【作者】 林康；

【导师】 李素霞；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2012， 硕士

【摘要】 粘质沙雷氏菌脂肪酶属于α/β水解酶,可广泛应用于食品、制药以及生物能源等领域。本课题研究了重组粘质沙雷氏菌ECU1010(Serratia marcescens ECU1010)脂肪酶LipA合成生物柴油的生产工艺、活性包涵体的固定化,并利用基因工程方法构建了脂肪酶LipB的突变体,对突变体的有机溶剂耐受性及三维结构进行了研究。通过单因素试验对生物柴油的酶法合成工艺进行了研究,重点考察了酶用量、醇油摩尔比、反应时间、反应温度等因素对脂肪酸甲酯(FAME)转化率的影响。以FAME为考察指标,通过响应面法(RSM)系统研究了酶用量、醇油摩尔比、反应时间、有机溶剂量等因素对转酯反应进程的影响,结果表明：以大豆油质量为基准,LipA用量10.8%(w/w),反应温度40℃,石油醚用量2.73mL/g,醇油摩尔比1.4,甲醇流加次数3次,每次流加间隔时间10.6h, FAME的转化率为84.2%,比优化前提高了10.7%。采用Plackett-Burman设计和Box-Behnken设计对影响S. marcescens LipA活性包涵体固定化的诸多因素进行考察和评价,并对筛选出的重要因素进行统计学优化,经RSM优化后的固定化条件为：戊二醛浓度0.13%(w/v),酶浓度16.5mg/mL,戊二醛与酶用量比5.5：1。在此条件下,固定化酶的酶活回收率比优化前提高了45%。通过实验研究了固定化后脂肪酶活性包涵体的部分酶学性质,对固定化酶的pH稳定性、热稳定性、金属离子和有机溶剂性耐受性、操作稳定性以及贮存稳定性进行了重点考察。通过与游离酶性质的对比,发现固定化酶在pH5.0～10.0范围内稳定性较游离酶高,且60℃温育1h后仍能保持48%以上的酶活。此外,固定化酶对亲水性有机溶剂、金属离子以及表面活性剂的耐受性增强,其操作稳定性和贮存稳定性也大有提高。利用定点突变技术,以重叠延伸PCR的方法将脂肪酶LipB上Gly33、Alal87以及Thr270分别突变成脂肪酶LipA对应位置上的Asp33、Val187和Ala270,并对LipB三个突变体进行不同浓度的有机溶剂处理,以考察三个突变体对有机溶剂的耐受情况,再通过同源建模和结构分析软件对三个突变体的结构进行模拟和分析,发现第33位Gly对脂肪酶LipB的有机溶剂耐受性起了重要作用。更多还原

【Abstract】 The extracellular lipases from Serratia marcescens ECU1010are a kind of α/β hydrolase fold protein; they are widely used in the food, medicine and bio-energy industries. In this paper, we investigated and optimized the production conditions of biodiesel using S. marcescens LipA as the catalyst. Through the single-factor experiment and response surface analysis, the optimal production conditions of biodiesel were obtained and the results were as follows:Based on the weight of soybean oil, enzyme dosage10.8%(w/w), reaction temperature40℃, petroleum ether2.73mL/g, substrate molar ratio1:4, adding methanol for3times, time interval10.6h. Under the optimal conditions, the conversion rate of FAME reached84.2%, which increased10.7%than prior to optimization.The immobilization conditions of lipase present in active inclusion bodies were studied by response surface methodology. Plackett-Burman design and Box-Behnken design were used to optimize the immobilization conditions, respectively. Through the experiment, we obtained the optimal immobilized conditions. The details were as follows:glutaraldehyde0.13%(w/v), enzyme16.5mg/mL, ratio of glutaraldehyde and enzyme5.5:1. Under this condition, the enzymatic activity was increased45%than prior to optimization.Comparative studies on the enzymatic properties of the free and immobilized enzymes were performed. The main properties studied in this work included the stability of pH and temperature, as well as in the presence of various metal ions and organic solvents. In addition, the operation stability and the storage stability were investigated. The immobilized lipase showed high stability in a broad pH range of5.0-10.0and remained over48%of its activity at60℃for1h. The tolerance to metal ions and water-miscible organic solvents was raised. The operation stability and the storage stability were also significantly improved.The mutants (G33D, A187V and T270A) of S. marcescens LipB were obtained through the site-directed mutagenesis technology and the tolerance of LipB mutants to various organic solvents were investigated. Based on homology modeling and structural analysis, the33rd amino acid (Gly) of LipB played an important role in the tolerance against organic solvents.更多还原