热稳定性脂肪酶生产菌的筛选及其酶学性质研究
Screening of a thermostable lipase producing bacterium and its enzyme characterization
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摘要: 采用平板透明圈法从不同来源土壤样品中分离得到31株脂肪酶生产菌株,经复筛得到一株脂肪酶产量较高的菌株Mhy-1。根据培养特征、生理生化分析及16S rDNA序列同源性分析,初步鉴定菌株Mhy-1为杀鲑气单胞菌(Aeromonas salmonicida),该菌的最适生长温度及初始pH分别为35℃和pH7.0,最适产酶温度及初始pH分别为30℃和pH9.0。采用硫酸铵盐析、DEAE-Sepharose fast flow阴离子交换层析从菌株Mhy-1的发酵上清液中分离纯化了脂肪酶,酶的分子量约为34ku,最适作用温度为45℃,最适作用pH8.0。该酶在pH7.09.0和温度3070℃的范围内稳定,在70℃条件下能维持80%以上酶活力,是一热稳定酶,纯化后的酶可用于生物柴油的催化合成。Abstract: Thirty-one lipase producing strains were isolated from the soil samples collected in Gansu province by the method of Tween- 80 nutrient agar plate method. One strain Mhy- 1 was selected for the high growth and production of lipase. The strain Mhy- 1 was preliminarily identified as Aeromonas salmonicida by morphological characteristics, physiological and biochemical, and sequence analysis of 16 S rDNA.The optimum temperature and pH for cell growth were 35℃ and pH7.0.The optimum temperature and pH for the lipase production were 30℃ and pH9.0.A lipase was purified from the culture supernatant of the strain Mhy- 1 by means of ammonium sulfate precipitation, column chromatography on DEAE-Sepharose fast flow.The enzyme appeared to be a single peptide of 34 ku on SDS- PAGE. The optimal temperature and pH of for the enzyme reactivity were 45℃ and 8.0respectively.The enzyme was stable at temperatures 30~70℃ and pH7.0~9.0.The purified enzyme could be used to catalyze the production of biodiesel.