Molecular Modification Of L-amino Acid Deaminase And Biosynthesis Of ?-ketoisocaproate

As a precursor of L-leucine,?-ketoisocaproate is widely used in food,feed and pharmaceutical industries.However,the most common method to produce?-ketoisocaproate is chemical synthesis,which requires expensive catalysts or special starting substances and subsequently results in the high price of?-ketoisocaproate.Microbial bioconversion is an alternative process by converting L-leucine to?-ketoisocaproate with the L-amino acid deaminase?L-AAD?.But few studies reported the L-AAD property,location on the membrane and important residues before.In this study,we achieved the whole-cell bioconversion to produce?-ketoisocaproate by expressing L-AAD from Proteus vulgaris in Escherichia coli BL21?DE3?.In addition,the catalyst property and location of L-AAD were also analyzed.To improve?-ketoisocaproate titer,we optimized fermentation conditions and expressional regulatory elements,and increased thermal stability of L-AAD by the semi-rational design.Major results were outlined as follows:?1?L-AAD from P.vulgaris was expressed in E.coli BL21?DE3?and the purification conditions?detergent,ammonium sulfate,stabilizer?were optimized.The detergent-protein complex was achieved with the specific activity of 1.36?mol·min^-1·mg^-1.The optimum pH is8.5,and the optimal temperature is 35°C.Most ions could inhibit the catalyst activity,and the catalyst activity decreased by 60%with the presence of Zn²⁺,Cu²⁺,Al³⁺and Cu²⁺.And no catalyst activity could be detected when 2000?mol·L^-1 H₂O₂ existed.The purified L-AAD could generate H₂O₂ with O₂ as the typical deamination without membrane.But no hydrogen peroxide was detected when cell membrane was in the reaction solution.The 3-D structure of L-AAD was modeled by Modeller 9.9 and the catalyst center was identified with Autodock.Eight residues?Y97,Q99,Q278,Q280,R315,M411,G437,W438?were identified important in the catalyst center.?2?The transcriptional levels of important transport genes were analyzed,which indicated that the L-AAD transport pathway was the twin-arginine translocation system.The L-AAD was identified to be located on the cell membrane by the combined efforts of bioinformation,Western blot and catalyst product.The surfactants and organic solvents were used to change the membrane property.We found that the biocatalyst activity increased by 37.03%when the whole cell was pretreated by 1%toluene.The further analysis suggested that the changes on membrane property could affect L-AAD catalyst activity by altering the membrane permeability and proton potential.?3?The whole-cell bioconversion conditions were optimized to produce?-ketoisocaproate.To reduce costs,lactose was used to induce the protein expression.The optimal whole-cell bioconversion condition was as:0.8 g·L^-1 cells,20?mol?L^-1 transporter inhibition CCCP,100mmol?L^-1 L-leucine,pH 7.5,35°C for 16 h.Afterwards the batch and feeding conditions were further optimized.In the batch conversion,under the optimal substrate concentration of 52.47g·L^-1,the?-ketoisocaproate titer reached 50.02 g·L^-1 with the bioconversion rate of 95.97%.Under the feeding condition,the?-ketoisocaproate yield reached 69.06 g·L^-1?bioconversion rate 50.31%?at 6.55 g·L^-1·h^-1 fed rate.Furthermore,the recycle ability of immobilized cells increased by 19.24%compared to free cells in repeated-batch conversion experiments.In addition,the immobilized cells had greater pH and thermal stability,and the biocatalyst activity of immobilized cells was 2.2-fold higher than that of free cells under the high temperature condition?45°C?.?4?In order to improve the L-AAD expression level,the mRNA structure of coding or non-coding region were designed to study the translation initiation rate based on thermodynamic equations.Additionally,the transcriptional level was regulated by different plasmid copy number.The biocatalyst activity increased by 24.89%through the N-terminal codon replacement strategy(the minimal fold energy changed from-4.3 kcal·mol^-1 to-3.4kcal·mol^-1).The maximum production titer reached 80.29 g·L^-1.The bioconversion rate was87.42%which increased by 16.19%compared with the control strain.Through the sequence optimization of the ribosome bind sites,the production titer of mutant BL21/RBS6 achieved81.41 g·L^-1 with the bioconversion rate of 88.66%.The maximum biocatalyst activity was achieved by L-AAD expressed on the plasmid pACYCDuet-1?p15A origin?through the optimization of plasmid copy number.The optimized titer of?-ketoisocaproate reached 76.47g·L^-1.Combined the optimal transcription condition and the translation condition,the production titer further increased to 86.55 g·L^-1?bioconversion rate of 94.25%?when the optimal RBS sequence was expressed on the plasmid pACYCDuet-1.?5?Based on the molecular dynamic simulation and protein folding free energy calculation,seven residuals with the high flexibility were subjected to site-saturation mutagenesis libraray.Two residuals D340 and L363 were screened as key points to affect the enzyme thermal stability via high-throughput screening.The half-life increase by 42%and 12%for mutants D340N and L363N at 37°C,respectively.However,D340N and L363N could not increase the stability of loop and the overall enzyme based on the molecular dynamics simulation without the membrane.We observed that these two residuals were in the insertion sequence of the membrane binding region and related to the interaction with the cell membrane.It was suggested that D340N and L363N increased the enzyme stability by enhancing the interaction relationship between protein and membrane by the analysis of amino acid types and charges.