NDLI: High-level heterologous expression of an alkaline lipase gene from Penicillium cyclopium PG37 in Pichia pastoris

Content Provider	Springer Nature Link
Author	Tan, Zhongbiao Li, Jianfang Wu, Minchen Tang, Cunduo Zhang, Huimin Wang, Junqing
Copyright Year	2011
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.
Starting Page	2767
Ending Page	2774
Page Count	8
File Format	PDF
ISSN	09593993
Journal	World Journal of Microbiology and Biotechnology
Volume Number	27
Issue Number	12
e-ISSN	15730972
Language	English
Publisher	Springer Netherlands
Publisher Date	2011-04-30
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Expression Alkaline lipase gene Penicillium cyclopium Pichia pastoris Optimization Enzymatic properties Environmental Engineering/Biotechnology Applied Microbiology Microbiology Biotechnology Biochemistry
Content Type	Text
Resource Type	Article
Subject	Physiology Medicine Applied Microbiology and Biotechnology Biotechnology

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