纹藤壶附生菌的分离鉴定及蛋白酶菌株的产酶性质研究
|
摘要
目的:研究纹藤壶附生菌的分离鉴定及蛋白酶菌株的产酶性质。方法:黄渤海潮间带海域纹藤壶中分离到54株菌株,经酪蛋白酶实验,筛选得到4株高产蛋白酶的菌株,并对其进行菌种鉴定、产蛋白酶的发酵条件优化及产酶活性测定。结果:经筛选,得到4株高产蛋白酶的菌株,编号分别为X8、X18、X21、X48。通过16S rDNA序列分析,结合透射电镜及形态学观察、生理生化测试结果,初步鉴定X8为河豚毒素假交替单胞菌、X18为乔治亚海杆菌、X21为杀鱼假交替单胞菌、X48为康氏菌。通过测定菌株的生长曲线,确定菌株X8发酵量最高的培养时间为18h、确定菌株X18发酵量最高的培养时间为18h、确定菌株X21发酵量最高的培养时间为21h、确定菌株X48发酵量最高的培养时间为18 h。在最适培养时间的基础上,通过单因素多水平试验设计,可得产蛋白酶菌株X8的最佳发酵条件:可溶性淀粉5. 0‰、初始p H 7. 0、培养基盐度10‰、培养温度25℃; X18最佳发酵条件:可溶性淀粉5. 0‰、初始p H 7. 0、培养基盐度15‰、培养温度30℃; X21最佳发酵条件:蔗糖5. 0‰、初始p H 7. 0～8. 0、培养基盐度15‰、培养温度25℃; X48最佳发酵条件:可溶性淀粉5. 0‰、初始p H 7. 0、培养基盐度15‰、培养温度25℃。结论:在优化发酵条件下,X8、X18、X21、X48菌株产蛋白酶酶活力分别达到707. 06、240. 00、441. 18、328. 22 U/m L。该研究为探索潜在的高产蛋白酶菌株提供了科学依据。
Objective To study isolate and identify epiphytic bacteria from barnacle and enzymatic properties of protease-producing strains. Method Totally 54 strains were isolated from barnacles in the contaminated coastal waters of Shandong province. Result Totally 4 strains with high protease production were screened by casein test. The strains were identified,the fermentation conditions of protease production were optimized and the enzyme activity was tested. Totally 4 strains of protease-producing strains were screened and identified as X8,X18,X21 and X48,respectively. X8 was identified as Pseudoalteromonas tetraodonis,X18 as Marinobacterium georgiense,X21 as Pseudoalteromonas piscicida and X48 as Kangiella japonica by 16 S rDNA sequence analysis,transmission electron microscopy,morphological observation and physiological and biochemical characteristics test. By measuring the growth curve of the strain,it was determined that the highest fermentation time of strain X8 was 18 hours,the highest fermentation time of strain X18 was 18 hours,the highest fermentation time of strain X21 was 21 hours,and the highest fermentation time of strain X48 was 18 hours. On the basis of optimum fermentation time and single factor experiment,the optimum fermentation conditions of protease production by X8 strain were soluble starch 5. 0‰,initial pH 7. 0,medium salinity 10‰ and culture temperature 25℃. The optimum fermentation conditions of protease production by X18 strain were soluble starch 5. 0‰,initial pH 7. 0,medium salinity 15‰ and culture temperature 30℃. The optimum fermentation conditions of protease production by X21 strain were sucrose 5. 0‰,initial pH 7. 0 ～ 8. 0,medium salinity 15‰ and culture temperature 25℃. The optimum fermentation conditions of protease production by X48 strain were soluble starch 5. 0‰,initial pH 7. 0,medium salinity 15‰ and culture temperature25℃. Conclusion Under the optimum fermentation conditions,the protease activity of strains X8,X18,X21 and X48 reached 707. 06 U/mL,240. 00 U/mL,441. 18 U/mL and 328. 22 U/mL, respectively. This study provided a preliminary basis for exploring potential protease-producing strains.
