A Study Of Cloning,Expression And Synthetic Application Of Monoamine Oxidase From Pseudomonas Monteilii ZMU-T01

Objective:To clone and express a monoamine oxidase（MAO）from Pseudomonas monteilii ZMU-T01.To synthesize chiral 2-methyl-1,2,3,4-tetrahydroquinolines via asymmetric oxidative resolution reaction with high enatioselectivity using prepared MAO.Methods:The MAO gene sequences were obtained by searching the complete genome sequence of Pseudomonas monteilii SB3101 in NCBI databases.The MAO genes were amplified from Pseudomonas monteilii ZMU-T01 genome DNA by PCR and ligated to vector pET32a（+）by double restriction enzyme reaction,resulting in the recombinant expression plasmids pET32a（+）-MAO.The recombinant expression plasmids were then transformed into E.coli BL21（DE3）cells and MAO proteins expressed by IPTG induction.The catalytic activity of the engineered strains were evaluated by using 2-methyl-tetrahydroquinine as substrate and the strain with the highest activity was selected to explore the substrate scope using a series of 2-methyl-tetrahydroquinine derivatives.Results:（1）Five MAO genes were obtained from the Pseudomonas monteilii SB3101complete genome DNA.The obtained MAO genes were amplified from Pseudomonas monteilii ZMU-T01 genome DNA and sequenced.（2）Five genetically engineered MAO strains were constructed with pET32a（+）/E.coli BL21（DE3）as the enzyme expression system,resulting in four engineered strains having correctly expressed the expected enzyme.Activity assay was carried out using the engineered recombinant MAO strains and the results revealed that only the recombinant MAO5 showed activity to catalyze the substrate 2-methyl-tetrahydroquinine.Sequence alignment revealed that the gene of the MAO5 shared only 14.7%identity as compared to the well-studied MAO-N from Aspergillus niger.（3）To improve the catalytic efficiency,several expression systems were tested to heterologously express MAO5:1)pET32a（+）、pET22b（+）and pTrc99a as expression vector with the host of E.coli BL21（DE3）;2)pPICZ-A and pPICZα-A as expression vector with the host of Pseudomonas pastoris X-33.In addition,the chaperone plasmid pG-KJE8 was used to co-express MAO5 in E.coli BL21（DE3）.Activity assay found that the strain,which expressed the MAO5 using the vector pET28b（+）in E.coli along with the chaperone plasmid pG-KJE8 as the co-expression system,showed relatively high activity as compared with other engineered strains.（4）The effect of pH and temperature on activity of MAO5 was also investigated.The results indicated that the highest activity of MAO5 was at pH 7.5 in Tris-H₂SO₄ buffer,37?C.The substrate selectivity of MAO5 tested using the 10 2-methyl-tetrahydroquinolines derivatives bearing various substituents on phenyl ring indicated that the MAO5 displayed excellent enantioselectivty towards the（R）-2-methyl-tetrahydroquinolines with an ee value of higher than 99%and an E value of greater than 200.（5）To understand the resolution process of the 2-methyl-tetrahydroquinoline derivatives,kinetic resolution and dynamic kinetic resolution experiments were performed using the（rac）-2-methyl-tetrahydroquinoline（4 mM）as substrate and the cell pellet of the MAO5strain as biocatalyst.An analytical yield of 49%and ee value of higher than 99%were obtained for（R）-2-methyl-tetrahydroquinoline during the kinetic resolution process,while a result of NH₃?BH₃ improving the yield of（R）-2-methyl-tetrahydroquinoline to 83%and ee to 98%was obtained during the dynamic kinetic resolution of（rac）-2-methyl-tetrahydroquinoline.Conclusion:（1）The monoamine oxidase（MAO5）from Pseudomonas monteilii ZMU-T01 was heterologously expressed in E.coli BL21（DE3）for the first time.The expression conditions and expression system of MAO5 were optimized.（2）A new monoamine oxidase,the MAO5,was found in the Pseudomonas monteilii ZMU-T01 strain.（3）The genetically engineered MAO5 strain was used as biocatalyst for the deracemization of the phenyl-substuted racemic 2-methyl-tetrahydroquinoline derivatives to yield unreacted the（R）-enantiomer with 97->99%ee.（4）The enzyme MAO5,being a new type of monoamine oxidase from the Pseudomonas monteilii ZMU-T01 strain,not only enriches the enzyme family of the monoamine oxidases,but also provides a green and efficient approach for synthesis of optically pure 2-methyl-1,2,3,4-tetrahydroquinoline derivatives.