产蛋白酶海洋细菌的筛选及产酶工艺优化
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摘要
[目的]筛选产蛋白酶的海洋细菌资源。[方法]分别采用2216E培养基和酪蛋白固体培养基,对采自宁波洋沙山和象山海域的海水进行微生物的分离与纯化,采用平板透明圈法初筛和酶活力测定法复筛,采用16S r DNA序列分析进行菌株鉴定,并进行产酶工艺优化研究。[结果]纯化出15株产蛋白酶海洋细菌,从中筛选出1株产蛋白酶活力最高的菌株PB02,经鉴定为交替假单胞菌(Pseudoalteromonas sp.)。该菌株最适培养温度为20℃,最适装液量为10%,最适接种量为0.5%,最适转速为100 r/min,最适培养基pH为7。酶促反应最适温度为40℃,最适pH为10。[结论]该研究为蛋白酶高产菌株的改造和定向进化奠定了资源基础。
[Objective]To screen the protease-producing marine bacterium.[Method]Firstly,2216 E medium and casein solid medium were applied to isolate the protease-producing marine bacteria from Ningbo Yangshashan and Xiangshan Sea. The protease-producing capacity was got by the screening of flat transparent circle method and rescreening of Folin phenol reagent colorimetric method. The strain was identified with the 16 S r DNA sequence analysis.[Result]A total of 15 strains of protease-producing marine bacteria were isolated and the strain of PB02 with the highest protease-producing capacity was got. According to the 16 S r DNA sequence analysis,the strain belonged to the Pseudoalteromonas sp.. The optimum culture temperature 20 ℃,the optimum loading volume 10%,the optimum inoculum size 0. 5%,the optimum speed 100 r/min and the optimum medium pH 7 were obtained by the optimization of fermentation conditions. With the research on the properties of protease,the optimum reaction temperature was 40 ℃ and the optimum pH was 10.[Conclusion]The research laid a good foundation for the selection and directed evolution of highly protease-producing strains.
[Objective]To screen the protease-producing marine bacterium.[Method]Firstly,2216 E medium and casein solid medium were applied to isolate the protease-producing marine bacteria from Ningbo Yangshashan and Xiangshan Sea. The protease-producing capacity was got by the screening of flat transparent circle method and rescreening of Folin phenol reagent colorimetric method. The strain was identified with the 16 S r DNA sequence analysis.[Result]A total of 15 strains of protease-producing marine bacteria were isolated and the strain of PB02 with the highest protease-producing capacity was got. According to the 16 S r DNA sequence analysis,the strain belonged to the Pseudoalteromonas sp.. The optimum culture temperature 20 ℃,the optimum loading volume 10%,the optimum inoculum size 0. 5%,the optimum speed 100 r/min and the optimum medium pH 7 were obtained by the optimization of fermentation conditions. With the research on the properties of protease,the optimum reaction temperature was 40 ℃ and the optimum pH was 10.[Conclusion]The research laid a good foundation for the selection and directed evolution of highly protease-producing strains.
引文
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[6]刘婷,张天斌,林元山.产中性蛋白酶菌株的筛选及其发酵条件的优化[J].湖南农业科学,2009(5):102-104,107.
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[8]万琦,陆兆新,高宏.低温碱性蛋白酶菌株的筛选及产酶条件的研究[J].微生物学杂志,2002,22(5):16-18.
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[10]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[M].保定:河北农业大学,2009:20-47.
[11]方春玉,周健,邓静,等.泸型大曲黑曲霉产酸性蛋白酶条件的优化及其酶学性质的研究[J].食品与发酵科技,2011,47(2):13-19.
[12]曲均革,姚晓敏,朱鹏,等.产纤维素酶海洋细菌的筛选鉴定和产酶条件优化[J].上海海洋大学学报,2012,21(6):1054-1056.
[2]路英华.蛋白酶的研究进展[J].生命科学信息,1991,8(2):8-10.
[3]NASCIMENTO W C A,MARTINS M L L.Production and properties of an extracellular protease from thermophilic Bacillus sp[J].Brazilian journal of microbiolgy,2004,35:91-96.
[4]RAI S K,ROY J K,MUKHERJEE A K.Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp.nov.AS-S24-Ⅱ[J].Applied microbiology and biotechnology,2010,85(5):1437-1450.
[5]张晓.海洋产蛋白酶菌的分离、培养及其防污活性研究[M].青岛:中国海洋大学,2013:12.
[6]刘婷,张天斌,林元山.产中性蛋白酶菌株的筛选及其发酵条件的优化[J].湖南农业科学,2009(5):102-104,107.
[7]张锐.极端微生物产碱性蛋白酶菌株的筛选及发酵条件的研究[J].微生物学通报,2001,28(4):5-9.
[8]万琦,陆兆新,高宏.低温碱性蛋白酶菌株的筛选及产酶条件的研究[J].微生物学杂志,2002,22(5):16-18.
[9]宋明徽.产碱性蛋白酶海洋细菌筛选及发酵研究[M].大连:大连工业大学,2013:12-14.
[10]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[M].保定:河北农业大学,2009:20-47.
[11]方春玉,周健,邓静,等.泸型大曲黑曲霉产酸性蛋白酶条件的优化及其酶学性质的研究[J].食品与发酵科技,2011,47(2):13-19.
[12]曲均革,姚晓敏,朱鹏,等.产纤维素酶海洋细菌的筛选鉴定和产酶条件优化[J].上海海洋大学学报,2012,21(6):1054-1056.