【作者】 谭中标；

【导师】 邬敏辰；

【作者基本信息】 江南大学 ， 发酵工程， 2014， 博士

【摘要】 脂肪酶(EC3.1.1.3)是能够将长链脂酰甘油酯水解的一类酰基水解酶，不仅能在油水界面催化水解反应，还可以在疏水介质中催化转酯、酯化、酯交换等反应。根据脂肪酶的底物专一性，可以将其分为三脂酰甘油脂肪酶和单双脂酰甘油脂肪酶等。三脂酰甘油脂肪酶能将三脂酰甘油酯水解为脂肪酸和甘油，而单双脂酰甘油脂肪酶只能水解单脂酰甘油酯和二脂酰甘油酯。圆弧青霉(Penicillium cyclopium) PG37可产三脂酰甘油脂肪酶(PcLipI)，也能产单双脂酰甘油脂肪酶(PcMdl)。为了更好地利用这些脂肪酶资源，首先克隆了它们的cDNA序列，并实现了在毕赤酵母(Pichia pastoris) GS115中的异源表达。根据对重组酶酶学性质研究，发现它们的耐热性较差，通过基因工程手段改造了PcLipI以提高其热稳定性。PcMdl的底物专一性不同于PcLipI，通过实验验证其底物专一性后，运用计算机模拟分析了其底物专一性的分子基础。三脂酰甘油酯属于小分子化合物，但是它的水解在rePcLipC(重组热稳定性提高的改造PcLipI)和rePcMdl (重组PcMdl)共同作用时有更好的效果。通过RT–PCR技术获得了PcLipI成熟肽的cDNA序列，其长度为777bp，共编码258个氨基酸。PclipI在毕赤酵母GS115中实现了高效的异源表达。重组毕赤酵母GSL4-7高效表达rePcLipI的培养基初始pH为9.0，甲醇添加量为1.0%，诱导温度为26℃，在此条件下诱导108h产酶水平可达407.3U mL-1。rePcLipI被证实为糖基化蛋白，其表观分子量为31.5kDa。Hg2+、Fe2+和Cu2+抑制rePcLipI的酶活性。rePcLipI的最适pH为10.5，最适温度为25℃，在温度低于30℃和pH7.0~10.5范围内稳定，表明rePcLipI属低温碱性三脂酰甘油脂肪酶，且热稳定性差。rePcLipI的比酶活性为5,300U mg-1。借助同源建模、二硫键预测和分子动力学模拟的方法，预测出能提高PcLipI热稳定性的二硫键。通过定点突变技术获得突变的PcLipI基因，并将其在大肠杆菌BL21(DE3)和毕赤酵母GS115中表达。酶学特性分析的结果表明，大肠杆菌重组突变酶reE-PcLipV248C-T251C和毕赤酵母重组突变酶reP-PcLipV248C-T251C(即为rePcLipC)在35℃下的t1/2分别是对应重组酶reE-PcLip和reP-PcLip的4.5倍和12.8倍。同时，reE-PcLipV248C-T251C和reP-PcLipV248C-T251C比相应reE-PcLip和reP-PcLip的最适温度分别都提高了5℃。通过RT–PCR技术获得了PcMdl完整cDNA序列，提交至GenBank后获得的登陆号为：HM135194。成熟肽cDNA基因Pcmdl在毕赤酵母GS115中实现了异源表达。rePcMdl的表观分子量为39.0kDa。重组毕赤酵母GSM4-2高效表达rePcMdl的条件为：初始pH6.5、甲醇添加量1.5%、诱导温度28℃、诱导时间120h，在此条件下GSM4-2的产酶水平可达215.2U mL-1。rePcMdl经过了糖基化修饰。rePcMdl的最适反应温度为35℃，最适反应pH值为7.5，在35℃以下及pH6.5~9.5之间具有较好的稳定性。Fe3+和Hg2+抑制rePcMdl的酶活性。rePcMdl能催化1-单丁酰甘油酯和1,2-二丁酰甘油酯的水解，不能催化三丁酰甘油酯的水解。rePcMdl水解1-单丁酰甘油酯和1,2-二丁酰甘油酯的Vmax值分别为189.3U mg-1和344.9U mg-1，Km分别为29.1mmol L-1和42.6mmol L-1。借助同源建模、分子对接及分子动力学模拟等方法，对PcMdl底物专一性进行了分析。同源建模构建的活性状态PcMdl，其催化三联体Ser145-Asp199-His259暴露于溶剂中；同时Ser83和Ser145构成氧离子洞，能够稳定催化反应时形成的四面体中间产物。催化口袋由下列氨基酸构成：Tyr21、Phe112、Leu146、Pro174、Val201、Phe256、Val266和Asp267。活性状态下，催化口袋的口部不被盖子覆盖，暴露于溶剂中并有利于底物的进入。利用分子对接及分子动力学模拟，构建了PcMdl与三脂酰甘油酯类似物或二脂酰甘油酯类似物的对接模型。PcMdl中的Phe256将其残基侧链伸向底物结合沟，使得三脂酰甘油酯的sn-1部分进入困难，进而阻碍sn-1的羰基碳原子接近催化三联体中Ser145的Oγ；相反，二脂酰甘油酯能够顺利进入PcMdl底物结合沟并被催化水解。研究了rePcLipC和rePcMdl共同水解油脂的影响因素，同时考察了它们对橄榄油和三丁酰甘油酯的共同水解，并利用HPLC对部分水解产物进行了分析。通过单因素实验确定rePcLipC和rePcMdl共同水解三丁酰甘油酯的最佳温度为30℃，最佳初始pH10.5，最佳酶活性比例为3:1。在最佳温度30℃和恒定pH8.5下，用酶活性比例为3:1的rePcLipC和rePcMdl共同水解橄榄油，水解速度加快，rePcMdl同样能与南极假丝酵母脂肪酶B(CALB)共同水解橄榄油。rePcLipC和rePcMdl共同水解三丁酰甘油酯，相对于rePcLipC单独作用，随着酶解时间的延长，三丁酰甘油酯逐渐减少，单丁酰甘油酯和二丁酰甘油酯相对含量增加，但增加较少，说明rePcMdl加速了rePcLipC催化的水解产物的水解。更多还原

