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ZHU Yanbing,LI Hebin,NI Hui,LIU Jingwen,XIAO Anfeng,CAI Huinong. 2014. Purification and biochemical characterization of manganesecontaining superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3. Acta Oceanologica Sinica, 33(12):163－169

Purification and biochemical characterization of manganesecontaining superoxide dismutase from deep-sea thermophile Geobacillus sp. EPT3

深海嗜热菌Geobacillus sp. EPT3锰超氧化物歧化酶的纯化与生物化学鉴定

Received:March 15, 2013 Revised:September 13, 2013

DOI：10.1007/s13131-014-0534-2

Key words:Geobacillus sp. superoxide dismutase thermostability purification

中文关键词: Geobacillus sp. 超氧化物歧化酶热稳定性纯化

基金项目:The National Natural Science Foundation of China under contract No. 31371751; the Science and Technology Program of Xiamen, China under contract No. 201303120001; the Foundation for Innovative Research Team of Jimei University, China under contract No. 2010A006.

Author Name	Affiliation	E-mail
ZHU Yanbing	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Microbiology and Enzyme Engineering Technology of Fujian Province, Xiamen 361021, China
LI Hebin	Xiamen Medical College, Xiamen 361008, China
NI Hui	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Microbiology and Enzyme Engineering Technology of Fujian Province, Xiamen 361021, China
LIU Jingwen	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
XIAO Anfeng	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Microbiology and Enzyme Engineering Technology of Fujian Province, Xiamen 361021, China
CAI Huinong	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Microbiology and Enzyme Engineering Technology of Fujian Province, Xiamen 361021, China	jmswthermo@hotmail.com

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Abstract:

Thermostable SOD is a promising enzyme in biotechnological applications. In the present study, thermophile Geobacillus sp. EPT3 was isolated from a deep-sea hydrothermal field in the East Pacific. A thermostable superoxide dismutase (SOD) from this strain was purified to homogeneity by steps of fractional ammonium sulfate precipitation, DEAE-Sepharose chromatography, and Phenyl-Sepharose chromatography. SOD was purified 13.4 fold to homogeneity with a specific activity of 3 354 U/mg and 11.1% recovery. SOD from Geobacillus sp. EPT3 was of the Mn-SOD type, judged by the insensitivity of the enzyme to both KCN and H₂O₂. SOD was determined to be a homodimer with monomeric molecular mass of 26.0 kDa. It had high thermostability at 50℃ and 60℃. At tested conditions, SOD was relatively stable in the presence of some inhibitors and denaturants, such as β-mercaptoethanol (β-ME), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), urea, and guanidine hydrochloride. Geobacillus sp. EPT3 SOD showed striking stability across a wide pH range from 5.0 to 11.0. It could withstand denaturants of extremely acidic and alkaline conditions, which makes it useful in the industrial applications.

中文摘要:

热稳定SOD是一种在生物技术应用中有潜力的酶.本研究中,嗜热菌Geobacillus sp. EPT3分离自东太平洋一个深海热液区.经硫酸铵分级沉淀、DEAE-Sepharose层析及Phenyl-Sepharose层析,从该菌株中纯化得到热稳定超氧化物歧化酶(SOD),纯化倍数13.4,回收率11.1%,酶比活力达3354 U/mg.SOD对KCN和H₂O₂不敏感性,说明来自Geobacillus sp. EPT3的超氧化物歧化酶为Mn-SOD类型.SOD为同聚二聚体,亚基分子量为26.0 kDa.该酶在50℃和60℃下有高的热稳定性.在检测条件下,SOD在一些抑制剂和变性剂存在时相对稳定,例如β-ME、DTT、PMSF、尿素和盐酸胍.Geobacillus sp. EPT3超氧化物歧化酶在pH 5.0~11.0的广范围内有好的稳定性,能耐受极酸和极碱的变性条件,有利于它的工业应用.

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