【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。更多还原

【Abstract】 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.更多还原