High-level heterologous expression of an alkaline lipase gene from <Emphasis Type="Italic">Penicillium cyclopium</Emphasis> PG37 in <Emphasis Type="Italic">Pichia pastoris</Emphasis> |
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Authors: | Zhongbiao Tan Jianfang Li Minchen Wu Cunduo Tang Huimin Zhang Junqing Wang |
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Institution: | (1) School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu Road, 214122 Wuxi, Jiangsu, People’s Republic of China;(2) The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, 214122 Wuxi, Jiangsu, People’s Republic of China;(3) School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, 214122 Wuxi, Jiangsu, People’s Republic of China; |
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Abstract: | A 777-bp cDNA fragment encoding a mature alkaline lipase (LipI) from Penicillium cyclopium PG37 was amplified by RT–PCR, and inserted into the expression plasmid pPIC9 K. The recombinant plasmid, designated as pPIC9 K-lipI,
was linearized with SalI and transformed into Pichia pastoris GS115 (his4, Mut+) by electroporation. MD plate and YPD plates containing G418 were used for screening of the multi-copy P. pastoris transformants (His+, Mut+). One transformant resistant to 4.0 mg/ml of G418, numbered as P. pastoris GSL4-7, expressing the highest recombinant LipI (rLipI) activity was chosen for optimizing expression conditions. The integration
of the gene LipI into the P. pastoris GS115 genome was confirmed by PCR analysis using 5′- and 3′-AOX1 primers. SDS–PAGE and lipase activity assays demonstrated that the rLipI, a glycosylated protein with an apparent molecular
weight of about 31.5 kDa, was extracellularly expressed in P. pastoris. When the P. pastoris GSL4-7 was cultured under the optimized conditions, the expressed rLipI activity was up to 407 U/ml, much higher than that
(10.5 U/ml) expressed with standard protocol. The rLipI showed the highest activity at pH 10.5 and 25°C, and was stable at
a broad pH range of 7.0–10.5 and at a temperature of 30°C or below. |
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