高产脂肪酶菌株的筛选鉴定及酶学、转酯特性  

Lipase-producing strain: isolation, identification, enzymology and transesterication

作　　者：马抒晗 张玮佳[1] 吴茜[1] 姜腾飞[1] 乔代蓉[1] 曹毅[1] 

机构地区：[1]四川大学生命科学学院微生物与代谢工程四川省重点实验室,成都610065

出　　处：《应用与环境生物学报》2014年第4期602-608,共7页Chinese Journal of Applied and Environmental Biology

基　　金："十二五"国家科技支撑计划项目(2011BAD14B05;2013BAD10B01;2014BAD02B02);国家自然科学基金项目(31171447);四川省科技厅项目(2014GZX0005;2013HH0007;2013GZX0161-3;2013GZ0058;2013JCPT003)资助~~

摘　　要：从自然环境中筛选水解酶活高且酶学、转酯特性优良的产脂肪酶菌株,对脂肪酶工业化发酵生产及生物柴油制备的研究具有重要意义.采用罗丹明B平板初筛和摇瓶发酵复筛法,从70份含油脂丰富的样品中筛选产脂肪酶酶活较高的菌株进行16S rRNA鉴定,研究其酶学性质;用大孔树脂固定酶,在无溶剂体系中催化橄榄油制备生物柴油,研究其转酯特性.结果筛选到一株高产脂肪酶的菌株WZ10-3,通过p-NPP法测得其初始酶活为78.68 U/mL,经16S rRNA鉴定属于伯克霍尔德氏菌(Burkholderia sp.),与B.stabilis同源性达到99%.该菌在发酵48 h时达到产酶高峰,所产脂肪酶的最适作用温度为50℃,最适作用pH为7.0,70℃下的半衰期可达1 h,pH为7-9时稳定性良好.以大孔树脂NKA-9和HPD600为载体制备的2种固定化脂肪酶,催化橄榄油生产生物柴油的转酯率均可达到97%.综合表明,菌株WZ10-3脂肪酶的初始水解酶活高于大多数野生脂肪酶,热稳定性好且转酯特性优良,有很好的后续研究价值.For a better understanding of lipase industrial fermentation and biodiesel production, this study screened from natural environment lipase producing strains with high hydrolysis activity, stable enzymatic properties and excellent transesterification characteristics. The high lipase activity producing strain was isolated from 70 rich oil samples by Rhodamine B screening plates and shake flask fermentation, and was identified through 16 S rRNA sequence. After studying of enzymatic properties, the lipase was immobilized on the macroporous resins NKA-9 and HPD600. The transesterification characteristics were studied by producing biodiesel from olive oil catalyzed by immobilized lipases in solvent-free system. The high lipase activity producing strain WZ10-3 was measured by p-NPP method to have an initial enzyme activity of 78.68 U/mL. The 16 SrRNA sequence identified the strain as Burkholderia sp., of 99% homology with B. stabilis. Its lipase production reached a peak at 48 h of fermentation with the optimum reaction temperature of 50 ℃and the optimum pH of 7.0. The lipase had a half-life of 1 h under 70 ℃, and was stable under pH 7-9. The preparing biodiesel by lipases immobilized on the macroporous resins NKA-9 and HPD600 indicated that both immobilized lipases had 97% transesterification rate. The initial lipase activity of strain WZ10-3 is much higher than most other wild strains. Its good thermal stability and excellent transesterification characteristics prove the follow-up research value of the strain WZ10-3.

关 键 词：脂肪酶 菌株筛选与鉴定 酶学性质 转酯 伯克霍尔德氏菌 生物柴油 

分 类 号：Q93[生物学—微生物学]