Aminopeptidase is an important flavorsome especially in protein hydrolysate debittering by removing hydrophobic @entity535 residue at the N-terminal end. Besides, it is also applied to preparation of active peptides and analysis of protein sequence. In this study, @entity368 aminopeptidase from @entity3881 was cloned and expressed in @entity10056 , a widely used heterologous protein expression host. Then it was purified and characterized. After @entity1042 induction for 96   h, the aminopeptidase activity in culture supernatant reached 28.4   U   ml(-1) , which was 7.1 times that of wild strain @entity3881 Zj016. The optimal temperature and pH of the purified recombinant enzyme were 60     C and 8.5, respectively. The purified aminopeptidase was stable within 30-60     C and pH 8.0-9.0. It was intensively inhibited by @entity3411 , @entity141 , DL-dithiothreitol (DTT) and @entity1098 ( @entity1098 ), but activated by @entity515 . The Km toward leucine-p-nitroanilines (Leu-pNA) of the enzyme was 0.97   mM. The sequence analysis of aminopeptidase indicated three potential N-glycosylation sites and it was further verified via MALDI-TOF-MS analysis. Consequently, the N-glycosylated aminopeptidase exhibited higher thermostability and catalytic efficiency. The purified enzyme exhibited two bands through sodium dodecyl sulfate polyacrylamide gel electrophoresis ( @entity214 -PAGE) while a single band can be identified when the enzyme was deglycosylated. Circular dichroism spectroscopy indicated that the secondary structure of recombinant aminopeptidase was similar to the wild-type.

Characterization of an N-glycosylated @entity3881 XXXX aminopeptidase expressed in @entity10056 .

@entity368 :: ('CHEBI:25017', 'Chemical') :: ['leucine'] @entity535 :: ('CHEBI:33704', 'Chemical') :: ['amino acid'] @entity1042 :: ('MESH:D000432', 'Chemical') :: ['methanol'] @entity10056 :: ('4922', 'Species') :: ['Pichia pastoris'] @entity515 :: ('CHEBI:29334', 'Chemical') :: ['Co(2+)'] @entity3411 :: ('CHEBI:49786', 'Chemical') :: ['Ni(2+)'] @entity141 :: ('CHEBI:29108', 'Chemical') :: ['Ca(2+)'] @entity3881 :: ('1423', 'Species') :: ['Bacillus subtilis', 'B. subtilis'] @entity1098 :: ('MESH:D004492', 'Chemical') :: ['EDTA', 'ethylene diamine tetraacetic acid'] @entity214 :: ('CHEBI:8984', 'Chemical') :: ['SDS']
@entity368 :: (CHEBI:25017,Chemical) :: ['leucine']
