Semi-rational Design And Immobilization Of Lactobacillus Plantarum Glutamate Decarboxylase For Synthesis Of γ-aminobutyric Acid

γ-Aminobutyric acid（GABA）,a non-protein amino acid widely present in plants,microorganisms and mammals,has versatile physiological functions including anti-depressant,improving sleep,regulating brain function,lowering blood pressure,protecting liver and kidney.Compared with chemical synthesis methods,biocatalytical approaches for the GABA synthesis have the advantages of mild reaction conditions,green reaction solution,high catalytic efficiency,and sustainability,thus attracting increasing attention.However,at present,microbial cell biocatalysts have the problems of complex culture conditions,low yields of key enzymes,and complex intracellular enzyme systems.On the other hand,enzyme biocatalysts also have the problems of unsatisfactory activity,poor stability and difficulty in recycling,which restrict the application of biocatalysts in the research and practical production.In this study,Lactobacillus plantarum CACC 558 glutamate decarboxylase（LpGAD）was mined by genetic engineering technology.Then,semi-rational design of LpGAD was performed to enhance its catalytic activity.After that,cross-linked enzyme aggregates（CLEAs）of LpGAD mutant was prepared based on self-assembly system.Finally,a reaction system containing deep eutectic solvents（DESs）was established for efficient synthesis of GABA.The main results of this dissertation are listed as following:1.On basis of genetic engineering technology,a glutamate decarboxylase LpGAD,which can catalyze the decarboxylation of L-sodium glutamate to GABA,was excavated from L.plantarum cells,cloned and expressed in Escherichia coli BL21（DE3）.LpGAD is an intracellular enzyme encoded by 467 amino acids with a theoretical molecular weight of 53.4 k Da and a protein isoelectric point of 5.62.The study of enzymatic properties showed that the optimum pH of LpGAD was pH 5.0,and the enzyme activity was relatively high at 37-40°C.Recombinant LpGAD exhibited good stability in the range of pH 4.0-5.5 and temperature range of 20-35°C.Metal ions Fe²⁺and Ca²⁺could improve the activity of LpGAD,while Cu²⁺could reduce the activity of LpGAD,and the enzyme was not a metalloenzyme.The results of kinetic parameter studies showed that the values of K_m,k_cat,kcat/K_m and specific activity of LpGAD catalyzing the decarboxylation of L-sodium glutamate at pH 5.0and temperature 37°C were 21.7 m M,14.6 s^-1,0.7 s^-1m M^-1and 8.2 U/mg,respectively.2.Based on protein engineering technology,the interaction between LpGAD and the substrate was analyzed through protein homology modeling and molecular docking,and a mutant LpGAD R421F/Q164V with higher activity than the wild-type enzyme was obtained by semi-rational design including alanine scanning,single-point saturation mutation and combinatorial mutation.The catalytic activity of LpGAD R421F/Q164V mutant for biosynthesis of GABA was 7.25 times higher than that of LpGAD.The results of enzymatic properties showed that the optimal pH and temperature of LpGAD R421F/Q164V for decarboxylation of L-sodium glutamate were 5.0 and 40℃,respectively.The mutant had good stability at pH of 4.0-5.5 and temperature of 20-30°C.The results of kinetic parameter studies showed that the values of K_m,k_cat,k_cat/K_m and specific activity of LpGAD R421F/Q164V catalyzing the decarboxylation of L-sodium glutamate at pH 5.0 and temperature 37°C were13.3 m M,70.0 s^-1,5.3 s^-1m M^-1 and 61.5 U/mg,respectively.3.On basis of recombinant LpGAD mutant（mLpGAD）,elastin-like polypeptide（ELP）and self-assembly system Spy Tag/Spy Catcher,immobilized mLpGAD was prepared by self-assembly and chemical cross-linking reaction.The fusion enzyme mLpGAD-Spy Tag（mLpGAD-ST）containing a self-assembly tool and protein support Spy Catcher-ELP-Spy Catcher（SCELPSC）were prepared through genetic engineering technology.Under optimal conditions,the fusion enzyme mLpGAD-ST was mixed with the protein scaffold SCELPSC to fabricate the protein complex by self-assembly,and then glutaraldehyde was added for chemical cross-linking to obtain the enzyme cross-linked aggregate（CLEAs）.The CLEAs prepared under the set conditions had the highest activity with a protein recovery of 88%.The optimum reaction pH and temperature of CLEAs were 5.5 and 45℃,respectively,and the immobilized enzyme showed good pH and temperature stability at pH of 5.0-7.0 and temperature of35-50℃.Compared with mLpGAD-ST,CLEAs exhibited better pH and temperature stability.In addition,CLEAs had better storage stability than free enzymes,and the activity of CLEAs still retained about 74%of the activity after 10 days of storage.The kinetic parameters of the immobilized enzyme catalyzing the decarboxylation of L-sodium glutamate at pH 5.5 and temperature 35°C are K_m value 20.4 m M,k_catvalue 43.6 s^-1,k_cat/K_m value 2.1 s^-1m M^-1 and specific activity 19.7 U/mg,respectively.4.Immobilized enzyme CLEAs was used as catalysts,effects of deep eutectic solvents（DESs）on the catalytic performance of immobilized enzymes were studied,and the key factors of the catalytic reaction were optimized,and a reaction system containing DESs for the efficient synthesis of GABA catalyzed by CLEAs was constructed.The results showed that addition of Choline chloride/Urea（ChCl/U）could enhance the catalytic performance of the immobilized enzyme CLEAs.Under the reaction conditions:the addition amount of ChCl/U 8%,the pH value of citric acid-sodium citrate buffer 5.5,reaction temperature 30°C,substrate levels 400 m M,the immobilized enzyme CLEAs catalyzed the decarboxylation of L-sodium glutamate with the 98.5%of GABA in yield.Finally,a reaction system containing ChCl/U for the efficient synthesis of GABA catalyzed by the immobilized enzyme CLEAs was established by a fed-batch strategy and pH regulation,with about 1107m M of the cumulative concentration and 92%of yield of the product by supplementing twice L-sodium glutamate.