【Abstract】 Lipases (EC3.1.1.3) catalyze the hydrolysis of long-chain acylglycerols at the oil–waterinterface, as well as the esterification, transesterification and interesterification in organicsolvents. According to the substrate specificity, they consist of triacylglycerol hydrolases,mono-and diacylglycerol hydrolases, and so on. Triacylglycerol hydrolases catalyze thehydrolysis of triacylglycerol, while mono-and diacylglycerol hydrolases catalyze thehydrolysis of mono-and diacylglycerol but not triacylglycerol. Penicillium cyclopium PG37strain can produce a triacylglycerol lipase (PcLipI) together with a mono-and diacylglycerollipase (PcMdl). In order to widen the application fields of PcLipI and PcMdl, the expressionsof PclipI and Pcmdl in Pichia pastoris GS115were firstly carried out. The recombinantPcLipI (rePcLipI) showed low thermostability. It is urgent to enhance the thermostability ofPcLipI by genetic engineering. The PcLipI and PcMdl showed different substrate specificities,it is necessary to understand these specificities especially in PcMdl substrate specificity. Inthis work, the synegistic reaction of rePcLipC(recombinant engineered thermostable PcLipI)and re (recombinant PcMdl) toward lipid was also investigated. The PG37strain is poor inproductive level, meanwhile the PcLipI has low thermostability.A777-bp cDNA fragment encoding a258-aa mature PcLipI from P. cyclopium PG37was amplified by RT–PCR, and expressed in P. pastoris GS115. One transformant resistant to4.0mg mL-1of G418, numbered as P. pastoris GSL4-7, expressing the highest rePcLipIactivity. When the P. pastoris GSL4-7was cultured under the optimized conditions, initial pH9.0, adding methanol of1.0%, inductive temperature26°C, the expressed rePcLipI activitywas up to407.3U mL-1. The rePcLipI was verified as a glycosylated protein with an apparentmolecular weight of about31.5kDa. The rePcLipI activity was significantly inhibited by Hg2+,Fe3+and Cu2+. The rePcLipI showed the highest activity at pH10.5and25°C, and was stableat a broad pH range of7.0–10.5and at a temperature of30°C or below. The rePcLipI belongsto an alkaline cold-active lipase and shows low thermostability. The specific activity ofrePcLipI was5,300U mg-1.By the methods of homologously modeling, disulfide bridge prediction, and moleculardynamics simulation, cysteine mutants of PcLipI predicted to have better thermostabilitiesthan the wild-type. The mutants were first expressed in Escherichia coli BL21(DE3) and then,for further investigation, in P. pastoris GS115. Based on the analysis of recombinant mutantPcLipIs, reE-PcLipV248C-T251C(expressed in E. coli) and reP-PcLipV248C-T251C(expressed in P.pastoris) both had enhanced thermostabilities with half-lives at35°C about4.5-and12.8-foldlonger than that of the parent PcLipI expressed in E. coli and P. pastoris, respectively. Thetemperature optima of reE-PcLipV248C-T251Cand reP-PcLipV248C-T251Cwere each5°C higherthan those of the parent PcLipI expressed in E. coli and P. pastoris.A full cDNA gene which encodes the mono-and diacylglycerol lipase from P. cyclopiumPG37was cloned, then the sequence was submitted to