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

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

doi:10.1186/1471-2180-13-250

Jose Antonio Gama Salgado, Marcelo Fernandez-Lahore + Show 1 more

Open Access

https://doi.org/10.1186/1471-2180-13-250

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Journal: BMC Microbiology	Publication Date: Nov 9, 2013
Citations: 35	License type: cc-by

Affiliation: Constructor University

Abstract

BackgroundExtracellular 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.ResultsThe 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.ConclusionA 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.

Highlights

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
Cloning and gene comparison of the cDNA encoding the acidic proteinase After obtaining the partial DNA sequence of MCAP, specific primers were designed for the amplification of 3′-RACE and 5′-RACE of aspartic proteinase gene from the first-strand cDNA of M. circinelloides by SMARTTM RACE PCR
The full-length cDNA of the aspartic proteinase from M. circinelloides was amplified from the 5′ first-strand, while the full-length MCAP encoding the aspartic proteinase was amplified from genomic DNA of M. circinelloides

Summary

Introduction

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. A milk coagulant from the abomasum of milkfed calves and lambs, is the industrial gold standard in cheese manufacturing [1]. Due to increased demand in cheese products, animal-derived milk coagulants are not sufficient to cover the production. The demand has prompted increased research efforts in the manufacture of recombinant and microbial rennin [3]. The rennin of microbial origin might be contaminated by other enzymes. Microbial APs such as rhizopuspepsin and penicillopepsin have been reported to be either intracellular or extracellular enzymes with most of them having been cloned and purified. Acid proteinase from Metschnikowia reukaufii [5] has been cloned and expressed in Escherichia coli while three clt genes encoding milk-clotting proteinases from Myxococcus xanthus have been cloned and expressed in E. coli, Saccharomyces cerevisiae and P. pastoris [3]

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