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Open Access Research article

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

Jose Antonio Gama Salgado, Martin Kangwa and Marcelo Fernandez-Lahore*

Author Affiliations

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

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BMC Microbiology 2013, 13:250  doi:10.1186/1471-2180-13-250

Published: 9 November 2013

Abstract

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 SMARTâ„¢ 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.

Keywords:
Aspartic proteinase; Mucor circinelloides; Pichia pastoris; SMARTâ„¢ RACE PCR (Rapid Amplification of cDNA Ends); Alpha factor secretion signal; Milk clotting units (MCU)