Purification and characterisation of a leucine aminopeptidase from Lactococcus lactis subsp. lactis cultured in skim milk

Ye, Xiu Juan; Ng, T.B.

doi:10.1016/j.idairyj.2011.02.007

Cited by 6 publications

(4 citation statements)

References 10 publications

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“…BAAP was inhibited by reducing agent β‐ME, DTT EDTA and Bestatin at 1 m m , the residual enzyme activity was 13.1%, 21.3%, 29.5% and 10.6%, respectively. Amongst them, Bestatin was considered to inhibit leucine aminopeptidase specifically (Ye & Ng, 2011). The influence of STI and Pepstatin was not significant under the tested conditions.…”

Section: Resultsmentioning

confidence: 99%

An arginine aminopeptidase from marine Bacillus axarquiensis SWJSX8 and its application in improving pumpkin seed protein hydrolysis

Lei

Chen

et al. 2021

Int J of Food Sci Tech

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Summary An arginine aminopeptidase from Bacillus axarquiensis SWJSX8 (BAAP) was developed for the first time. The enzyme was purified 16.6‐fold with specificity activity of 267.2 U/mg protein and a total yield of 10.5% and was estimated to be 67.0 KDa by SDS‐PAGE. It exhibited the maximum activity at 55 °C and pH 8.5 and was activated by 15–20% NaCl with 50% improvement. BAAP was a metalloaminopeptidase and showed better preference for Arg‐pNA, Leu‐pNA and Lys‐pNA amongst the substrates tested. The introduction of BAAP improved the hydrolysis degree of pumpkin seed protein hydrolysates (PSPHs) by 5.7–8.2% and significantly increased the contents of small peptides and free amino acids, while decreased the bitter intensity score of the PSPHs hydrolysed by Alcalase 2.4 L. The antioxidant capacity of the PSPHs had also been improved. The newly developed BAAP has great potential to be used in hydrolysing proteins that are rich in arginine.

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Section: Resultsmentioning

confidence: 99%

An arginine aminopeptidase from marine Bacillus axarquiensis SWJSX8 and its application in improving pumpkin seed protein hydrolysis

Lei

Chen

et al. 2021

Int J of Food Sci Tech

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“…LAPs are generally most active across neutral and basic pH levels but exceptions have been reported [ 40 ]. Activity of rMHJ_0461 against leucine-, methionine- and phenylalanine AMC-coupled substrate in the presence of Mn 2+ was tested using a range of buffered substrates from pH 4 to pH 10.…”

Section: Resultsmentioning

confidence: 99%

MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

et al. 2015

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Aminopeptidases are part of the arsenal of virulence factors produced by bacterial pathogens that inactivate host immune peptides. Mycoplasma hyopneumoniae is a genome-reduced pathogen of swine that lacks the genetic repertoire to synthesize amino acids and relies on the host for availability of amino acids for growth. M. hyopneumoniae recruits plasmin(ogen) onto its cell surface via the P97 and P102 adhesins and the glutamyl aminopeptidase MHJ_0125. Plasmin plays an important role in regulating the inflammatory response in the lungs of pigs infected with M. hyopneumoniae. We show that recombinant MHJ_0461 (rMHJ_0461) functions as a leucine aminopeptidase (LAP) with broad substrate specificity for leucine, alanine, phenylalanine, methionine and arginine and that MHJ_0461 resides on the surface of M. hyopneumoniae. rMHJ_0461 also binds heparin, plasminogen and foreign DNA. Plasminogen bound to rMHJ_0461 was readily converted to plasmin in the presence of tPA. Computational modelling identified putative DNA and heparin-binding motifs on solvent-exposed sites around a large pore on the LAP hexamer. We conclude that MHJ_0461 is a LAP that moonlights as a multifunctional adhesin on the cell surface of M. hyopneumoniae.

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“…Many APs were reported to be metalloproteases, such as lysine aminopeptidase from Japanese flounder skeletal muscle , aminopeptidase from earthworm Eisenia foetida , and leucine aminopeptidase from Lactococcus lactis subsp. lactis .…”

Section: Resultsmentioning

confidence: 99%

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

Tian

Zhou

et al. 2014

J. Basic Microbiol.

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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.

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Purification and characterisation of a leucine aminopeptidase from Lactococcus lactis subsp. lactis cultured in skim milk

Cited by 6 publications

References 10 publications

An arginine aminopeptidase from marine Bacillus axarquiensis SWJSX8 and its application in improving pumpkin seed protein hydrolysis

An arginine aminopeptidase from marine Bacillus axarquiensis SWJSX8 and its application in improving pumpkin seed protein hydrolysis

MHJ_0461 is a multifunctional leucine aminopeptidase on the surface of Mycoplasma hyopneumoniae

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

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