| Whole-cell bioconversion,drawed extensive attention recently in the fields of both lab research and industrial manufacture for its merits of low energy consumption,convenient operation and highly specifity.While Bacillus subtilis appears to be a more appropriate whole-cell biotransformation tool in favour of demanding food and medicine industries for its noticeable character of safety compared with Escherichia coli.Nevertheless,deficiencies of B.subtilis in biotransformation process as following rises cost of industrialization: the low expression of enzymes;the degradation of substrate or product caused by side reaction;the transformation efficiency influenced by membrane permeability rate of the substrate and so on.In order to solve these problems,we chose LGOX as the reporter protein to study B.subtilis whole-cell biotransformation combined with endogenous glutamate metabolic pathways and transport system through the screening of exogenous enzyme.The main contents and results were as follows: 1.Analysis of catalytic properties and functional verification of LGOX from different strainsL-glutamic acid oxidase?LGOXstr?from Streptomyces sp.X-119-6 and L-glutamic acid oxidase?LGOXkit?from Kitasatospora setae KM-6054 were successfully expressed heterologous in E.coli BL21.The LGOXstr and LGOXkit was and purified by HisTrapTMFF affinity chromatography column and with the recombinant LGOX activity was 101.51 U×mg-1 and 45.98 U×mg-1,respectively.The enzymatic properties of LGOXstr and LGOXkit were shown that the optimal reaction temperature were 50? and 40?.And the optimal reaction pH were 6.5 and 6.0,respectively.In our research,LGOX activity increasd with Mn2+ ion addition.Last,the two LGOXs substrate specificity showed both strong substrate specificity for L-Glu but weak effect on glutamine or aspartic acid by LGOXstr and on histidine or glutamine by LGOXkit.2.Induced expression of recombinant B.subtilis preliminary verfication of whole-cell transformationThe LGOX gene of Streptomyces sp.X-119-6 and Kitasatospora setae KM-6054 were cloned to construct the expression plasmids pHYBSLGOXstr and p HYBSLGOXkit.The plasmids were transformed into B.subtilis WB600,two recombinant LGOX expression strains WB602 and WB603 were constructed respectively.The LGOX expression under control of xylose isomerase promoter,with the highest intracellular enzyme production level from WB602.Then the highest enzyme activity was 0.57 U×mL-1 under the optimal induction conditions: after 8 h of fermentation?the logarithmic growth stage?was added with 12 g×L-1 xylose for 12 h.40 g×L-1 glutamic acid could be catalyzed into 25.8 g×L-1 ?-KG by 20 g×L-1 whole cell.3.Effects of endogenous glutamate metabolic pathway transformation on B.subtilis on whole-cell transformation systemMutant strain WB605 with glnA gene inactived was constructed,and recombinant plasmid pHYBSLGOXstr were transformed into WB605 to obtain recombinant strains WB606.Mutant strains WB609 and WB610 retained?has plasmid pHYBSLGOXstr?with amyE gene inactived was constructed also.Whole-cell biocatalysis 10 g×L-1 glutamate shown that the conversion of recombinant strain WB606 increased from 87.9% of the original strain WB602 to 95.4%,and the conversion of WB610 was 91.2%.The ?-KG production of the mutant strains WB605 and WB609,which achieved the integration of the LGOXstr gene,increased from 0.08 g×L-1 to 0.32 g×L-1.It could contribute to improve the whole-cell transformation rate by changed endogenous glutamate metabolic pathway with achieving knockout mutations of glnA gene.4.Effects of substrate transport system on whole cell transformation systemCo-expression vector pHYBSLGOCstrGltP of LGOX and glutamte transporter?GltP?was constructed,and was transformed into B.subtilis WB600 to obtain recombinant strain WB611.Verified by L-glutamic acid assay medium,the ratio of L-glutamic acid was increased from 0.19 mmol×?h×g DCW?-1 of WB602 to 0.24 mmol×?h×g DCW?-1 expressing the transgenic recombinant strain,and increased by 26.3%.The recombinant strain WB602,which was not expressed GltP,and WB611 with overexpressing GltP,was subjected to whole cell catalysis 40 g×L-1 L-glutamic acid,respectively.Results showedn that the average conversion rate increased from 1.55 g×L-1×h-1 to 1.89 g×L-1h-1,increased by 21.9%,when reaction reaches equilibrium.It is indicated that overexpression of glutamate transporters can promote whole cell catalytic rates.In this paper,we first screened LGOX with excellent enzymatic properties,followed study genetic transformation by optimization of allogeneic gene expression,cell endogenous substrate pathway and cell substrate transport system.The conversion rate of ?-KG was increased by 8.5% compared with that before transformation,and the average transformation rate was improved 21.9% when the biotransformation was balanced.This study provides experimental basis for the construction and improvement of whole cell transformation system involved in intracellular endogenous metabolism. |
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