Study On The Production Of Isoprene By Immobilized Enzyme

Isoprene is an important part of rubber production,which can be synthesized by microbial fermentation by methoxyvaleric acid(MVA)pathway.Because of its green and sustainable advantages,the production of isoprene has received more and more attention.However,due to the cumulative pollution of toxic intermediates and the lack of nutritional resources,the production and cell growth of isoprene have been seriously affected,resulting in low production of isoprene in microbial fermentation.However,the use of outfield to enhance microbial fermentation to increase the yield of isoprene has become an ideal solution to overcome the problems of toxic intermediate accumulation and low target product yield in current fermentation methods.Microbial fermentation is generally through the introduction of exogenous mevalonate(MVA)pathway and isoprene synthase gene to achieve intracellular reaction to synthesize isoprene.Four main enzymes,including mevalonate kinase(MVK),mevalonate phosphate kinase(PMK),mevalonate diphosphate decarboxylase(MVD)and isopentenyl diphosphate heteroenzyme(IDI),are the basis of the downstream metabolic process of MVA,while isoprene synthetase(ISPS)plays a very important role in the synthesis of isoprene.By using 3-aminopropyltriethoxysilane(APTES),we successfully modified SBA-15 and added amino functional groups to its surface to form NSBA-15 as a carrier.In addition,we also used the adsorption-cross-linking method to effectively combine MVK,PMK,MVD,IDI and ISPS to obtain the PEGDGE-NSBA-15@MVK-PMK-MVD-IDI-ISPS five-enzyme system.The immobilization conditions were further optimized as follows: carrier amount 0.1g,immobilization temperature 40 ℃ and cross-linking time 2h(during immobilization,the concentration ratio of enzyme was 1: 1: 1: 2: 16).On this basis,isoprene was produced by immobilized enzyme.At the same time,the optimum reaction conditions were optimized,the optimum catalytic p H of immobilized enzyme was 6.5and the optimum catalytic temperature was 50 ℃.Under the optimal catalytic conditions,after immobilized enzyme treatment,the production effect of isoprene was considerable within 48 hours,reaching 451mg/L,while the formation effect of free enzyme was only 191mg/L,and the production effect of isoprene was improved obviously.In addition,the storage stability and reusability of the immobilized enzyme were also excellent.After 30 days of storage,the remaining enzyme activity was85.82%.After participating in five reactions,the enzyme activity could be maintained at 94.14%.In addition,the effect of NSBA-15 immobilized enzyme on isoprene synthesized by MVA metabolic fermentation bacteria(PT-P)was also studied.The strain PT-P was constructed by genetic engineering technology,and its fermentation temperature,initial p H,initial glucose concentration and the amount of immobilized enzyme were optimized.The optimum fermentation conditions were as follows: fermentation temperature 40 ℃,initial p H6.5,initial glucose concentration 5g/L and 0.5 g immobilized enzyme.Under the optimum fermentation conditions,the immobilized enzyme enhanced PT-P fermentation for 48 h.Compared with PT-P fermentation alone,the conversion rate of MVA was as high as 50.86%,and the yield of isoprene increased by about 30%,reaching 234.47mg/L.And the immobilized enzyme showed strong reuse characteristics in the fermentation process,and the enzyme activity remained93.85% after participating in the fermentation reaction for 5 times.