Objective To study isolate and identify epiphytic bacteria from barnacle and enzymatic properties of protease-producing strains. Method Totally 54 strains were isolated from barnacles in the contaminated coastal waters of Shandong province. Result Totally 4 strains with high protease production were screened by casein test. The strains were identified,the fermentation conditions of protease production were optimized and the enzyme activity was tested. Totally 4 strains of protease-producing strains were screened and identified as X8,X18,X21 and X48,respectively. X8 was identified as Pseudoalteromonas tetraodonis,X18 as Marinobacterium georgiense,X21 as Pseudoalteromonas piscicida and X48 as Kangiella japonica by 16 S rDNA sequence analysis,transmission electron microscopy,morphological observation and physiological and biochemical characteristics test. By measuring the growth curve of the strain,it was determined that the highest fermentation time of strain X8 was 18 hours,the highest fermentation time of strain X18 was 18 hours,the highest fermentation time of strain X21 was 21 hours,and the highest fermentation time of strain X48 was 18 hours. On the basis of optimum fermentation time and single factor experiment,the optimum fermentation conditions of protease production by X8 strain were soluble starch 5. 0‰,initial pH 7. 0,medium salinity 10‰ and culture temperature 25℃. The optimum fermentation conditions of protease production by X18 strain were soluble starch 5. 0‰,initial pH 7. 0,medium salinity 15‰ and culture temperature 30℃. The optimum fermentation conditions of protease production by X21 strain were sucrose 5. 0‰,initial pH 7. 0 ～ 8. 0,medium salinity 15‰ and culture temperature 25℃. The optimum fermentation conditions of protease production by X48 strain were soluble starch 5. 0‰,initial pH 7. 0,medium salinity 15‰ and culture temperature25℃. Conclusion Under the optimum fermentation conditions,the protease activity of strains X8,X18,X21 and X48 reached 707. 06 U/mL,240. 00 U/mL,441. 18 U/mL and 328. 22 U/mL, respectively. This study provided a preliminary basis for exploring potential protease-producing strains.
引文
[1]Niyonzima F N,More S.Detergent-compatible proteases:microbial production,properties,and stain removal analysis[J].Prep Biochem Biotech,2015,45(3):233-258.
[2]方海红,胡好远,黄红英,等.微生物碱性蛋白酶的研究进展[J].微生物学通报,2002,29(2):57-59.
[3]张力元.海洋来源产蛋白酶的菌株筛选及其产酶条件的优化研究[D].河北保定:河北农业大学,2014.
[4]葛艳辉,等.聚磷菌胞内聚合物的染色条件优化及染色方法比较[J].环境科学与技术,2014,32(2):1-6.
[5]高信曾.电子显微镜的原理和技术[J].山西农业科学,1980(1):28-29、13.
[6]刘威,任瑞晨.透射电子显微镜在矿物加工与利用中的应用[J].微计算机信息,2010,26(12-1):141-143.
[7]Chen W S,et al.Revealing the structures of cellulose nanofiber bundles obtained by mechanical nanofibrillation via TEMobservation[J].Carbohydr Polym,2015(117):950-956.
[8]Srot V,et al.Influence of TEM specimen preparation on chemical composition of Pb(Mg1/3Nb2/3)O3-PbTiO3single crystals[J].Micron,2014(62):37-42.
[9]Buchanan R E,Gibbens N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:274-325.
[10]黄文芳.粘质沙雷氏菌的研究Ⅰ-Serratiamarcescens9-2菌株分离、分类鉴定和形态特征[J].华南师范大学学报(自然科学版),2003(3):108-111.
[11]Felsenstein J.Confidence-limits on phylogenies-an approach using the bootstrap[J].Evolution,1985,39(4):783-791.
[12]郑朝成,周立祥.污泥中一株产耐高温蛋白酶菌株的分离、鉴定及酶学特性[J].环境科学学报,2012,32(3):577-583.
[13]Laemmli U K.cleavage of structural proteins during assembly of head of Bacteriophage-T4[J].Nature,1970,227(5259):680-685.
