| ?-ketoisovalerate?KIV?is vital in the field of biosynthesis,which has been widely used in the areas such as food,feed,pharmaceutical and chemical synthesis.At present,KIV is mainly synthesized by chemical method,microbial fermentation method and enzyme catalytic conversion method.The chemical method requires toxic materials as initial precursors,harsh reaction conditions and complex-step chemical reaction.The microbial fermentation method has many disadvantages such as long fermentation cycle and low yield.Compared with them, the enzyme catalytic conversion method has the advantages such as high substrate conversion,high yield,easy separation of products and little pollution to environment.In this study,E.coliBL21?DE3?was used as host strain to express L-amino acid deaminase?PmLAAD?from Proteus mirabilis,resulting in the recombinant strain E.coli BL21?pET-28a-PmLAAD?,and the recombinant strain was employed to synthesis KIV from the substrate L-Valine?L-Val?.The efficiency of L-amino acid deaminase synthesis of KIV was further improved with protein engineering,optimization of fermentation and whole-cell transformation conditions,and optimization of transformation conditions on 5 L fermentor.The major results of this paper are listed below:?1?The recombinant strain E.coli BL21?pET-28a-PmLAAD?,a strain containing the L-amino acid deaminase gene from P.mirabilis was successfully constructed.The recombinant strain was employed to produce KIV from L-Val.It was found that the conversion is lower than95%,when using over 20 g·L-11 L-Val as substrate.Effects of L-Val concentration,whole-cell catalyst concentration and initial product addition on PmLAAD whole-cell transformation were investigated.The results showed that the substrate had little influence on the transformation,and the increasing catalyst concentration did not increase the KIV yield.However,the whole cell transformation reaction decreased with the increasement of the initial product addition,which indicates that the product inhibition was the rate-limiting step of whole cell transformation.?2?L-amino acid deaminase was engineered to relieve product inhibition for improving transformation efficiency of substrate.The protein structure of PmLAAD was obtained by homologous modeling using the online website SWISS MODEL.Based on the analysis of the structure,mutation candidates,which are located on the Loop regions around the product-binding sites were selected.The candidate residues were saturated and mutated.We obtained four mutants?S98A?T105A?S106A and L341A?with higher yield than wild type using high-throughput screening.The highest yield of single mutant?S98A?is 46.9 g·L-1.Combinatorial mutant was constructed and mutant Pm LAADM4?S98A/T105A/S106A/L341A?was achieved with the yield of 66.7 g·L-1.The mutant PmLAADM4 achieved a 6.2-fold higher catalytic efficiency and an almost 6.7-fold reduction in product inhibition than the wild-type enzyme.Effects of 70 g·L-11 L-Val and 40 g·L-11 initial product addition on whole-cell transformation were investigated,and the results showed the mutants had higher yield,conversion,activity and production rate of KIV than wild type.Docking experiments suggested that S98A weakened the interactions between the product and enzyme,and other mutations?T105A,S106A and L341A?enhanced the product release,thereby relieving LAAD product inhibition.?3?The enzyme production conditions and the whole-cell transformation conditions of the best mutant were optimized.The optimum enzyme production conditions of L-amino acid deaminase were determined as follows:the optimum expression plasmid was pACYCDuet-1,the optimum culture medium was TB,the optimum seed solution OD600 was 0.8,optimal induction time was 12 h,the optimum inducer IPTG concentration was 0.4 mmol·L-1,and the optimum induction temperature was 25 oC.The optimum whole-cell transformation conditions of L-amino acid deaminase were determined as follows:temperature 30 oC,pH 8.5.The maximum yield of KIV prepared by mutant PmLAADM4 was 86.5 g·L-1.We further optimized the whole-cell transformation conditions of PmLAADM4 on 5 L fermentor.The optimum whole-cell catalysis concentration was 10 g·L-1,the optimal agitation speed during the whole cell transformation was 600 rpm,the optimal aeration was 1.5 vvm and the optimum substrate loading was 100 g·L-1,After 18 h,the concentration of KIV reached 98.5 g·L-1,and the conversion rate reached 99%. |
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