0.54 U/mL of the extracellular FRAPD-TGm1. A script was developed to shuffle the codon of pelB and calculate the corresponding mRNA folding energy. A 1.8-fold increase in the extracellular expression of FRAPD-TGm1 was achieved by the Top-9 pelB sequence derived from the coding sequences with the lowest mRNA folding energy. Last, deleting the gene of Braun's lipoprotein further increased the extracellular yield of FRAPD-TGm1 by 31.2%, reached 1.99 U/mL.ConclusionsThe stabilized FRAPD-smTG here could benefit the enzyme application in food and non-food sectors, while the E. coli system that enables secretory expression of active smTG will facilitate the directed evolution for further improved catalytic properties. The combined strategy (N-terminal modification, co-expression with chaperones, mRNA folding energy optimization of signal peptide, and lipoprotein deletion) may also improve the secretory expression of other functional proteins in E. coli.

Dataset Information

High-level expression of a novel thermostable and mannose-tolerant ?-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli.

Publications

High-level expression of a novel thermostable and mannose-tolerant β-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli.

Similar Datasets

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