牦牛瘤胃中产脂肪酶微生物的分离与鉴定

【背景】脂肪酶是一类特殊的酯键水解酶,广泛应用于工业化生产中,微生物是工业脂肪酶的主要来源。瘤胃中微生物种类繁多、数量庞大,已有关于瘤胃微生物产纤维素酶的报道,尚无产脂肪酶瘤胃微生物的分离筛选报道。【目的】从牦牛瘤胃中分离筛选出能够产脂肪酶的微生物,并进行菌株鉴定及其酶学性质的研究。【方法】以橄榄油为唯一碳源,通过中性红油脂平板进行初步筛选后,用改进铜皂-分光光度法测定酶活力进行复筛;再经形态学观察、生理生化实验和16S rRNA基因序列分析进行菌种鉴定;研究3种脂肪酶的最适作用温度、pH值及金属离子、有机溶剂和表面活性剂对酶活力的影响。【结果】筛选出6株酶活力较高的菌株,其中3株为液化沙雷氏菌,2株为白地霉,1株为卷枝毛霉。脂肪酶的酶学性质研究表明:液化沙雷氏菌、白地霉和卷枝毛霉所产脂肪酶的最适作用温度为45、35和40°C;最适pH为8.0、7.0和7.0;Ca2+和Mg2+对3种脂肪酶均有激活作用;Zn2+对3种脂肪酶有不同程度的抑制作用,EDTA、SDS可使3种脂肪酶失活;3种脂肪酶对丙三醇的耐受力较高,卷枝毛霉脂肪酶对甲醇、乙醇、丙酮的耐受力较高。【结论】从牦牛瘤胃中分离出3种产脂肪酶的微生物,且证实瘤胃微生物在脂肪酶研究方面具有较高的价值。

Isolation and identification of lipase produced by microbes in yak rumen

Background] Lipase is a special ester bond hydrolase and widely used in industries. Microorganisms are the main source of lipase. Many microorganisms in the rumen are of great diversity. Although it has been reported about the production of cellulase by rumen microorganisms, there is no report about lipase produced by rumen microorganisms. Objective] The purpose of this study was to isolate and screen lipase produced by microorganisms from the rumen of yak, and to identify the strains and characterize the enzymes. Methods] Olive oil was used as the sole carbon source in neutral red fat medium for preliminary screening, then the obtained strains were rescreened by the improved copper soap-photometric. Finally, the rescreened strains were identified by morphological observation, physiological and biochemical experiments and 16S rRNA gene sequence analysis. The lipases produced by the isolated microorganisms were tested by temperature, pH, metal ions, organic solvents and surfactant, accordingly we had found the optimum conditions. Results] Six strains with high enzyme activity were obtained. Three were Serratia liquefaciens, two were Geotrichum candidum, and one was Mucor circinelloides. Enzymatic properties showed that the optimal reaction temperature of three lipases (Serratia liquefaciens, Geotrichum candidum and Mucor circinelloides lipase) was 45, 35 and 40 °C, and the optimal reaction pH of them was 8.0, 7.0 and 7.0 respectively. The activities of these lipases were stimulated by Ca2+ and Mg2+ ions and inhibited by Zn2+ ions. Lipases were inactivated by EDTA and SDS. These lipases tolerated glycerol, while the lipase of Mucor circinelloides showed a better tolerance to methanol, ethanol and acetone than others. Conclusion] We isolated three lipases produced by microorganisms from the rumen of yak. This study showed that rumen microorganisms are of high value in lipase research.