1株枯草芽胞杆菌的鉴定及其产脂肪酶特性  

Identification of a Bacillus subtilis strain and characteristics of lipase production

作　　者：张震[1] 张岩[1,2] 杨朋坤[1] 任双慧[1,2] 高雅欣 孙玉娇 王永芬 ZHANG Zhen;ZHANG Yan;YANG Pengkun;REN Shuanghui;GAO Yaxin;SUN Yujiao;WANG Yongfen(College of Food and Bioengineering,Henan Institute of Animal Husbandry Economics,Zhengzhou,Henan 450046,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou,Henan 450046,China)

机构地区：[1]河南牧业经济学院食品与生物工程学院,河南郑州450046 [2]河南农业大学动物医学院,河南郑州450046

出　　处：《西北农林科技大学学报（自然科学版）》2024年第4期66-74,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金青年基金项目(31802111);河南省科技攻关项目(232102110084,212102110004,212102110007,202102110235);河南省青年骨干教师资助项目(2020GGJS257);中原院士基金项目(豫财行[2020]140号)。

摘　　要：【目的】对1株产脂肪酶细菌MY016进行鉴定并对其产脂肪酶特性进行研究,为后期规模化发酵生产脂肪酶及其在畜牧业中的实际应用奠定基础。【方法】采用形态学观察、生理生化鉴定、16S rDNA测序分析,对实验室分离保存的1株产脂肪酶细菌MY016进行鉴定;通过菌落和芽胞计数、生长曲线测定,对MY016的生长特性进行分析;通过产脂肪酶量和酶活检测、脂肪酶基因扩增和系统进化分析,对MY016菌株的产脂肪酶特性进行探讨。【结果】产脂肪酶细菌MY016经形态观察、生理生化鉴定和16S rDNA分析,被鉴定为枯草芽胞杆菌。MY016在培养2 h后进入对数生长期,生长迅速;培养10 h其菌液600 nm吸光度最高达到4.7,随后进入稳定期;12 h时活菌数约为5.3×10^(9)CFU/mL;培养22 h后开始二次生长,吸光度最高达5.2;培养46 h后进入衰退期。芽胞观察和计数结果显示,MY016菌株在接种16 h后开始形成芽胞,随着培养时间延长,芽胞形成率逐渐升高,直至72 h完全形成芽胞,芽胞数最高为1.9×10^(9)CFU/mL。产脂肪酶能力检测结果显示,MY016菌株在培养36 h时产酶量达到最大,为246.918 mg/L,然后总酶量逐步降低;48 h时总酶活达到最高,约为281.883 U/L。成功扩增了MY016菌株的脂肪酶基因,并构建系统进化树,结果显示MY016脂肪酶基因与其他芽胞杆菌的脂肪酶基因亲缘关系最近,表明其脂肪酶基因进化保守。【结论】产脂肪酶枯草芽胞杆菌MY016生长迅速、产酶量稳定,总酶活较高,适合后期规模化生产和在畜牧业中推广应用。【Objective】This study identified a strain of lipase producing bacteria and studied its lipase producing characteristics to provide basis for following large-scale fermentation production of lipase and practical application in animal husbandry.【Method】A lipase producing bacterium MY016 preserved in laboratory was identified using physiological observation,biochemical identification and 16S rDNA sequencing analysis.The growth characteristics of MY016 were analyzed through nutrient and spore counting and growth curve measurements.The lipase production characteristics of MY016 were analyzed through lipase production and activity detection,lipase gene amplification and phylogenetic analysis.【Result】The lipase producing bacteria MY016 was identified as Bacillus subtilis by morphological observation,physiological and biochemical identification and 16S rDNA analysis.MY016 entered a logarithmic growth phase 2 hours after cultivation and grew rapidly.The absorbance value of the bacterial solution reached a maximum of 4.7 at 600 nm after 10 hours.The growth then entered a stable phase,with viable bacterial count of 5.3×10^(9)CFU/mL after 12 hours.After 22 hours,secondary growth began with a maximum absorbance value of 5.2,and then it entered a decline period after 46 hours.Observation and spores counting showed that MY016 began to form spores 16 hours after inoculation.With the prolongation of cultivation time,the spore formation rate of MY016 increased until complete spore formation was achieved at 72 hours,with a maximum spore count of 1.9×10^(9)CFU/mL.The results of lipase production capacity showed that MY016 reached its maximum enzyme production of 246.918 mg/L after 36 hours cultivation,followed by a gradual decrease in total enzyme activity.At 48 hours,the total enzyme activity reached its maximum at approxi-mately 281.883 U/L.The lipase encoding gene of MY016 was successfully amplified and a phylogenetic tree was constructed.It had the closest genetic relationship with other lipase encoding genes of Ba

关 键 词：枯草芽胞杆菌 菌株鉴定 脂肪酶 

分 类 号：Q939.124[生物学—微生物学]