title: Cloning and expression of an active aspartic proteinase from Mucor circinelloides in Pichia pastoris.
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 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.
  
  
  CONCLUSIONS
  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.
authors.forename: Jose Antonio
authors.initials: JA
authors.lastname: Gama Salgado
authors.forename: Martin
authors.initials: M
authors.lastname: Kangwa
authors.affiliation: School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, Bremen 28759, Germany. m.fernandez-lahore@jacobs-university.de.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Fernandez-Lahore
mesh_terms: D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009089:Mucor / Q000201:enzymology* / Q000235:genetics; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D011994:Recombinant Proteins; D057055:Aspartic Acid Proteases