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Research On The Characteristics Of A Recombinant Aspergillus Sojae Leucine Aminopeptidase1(sLAP1) Expressed In Pichia Pastoris

Posted on:2015-02-22Degree:MasterType:Thesis
Country:ChinaCandidate:W Q HuangFull Text:PDF
GTID:2180330422482441Subject:Biochemistry and Molecular Biology
Abstract/Summary:PDF Full Text Request
High efficient protein utilities attract more and more attention. In food industry,proteins are hydrolyzed by proteases to be small peptides and free amino acids to increasetheir absorption. However, many food protein hydrolysates show bitter taste, which directlyaffects their application value. Research found that the bitter taste mainly comes from lowmolecular weight peptides with terminal hydrophobic amino acids. Leucine aminopeptidasesare exopeptidases, which can selectively remove the hydrophobic amino acid residues fromthe N-terminal of the peptide chains or proteins and efficiently reduce the bitter taste ofprotein hydrolysates.In this study, the full lenght leucine aminopeptidase1(slap1) gene of Aspergillus sojaewas obtained by reverse transcription PCR. We have successfully constructed therecombination Pichia pastoris strain pPIC9K-slap1-KM71and harvested the solublerecombinant rSLAP1. In triangle flask fermentation,the productivity of rSLAP1was766.9U/ml. The rSLAP1was purified by Sephadex G-75column gel permeationchromatography. Experiments were carried out to investigate the enzyme characteristics of therSLAP1and its potential utilization in the debitterizing of alkaline and neutral proteasehydrolysates. The optimal temperature and pH of rSLAP1were respectively70℃and8.0inpotassium-phosphate buffer, and the activity was significantly stimulated by Co2+, followedby Mn2+. The substrate preference of rSLAP1was tested by different aminoacid-p-nitroanilide substrates,results showed its highest activity to Lue-pNA and Arg-pNAand lowest activity to Ala-pNA and Pro-pNA. The potential application of sLAP1wasevaluated through combination with two endoproteases Alcalase and a recombinant neutralprotease rNpI to hydrolysis the casein or soy protein. Results showed that rSLAP1canefficiently improved the hydrolysis degree of Alcalase soy bean hydrolysates23%, andimproved casin hydrolysates137.9%and133.9%. The bitter taste of Alcalase and rNpIhydrolysates was significantly removed by further rSLAP1hydrolysis. All these resultsshowed the great utility potential of this recombinant leucine-aminopeptidase rSLAP1.
Keywords/Search Tags:Aspergillus sojae, rSLAP1, Enzyme characteristics, Protein hydrolysis, Enzymatic debittering
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