Identification, Fermentation Optimization And Determination Of Enzymatic Characterization For Aminopeptidase Production Of Bacillus Methylotrophicus CC

As an important exopeptidase, aminopeptidase have very significant industrial applications. They can remove groups which cause bitterness and hence reduce the bitterness of polypeptides effectively and environmental friendly by related Hydrolysis reactions. Therefore, these enzymes play an important role in many aspects including food, industry and medicine.In this paper, the strains which have a high producity of aminopeptidase were obtained from fermented soya beans. Besides, compared to a similarity of100%of16S rDNA, these strains have the closest relationship to Eosinophilia methyl bacillus by many identifications including morphological specificity, cultural characteristic, physiological and biochemical property, thus we identified them as Bacillus. Therefore, the strainswere named as Bacillus methylotrophicus CC.Aminopeptidase can hydrolyse polypeptide chain to amino acids from N-terminal,so it is applied in the fields of food, industry and medicine. This paper optimized fermentation conditions for aminopeptidase production of Bacillus methylotrophicus CC byOne-factor experimental design, Orthogonal experimental design and Response surfaceexperimental design. The result showed that the optimal compositions of media were1%sucrose,1.5%soybean cake meal,0.1%sodium chloride,0.05%potassium d-ihydrogen phosphate,0.02%magnesium sulfate heptahydrate and0.01%cobalt chloride; theoptimal culture conditions were pH value8.5, temperature37℃, rotating speed180r/min, inoculum6%, culture time15h. Under above-mentioned conditions, the highest yield of aminopeptidase reached469.35U/ml and increased by4.65times, compared with83.09U/ml before optimization.After the study of the enzymatic properties of aminopeptidase, we have the following conclusions: Firstly, the strain got into its logarithmic period after4hours’ growth and reached the maximum at the17thhour while the best time for producing ammoniapeptide is the15thhour and the highest enzyme activity is474.52U/ml.Secondly,the optimum temperature is50℃and the enzymatic activity are62.78%,56.50%and62.78%after under50℃for60min,60℃for30min and70℃for15min,respectively,indicating that aminopeptidase have better thermal stability.Thirdly, the optimum pH is9.0and the enzyme can remain high enzyme activity during the range from pH8to10indicating that aminopeptidase have better pH stability. Lastly, as for the mentalions, Co2+, Zn2+show obvious promotions to enzyme activity while Fe2+、Mg2+、Ca2+show obvious inhibitions, besides, K+did not show any significant effect. As for theinhibitions, PMSF,1,10-Phenantroline, EDTA and IAA show different inhibitory effects and these enzymes are the most sensitive to DTT, besides, the ammonia peptide enzyme has better substrate specificity to argini ne and leucine. The enzyme had molecular weight of about53KD evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.In this study, we sucessfully identified the strain of the species relationship and o ptimized fermentation conditions and fermentation medium for aminopeptidase production of Bacillus methylotrophicus CC by molecular identification. By doing it, we enhanced the yield of aminopeptidase and studied their biochemical properties deeply, thuswe expect to create the basis for the industrialization of the aminopeptidase and provide scientific basis for mass production.