【作者】 彭仁；

【导师】 魏东芝；

【作者基本信息】 华东理工大学 ， 生物化工， 2010， 博士

【摘要】 脂肪酶(lipase,EC3.1.1.3)是普遍存在于自然界中的酶,它被广泛地运用于精细化工、洗涤、医药、食品、造纸、皮革加工、纺织和饲料工业等领域。从催化特性来看,脂肪酶具有高度的区域选择性和立体异构选择性,且反应不需要辅酶、反应条件温和、副产物少。脂肪酶的另一显著特点是它能在异相系统(即油-水界面)或有机相中作用。在水相中,脂肪酶通常催化水解反应的进行,而在有机相中,它却能催化各种合成反应：如酯化、酯的醇解、酯的氨解和酯的酸解等。然而有机溶剂会使酶变性或使酶活力下降,因此寻找耐有机溶剂的脂肪酶,使其在有机溶剂或含有机溶剂的环境中具有较高的催化活性,已成为脂肪酶研究领域的一个重要方向。本论文从自然界中成功筛选到产耐有机溶剂脂肪酶的菌株,对其产酶条件进行了研究,此外提纯和表征了该酶,并克隆和共表达了该脂肪酶及其折叠酶基因,最后用固定化的重组CS-2脂肪酶催化催化合成了乙酸丁酯。论文内容分为五部分。第一部分：产耐有机溶剂脂肪酶的菌株的筛选和鉴定。通过富集培养、粗筛和复筛,得到一株产耐有机溶剂脂肪酶的菌株CS-2。其产酶活性为1.63 U/ml,它在25%(v/v)的甲苯中30℃下孵育30 min后残余酶活力达95.7%。菌株CS-2的菌落为圆形、有光泽、乳黄色。菌株形状为杆状,革兰氏阴性,不产芽孢,具夹膜,单端生鞭毛。在25℃、37℃、45℃均能生长。不产脲酶,不产H2S,能利用柠檬酸盐、D-乳糖、D-果糖、D-葡萄糖、甘露醇和甘油,但不能利用D-麦芽糖和蔗糖。能水解明胶、干酪素和吐温-80,但不能水解淀粉。菌株CS-2的16SrDNA基因序列长度为1499bp,在Genbank的序列登录号为GQ254065。根据菌株CS-2的形态特征、生理生化特征和16SrDNA序列分析,菌株CS-2命名为Pseudomonas aeruginosa CS-2。来自Pseudomonas aeruginosa CS-2的粗脂肪酶在有机溶剂中表现出一定的稳定性。第二部分：产酶条件的研究。Pseudomonas aeruginosa CS-2发酵24 h后,其生长达到最大,然后缓慢下降。脂肪酶的活性在18 h后快速增加,48 h时产酶活力最高。有机氮源比无机氮源对产脂肪酶更有利,其中蛋白胨和胰蛋白胨为氮源时产脂肪酶最高。阿拉伯胶、吐温-20、吐温-80、聚乙烯醇124能促进脂肪酶的产生,而CTAB和Triton X-100则起相反的作用。培养温度37℃为产脂肪酶的最佳温度,转速200r/min为产脂肪酶的最佳转速,初始pH 7.5为产脂肪酶的最佳pH。第三部分：建立了P. aeruginosa CS-2所产胞外脂肪酶的纯化过程,并对纯化的脂肪酶进行表征。经过超滤、有机溶剂沉淀和离子交换层析等步骤,获得了电泳纯的脂肪酶,纯化倍数达到25.5倍,总活力回收率为45.5%。SDS-PAGE和凝胶过滤法测得的该酶的分子量约为33.9 kDa和36 kDa,因此该脂肪酶为单一亚基。该脂肪酶的C端序列为KNASLWDPGRG,与NCBI蛋白质数据库中现有脂肪酶的C端序列无一致性。该酶催化的最适温度为50℃,在40-70℃范围内趋于稳定,能达到最大酶活的80%以上。CS-2脂肪酶热稳定性较高,在80℃处理30min后残余酶活力为62.7%。该酶最适作用pH值为8.0,在pH为7.0-9.0范围内趋于平稳,该酶在较宽的pH(4.0-10.0)范围内稳定性较高,孵育30min后,残留酶活力仍在80%以上,因此它属于碱性脂肪酶。Ca2+对酶有激活作用,其它测定的金属离子对酶则起抑制作用,并且激活或抑制作用强弱与金属离子浓度有关。CTAB、Tween20、Tween80和Triton X-100对CS-2脂肪酶有抑制作用,而阿拉伯胶和聚乙烯醇-124有激活作用。在不同链长的对硝基苯酚酯中,CS-2脂肪酶的最佳底物为对硝基苯酚棕榈酰酯。CS-2脂肪酶在乙腈中活性提高,在DMSO和一些非亲水有机溶剂中具有一定的稳定性。在第四部分：CS-2脂肪酶及其折叠酶基因的克隆和表达。CS-2脂肪酶及其折叠酶基因的序列长度为2025bp,其中CS-2脂肪酶基因不含有终止密码子。CS-2脂肪酶及其折叠酶基因在E. coli BL21(DE3)进行共表达,重组的CS-2脂肪酶在细胞中能以具有活性的可溶蛋白形式存在。据我们所知,不含有终止密码子的基因能够在细胞中以活性形式进行表达还未见报道。经亲和层析纯化后的脂肪酶活性为203.8U/mg,其中纯化倍数为10.2,得率为40.9%。亲和层析纯化后的酶在SDS-PAGE中显示两条明显的条带,分子量约为35.7kDa和38.3 kDa。CS-2脂肪酶基因的可溶性表达需要折叠酶基因共表达进行辅助。在第五部分：固定化的重组CS-2脂肪酶在正庚烷中催化合成乙酸丁酯。催化的最适反应条件如下：反应时间10h,水分活度0.02,反应温度为55℃,乙酸和正丁醇浓度分别为0.1mol/L和0.2mol/L,在反应8h时加入0.5g 4A分子筛作为吸水剂。在该优化条件下,底物转化率达到了98.2%,并且该固定化酶使用5次,底物转化率仅从98.2%降低到87.4%。因此该固定化酶催化合成乙酸丁酯时表现出较高的催化能力和良好的重复使用性。更多还原

【Abstract】 Lipase (EC3.1.1.3) is ubiquitous in nature, which is used in a wide range of field such as fine chemical, detergent, pharmaceutical, food, leather processing, paper manufacture, textile, feed industry. In the term of catalytic feature, lipases have shown many advantages:high regiospecificity and stereoselectivity; no requirement of coenzyme; mild reaction condition and little by-product. Another important property of lipases is that they play a catalytic role in heterogeneous system or organic phase. Usually, numerous lipases catalyze the hydrolysis of esters to the corresponding acids and alcohols in water, while in organic phase esterification, transesterification, aminolysis, acidlysis