title: Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization.
abstract: A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca(2+) and inhibited by Zn(2+), Mn(2+), Al(3+), and Cu(2+). The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China, kangchao61@hotmail.com.
authors.forename: Chao
authors.initials: C
authors.lastname: Kang
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Yu
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001515:Beer / Q000382:microbiology*; D019149:Bioreactors / Q000382:microbiology; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D005285:Fermentation; D010843:Pichia / Q000235:genetics; D000085822:Prolyl Oligopeptidases; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D012697:Serine Endopeptidases / Q000096:biosynthesis*; D013696:Temperature
chemical_list: D004271:DNA, Fungal; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases