【作者】 李明伟；

【导师】 范立强；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 γ-氨基丁酸是广泛存在于自然界中的一种非蛋白质氨基酸,它既是中枢神经系统中参与多种生理反应的重要的抑制性神经递质,也是合成生物基材料尼龙-4的前体原料,广泛用于食品、医药、饲料与化工领域。谷氨酸脱羧酶(Glutamate decarboxylase,GAD)能够专一性地催化L-谷氨酸生成γ-氨基丁酸,是生物催化法制备γ-氨基丁酸的唯一关键酶。但该酶存在两大缺陷:一是最适反应pH低(3.6～5.4之间),pH 6.0以上酶极易失活;二是热稳定性差,酶易热失活,大大限制了 GAD的工业应用。本论文一方面通过定点饱和突变和高通量筛选法获得了大肠杆菌来源谷氨酸脱羧酶的pH稳定范围与热稳定性提高的菌株,比较了野生酶与突变酶的酶学性质;另一方面,构建了基于冰核蛋白INP的谷氨酸脱羧酶表面展示重组菌,比较了胞内表达与胞外展示重组菌的表观酶活力和在全细胞催化制备γ-氨基丁酸的可行性。具体内容如下:首先,构建了大肠杆菌来源谷氨酸脱羧酶B(GadB)的野生型重组菌、C末端缺失突变重组菌与N端定点饱和突变重组菌,并在大肠杆菌中成功表达。其次,比较了野生型GadB、C端缺失突变GadB与热稳定突变GadB的酶学性质,野生型GadB、C端缺失突变GadB(GadBΔHT)和热稳定突变体M3、M4、M5的最适反应温度分别为30℃、32℃、37℃和35℃;最适反应pH除GadBΔHT为4.8外,其它均为3.8;热稳定突变体M3、M4、M5在40、50、60℃的热稳定性均较野生型GadB显著提高;突变体M4的T1550、Tm、半衰期分别为54.5℃、51.6℃、160.05 min,较野生型 GadB(40.2℃、40.8℃、24.24min)分别提高了 14.5℃、10.8℃和 5.62 倍;同源建模分析显示了热稳定突变体M3、M4、M5热稳定性提高的主要原因是GadB六聚体的分子链内与链间氢键增加。最后,成功构建了野生型GadB及其突变株的细胞表面展示重组菌。重组菌全细胞催化比较显示:表面展示菌的表观酶活明显高于胞内表达重组菌;细胞添加量为10 g/L转化制备γ-氨基丁酸,在前6 h内,野生型GadB的最大产物生成量和摩尔转化率分别为254.80 g/L和82.36%,而表面展示菌的为270.80 g/L和87.4%,分别提高16 g/L和5%左右;反应至12h时,表面展示菌催化生成的γ-氨基丁酸较胞内表达菌多达27g/L;24h时,展示表达菌的摩尔转化率近98%,达303.98 g/L,比胞内表达重组菌高出24.13 g/L。表面展示热稳定突变株M4的重组菌转化能力最强,在12 h内就能完成3 M L-谷氨酸的完全转化,较表面展示菌催化能力快2 h。更多还原

【Abstract】 γ-aminobutyric acid,a non-protein amino acid widely exits in nature,is an important inhibitory neurotransmitter involved in a variety of physiological responses in the central nervous system.It is also a raw material for synthesizing the precursor of nylon 4,and widely used in food,medicine,feed and chemical fields.Glutamate decarboxylase(GAD)can specifically catalyze the production of γ-aminobutyric acid from L-glutamic acid,which is the only key enzyme for the preparation of y-aminobutyric acid by biocatalysis.However,the enzyme has two main defects:First,the optimum reaction pH is low(3.6～5.4),and the enzyme easily inactivated at pH above 6.0;Second,the enzyme has the poor thermal stability,and is easy to lose its activity.These drawbacks greatly limit the industrial applications of GAD.In this study,recombinant glutamate decarboxylase mutants from Escherichia coli strains with high pH stability and thermal stability were obtained by site-directed saturation mutation and high through-put screening.The properties of wild-type GAD and its mutants were compared.In addition,recombinant GAD surface display expression Escherichia coli on the help of the ice nucleoprotein(INP)was constructed.The whole cell apparent activities and their GABA converting abilities of intracellular GAD expression and cell surface display GAD expression recombinant bacteria were studied.The details are as follows:Firstly,the recombinant strain with wild type glutamate decarboxylase B(GadB)from E.coli,recombinant strain with GadB the C-terminal deletion mutant and recombinant strain with N-terminal site-directed mutant were constructed.Recombinant GadB and its mutants were successfully expressed in Escherichia coli.Secondly,the enzyme characters of wild-type GadB,C-terminal deletion mutant GadB(GadBΔHT)and thermostable mutant GadB(M3,M4,M5)were compared.The optimal reaction temperature of wild type GadB,M3,M4,M5 is 30,32,37,35℃,respectively.Except for GadBΔHT which has the optimum reaction pH of 4.8,the optimum reaction pH of wild-type GadB,M3,M4 and M5 were all 3.8.The mutants(M3,M4 and M5)showed higher thermostability than wild-type GadB at 40℃,50℃ and 60℃ with M4 the best.The T1550,Tm value and half-life of M4 were 54.5℃,51.6℃ and 160.05 min,respectively,which was 14.3℃,10.8℃ and 5.62 times higher than that of wild type GadB(40.2℃,40.8 ℃,24.24 min),respectively.Homology modeling analysis showed that the increased hydrogen bonds both inside a subunit chain and between subunit chains of GadB hexamers may be main cause of increased thermal stability of M3,M4 and M5.Finally,the recombinant bacteria with wild-type GadB or its mutants surface display expression were successfully constructed.The apparent activity of recombinant bacteria with GAD surface display expression was significantly higher than that of GadB intracellularly expression ones.In a 100 mL GABA preparation experiment with 10 g/L recombinant cells addition,the GABA yield and L-Glu molar conversion rate of wild type GadB intracellular expression bacteria were 254.8 g/L and 82.36%,respectively,at 6 h,while those of wild type GadB cell surface display expression ones were 270.8 g/L and 87.4%,respectively,which were 16 g/L and 5%increase,respectively.At 12 h,the yield of GABA produced by surface display expressed GadB was 27 g/L more than the intracellular expression one.At 24 h,the molar conversion rate was nearly 98%,and the GABA yield was 303.98 g/L.Bacteria with cell surface display M4 expression showed higher conversion speed,which can completely convert 3 M L-Glu within 12 h.更多还原