and alcoholysis can be catalyzed by lipases. However, organic solvents often give rise to denature of lipase and decreased activity. Therefore, screening of organic solvent-tolerant lipases with high activity in the presence of organic solvents has become a focus of researches on lipase. In the present paper, a novel strain producing an organic solvent-tolerant lipase was successfully screened, and the fermentation condition was optimized. Furthermore, the lipase was purified and characterized. The lipase and it cognate foldase genes were cloned and co-expressed. Finally, the use of the immobilized recombinant lipase for the synthesis of butyl acetate was attempted.The paper was divided into five parts.The screening and identification of a strain producing an organic solvent-tolerant lipase were included in the first part. A novel strain producing an organic solvent-tolerant lipase was obtained after enrichment cultivation, initial screening and the second screening. The activity of lipase in the culture was 1.63 U/ml, with the residual activity of 95.7% after incubating at 30℃for 30 min in the presence of benzene (25%v/v).The colony of strain CS-2 was of round shape, bright, milky yellow. Strain CS-2 was rod shaped, Gram negative and it had capsule, single-end flagella and no spore. It can grow at 25℃,37℃and 45℃and can not produce urease and sulfide hydrogen. It can utilize citrate, D-lactose, D-fructose, D-glucose, mannitol and glycerol, but not D-maltose and sucrose. It can hydrolyze gelatin, casein and tween-80, but not starch. The length of 16S rDNA sequence of strain CS-2 was 1499bp, and the sequence data were submitted to Genbank with the accession number GQ254065. According to the morphological trait, biochemical feature and 16S rDNA sequence, strain CS-2 was name after Pseudomonas aeruginosa CS-2. The crude lipase from Pseudomonas aeruginosa CS-2 showed stability in some organic solvents.The optimization of fermentation condition was included in the second part. The maximum growth was achieved at 24 h, and then the growth was decreased slowly. Substantial lipase production started at 18 h and reached to maximum at 48 h. Organic nitrogen source was preferred over inorganic nitrogen resource. Peptone and tryptone was determined to be best organic nitrogen source for CS-2 lipase production. Gum arabic, Tween-20, Tween-80 and Polyvinyl alcohol 124 enhanced the lipase production, whereas CTAB and Triton X-100 played a negative role in lipase production. The optimal temperature, rotary shaking speed initial pH for lipase production was 37℃,200r/min and 7.5, respectively.The purification and characterization of the extra-cellular lipase secreted by Pseudomonas aeruginosa CS-2 were included in the third part. The purified lipase was obtained by ultra-filtration, acetone precipitation and ion exchange chromatography. CS-2 lipase was purified about 25.5-fold with an overall yield of 45.5%. Molecular