鲅鱼副产物中产蛋白酶微生物的分离与表征

Isolation and characterization of microorganisms producing proteinase from by-products of Scomberomorus niphonius

中文关键词:  鲅鱼副产物  蛋白酶活性  产酶能力  枯草芽孢杆菌  蜡样芽孢杆菌

英文关键词:by-products of Scomberomorus niphonius  proteinase activity  production capacity of proteinase  Bacillus subtilis  Bacillus cereus

中文摘要:

      目的 以鲅鱼副产物为实验材料, 定向筛选高产蛋白酶的野生菌株。方法 以脱脂牛奶培养基对鲅鱼副产物中的产蛋白酶微生物进行初筛, 采用福林酚方法对各菌株产蛋白酶的活力进行测定, 复筛出具有高产碱性蛋白酶和中性蛋白酶的野生菌株, 并通过分子生物学方法(16S rDNA测序分析)对高产蛋白酶菌株进行物种鉴定。进一步通过测定高产菌发酵过程中的菌株浓度和蛋白酶活力, 分析菌株随发酵时间的生长情况和产酶动力学。结果 采用选择性培养基初步筛选获得8株具有蛋白酶活性的菌株, 进一步复筛发现两株菌Z3和Z8菌株产蛋白酶的能力较强[碱性蛋白酶活力分别为(393.87±19.69)、(358.87±17.94) U/mL; 中性蛋白酶活力分别为(344.87±17.24)、(291.20±14.56) U/mL]。经分子生物学测序比对分析菌株Z3和Z8分别鉴定为枯草芽孢杆菌(Bacillus subtilis)和蜡样芽孢杆菌(Bacillus cereus)。对两株菌的生产情况和产酶能力进行研究, 发现两株菌的产酶模式均为部分生长偶联型, 枯草芽孢杆菌Z3菌株合成碱性蛋白酶和中性蛋白酶的能力优于蜡样芽孢杆菌Z8。结论 本研究从鲅鱼副产物中定向分离得到2株产蛋白酶的野生菌株, 在基础发酵培养基上展现出较好的产蛋白酶的能力。

英文摘要:

      Objective To screen the wild strain with high proteinase yield using the by-product of Scomberomorus niphonius as experimental material. Methods The proteinase-producing microorganisms from the by-products of Scomberomorus niphonius were selected by using the skim milk medium. The ability of producing proteinase for each strain was measured by Folinol-Ciocalteu method, and wild strains with high-yield of alkaline proteinase and neutral proteinase were screened. The species of high-yield abilities of producing proteinase strain was identified by molecular biological method (16S rDNA sequencing analysis). Further, the growth and production of proteinases for each strain were analyzed by measuring the concentration of bacteria and the activity of proteinase during the fermentation process. Results Eight microbial strains with potential activity of proteinase were obtained by preliminary screening using selective medium from the by-products of Scomberomorus niphonius. Further, it was found that the 2 strains of Z3 and Z8 performed more excellent abilities of producing proteinase. The activity of alkaline proteinase from strain Z3 and Z8 was (393.87±19.69) and (358.87±17.94) U/mL respectively; and the activity of neutral proteinase was (344.87±17.24) and (291.20±14.56) U/mL for strain Z3 and Z8. Using molecular biology identification methods, it was determined that the strains of Z3 and Z8 were identified as Bacillus subtilis and Bacillus cereus, respectively. The production situation and enzyme production capacity of the 2 strains were studied. It was found that the enzyme production mode of the 2 strains was partial growth-related type. Bacillus subtilis Z3 had a better ability to synthesize alkaline proteinase and neutral proteinase than Bacillus cereus Z8. Conclusion In this study, 2 wild strains producing proteinase are obtained by directional isolation from by-products of Scomberomorus niphonius and shows good abilities of producing proteinase on basic fermentation medium.