Rhizopus chinensis lipase: Gene cloning,expression in Pichia pastoris and properties |
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| Institution: | 1. Gwangju Bio/Energy R&D Center, Korea Institute of Energy Research (KIER), Gwangju 61003, Republic of Korea;2. Department of Food Science and Technology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 34134, Republic of Korea;3. Department of Chemical Engineering and Materials Science, Graduate Program in System Health Science & Engineering, Ewha Womans University, Seoul 03760, Republic of Korea;4. Department of Biotechnology, The Catholic University of Korea, Bucheon-si, Gyeonggi-do 14662, Republic of Korea;5. School of Chemical and Biological Engineering, Seoul National University, Seoul 08826, Republic of Korea;1. School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Shenzhen, 518107, China;2. School of Food Science and Engineering, South China University of Technology, Guangzhou, 510640, China;1. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China;2. Department of Natural Sciences, Northeastern State University, Broken Arrow, OK 74014 USA;2. Wuliangye Yibin Co. Ltd., Yibin, 644007, Sichuan Province, PR China |
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| Abstract: | Lipases are the most attractive enzymes for use in organic chemical processes. In our previous studies, a lipase from Rhizopus chinensis CCTCC M20102 was found to have very high ability of esterification of short-chain fatty acids with ethanol. In this study, we reported the cloning and expression of the lipase gene from R. chinensis in Pichia pastoris and characterization of the recombinant lipase. The lipase gene without its signal sequence were cloned downstream to the alpha-mating factor signal and expressed in P. pastoris GS115 under the control of AOX1 promoter. In the induction phase, two bands of 37 kDa and 30 kDa proteins could be observed. The amino-terminal analysis showed that the 37-kDa protein was the mature lipase (30 kDa) attached with 27 amino acid of the carboxy-terminal part of the prosequence (r27RCL). The pH and temperature optimum of r27RCL and mRCL were pH 8.5 and 40 °C, and pH 8 and 35 °C, respectively. The stability, reaction kinetics and effects of metal ions and other reagents were also determined. The chain length specificity of r27RCL and mRCL showed highest activity toward p-nitrophenyl hexanoate or glyceryl tricaproate (C6) and p-nitrophenyl acetate or glyceryl triacetate (C2), respectively. This property is quite rare among lipases and gives this new lipase great potential for use in the field of biocatalysis. |
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