Screening Of Strains With High Yield Of Aminopeptidase And Enzymatic Properties

It is rich in natural resources in Changbai Mountain area of Jilin Province,where the fermented pickles contain a large number of undeveloped lactic acid bacteria resources.Lactic acid bacteria are with strong protease hydrolysis system,so they can secrete a variety of proteases,including aminopeptidase which has been studied more in recent years.Although many microorganisms can secrete aminopeptidase,most strains are not suitable for large-scale production of aminopeptidase due to low enzyme production capacity or potential safety hazards.In order to find lactic acid bacteria strains producing high level of aminopeptidase,more than 300 naturally fermented pickles,including spicy cabbage and pickled cabbage,etc.were collected from Yanji city,Jilin Province,and then 170 strains of lactic acid bacteria were isolated.The acid production ability,low temperature tolerance,salt tolerance,acid tolerance and bile salt tolerance were tested,and the effects of temperature and salt concentration on the self solubility of different strains were also evaluated.Meanwhile,three strains of Lactobacillus plantarum（No.91,No.106 and No.120）with high aminopeptidase production were screened using Glu-ρ-NA,Leu-ρ-NA,Leu-ρ-NA as substrate,and the extraction method of aminopeptidase was optimized.Furthurmore,the optimum reaction temperature,temperature stability,optimum p H,p H stability,the effect of metal ions on peptidase,the effect of protein inhibitors on peptidase and the effect of salt concentration on peptidase were also determined.See below for details:（1）More than 300 samples of naturally fermented pickles were collected from Yanji city,Jilin Province.170 strains of lactic acid bacteria were isolated by Gram staining,microscopic examination and Catalase test.（2）160 strains with strong activity were screened from 170 strains of lactic acid bacteria.The acid production test,low temperature resistance test,salt resistance test,acid resistance test and bile salt resistance test were carried out respectively,and then the strains with excellent performance were selected for protein hydrolysis test to screen the strains with protein hydrolysis ability.Through the above experiments,a total of 10strains with excellent performance were selected for protein hydrolysis test.Finally,7strains with protein hydrolysis ability were obtained.According to the activity of the strains,No.91,No.106 and No.120 with the highest activity were selected to extract aminopeptidase.16S r DNA results showed that the above strains were Lactobacillus plantarum.The self solubility of strain 91 was 73.07%at 10℃,that of strain 106 was55.61%at 4℃,and that of strain 120 was 55.80%at 37℃.When different concentrations of Na Cl were added to the autolysis reaction,the autosolubility of the three strains increased to varying degrees when the concentration of Na Cl was 1%.（3）It was found that the crude enzyme solution with the highest enzyme activity could be obtained by repeatedly centrifuging and blowing for 4 times after incubation with lysozyme for 4 hours.（4）Study on enzymatic properties of aminopeptidase.In this experiment,the optimum reaction temperature of aminopeptidase was 45℃,and the optimum p H was7.0;The enzyme activity of aminopeptidase produced by strain 91 was increased upon the exposure to some metal ions(Ca²⁺,Ba²⁺,K⁺,Fe²⁺,Cu²⁺).Eight metal ions(Na⁺,Ca²⁺,Ba²⁺,K⁺,Fe²⁺,Cu²⁺,Mn²⁺,Mg²⁺)inhibited the peptidase produced by strain 106and promoted the activity of anminopeptidase produced by strain 120.In conclusion,three strains with high-yield of aminopeptidase were selected and their enzymatic properties were studied so as to provide support for the application of anminopeptidases in food industry.