Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris

BMC Microbiology

Marcelo Fernandez-Lahore¹  Martin Kangwa¹  Jose Antonio Gama Salgado¹ 

[1] School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, Bremen 28759, Germany

关键词: Milk clotting units (MCU);    Alpha factor secretion signal;    SMART™ RACE PCR (Rapid Amplification of cDNA Ends);    Pichia pastoris;    Mucor circinelloides;    Aspartic proteinase;   

Others  :  1142771
DOI  :  10.1186/1471-2180-13-250

 received in 2013-08-11, accepted in 2013-11-04,  发布年份 2013

【 摘 要 】

Background

Extracellular aspartic proteinase (MCAP) produced by Mucor circinelloides in solid state fermentations has been shown to possess milk clotting activity and represents a potential replacement for bovine chymosin in cheese manufacturing. Despite its prospects in the dairy industry, the molecular characteristics of this enzyme remain unknown. This work focuses on MCAP cloning and optimization of heterologous expression in Pichia pastoris, and characterization of the enzyme.

Results

The cloning of cDNA sequence encoding MCAP from M. circinelloides was performed using a fragment of approximately 1 kbp as a probe. The fragment was amplified using non-specific primers designed from the NDIEYYG and KNNYVVFN consensus motifs from aspartic proteinases of different fungi. Gene specific primers were designed to amplify a full-length cDNA using SMART™ RACE PCR. MCAP was expressed in P. pastoris under the control of the constitutive GAP promoter. It was shown that P. pastoris secreted non-glycosylated and glycosylated MCAPs with molecular weights of 33 and 37 kDa, respectively.

Conclusion

A novel MCAP was expressed in P. pastoris and efficiently secreted into the culture medium. The expression of the heterologous proteins was significantly increased due to advantages in codon usage as compared to other expression systems. The results suggest that P. pastoris could be exploited as a safe production platform for the milk clotting enzyme.

【 授权许可】

   
2013 Gama Salgado et al.; licensee BioMed Central Ltd.

【 图 表 】

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