Heterologous Expression And Application Of L-glutamate Oxidase And L-amino Acid Oxidase

L-glutamate oxidase is a flavin protease.It can specifically convert L-glutamic acid or L-glutamate sodium to a-ketoglutarate,ammonia and hydrogen peroxide.The convertion reacts mildly without cofactor and the stereo specificity of the reaction is strong.The hydrogen peroxide electrolysis generated during the reaction can produce weak electron flow which is often used as a biosensor for disease research,food safety testing,fermentation processes,and so on.For nearly half a century,researchers have been focusing on the screening of wild strains,optimizing of induction conditions,and simplifying of purification processes of L-glutamate oxidase.Studies on the heterologous expression and application of L-glutamate oxidase are relatively lagging.L-amino acid oxidase,like L-glutamate oxidase,is also a flavin protease with FAD or FMN binding subunits which could catalyze the oxidative deamination of L-amino acids to produce alpha-keto acids,ammonia and hydrogen peroxide.The catalytic mechanism of the enzyme on the L-glutamic acid is basically the same as that of L-glutamate oxidase,but the studiea of their functions is totally different.L-amino acid oxidase is widely distributed in various organisms,but the studies on it mainly focus on collecting L-amino acid oxidase from snake venom.Most of the articles only report the hemolysis,antibacterial,antiviral,cancer inhibition and apoptosis-inducing funtions of various kinds of L-amino acid oxidase.For the hydrogen peroxide is generated during the oxidation process of L-amino acid oxidase and it has the stereo specificity,it was reported in some articles that Chiral electrodes were made by using ketonic acid,ammonia and hydrogen peroxide generated by L-phenylalanine---the catalysis substrates of L-amino acid oxidase.In view of the many functions of L-amino acid oxidase,over the past decade,an increasing number of researchers have started cloning and heterologous expressing of L-amino acid oxidase from various sources,then exploring the functions of recombinant enzyme.In this study,L-glutamate oxidase and L-amino acid oxidase were used as research objects,and then they were synthesized and cloned into two kinds of enzyme genes which were then efficiently expressed in E.coli and yeast expression systems.The activity characterization analysis was conducted and the reaction conditions of glutamic acid(salt)to alpha ketosglutaric acid were optimized.Besides,these two enzymes were subsequently immobilized and displayed on the yeast cell wall.The enzyme membrane was prepared by immobilization technology,which had been successfully ultilized on biosensor instruments to determine the concentration of glutamic acid in soy sauce.All the contents are described asfollows:1.Cloning,expression and characterization of L-glutamate oxidase gene lgoxIn this study,the L-glutamate oxidase gene(GenBank:EFE71695.1)from Streptomyces ghanaenis ATCC 14672 was synthesized.Firstly,the protein was expressed in E.coli and then characterized.After IPTG induction,most of the recombinant enzyme existed in the supernatant in soluble form.The optimum reaction temperature and pH of the enzyme are 30? and 6,respectively.The enzyme is stable at 30 and 40?.2 mmol/L Mg2+ exhibited a simulating effect and enhanced LGOX activity by 15%.Then the enzyme is expressed in Pichia pastoris expression system.Here,pHBM905BDM vector was used to construct multi-copy plasmids of the gene in vitro which were electrotransformed into Pichia pastoris strains.The results indicated that LGOX protein levels and enzyme activity increased with increasing lgox copy number.The total enzyme activities of LGOX of the multi-copy expression strains P-LGOX2,P-LGOX3 and P-LGOX4 were 1.75,2.41 and 2.75 fold higher than that of the single-copy strain P-LGOX1,respectively.Strain P-LGOX4 was used for subsequent high-density fermentation experiment.Two kinds of fed fermentation technologies,DO-stat and index feeding,were tried respectively.By using DO-stat feeding,the wet weight of the cells and the highest enzyme activities reached 386 g/L and 150.1 U/ml at the end of the fermentation,respectively.By using glycerol and methanol index feeding,the total enzyme activity and unit production efficiency finally reached 247.8 U/mL and 2581 U/L/h at the end of fermentation,respectively.Compared with shake flask experiments,all these have been greatly improved.Then the recombinant