Alkaline Protease Gene Cloning from the Marine Yeast Aureobasidium pullulans HN2-3 and the Protease Surface Display on Yarrowia lipolytica for Bioactive Peptide Production
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The alkaline protease genes (cDNAALP2 gene and ALP2 gene) were amplified from complementary DNA (cDNA) and genomic DNA of the marine yeast Aureobasidium pullulans HN2-3, respectively. An open reading frame of 1,248 bp encoding a 415-amino acid protein with a calculated molecular weight of 42.9 kDa was characterized. The ALP2 gene contained two introns, which had 54 and 52 bp, respectively. When the cDNAALP2 gene was cloned into the multiple cloning sites of the surface display vector pINA1317-YlCWP110 and expressed in cells of Yarrowia lipolytica, the cells displaying protease could form a clear zone on the double plate containing milk protein and had protease activity. The cells displaying alkaline protease were also found to be able to produce bioactive peptides from different sources of proteins. The peptides produced from single-cell protein of marine yeast strain G7a had the highest angiotensin-converting enzyme inhibitory activity, while the peptides produced from spirulina protein had the highest antioxidant activity. This is the first report that the yeast cells displaying alkaline protease were used to produce bioactive peptides.
KeywordsAlkaline protease gene Marine yeasts A. pullulans Surface display Bioactive peptides
This work was supported by Hi-Tech Research and Development Program of China (863). The grant no. is 2006AA09Z403.
- Adams A, Gottschling DE, Kaiser CA, Stearms T (1998) Yeast immunofluorescence. In: Burke D, Dawson D (eds) Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. Cold Spring Harbor Laboratory, New York, p. 100Google Scholar
- Gobbetti M, Ferranti P, Smacchi E, Goffredi F, Addeo F (2000) Production of angiotensin-I-converting-enzyme-inhibitory peptides in fermented milks started by Lactobacillus delbrueckii subsp. bulgaricus SS1 and Lactococcus lactis subsp. cremoris FT4. Appl Environ Microbiol 66:3898–3904PubMedCrossRefGoogle Scholar
- Inamura H, Nakai T, Muroga K (1985) An extracellular protease produced byVibrio anguillarum. Bull Jpn Soc Sci Fish 51:1915–1920Google Scholar
- Ni XM, Chi ZM, Liu ZQ, Yue LX (2008a) Screening of protease-producing marine yeasts for production of the bioactive peptides. Acta Oceanol Sin 27:116–125Google Scholar
- Okamoto H, Fujiwara T, Nakamura E, Katoh T, Iwamoto H, Tsuzuki H (1997) Purification and characterization of a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051; application to the recovery of bioactive peptides from fusion proteins by sequence-specific digestion. Appl Microbiol Biotechnol 48:27–33PubMedCrossRefGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, New York, pp 367–370 (Chinese translated edn.)Google Scholar
- Yang Y, Li J (2003) Isolation and properties of angiotensin converting enzyme from rabbit lung. Food Ferment Ind 8:53–56Google Scholar