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Cloning a novel endo-1,4-?-D-glucanase gene from Trichoderma virens and heterologous expression in E. coli.

ABSTRACT: Endo-1,4-?-D-glucanase (EG), as a key constituent of cellulase taking the responsibility of cutting ?-1,4 glycosidic bonds, plays the essential role in the process of degrading cellulose by cellulase. Cloning and expressing the EG gene is important to the cellulase research and application. In this work, a novel EG gene was cloned from Trichoderma virens ZY-01, which was a cellulase secreting microbe isolated by our laboratory. The DNA sequence showed that the length of the cloned EG is 1069 bp, which had 95.2% similarity to the EG IV from T. viride AS 3.3711. Further, the expression vector pET-32a-EG was constructed and was successfully heterologously expressed in Escherichia coli. The expression product was purified with Ni2+ affinity chromatography and its enzymatic properties were investigated. The SDS-PAGE showed the target protein is 39 kDa, which is consistent with the translated result from the DNA sequence. The kinetic parameter for the expression product was Km = 13.71 mg/mL and Vmax=0.51 ?mol/min·mL. The optimal reaction pH and temperature was pH = 7.0 and T = 40 °C, which is similar to the native EG produced by Trichoderma virens ZY-01. It provides the foundation for the endo-1,4-?-D-glucanase further evolution and application.

SUBMITTER: Zeng R

PROVIDER: S-EPMC5103005 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Cloning a novel endo-1,4-β-D-glucanase gene from Trichoderma virens and heterologous expression in E. coli.

Zeng Rong R Hu Qiao Q Yin Xiao-Yan XY Huang Hao H Yan Jia-Bao JB Gong Zhi-Wei ZW Yang Zhong-Hua ZH

AMB Express 20161109 1

Endo-1,4-β-D-glucanase (EG), as a key constituent of cellulase taking the responsibility of cutting β-1,4 glycosidic bonds, plays the essential role in the process of degrading cellulose by cellulase. Cloning and expressing the EG gene is important to the cellulase research and application. In this work, a novel EG gene was cloned from Trichoderma virens ZY-01, which was a cellulase secreting microbe isolated by our laboratory. The DNA sequence showed that the length of the cloned EG is 1069 bp, ...[more]

PMID: 27830495

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Project description:The food enzyme containing cellulase (EC 3.2.1.4), endo-1,3(4)-β-glucanase (EC 3.2.1.6) and endo-1,4-β-xylanase (EC 3.2.1.8) is produced with the non-genetically modified Trichoderma reesei strain AR-256 by AB-Enzymes GmbH. The food enzyme is considered free from viable cells of the production organism. It is intended to be used in seven food manufacturing processes: baking processes, cereal-based processes, brewing processes, fruit and vegetable processing for juice production, wine and wine vinegar production, distilled alcohol production and grain treatment for production of starch and gluten fractions. Since the residual amounts of total organic solids (TOS) are removed during grain treatment and distilled alcohol production, dietary exposure was estimated for the remaining five processes and amounted up to 3.92 mg TOS/kg body weight (bw) per day. The toxicity studies were carried out with an endo-1,4-β-xylanase from T. reesei ■■■■■, considered by the Panel as a suitable substitute, because the genetic differences between the strains are well characterised and of no concern. Additionally, several strains derived from the production strain are considered safe by EFSA and the manufacturing of both food enzymes is similar. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity rat study. The no observed adverse effect level of 939 mg TOS/kg bw per day, the highest dose tested, compared with the estimated dietary exposure, resulted in a margin of exposure above 239. In the search for the similarity of the amino acid sequences to known allergens, one match (salmon) was found. The Panel considered that, under the intended conditions of use (except distilled alcohol production), the risk of allergic reactions by dietary exposure cannot be excluded, in particular for individuals sensitised to salmon. The Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Dataset Information

Cloning a novel endo-1,4-?-D-glucanase gene from Trichoderma virens and heterologous expression in E. coli.

Publications

Cloning a novel endo-1,4-β-D-glucanase gene from Trichoderma virens and heterologous expression in E. coli.

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