enzyme from Pichia pastoris was characterized.The optimal temperature and pH of the enzyme were 40 ? and 6.5,respectively.Compared with the recombinant enzyme from E.coli,the thermal stability was further improved.These are more conducive to the industrial application of glutamate oxidase.By glycosidase Endo H and SDS-PAGE gel electrophoresis identification,it was found that yeast-produced recombinant enzyme was glycosylated,which could explained its better thermal stability.The conversion reaction conditions of glutamate oxidase were optimized,and catalase was added.The concentration of a-ketoglutaric acid reached 106 g/L.The conversion efficiency was 95.5%.2.Cloning,expression and characterization of L-glutamate oxidase gene lgoxIn tins study,the L-glutamate oxidase gene(GenBank:EFE71695.1)from Streptomyces ghanaenis ATCC 14672 was synthesized.Firstly,the protein was expressed in E.coli and then characterized.After EPTG induction,most of the recombinant enzyme exists in the supernatant in soluble form.So the gene was fused with soluble labels(c17,mCherry,sfGFP).SDS-PAGE showed that soluble labels c17,mCherry and sfGFP had good solubility promoting effect on kc-LAAO protein.After removal of the soluble label,the enzyme activity experiment showed that the optimal reaction temperature and pH of the enzyme were 20 and 5.5,respectively.Thermal stability was good at 30 and the activity would drop sharply when exceeding that temperature.Then we expressed the enzyme in Pichia pastoris.Here,we used the pHBM905BDM vector to realize the multi-copy quality of the gene in vitro.The activity of kc-LAAO in the supernatant of P-kc-LAAO2,P-kc-LAA03 and P-kc-LAA04 was 1.89,2.47 and 3.06 times higher than that of P-kc-LAAO1.Four copies of recombinant strains were used to carry out high-density fermentation experiments.DO-stat feeding process was used.At the end of fermentation,the wet weight and enzyme activity of the recombinant strains reached 280 g/L and 120.8 U/mL,respectively,which were greatly improved compared with shaking flasks.Then the activity of recombinant enzyme expressed in Pichia pastoris was characterized.The optimum reaction temperature and pH of the enzyme were 20 ? and 6,respectively.Compared with recombinant enzyme derived from E.coli,the thermal stability of recombinant enzyme was further improved,which was very similar to L-glutamate oxidase.Similarly,the glycosylation of yeast-derived kc-LAAO recombinase was confirmed by Endo H and SDS-PAGE experiments.The conversion conditions of amino acid oxidase were optimized.Catalase was added.The concentration of the product was 103 g/L.The conversion efficiency was 85.8%and the space-time conversion rate was 3.46 g/L/h.3.Surface display and immobilization of L-glutamate oxidase and L-Amino Acid OxidaseIn this study,S.cerevisiae cell wall binding protein SED1 was used as an anchor protein.HA tag was introduced to facilitate the localization and expression of the gene,and L-amino acid oxidase was used as a display protein.Western blot and fluorescence confocal photographs showed that L-amino acid oxidase was successfully displayed on the surface of Pichia pastoris cells.Flow cytometry analysis indicated that 77.95%of L-amino acid oxidase was expressed on the cell walls.The methods and conditions for the immobilization of Escherichia coli and Pichia pastoris cells were studied.By comparing the catalyzed reaction between the immobilized cells containing the enzyme and the whole cells containing the enzyme,it was found that the immobilized cells showed better temperature tolerance and pH tolerance than the other.Frequency studies found that immobilized cells could be used more often than whole cells.The successful catalysis of immobilized cells on substrates laid the foundation for the industrial application of L-glutamate oxidase.4.Application of L-glutamic acid sodium in soy sauce with immobilized cell technologyIn this study,L-glutamate oxidase and L-amino acid oxidase from E.coli were immobilized on a membrane of a specific shape,with which a hydrogen peroxide electrode was used to determine the content of sodium glutamate in various brands of soy sauce.As a result,the enzyme membrane has good stability and high precision,and the whole test process takes less than 1min.It is an efficient,high-precision,convenient and rapid quantitative analysis method.The prepared enzyme membrane is placed at 4? and can be reused for 3 months with good detection performance and accuracy.