Gene Identification, Molecular Modification and Efficient Extracellular Expression of...

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Title
Gene Identification, Molecular Modification and Efficient Extracellular Expression of Thermobifida fusca Cutinase
Speaker
Jing Wu, Jiangnan University
Date & Time
Monday, January 8, 2018 - 14:00 to 15:00
Location
Room 264, Geography Building, 3663 Zhongshan Road North, Shanghai

Abstract:
Cutinase is a multifunctional enzyme which has a broad application in the textiles, detergents and ester synthesis. In this study, the gene encoding T. fusca cutinase, Tfu_0883 and Tfu_0882, was identified by the combined technology of inducible expression of the enzyme, peptide fingerprinting, database comparison as well as gene recombination. On the basis of analyzing the crystal structure of cutinase, the mutants of I218A and Q132A/T101A with improved hydrolysis capacity of the polyester were obtained. In addition, the cellulose-binding domain was screened from database for fusion with cutinase. The catalytic efficiency of the constructed fusion protein toward cotton was improved by more than 3 times when combined with pectinase. In order to increase the production of cutinase, different E.coli secretion systems have been used. When expressed by type II secretion system, extracellular cutinase activity was only 10-15% of total enzyme activity. With type I secretion system, extracellular secretion of cutinase can increased to 45-55%. Moreover, when the cutinase expressed without mediation of any signal peptide, the extracellular "secretion" of the enzyme was found to be 92.5% of the total expression, which was unexpected. The fermentation results in a 3-L fermenter showed that the cutinase activity in the medium reached 1063.5 U·mL-1, and the productivity was 40.9 U·mL-1·h-1, which was 2.4 and 6.3 times higher than that of type I and II secretion system, respectively. Furthermore, the mechanism of extracellular "secretion" of cutinase expressed without mediation of signal peptide was explored.

Biography:

Dr. Wu obtained bachelor’s and master’s degree of Fermentation Engineering from Wuxi Institute of Light Industry, and doctoral degree of Microbial and Biochemical Pharmacy from China Pharmaceutical University. She performed post-doctoral training of Biochemistry in University of Michigan, and is now a full professor in State Key Laboratory of Food Science and Technology, Jiangnan University. Dr. Wu’s work focuses on the development of basic theories and key technologies for gene identification, functional reconstruction and high-yield production of enzymes utilized in food industry. So far, she has published more than 160 papers and about 110 of them were collected by SCI. She has chief-edited 3 scientific books, obtained 46 authorized patents (3 international). Dr. Wu received the First Prize of Progress in Science and Technology awarded by the Ministry of Education (ranking No.1). Moreover, she was elected to the Young Scientific and Technological Innovation Leader program by the Ministry of Science and Technology and the Excellent Talents in New Century Program by the Ministry of Education, and received the National Outstanding Researcher in Science and Technology Award. In addition, she was supported by National Science Fund for Distinguished Young Scholars and Yangtze River Scholar Bonus Schemes.

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