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Nasir Ali

Nasir Ali

Xiamen University, China

Title: Heterogeneous expression and functional characterization of β-glucosidase gene from AspergillusnigerBE-2 and construction of it’s hyper-expression system in Trichoderma orientalis EU7-22 by (cbh1) promoter optimization

Biography

Biography: Nasir Ali

Abstract

The cDNA gene (AnBgL1), encoding GH3 family β-glucosidase (EC3.2.1.21) from Aspergillus niger BE-2 (abbreviated to AnBgL1) was amplified and inserted into the yeast expression pPIC9K vector at the site of Bln I (Avr II) and Not1. The recombinant expression vector designated as pPIC9K-AnBgL1 was transformed into Pichia pastoris GS115. The transformants were screened on a MD plate which inoculated on geneticin G418-containing YPD plates. The transformants expressed the high β-glucosidase activity of 22.6 U/ml. SDS-PAGE assay demonstrated that the AnBgL1 was extracellularly expressed with an apparent M.W. of 90.0 kDa. The purified AnBgL1 displayed the maximum activity at pH 6.0 and 60° C. It was highly stable at a broad pH range of 4.0-7.5, and at a temperature of 60° C. The Km and Vmax, towards p-NPG at pH 5.5 and 60° C were 1.45 mg/ml and 2,365 U/mg, respectively. The AnBgL1 displays high similarity to the β-glucosidases of A. niger (FN430671) and A. niger (DQ655704), the members of the GH3 family. The β-glucosidase gene (Bgl1) from A. niger was cloned and recombined with cbh1 optimized promoter (pcbh1) and terminator trpC. The expression cassette was ligated to the binary vector to form pUR5750-Bgl1 and then transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT) using hygromycin B resistance gene as the screening marker. Bgl-1 transformants was screened. The enzyme activities of filter paper (FPA) and β-glucosidase (BG) of transformants increased by 8.5% and 15.2% under induction condition, respectively compared with the host strain EU7-22. The results showed that the cbh1 promoter (pcbh1) has successfully driven the over-expression of Bgl1 gene in T. orientalis under glucose repression condition.