GenBank under the accession numberof HM135194. Based on the principle of the nest PCR, a cDNA gene, Pcmdl, encoding themature peptide of the mono-and diacylglycerol lipase from P. cyclopium PG37was cloned, then expressed in P. pastoris GS115. One transformant resistant to4.0mg mL-1of G418,numbered as P. pastoris GSM4-2, expressed the highest recombinant PcMdl (rePcMdl)activity. When the P. pastoris GSM4-2was cultured under the optimized conditions, initial pH6.5, adding methanol of1.5%, inductive temperature28°C, the expressed rePcLipI activitywas up to215.2U mL-1. The rePcMdl was also verified as a glycosylated protein with anapparent molecular weight of39.0kDa. The rePcMdl showed the highest activity at pH7.5and35°C, and was stable at pH range of6.5–9.5and the temperature below35°C. TherePcMdl activity was significantly inhibited by Hg2+and Fe3+. The rePcMdl was confirmed tobe strictly specific for1-monobutyrin (189.3U mg-1) and1,2-dibutyrin (344.9U mg-1), butnot tributyrin (0U mg-1).Using the methods of homology modeling, molecular docking, and molecular dynamicssimulation, the substrate specificity of PcMdl were investigated. The α-helix form lid in openconformation uncovers the catalytic center and leads to the catalytic triad Ser145-Asp199-His259exposing to solvent. The residue Ser83, together with the Ser145, constitutes the oxyanion holewhich stabilizes the tetrahedral intermediates. The catalytic pocket in open conformation,organized by residues Tyr21, Phe112, Leu146, Pro174, Val201, Phe256, Val266, and Asp267, exposesto solvent. Stereographic view of PcMdl docked with substrate (tri-or diacylglycerol)analogue indicated that the residue Phe256played an important role in conferring the substrateselectivity. The Phe256projected its side chain towards the substrate binding groove and madethe sn-1moiety difficult to insert in. On the contrary, sn-1moiety hampered the phosphorusatom (taking the place of carboxyl carbon) from getting to the Oγof Ser145and caused thefailure of triacylglycerol hydrolysis.The factors affecting lipid hydrolysis by rePcLipC(reP-PcLipV248C-T251C) and rePcMdlwere determined. The hydrolysis of olive oil and tributyrin was investigated, meanwhile partof the hydrolysis products were analyzed by HPLC. The rePcLipCand rePcMdl have optimalsynergistic initial pH10.5, temperature30°C, and dosage ratio3:1by the active unit.Comparing with the hydrolysis by CALB or rePcLipCalone, rePcMdl could increase thehydrolysis rate by synergy with CALB or rePcLipC. With the addition of rePcMdl, there weremore monobutyrin and dibutyrin released from tributyrin than that by rePcLipCalone. Theresult demonstrates there is synergistic interaction between rePcMdl and rePcLipCwhichaccelerates the hydrolyzing of tributyrin.更多还原