Study On The Enzymatic Synthesis Of Non-natural Amino Acids In Organic/Water Phases,and Screening,Identification And Degradation Characteristics Of Toluene-Degrading Bacteria

Non-natural amino acids,used as small molecular probes and important drug precursors,have been widely applied in biological research,chiral pesticides,food additives,and chiral drugs.The existing chemical synthesis has many disadvantages,such as complex process,low yield,high cost and serious pollution.Transaminase has wide substrate specificity,high catalytic speed,high enantioselectivity and stereoselectivity and no need for cofactor regeneration etc.These characteristics make the enzyme plays an important role in the synthesis of non-natural amino acids.In this project,we constructed a multi-enzyme reaction system mainly focused on Escherichia coli aromatic aminotransferase(AroAT),and asymmetric synthesis of amino acids in aqueous/organic two-phase system.It aims to eliminate/reduce the obstacles of aminotransferase,optimizing the reaction conditions and process,and improving the biocatalytic efficiency of the enzyme to the maximum.The research is carried out as following:1)Purification and characterization of Escherichia coli aromatic amino acid aminotransferase(AroAT)and coupling enzyme Acidaminococcus fermentans alpha-ketoglutarate dehydrogenase(HGDH).And determination the kinetic constants of aminotransferase towards non natural amino acids in aqueous media.From the results we can see,AroAT was highly capable of producing derivatives of L-phenylalanine,in this case,L-2-fluoro-phenylalanine,L-4-fluoro-phenylalanine and L-4-methoxyphenylalanine.The ortho-substituted derivative of phenylpyruvate:2-fluoro-phenylpyruvate appeared to be a good substrate with a slight loss of activity(25%)compared to the natural substrate:4-hydroxyphenylpyruvate.Moreover,replacement of the hydroxy group of 4-hydroxyphenylpyruvate at the p-position of the phenyl ring by a methoxy group(4-OCH3-pp)decreased the activity by 31%while replacing with a fluoro group reduced the activity by 61%.2)Tolerance of HGDH and AroAT to organic solvents.It was found that adding double concentration of HGDH in enzyme coupling system can compensate for the decrease of activity caused by 10%ethanol.And the overall catalytic efficiency(kcat/Km)of the AroAT towards natural substrate 4-hydroxyphenylpyruvate increased 50%,while towards 4-OCH3-pp decreased 15.4%.3)A multi-enzyme system is constructed to asymmetric synthesis of L-tyrosine in the presence of 10%ethanol,GDH,HGAH with a yield of 85.3%,ee>99%.After the completion of the construction of the above multi-enzyme system and the application of this system in the organic/water two-phase solvent,I conducted the next research work.In order to further study the application of microorganisms in the field of biodegradation and to study the enzymes involved in the synthesis and decomposition of toluene,the toluene-degrading bacterium were screened and identified,and their degradation characteristics were preliminary studied.Toluene plays a crucial role as a synthetic raw material in the chemical and pharmaceutical industries.Meanwhile,toluene is also a common pollutant that can contaminate soil and water and affect people's health.The traditional way to degrade toluene is time-consuming and laborious,and it also causes secondary pollution.Microbial degradation of toluene is not only effective and inexpensive,it has great value in application and research prospects.In this project,several bacterium that can degrade toluene were separated from the sewage outlet of chemical plants.Three strains with high efficiency(L1,L2,and L3)were selected by re-screening of the degradation speed and concentration of toluene.L3 stain was isolated and identified,and the effects of various conditions on the ability of the strain to degrade toluene were studied,and the genes involved in the degradation of toluene were cloned.The expression vector of catechol 2,3-dioxygenase was constructed using pET23a,and its enzymatic properties were preliminary studied after purification.The research is carried out as following:1)Seven strains that have capable of degrading toluene were isolated from the sewage outlet of a chemical plant.Three strains could degrade toluene better than other strains,which numbered L1,L2,and L3,respectively.PCR was performed using the bacterial genome as a template and 16S rDNA universal primers.The PCR product was sequenced and subjected to Blast alignment.The similarity with the genus Bacillus was found to be high.Further morphological identification and physiological and biochemical tests were performed on the strains,suggesting that L1 is Brevibacillus and L3 is Lysinibacillus fusiformis.2)By comparing the speed of degradation of toluene,L3 could completely degrade 433 mg/L toluene within 24 h.However,L1 strain could not degrade 433 mg/L toluene after 48 hours.Therefore,the degradation effect of L3 was better.L3 was chosen as a subject for further study of high-efficiency toluene degrading bacteria.The effect of physical and chemical factors on the ability of the strain to degrade toluene was studied by changing the pH of the culture medium,the culture temperature,the amount of bacterial seed and the initialtoluene concentration.The test found that when the temperature is 30?,pH 8.0 degradation efficiency reach to the best.There is a positive correlation between the inoculum concentration and the rate of degradation,however above 9%the change is not significant due to saturation.L3 can rapidly degrade 433 mg/L toluene,but the degradation rate becomes very slow when the toluene concentration is above 541.25 mg/L.At 30 h,L3 can degrade 93.87%of toluene with a concentration of 541.25 mg/L,and can degrade 80.49%of toluene with a concentration of 649.5 mg/L.3)The genes for the degradation of toluene were cloned.The expression vector of catechol 2,3-dioxygenase was constructed using pET23a.The protein concentration was 8.3 mg/mL after purification by His-tag.The thermo-stability of the purified C230 were found to be in general,and the optical temperature was 30?.The stability of the enzyme under alkaline conditions is higher than that under acidic conditions,and the optical pH is 8.0.Mg2+and Fe2+ had a significant improvement in enzyme activity;Cu2+ reduced enzyme activity;Ca2+ and Fe3+ did not affect enzyme activity.As a metal ion chelator,EDTA has little effect on enzyme activity.The enzyme is stored well at 4?.