mass of the native lipase was about 33.9 kDa and 36 kDa, as determined by SDS-PAGE and size chromatography. Therefore, this suggested the lipase was a monomer. The C terminal of the lipase was KNASLWDPGRG. No protein was identical with the C-terminal sequence of CS-2 lipase using blastp. The temperature optimum of the CS-2 lipase was 50℃, and was stable in a temperature range of 40-70℃with more than 80% of the maximum activity. CS-2 lipase showed good thermostability. It remained 62.7% residual activity after incubated at 80℃for 30min. CS-2 lipase showed an optimum activity at pH 8.0, and was stable over a pH range of 7.0-9.0. It kept over 80% residual activity after incubated in a pH range of 4.0-10.0, which showed the lipase exhibited pH stability. These results confirmed the lipase was an alkaline lipase. Ca2+ increased the lipase activity, while other metal ions inhibited the activity. The activation and inhibition of activity were related to the concentration of metal ions. CTAB, tween 20, tween 80 and triton X-100 inhibited the lipase activity. On the contrary, gum Arabic and polyvinyl alcohol 124 enhanced lipase activity. Among the ester of different fatty acids with longer chain,p-nitrophenyl palmitate was the best substrate as compared to other esters. The lipase was activated in the presence of acetonitrile, and it showed stability in DMSO and some immiscible organic solvents to some extent.The clone and expression of the genes of CS-2 lipase and foldase in E.coli were included in the fourth part. The length of CS-2 lipase and foldase gene was 2025bp, and there was no stop codon in the CS-2 lipase gene. The genes of CS-2 lipase and foldase were co-expressed in E. coli BL21 (DE3). The CS-2 lipase was soluble in recombinant cell.To our knowledge, there was no report that a gene without a stop codon can be expressed as an active form. The activity of 203.8 U/mg was achieved after affinity chromatography, in which purification fold was 10.2 and yield rate was 40.9%. The purified enzymes were stained in SDS-PAGE with two bands and their molecular mass was estimated to be 35.7 kDa and 38.3 kDa, respectively. The expression of CS-2 lipase gene in soluble state should be aided by the co-expression of its foldase gene.The synthesis of butyl acetate in heptane by the immobilized recombinant CS-2 lipase was included in the fifth part. The optimum reaction condition was on the following:reaction time (10h); water activity (0.02); reaction temperature (55℃); the concentration of acetic acid and n-butanol (0.1mol/L and 0.2mol/L); the addition of 4A molecular sieve (0.5g) at 8h of reaction time. The conversion of 98.2% was achieved in the optimum reaction condition. The conversion of substrate decreased only from 98.2% to 87.4% after five cycles in use of the immobilized lipase.Therefore, the immobilized lipase showed good catalytic capability and repeated use for the synthesis of butyl acetate.更多还原