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荧光假单胞菌胞外蛋白酶的纯化及特性研究 被引量:10

Purification and Properties of Extracellular Protease from Pseudomonads fluorescens
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摘要 采用硫酸铵沉淀、DEAE-Sepharose FF离子交换层析、Sephacryl S-100 HR凝胶过滤层析方法对从原料乳中分离出一株荧光假单胞菌胞外耐热蛋白酶进行纯化及特性研究。将纯化后的单一蛋白酶进行SDS-PAGE分子质量测定,并考察最适温度、最适pH值、热稳定性、金属离子对其酶活性的影响及对该蛋白酶的氨基酸种类分析。纯化后蛋白酶的分子质量为47kD,经纯化后蛋白酶比活力提高了近61.38倍;最适温度和pH值分别为30℃和7.0;二硫苏糖醇对蛋白酶活性具有一定的抑制作用,Fe2+可以促进蛋白酶活性的提高;该酶具有较强耐热性,原酶液经130℃热处理3min后,残留酶活仍超过原酶液的47.67%;该蛋白酶氨基酸组成中甘氨酸(Gly)、丙氨酸(Ala)和谷氨酸(Glu)的含量占明显优势,其中Gly含量最高,物质的量分数高达42%。 The aim of this study was to purify and characterize heat-stable extracellular protease produced by Pseudomanas fluorescens isolated from raw milk.Crude protease was purified by ammonium sulfate fractionation,DEAE-Sepharose FF column chromatography and Sephacry S-100 column chromatography.The molecular weight,optimum pH and temperature,heat stability and amino acid sequence of purified protease as well as the effect metal ions on protease activity were analyzed.The purified enzyme is a monomer with a molecular weight of 47 kD as evaluated by SDS-PAGE.The specific activity of the crude protease was increased by 61.38 fold when compared with the purified protease.The optimum pH and temperature were 7.0 and 30 ℃,respectively.The purified protease was partially inhibited by DL-dithiothreitol but activated slightly by Fe2+.The protease revealed typical heat-stable behavior.After treatment at 130 ℃ for 3 min,more than 47.67% of the original activity was remained.The dominant amino acids in the purified protease were glycine,alanine and glutamic acid and glycine was the most dominant amino acid with a molar percentage of 42%.This study will contribute to further understanding of the properties protease from Pseudomonas fluorescens and the possible relationship between protease type and ultra high temperature-treated milk gelation.
出处 《食品科学》 EI CAS CSCD 北大核心 2012年第9期206-210,共5页 Food Science
基金 农业部公益性行业科研专项(200903043) 国家自然科学基金项目(31071575)
关键词 荧光假单胞菌 蛋白酶 纯化 特性 牛乳 腐败 Pseudomonads fluorescens protease purification characterization milk spoilage
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