[14]杨国钊,等.嗜水气单胞菌生长曲线的测定及药物敏感性试验[J].畜牧兽医科技信息,2011(9):27-30.
[15]王亚敏.养殖刺参(Apostichopus japonicus)肠道益生菌的筛选[D].大连:大连水产院,2008(6):32-33.
[16]赵海霞,赵培培,陈惠,等.一株产胶原蛋白酶沙雷氏菌的分离及鉴定[J].中国饲料,2012(1):9-11、14.
[17]冯爱娟,叶茂,陈萍柔,等.蛋白酶菌株的筛选与鉴定[J].中国调味品,2017,42(11):49-52.
[18]张力元.海洋来源产蛋白酶的菌株筛选及其产酶条件的优化研究[D].河北保定:河北农业大学,2014.
[19]袁艳超.蛋白酶高产菌株的筛选及其发酵芝麻粕制备芝麻多肽的研究[D].南昌:江西师范大学,2017.
[20]耿芳,等.土壤中高产蛋白酶菌株的筛选鉴定及发酵条件优化[J].中国酿造,2018,37(4):66-71.
[2]方海红,胡好远,黄红英,等.微生物碱性蛋白酶的研究进展[J].微生物学通报,2002,29(2):57-59.
[3]张力元.海洋来源产蛋白酶的菌株筛选及其产酶条件的优化研究[D].河北保定:河北农业大学,2014.
[4]葛艳辉,等.聚磷菌胞内聚合物的染色条件优化及染色方法比较[J].环境科学与技术,2014,32(2):1-6.
[5]高信曾.电子显微镜的原理和技术[J].山西农业科学,1980(1):28-29、13.
[6]刘威,任瑞晨.透射电子显微镜在矿物加工与利用中的应用[J].微计算机信息,2010,26(12-1):141-143.
[7]Chen W S,et al.Revealing the structures of cellulose nanofiber bundles obtained by mechanical nanofibrillation via TEMobservation[J].Carbohydr Polym,2015(117):950-956.
[8]Srot V,et al.Influence of TEM specimen preparation on chemical composition of Pb(Mg1/3Nb2/3)O3-PbTiO3single crystals[J].Micron,2014(62):37-42.
[9]Buchanan R E,Gibbens N E.伯杰细菌鉴定手册[M].8版.北京:科学出版社,1984:274-325.
[10]黄文芳.粘质沙雷氏菌的研究Ⅰ-Serratiamarcescens9-2菌株分离、分类鉴定和形态特征[J].华南师范大学学报(自然科学版),2003(3):108-111.
[11]Felsenstein J.Confidence-limits on phylogenies-an approach using the bootstrap[J].Evolution,1985,39(4):783-791.
[12]郑朝成,周立祥.污泥中一株产耐高温蛋白酶菌株的分离、鉴定及酶学特性[J].环境科学学报,2012,32(3):577-583.
[13]Laemmli U K.cleavage of structural proteins during assembly of head of Bacteriophage-T4[J].Nature,1970,227(5259):680-685.
[14]杨国钊,等.嗜水气单胞菌生长曲线的测定及药物敏感性试验[J].畜牧兽医科技信息,2011(9):27-30.
[15]王亚敏.养殖刺参(Apostichopus japonicus)肠道益生菌的筛选[D].大连:大连水产院,2008(6):32-33.
[16]赵海霞,赵培培,陈惠,等.一株产胶原蛋白酶沙雷氏菌的分离及鉴定[J].中国饲料,2012(1):9-11、14.
[17]冯爱娟,叶茂,陈萍柔,等.蛋白酶菌株的筛选与鉴定[J].中国调味品,2017,42(11):49-52.
[18]张力元.海洋来源产蛋白酶的菌株筛选及其产酶条件的优化研究[D].河北保定:河北农业大学,2014.
[19]袁艳超.蛋白酶高产菌株的筛选及其发酵芝麻粕制备芝麻多肽的研究[D].南昌:江西师范大学,2017.
[20]耿芳,等.土壤中高产蛋白酶菌株的筛选鉴定及发酵条件优化[J].中国酿造,2018,37(4):66-71.