Screening Of Esterifying Enzyme-Producing Bacteria From Medium And High Temperature Daqu

Through adding Glyceryl Tri-n-butyrate in the culture medium,15strains producing esterase are obtained after the initial screening in medium and high temperature Daqu.Then putting the4-Nitrophenyl acetate as substrate,the12#strain shows the highest catalytic activity of which the enzyme activity is0.77U/ml;putting ethanol and caproic acid as substrate,the10#strain shows the highest catalytic activity of which the enzyme activity is5.3U/ml. The strains which have relatively higher esterase measured with the above two methods are cultured for72h in39℃,150r/min and its supernatant centrifugatied in12000r/min4℃for20min was analysied by GC, the results show that these strains can produce a certain amount of acids,alcohols and esters.Follow-up study Select of10strains. Through analysis of the strain10#’s morphological, physiological, biochemical and16S rDNA sequence, the results showed that:the colonies of bacteria10#were yellow and the fold uplift, Gramposi tive. the result of16S rDNA sequence analysis show that the homology of10#bacteria and Bacillus licheniformis strain can reach99%, preliminarily identified it as Bacillus licheniformis strain.The sequence KC182795.1was applicated in Genbank. The10#strains was mutagenized by using low temperature plasma and three strains with high esterifying enzyme activity was screened as parents.The parent protoplast was inactivated for genome shuffling. After the fused strains were purified by selection, the enzyme activity of fusants was determinated by fermentation, the strains of higher enzyme activity were screened to passage and their stability of enzyme activity were detected.The highest and stable enzyme activity of6#fusant can reach10.7U/ml,which has been improved by102%compared with the5.3U/ml of original strain G2.The fermentation broth of the6#fusant was analyzed by GC, the results show that the strain can produce a certain amount of acids,alcohols and esters, and the acetoin of the fermentation broth can reach2565mg/L.Investigated the effects of the nitrogen source, carbon source, initial pH value in the influence of the enzyme activity of the fusants6#.And find that glucose concentration, the rotational speed, and the fermentation time have greater impact on bacterial enzyme activity. Select three factors using Design-Expert software to do the analysis of the response surface analysis. The results show that the model have reasonable fit. The glucose concentration35.23g/L, speed75r/min, fermentation time29h after the actual correction software to analyze the optimal value. Fermentation under optimal conditions, the actual measured activity is15.3U/mL,42.9%higher than that before optimization.Mixed fermentation the fusants6#,the aroma of yeast and the caproic acid bacteria. Investigated the influence of inoculum size and fermentation temperature to the fermentation produce esters. Found that ethyl caproate content can reach801.1mg/mL when the inoculation amount is8%,and same circumstances the ethyl acetate can reach744.6mg/mL when the inoculation amount is2%.When the fermentation temperature of35℃the ethyl hexanoate content can reach978.3mg/mL,and the ethyl acetate content can reach698.9mg/mL when the fermentation temperature of30℃.Mixing the fusants6#and aroma of yeast and Aspergillus niger to production bran song,found that fusant6#have a significant role in promoting the Okuma sample esterifying capacity after determination the esterification capacity in the different combinations.