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Influences of N-linked glycosylation on the biochemical properties of aspartic protease from Aspergillus glaucus MA0196
- Lim, Lihui, Senba, Hironori, Kimura, Yukihiro, Yokota, Satoko, Doi, Mikiharu, Yoshida, Ken-Ichi, Takenaka, Shinji
- Process biochemistry 2019 v.79 pp. 74-80
- thermal stability, Komagataella pastoris, decolorization, polyacrylamide gel electrophoresis, industry, molecular weight, Aspergillus glaucus, sequence analysis, meat, mutagenesis, tuna, water content, hydrolysis, aspartic proteinases, detergents, meat processing, glycosylation, hemoglobin, pH, mutants, heme proteins, water activity
- Aspergillus glaucus MA0196 produces a highly glycosylated aspartic protease (PepA_MA0196) that shows hydrolytic and decolorization activities toward hemoglobin. Sequence analysis of PepA_MA0196 indicated two potential N-glycosylation consensus sites, at Asn131 and Asn275. To elucidate the role of N-glycosylation in the biochemical properties of PepA_MA0196 and the resulting effects, recombinant PepA_MA0196 and three mutant proteases deglycosylated via site-direct mutagenesis were heterologously expressed in Pichia pastoris. After hydrolysis of the enzymes by PNGase F or Endo H, the molecular masses on SDS-PAGE of the enzymatically deglycosylated wild-type PepA_MA0196 and recombinant and single-mutant proteases were identical to that of the double-mutant protease (42.2 kDa). PepA_MA0196 and its recombinant were stable over a broader pH range and more thermostable than the deglycosylated mutant proteases, especially the double-mutant protease. In addition, PepA_MA0196 retained a higher activity at low water activity than did the double-mutant protease. The commercial applications of the glycosylated protease from strain MA0196 include hydrolysis of the heme proteins in dried tuna meat under low water content. Hemoprotein lysis is also a property of interest to the meat processing and detergent industries.