脂肪酶产生菌的筛选鉴定、产酶条件优化及酶学性质研究

发布时间：2018-01-10 02:07

  本文关键词：脂肪酶产生菌的筛选鉴定、产酶条件优化及酶学性质研究　出处：《湖北工业大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 脂肪酶 铜绿假单胞菌 发酵工艺优化 分离纯化 酶学性质

【摘要】：脂肪酶(E.C.3.1.1.3),又称三酰甘油酰基水解酶。在水相中,能将甘油三脂水解生成甘油一酯、二酯或者直接生成甘油和脂肪酸。在非水相体系中,脂肪酶具有良好的脂化、转脂、醇解和胺解等特性,该特性可使脂肪酶广泛应用在药物合成、造纸、纺织、食品、化妆品等行业。本文以橄榄油为唯一碳源选育出了一株高产脂肪酶菌株,优化了发酵产酶工艺条件,研究了酶的分离纯化及酶学性质,并探讨了菌株及其所产脂肪酶在生物降解餐馆废水中的应用。其主要研究内容如下:利用油脂同化平板和三丁酸甘油酯平板从生活废渣中筛选出了一株产脂肪酶的菌株HFE733,其酶活为1.93 U/mL。该菌株经形态学、生理生化及系统发育学方法鉴定为铜绿假单胞菌(Pseudomonas aeruginosa)。对出发菌株HFE733进行紫外及硫酸二乙酯诱变,最终得到最优突变菌株D13。该菌株经摇瓶发酵测得脂肪酶酶活为5.03 U/mL,比出发菌株HFE733的酶活提高了160.62%。并通过遗传稳定性试验证明,D13菌株有良好的遗传稳定性。将其命名为HFED13。采用单因素试验确定了HFED13菌株发酵产脂肪酶的最适发酵条件为:发酵温度30℃,发酵周期60 h,初始pH 7.0,接种量5%,装液量50 mL/250 mL,摇床转速220 r/min。通过Plackett-Burman设计试验、最陡爬坡试验、响应面法最终得到发酵培养基的最佳配方为:酵母膏34 g/L、橄榄油5.92 m L/L、蔗糖12.5 g/L、硫酸铜0.4 g/L、硫酸锰0.2 g/L。脂肪酶酶活可达到9.27 U/mL,试验结果与预测值基本吻合,且比优化前酶活提高了84.29%。通过硫酸铵盐析、Sephandex G-25凝胶过滤脱盐、DEAE-cellulose A-52离子交换层析的方法,对HFED13菌株发酵所产脂肪酶进行了分离纯化。经SDS-PAGE电泳分析,纯化后的脂肪酶为单一蛋白,其分子量为51.02 kDa,纯化倍数为9.97,回收率为31.54%,比酶活为79.25 U/mg。纯化的脂肪酶最适pH为7.0~8.5,最适反应温度为40℃,该酶在pH6.5~9.0范围内保持稳定。不同的金属离子和化学试剂对该脂肪酶具有不同的影响。其中,Fe3+、Al3+、β-巯基乙醇、半胱氨酸和二硫苏糖醇对该酶有一定的促进作用,而Co2+、Cu2+、Tween 80和Triton X-100对该酶有一定的抑制作用,10 mM的十二烷基硫酸钠完全抑制了该酶的活性。该菌株及其所产脂肪酶对餐馆废水有很好的降解作用,通过废水和发酵液的混合处理,经过六天的培养,废水中油脂含量和COD(化学需氧量)分别由4296.00 mg/L和11449.42 mg/L下降到195.79 mg/L和1243.97mg/L,下降了95.44%和89.14%,基本达到了排放标准。
[Abstract]:Lipase E.C.3.1.1.3, also known as triacylglycerol hydrolase. In aqueous phase, triglyceride can be hydrolyzed to glycerol monoester. In non-aqueous phase, lipase has good characteristics of lipase, such as lipid, fat, alcoholysis and amination, which make lipase widely used in drug synthesis, papermaking and textile. In this paper, a high yield lipase strain was selected with olive oil as the sole carbon source, the fermentation conditions were optimized, and the purification and properties of the enzyme were studied. The application of the strain and its lipase in biodegradable restaurant wastewater was also discussed. A lipase producing strain HFE733 was isolated from the domestic waste residue by using the oil assimilation plate and the tributyrate triglyceride plate. The enzyme activity was 1.93 U / mL. The strain was morphologically. Physiological, biochemical and phylogenetic methods were used to identify Pseudomonas aeruginosa as Pseudomonas aeruginosa. The original strain HFE733 was mutagenic by UV and diethyl sulfate. The optimal mutant strain D13 was obtained. The lipase activity of this strain was 5.03 U / mL by shaking flask fermentation. The enzyme activity of the original strain HFE733 was 160.62% higher than that of the original strain HFE733, and proved by genetic stability test. D13 strain had good genetic stability. It was named HFED13. The optimum fermentation conditions for lipase production of HFED13 strain were determined by single factor test: fermentation temperature was 30 鈩，

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