An Aminopeptidase From Marine Bacillus Licheniformis SWJS33 And Its Application

Aminopeptidases are a kind of extroproteases that attack the peptide chain at the N-terminus.They could efficiently improve the degree of hydrolysis(DH),improve the content of free amino acid(FAA),and modify the taste of hydrolysates by used together with endoproteases.The widely application of aminopeptidase has not been realized due to the limited commercially available aminopeptidases.This paper was aimed at screening safety aminopeptidase-producing strain from deep-sea sediments,optimizing the aminopeptidase fermentation process by using soybean meal as substrate,purifying and characterizing the aminopeptidase,studying the hydrolysis specificity of the enzyme and applying the enzyme in hydrolyzing peanut protein isolate and zein.(1)Among the aminopeptidase-producing strains isolated,Bacillus licheniformis SWJS33 finally selected due to its safety and better performance in soy protein isolate hydrolysis.The fermentation conditions were as follow: Glucose 5 g,yeast extract 10 g,KH2PO4 0.5 g,CaCl2·2H2O 1 g,(NH4)2SO4 1 g,MgSO4·7H2O 0.3 g and NaCl 20 g dissolved in 1 L artificial seawater;temperature 37 oC,original pH 7.2,medium volume 25/250 mL,shaking speed 180 rpm and fermentation time 40 h.The maximum aminopeptidase activity obtained was 1.90 LAP/mL.(2)SWJS33 could directly use soybean meal as substrate to produce aminopeptidase as soybean meal provided both carbon and nitrogen source.Supplement of 3 g/L glucose or yeast extract 15g/L improved the aminopeptidase production by 73.7% and 53.7%,respectively.Addition of yeast extract shortened the lag phase of the strain and improved the specific growth rate and specific aminopeptidase synthesis rate.The production of aminpeptidase in 2.5L fermentor reached 4.95 LAP/mL after optimization,improved 1.6 times compared with the fermentation in shake flask.The scale-up fermentation was realized in 20 L fermentor,and the aminopeptidase activity was 4.35 LAP/mL.(3)Ammonium sulfate precipitation,DEAE-Sepharose fast flow column and Superdex 200 pg column were applied to purify the aminopeptidase secreted by SWJS33(BLAP).The purified protein showed a single band with an apparent molecular weight of 100 kDa on SDS-PAGE.The identification by tandem mass spectrometry showed that BLAP matched the aminopeptidase from Bacillus licheniformis ATCC14580(gi|52079505)most.The enzyme was most active at 60 oC and pH 8.0–8.5.It had good stability at temperature lower than 70 oC,and in pH 6.0–10.0.The presence of NaCl enabled 50% improvement of enzyme activity with 10–15% NaCl being the best.The observed inactivation by EDTA and bestatin and activation by Co2+ and Ag+ indicated that the obtained enzyme was a metalloaminopeptidase.BLAP showed high preference for Leu-pNA,and the Km and Vmax values for Leu-pNA were 1.85 mmol L-1 and 1.69 mmol(L·min)-1.(4)The hydrolysis specificity of BLAP was evaluated using a series of synthetic peptides and soybean protein isolate.The aminopeptidase showed high specificity for dipeptides with Leu,Val,Ala,Gly,and Phe at the N-terminus,and the specificity was significantly affected by the nature of the penultimate residue.In the hydrolysis of soy protein isolate,BLAM preferred peptides with Leu,Glu,Gly and Ala at the N-terminus by free amino acid analysis and preferred peptides with Leu,Ala,Ser,Trp and Tyr at the N-terminus by UPLC-MS/MS.Considering the above results,we predicted that BLAP preferred peptides with Leu at the N-terminus most,followed by peptides with Gly,Ala,Glu and Val at the N-terminus.(5)The application of BLAP could improve the DH of PPI hydrolysates(BPHs)and zein hydrolysates(BZHs),and the improvement for the latter was more significant.The specificity of BLAP had great influence on the FAA composition and releasing regularity of hydrolysates,it increased the percentages of Leu and PreII in FAA for both BPHs and BZHs.The introduction of BLAP improved the antioxidant activity of BPHs and BZHs,and promoted the ACE-inhibitory activity of BZHs.For fractions with MW<3 kDa,the contents of Glu,Val and Leu in BPHs,Glu,Ala and Leu in BZHs were increased compared with the raw materials.Differences were existed between the peptides identified from hydrolysates catalyzed by Alcalase 2.4L and Alcalase 2.4L+BLAP,while the major subunits hydrolyzed were nearly the same.The influence of BLAP on the peptide composition of hydrolysates was greatly affected by the endoprotease used together.