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Purification and Characterization of the Recombinant Multifunctional Cellulase from Volvariella volvacea

Author:
Zhao, Li-Chao, Guo, Li-Qiong, Xiao, Hang, Zheng, Lan-Juan, Wang, Yan, Lin, Jun-Fang
Source:
Food biotechnology 2012 v.26 no.2 pp. 164-179
ISSN:
1532-4249
Subject:
Volvariella volvacea, endo-1,4-beta-glucanase, gel chromatography, gene expression, genes, heat, hydrolysis, ion exchange, manganese, metal ions, pH, polyacrylamide gel electrophoresis, post-translational modification, temperature, ultrafiltration, xylan
Abstract:
The recombinant multifunctional cellulase (MFCase) produced from strain TVM5186 of Volvariella volvacea was purified 270-fold by ultrafiltration, DEAE-Sepharose Fast Flow ion-exchange resin chromatography, and Sephadex G-100 gel filtration, after which the enzyme was characterized. The SDS-PAGE of the transformants showed a prominent band approximately 46 kDa corresponding to the expected size of MFCase (45.8 kDa). The characteristics of the recombinant MFCase were consistent with that expressed by the original gene, indicating that Volvariella volvacea is a foreign gene expression system with correct posttranslational protein processing. The optimum temperatures and pH of the recombinant MFCase for the hydrolysis of xylan and CMC were 55°C and 50°C, and 5.5 and 6.0, respectively. The stability results showed that recombinant MFCase was sensitive to high temperature. The relatively activities remained at 75.6% and 74.4% after heating for 1 h at 50°C, after which its activity decreased. While the pH stability tests showed that the recombinant MFCase is active over a wide range of pH (between 4.0 and 9.0), especially under alkaline conditions, the effects of various metal ions on the activity of the recombinant MFCase were different. With xylan as the substrate, the effects of most metal ions were positive. Only Mn²⁺ had a significantly negative effect (74.3% of CK). When the substrate was CMC, the recombinant MFCase was insignificantly inhibited by most metal ions. But Mn²⁺ also had a significantly negative effect (54.4% of CK). Kinetic studies revealed that MFC has a higher affinity for xylan than CMC.
Agid:
265496