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| High Cell-density Fermentation and Shaking Flask Optimization of Geotrichum candidum Lipase Production in Multi-copy Pichia pastoris |
| ZHI Xiao-yan, WANG Xiao-feng, SUN Yong-chuan, KE Feng, DAI Min, YAN Yun-jun |
| Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China |
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Abstract Objective: To effectively express Geotrichum candidum lipase (GCL) in Pichia pastoris, optimize the culture conditions of the recombinant strain in shaking flask and investigate its high-level expression in a 3L bioreactor. Methods: The inducible secretion vector pPIC9K-gcl was transformed into P. pastoris GS115 by electroporation. Clones with high lipase activity were screened by olive oil-rhodamine B plates and shaking flask cultivation. Real-time PCR based TaqMan probe was applied to determine the copy number of G. candidum lipase gene in the target clones. Then six key factors of shaking flask cultivation were optimized, including induction time, methanol concentration per 24h, culture medium initial pH, inoculation concentration, culture medium volume in shake flask and induction temperature. Based on the results of shaking flask cultivation, fed-batch fermentation was implemented in a 3L bioreactor. Results: A clone GS115/pPIC9K-gcl 78# which possessed three copies of G. candidum lipase gene was obtained. Its initial lipase activity was up to 220 U/ml. The results also showed that when the cultivation conditions were 96 h after methanol induction, 1% of methanol addition per 24 h, 2% of inoculation concentration, initial pH 7.0 of culture medium, 50 ml of culture medium volume, and induction temperature at 25 ℃, the lipase activity reached 735 U/ml. In the 3L bioreactor, lipase activity achieved 3360 U/ml and the total protein concentration reached 4.30 g/L after 176.5 h cultivation. Moreover, the cell viability was above 96% during the high cell-density fermentation. Conclusion: Pichia pastoris GS115 is a suitable host for high-level expression of G. candidum lipase. Multiple gene copies had a positive effect on expression level of GCL. Single factor optimization can effectively enhance lipase expression. This high cell-density fermentation would offer a solid basis for large-scale production of GCL.
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Received: 16 November 2011
Published: 25 February 2012
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