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

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16SrDNA序列同源性分析，初步鉴定菌株Mhy一1为杀鲑气单胞菌（Aeroraonas 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45qC，最适作用pH8．0。该酶在pH7．0—9．0和温度30—70℃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。

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 16S 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 34ku on SDS- PAGE. The optimal temperature and pH of for the enzyme reactivity were 45℃ and 8.0 respectively.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.