Characterization of an N‐glycosylated Bacillus subtilis leucine aminopeptidase expressed in Pichia pastoris

Abstract

Aminopeptidase is an important flavorsome especially in protein hydrolysate debittering by removing hydrophobic amino acid residue at the N-terminal end. Besides, it is also applied to preparation of active peptides and analysis of protein sequence. In this study, leucine aminopeptidase from Bacillus subtilis was cloned and expressed in Pichia pastoris, a widely used heterologous protein expression host. Then it was purified and characterized. After methanol induction for 96 h, the aminopeptidase activity in culture supernatant reached 28.4 U ml(À1) , which was 7.1 times that of wild strain B. subtilis 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 Ni(2β) , Ca(2β) , DL-dithiothreitol (DTT) and ethylene diamine tetraacetic acid (EDTA), but activated by Co(2β) . 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 (SDS-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.

Quantitative N-Glycoproteomic Analysis of Cattle-Yak and Yak Longissimus Thoracis

“…Of these, only one N-glycosylation site showed a significant difference in abundance levels between CYLT and YLT, N 577 (CYLT:YLT = 3.63). Previous studies found that N-glycosylated aminopeptidases exhibited higher thermal stability and catalytic efficiency . This study demonstrated that N-glycosylation would promote aminopeptidase catalysis to increase the level of protein degradation in CYLT.…”

“…Previous studies found that Nglycosylated aminopeptidases exhibited higher thermal stability and catalytic efficiency. 49 This study demonstrated that Nglycosylation would promote aminopeptidase catalysis to increase the level of protein degradation in CYLT.…”

Halotolerant aminopeptidase M29 from Mesorhizobium SEMIA 3007 with biotechnological potential and its impact on biofilm synthesis

“…Furthermore, in certain cases, along with the metagenomic approach 29 , it is possible to identify new enzymes with high biotechnological potential in a sea of bioinformatics data. Some thermo-tolerant 52 , solvent-tolerant 20 and halo-tolerant 53 aminopeptidases have been reported, however, none display all three features simultaneously (see Table 4 ).…”

A novel aminopeptidase with potential debittering properties in casein and soybean protein hydrolysates

“…Aminopeptidase activity was determined according to Xi et al (2015) using 2 mM Arg-pNA as substrate suspended in 50 mM Tris-HCl, pH 7.0 at 40 °C for 10 min. The concentration of released p-nitroaniline was calculated using absorbance readings at 405 nm in a microplate reader (TECAN, Ma ¨nnedorf, Netherland).…”