Journal Article

Characterization of Trichoderma reesei Endoglucanase Ⅱ Expressed Heterologously in Pichia pastoris for Better Biofinishing and Biostoning

Halder, Umesh Chandra, Sen, Soumitra Kumar, Samanta, Sutanu, Basu, Asitava

2012

Overview

The endoglucanase Ⅱ of Trichoderma reesei is considered the most effective enzyme for biofinishing cotton fabrics and biostoning denim garments. However, the commercially available preparation of endoglucanase Ⅱ is usually mixed with other cellulase components, especially endoglucanase I, resulting in hydrolysis and weight loss of garments during biofinishing and biostoning. We thus isolated the endoglucanase Ⅱ gene from T. reesei to express this in Pichia pastoris, under the control of a methanol-inducible AOX1 promoter, to avoid the presence of other cellulase components. A highly expressible Mut+ transformant was selected and its expression in BMMH medium was found most suitable for the production of large amounts of the recombinant protein. Recombinant endoglucanase Ⅱ was purified to electrophoretic homogeneity, and functionally characterized by activity staining. The specific activity of recombinant endoglucanase Ⅱ was found to be 220.57 EU/mg of protein. Purified recombinant endoglucanase Ⅱ was estimated to have a molecular mass of 52.8 kDa. The increase in molecular mass was likely due to hyperglycosylation. Hyperglycosylation of recombinant endoglucanase Ⅱ secreted by P. pastoris did not change the temperature or pH optima as compared to the native protein, but did result in increased thermostability. Kinetic analysis showed that recombinant endoglucanase was most active against amorphous cellulose, such as carboxymethyl cellulose, for which it also had a high affinity.

• Publisher: Springer-Verlag,The Microbiological Society of Korea,Microbiological Society of Korea (MSK),한국미생물학회
• Subject: Biomedical and Life Sciences / biotechnology / Carboxymethylcellulose / Cellulase / Cellulase - chemistry / Cellulase - genetics / Cellulase - isolation & purification / Cellulase - metabolism / Cellulose / chemistry / Cloning, Molecular / Cotton / Culture Media / Culture Media - chemistry / Denim / Efficiency / electrophoresis / endo-1,4-beta-glucanase / Endoglucanase / enzyme kinetics / Enzyme Stability / Enzymes / enzymology / Fabrics / Gene Expression / genes / Genetic engineering / genetically engineered microorganisms / genetics / glycosylation / growing media / Hydrogen-Ion Concentration / Hydrolysis / hyperglycosylation / Hypocrea jecorina / isolation & purification / Kinetics / Komagataella pastoris / Life Sciences / metabolism / methanol / methanol inducible promoter / Microbiology / Molecular Weight / pH effects / Pichia / Pichia - genetics / Pichia pastoris / Plasmids / promoter regions / Promoters / Proteins / recombinant proteins / Recombinant Proteins - chemistry / Recombinant Proteins - genetics / Recombinant Proteins - isolation & purification / Recombinant Proteins - metabolism / Substrate Specificity / Temperature / Temperature effects / Textile industry / Thermal stability / thermostability / Trichoderma / Trichoderma - enzymology / Trichoderma - genetics / TRICHODERMA LONGIBRACHIATUM / Trichoderma reesei / weight loss / 생물학