title: High expression of a neutral endo-β-glucanase gene from Humicola insolens in Trichoderma reesei.
abstract: The neutral endo-β-glucanase gene cel5A from Humicola insolens was cloned and connected with the cellobiohydrolase 1 promoter from Trichoderma reesei to construct a recombinant plasmid pCB-hEG with the hygromycin B resistance marker. The plasmid was introduced into conidia of T. reesei using the Agrobacterium tumefaciens mediated transformation method. Eight transformants were obtained on screening plates with sodium carboxymethyl cellulose as the sole carbon source. Stable integration of the cel5A gene into the chromosomal DNA of T. reesei was confirmed by PCR. An obvious protein band (approximately 52 kDa) was detected by SDS-PAGE from fermentation broth, which showed that the cel5A gene in recombinant T. reesei successfully fulfilled efficient expression and extracellular secretion. After 96 h shaking-flask fermentation, the endo-β-glucanase activity at pH 6.5 from recombinant T. reesei reached 3,068 U/ml, which was 11 times higher than that of the host strain. In a 2 m³ fermenter, the endo-β-glucanase activity could be further increased to 8,012 U/ml after 96 h fermentation. The results showed a good prospect for application of neutral endo-β-glucanase in the textile industry.
authors.affiliation: Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
authors.forename: Bintao
authors.initials: B
authors.lastname: Gu
authors.forename: Liming
authors.initials: L
authors.lastname: Xia
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D019149:Bioreactors; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics; D003001:Cloning, Molecular; D005285:Fermentation; D005819:Genetic Markers / Q000235:genetics; D006921:Hygromycin B / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D013783:Textile Industry; D014170:Transformation, Genetic; D014242:Trichoderma / Q000235:genetics* / Q000378:metabolism
chemical_list: D005819:Genetic Markers; D006921:Hygromycin B; C407024:endoglucanase Cel5A; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase