Isolation,Purification And Identification Of Cell-envelope Proteinase From Probiotic Lactobacillus

The proteolytic system of Lactobacillus includes cell-envelope proteinase?CEP?,oligopeptide transport system and endopeptidases.Among them,the CEP plays an important role as a key enzyme of this system.It not only participates in the degradation of casein,but also promotes the formation of flavor and texture of the fermented product.It can also hydrolyze the food protein to release bioactive peptide,which is beneficial to consumers'health.At present,there are few reports on the isolation and purification of CEP from Lactobacillus,especially on its structure identification.In this study,the effect of different extraction methods on the extraction efficiency of CEP from four Lactobacillus were investigated.The influence of aqueous two-phase system on the isolation of CEP were studied,and the optimum conditions for the extraction of CEP via aqueous two-phase system were obtained by the single factor experiment and response surface methodology.The isolation and purification of CEP was carried out by ultrafiltration and Sephadex chromatography.The molecular weight of the CEP was measured by electrophoresis.Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry?MALDI-TOF-MS?was used to identify CEP to compare the enzyme production differences of different Lactobacilli.The findings are as follows:1.Different extraction methods have different extraction effects on CEP of four Lactobacillus.The Ca²⁺-free method is the optimal extraction method of Lactobacillus plantarum LP69 and L.rhamnosus LR22 CEPs.The optimum extractants of L.bulgaricus LB6 CEP and L.casei L61 CEP are guanidine hydrochloride and sodium dodecyl sulfate?SDS?,respectively.Considering the versatility and effect of the extraction method on the four strains,the four kinds of Lactobacillus cells were treated by Ca²⁺-free method,and the enzyme activities of the crude enzymes were 6.28 U/mL,8.98 U/mL,6.08 U/mL,47.59U/mL,respectively.Their correspondingly specific activities were 70 U/mg,90U/mL,200 U/mL,210 U/mL,respectively.2.The extraction conditions of four Lactobacillus CEP in aqueous two-phase system were optimized by the single factor experiment and response surface methodology.The optimal extraction conditions for L.plantarum LP69CEP were:30%?w/v??NH₄?₂SO₄,30%?w/v?PEG-1000,pH 7.0.Under these conditions,the CEP recovery and the purification factor were?90.83±0.78?%and 2.57,respectively.The optimal extraction conditions for L.rhamnosus LR22CEP were:30%?w/v??NH₄?₂SO₄,25%?w/v?PEG-1000,pH 6.7.Under these conditions,the CEP recovery and the purification factor were?91.05±0.56?%and 1.11,respectively.The optimal extraction conditions for L.bulgaricus LB6CEP were:30%?w/v??NH₄?₂SO₄,30%?w/v?PEG-4000,pH 7.0.Under these conditions,the CEP recovery and the purification factor were?86.67±0.43?%and 1.25,respectively.The optimal extraction conditions of L.casei L61 CEP were:40%?w/v?NaH₂PO₄,15%?w/v?PEG-1000,pH 6.8.Under these conditions,the CEP recovery and the purification factor were?92.94±0.43?%and 1.95,respectively.The results indicate that the two-aqueous system extraction method is feasible for separation of CEP from Lactobacillus.3.The CEP of 4 strains were further separated and purified by ultrafiltration and Sephadex G-100 gel chromatography.The purity and molecular weight were determined by electrophoresis.The CEP obtained by aqueous two-phase extraction were separated by three ultra-filtration membranes with different molecular weights?100K Da,50K Da,10K Da?.The highest activity of LB6CEP was found to be at a molecular weight of 50K Da¹00K Da.The components with the highest CEP activity of the other three stains were concentrated in the ultrafiltrate with the truncated molecular weight of 10K Da⁵0K Da.Through the Sephadex G-100 gel chromatography,the specific activity,CEP recovery and purification factor of LP69 CEP were 976 U/mg,45.32%and13.94,respectively.The specific activity,CEP recovery and purification factor of LR22 CEP were 410 U/mg,23.43%and 4.56,respectively.The specific activity,CEP recovery and purification factor of LB6 CEP were 1180 U/mg,33.58%and 5.90,respectively.The specific activity,CEP recovery and purification factor of L61 CEP were 5310 U/mg,21.12%and 25.29,respectively.The electrophoresis results of CEP from four Lactobacillus were all single bands,and their molecular weights were about 45K Da,80K Da,30K Da,and 38K Da,respectively.Lactobacillus CEP were analyzed by LC-MS/MS,and the molecular weight of the polypeptide was in the range of 600-1800 Da.The CEP of 4 strains had 6 identical peptides:SIEDTK,RMISVAR,TIDDLK,QSELKAATK,YISTK,FLEQQNK.In this study,the optimal extraction method of Lactobacillus CEP and the extraction of CEP by aqueous two-phase system were investigated,which provided technical basis for the subsequent development of biologically active functional products.The amino acid composition of different Lactobacillus CEP was obtained,which can provide a reference for the subsequent genetic engineering expression of CEP.