一株产胶原蛋白酶细菌的鉴定及产酶条件优化
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摘要
采用形态学特征、生理生化特征结合16S rDNA对一株产胞外胶原蛋白酶的菌株AL-13进行鉴定。获得安全、高效、高产量的生产胶原蛋白酶工艺。以胶原蛋白酶活性为指标,采用单因素实验优化温度、p H、接种量等产酶培养条件后,利用单因素结合响应曲面法优化产酶培养基。结果表明,AL-13鉴定为侧孢短芽孢杆菌(Brevibacillus laterosporus),最适产酶培养条件为:培养时间48 h、培养温度25℃、初始pH8.0、接种量6%(v/v),最适产酶培养基为:葡萄糖8.14 g/L、牛肉膏11.63 g/L、氯化钙0.17 g/L、磷酸氢二钾2.08 g/L、氯化钠9.48 g/L,在该产酶条件下胶原蛋白酶活力预测值为154.89 U/m L,验证结果显示酶活为(153.06±3.73) U/m L,此结果与预测值接近,相对误差为0.04%。本实验成功优化了一株产胶原蛋白酶细菌的产酶条件,产酶条件优化后的酶活(153.06 U/m L)较优化前(16.14 U/m L)提高了9.5倍。
A strain AL-13 producing extracellular collagenase was identified by morphological,physiological and biochemical characteristics and 16S rDNA sequencing.In order to obtain a safe,efficient and high-yield method for collagenase processing,with the activity of collagenase as an index,single factor experiments was used to optimize the culture conditions of temperature,pH and inoculation quantity for collagenase producing.Response surface methodology combined with single factors were used to optimize the enzyme-producing medium. The results showed that,AL-13 was identified as Brevibacillus laterosporus.The optimal enzyme production conditions were: Culture time 48 h,culture temperature 25 ℃,initial pH8.0,inoculum 6%( v/v). Optimal enzyme production medium were: Glucose 8.14 g/L,beef extract 11.63 g/L,CaCl_2 0.17 g/L,K_2 HPO_4 2.08 g/L,NaCl 9.48 g/L. Under the conditions of enzyme production,the predicted value of collagenase activity was 154.89 U/m L.And the obtained reliability enzyme activity was( 153.06 ± 3.73) U/m L under the optimized processing.The result was close to the predicted value,and the relative error was 0.04%. The enzyme production conditions of the collagenase-producing bacteria were successfully optimized,and the enzyme activity( 153.06 U/m L) optimized for enzyme production was increased by 9.5 times compared with that before optimization( 16.14 U/m L).
A strain AL-13 producing extracellular collagenase was identified by morphological,physiological and biochemical characteristics and 16S rDNA sequencing.In order to obtain a safe,efficient and high-yield method for collagenase processing,with the activity of collagenase as an index,single factor experiments was used to optimize the culture conditions of temperature,pH and inoculation quantity for collagenase producing.Response surface methodology combined with single factors were used to optimize the enzyme-producing medium. The results showed that,AL-13 was identified as Brevibacillus laterosporus.The optimal enzyme production conditions were: Culture time 48 h,culture temperature 25 ℃,initial pH8.0,inoculum 6%( v/v). Optimal enzyme production medium were: Glucose 8.14 g/L,beef extract 11.63 g/L,CaCl_2 0.17 g/L,K_2 HPO_4 2.08 g/L,NaCl 9.48 g/L. Under the conditions of enzyme production,the predicted value of collagenase activity was 154.89 U/m L.And the obtained reliability enzyme activity was( 153.06 ± 3.73) U/m L under the optimized processing.The result was close to the predicted value,and the relative error was 0.04%. The enzyme production conditions of the collagenase-producing bacteria were successfully optimized,and the enzyme activity( 153.06 U/m L) optimized for enzyme production was increased by 9.5 times compared with that before optimization( 16.14 U/m L).
引文
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[31]苏海玲,张海英,李红卫,等.蚕豆蛋白酶解工艺及响应面法的优化[J].中国食品学报,2013,13(5):39-46.
[2]田思涵.胶原蛋白的概况、性质与应用[J].化工管理,2018(5):160.
[3]Daviane M V,Aless A C,Rejane A B,et al.Collagen modification by Maillard reaction[J].Journal of Thermal Analysis and Calorimetry,2018,131(1):671-679.
[4]Steplewski A,Majsterek I,Mcadams E,et al.Thermostability gradient in the collagen triple helix reveals its multi-domain structure[J].Journal of Molecular Biology,2014,338(5):989-998.
