Asparagine as a nitrogen source for improving the secretion of mouse alpha-amylase in Saccharomyces cerevisiae protease A-deficient strains.
A modified chemically defined medium was achieved by using asparagine as a nitrogen source to increase the production of secreted mouse alpha-amylase in several Saccharomyces cerevisiae protease A-deficient (pep4) strains. The specific productivity (quantity) and the 53 kDa non-glycosylated active form (quality) of mouse salivary alpha-amylase in liquid medium containing asparagine was remarkably improved compared to media containing other nitrogen sources, including ammonium sulphate, glutamic acid, arginine, casamino acids, yeast extract and peptone. Similar improvement was also observed on starch solid agar regarding the clarity and size of the halo zone formed by alpha-amylase activity. Compared with ammonium sulphate, advantages of using asparagine as the nitrogen source in liquid or solid medium included increasing the cell mass of test strains, recovering the viability of protease-deficient strains to levels similar to the wild-type strain, and increasing the copy number of the mouse alpha-amylase expression vector in test strains. In turn, these advantages apparently contributed to the increase of secretion of mouse alpha-amylase in several test strains and especially in the protease A-deficient strains. In addition to demonstrating the use of modified chemically defined medium to improve the quality and quantity of secreted mouse alpha-amylase, this study also provides a new strategy to improve the secretion of heterologous proteins in protease A deficient strains.
Chen
D C
DC
Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China.
Wang
B D
BD
Chou
P Y
PY
Kuo
T T
TT
D000596:Amino Acids / Q000378:metabolism; D000645:Ammonium Sulfate / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D004274:DNA, Recombinant; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D009584:Nitrogen / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D003470:Culture Media; D004274:DNA, Recombinant; D029701:Saccharomyces cerevisiae Proteins; D001216:Asparagine; D000516:alpha-Amylases; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases; D009584:Nitrogen; D000645:Ammonium Sulfate