一株产乙酰酯酶细菌筛选、鉴定及酶学性质研究

【目的】利用筛选培养基,从肉牛瘤胃液中分离筛选产乙酰酯酶的细菌菌株,并研究菌株的产酶特征。【方法】利用厌氧培养技术,以木质素为唯一碳源,筛选并驯化所得菌株。根据菌株16S rDNA序列分析、革兰氏染色、伊红美蓝培养基培养、甲基红试验和柠檬酸盐利用试验,鉴定菌株。采用对-硝基苯乙酯测定酶活力。【结果】筛选得到产乙酰酯酶活力较高细菌菌株RB1,初步鉴定为Escherichia coli。菌株RB1的生长曲线表明,0 42 h为菌株的延迟期,42 60 h为菌株的对数期,60 66 h为菌株的稳定期,66 86 h为菌株的衰亡期。菌株所产乙酰酯酶最适温度为40°C,最适pH为8.0,在最适温度与pH条件下,培养基中添加玉米秸秆粉,乙酰酯酶最高酶活力达到0.52 U/mL。【结论】筛选获得产乙酰酯酶的细菌菌株RB1,其乙酰酯酶活力高于已报道的菌株,是一株具有研究和应用潜力的产乙酰酯酶的菌株。

Isolation, identification and enzyme characteristics of a strain producing acetylesterase

Objective] The aim of this study was to screen acetylesterase-producing strain and to investigate its characteristics. Methods] Anaerobic culture technique was used in screening and acclimatization of strain, and alkali lignin was a sole carbon source. The strain was identified by analyzing the sequence of 16S rDNA, Gram staining, Eosin Methylene Blue test, Methyl red test, and citrate utilization test. Acetylesterase was determined by ethyl p-nitrobenzoate. Results] Acetylesterase-producing strain RB1 was isolated from rumen fluid of beef. It was identified as Escherichia coli. The growth curve showed that 0?42 h was lag phase, 42?60 h was log phase, 60?66 h was stationary phase, and 66?86 h was death phase. The optimum temperature for the acetylesterase was 40 °C, and the optimum pH was 8.0. The highest enzyme activity was 0.52 U/mL under optimum temperature and pH, and corn stover powder as a carbon source. Conclusion] Strain RB1 was an acetylesterase-producing strain with application potential.