[5]Kuma R S,Sugihar A F,Suzuk I K,et al.A double-blind,placebo-controlled,randomized,clinical study on the effectiveness of collagen peptide on osteoarthritis[J].Journal of the Science of Food and Agriculture,2015,95(4):702-707.
[6]茅宇虹,杨文鸽.鱼皮胶原蛋白及胶原活性肽的研究[J].食品工业科技,2013,34(15):375-379.
[7]孙佳佳,王红英,钱斯日古楞,等.产胶原蛋白酶枯草芽孢杆菌的筛选[J].大连工业大学学报,2010,29(4):248-250.
[8]刘丽莉,杨协力.产骨胶原蛋白酶菌种的筛选与鉴定[J].食品与生物技术学报,2011,30(6):917-923.
[9]Petrova D,Vlahov S,Dalev P.Purification and characterization of a thermostable alkaline collagenase from Thermoactinomyces sp.E-21 strain[J].Biotechnology and Biotechnological Equipment,2001,15(2):31-38.
[10]Amira Abood,Asmaa M M,Azza M,et al.Purification and characterization of a new thermophilic collagenase from Nocardiopsis dassonvillei NRC2aza and its application in wound healing[J].International Journal of Biological Macromolecules,2018,116:801-810.
[11]何正文,王红英,李承华,等.产胶原蛋白酶嗜虫沙雷氏菌DL-12的紫外诱变育种[J].工业微生物,2015,45(6):43-47.
[12]Zhang X X,Li Y,Wang S Y,et al.Identification of a collagenase produced by Bacillus cereus R75E isolated from human colostrum[J].Applied Biochemistry and Microbiology,2015,51(5):511-521.
[13]杨光垚.胶原蛋白酶的纯化及其基因的克隆与表达研究[D].成都:四川大学,2004:10-30.
[14]麻金花.海参内脏中胶原降解蛋白酶的研究[D].厦门:集美大学,2016.
[15]郑杰,吴海涛,朱蓓薇,等.海参肠自溶水解物抗氧化活性的研究[J].大连工业大学学报,2011,30(5):313-317.
[16]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001:62-64.
[17]R.E.布坎南,N.E.吉本斯.伯杰细菌鉴定手册[M].北京:科学出版社,1984:732-735.
[18]上海市酿造科学研究所.SB/T10317-1999中华人民共和国行业标准蛋白酶活力测定法[S].北京:中国标准出版社,1999.
[19]孟甜,黄韬睿,李玉锋.添加剂对米曲霉产酶活力影响的研究[J].中国调味品,2013,38(12):95-98.
[20]牛放,高俊明,段雪薇,等.响应面法优化野菊花多糖的水解工艺[J].食品工业科技,2019(1):156-161.
[21]端木琳,徐飞.典型海水温度年计算方法及其应用[J].制冷学报,2016,37(3):6-11.
[22]Yang Z,Xu D,Du C.Studies on fermentation conditions of antimicrobial substances produced by Brevibacillus laterosporus BL-21[J].Agricultural Science&Technology,2014,15(11):1852-1856.
[23]刘寅,张永光,张汝兵,等.响应面法优化产酸丙酸杆菌丙酸发酵条件的研究[J].食品工业科技,2010,31(5):167-170.
[24]Francisco B P,Pedro M R G,JoséA T,et al.Optimization of low-cost medium for very high gravity ethanol fermentations by Saccharomyces cerevisiae using statistical experimental designs[J].Bioresource Technology,2010,101:7856-7863.
[25]于雷,雷霆,裴晓林,等.L-乳酸发酵培养基中氮源的优化[J].食品科技,2007(6):49-53.
[26]Pantoliano M W,Whitlo W M,Wood J F,et al.The engineering of binding affinity at metal ion binding sites for the stabilization of proteins:Subtilisin as a test case[J].Biochemistry,1988,27(22):8311-8317.
[27]赵春霞.海洋细菌与碳及营养盐的相互作用[D].青岛:中国海洋大学,2006.
[28]Sin H N,Yusof S,Hamidn S A,et al.Optimization of hot water extraction for sapodilla juice using response surface methodology[J].Journal of Food Engineering,2006,75(3):352-358.
[29]刘同方,于华忠,陈雁梅,等.响应面法优化二氢杨梅素半合成杨梅素[J].林产化学与工业,2015,35(4):125-130.
[30]卫功庆,刘少华,范宁,等.响应曲面法优化鹿角盘胶原蛋白酶解提取工艺研究[J].西北农林科技大学学报:自然科学版,2015,43(6):205-214.
[31]苏海玲,张海英,李红卫,等.蚕豆蛋白酶解工艺及响应面法的优化[J].中国食品学报,2013,13(5):39-46.