Isoprene Production By Immobilized Enzyme Via MVA Pathway In Vitro

Isoprene is an important source for the industrial production of synthetic rubber,and the synthesis of isoprene by microbial fermentation,via the mevalonate(MVA)pathway,is a green and sustainable approach that is receiving increasing attention and research.Microbial cell-based strategies for isoprene synthesis face a major problem: the production of toxic intermediates or nutrient limitations that may affect the yield of target products and cell growth,and complex and uncontrollable intracellular reactions that limit its development for isoprene synthesis.Outfield enhancement of fermentation for isoprene production to break the bottleneck problems such as low yield that currently exist with fermentation alone is the key to facilitate the production of isoprene by fermentation.In this thesis,the constructed recombinant Escherichia coli containing five proteins of MVA downstream pathway was cultured,and the free form of pure enzyme was obtained through the steps of induced expression,crushing collection,purification and ultrafiltration.the five key enzymes downstream of MVA were immobilized by two immobilization methods,adsorption and adsorption-crosslinking coupling,using mesoporous molecular sieve SBA-15 as the carrier,and the highly stable and active immobilized enzymes SBA-15-R and PEGDGE-SBA-15-R were produced through the regulation of the five enzyme ratios and the investigation of the immobilization methods.The effects of p H,temperature,crosslinker selection,crosslinker concentration,crosslinking time and other conditions were investigated to obtain the optimal production process conditions.Under the optimal production process conditions,the yield of isoprene synthesis by immobilized enzymes was above 200 mg/L,and the yield of PEGDGE-SBA-15-V was 453 mg/L,which was higher than that of free enzyme at 191mg/L.In addition,the immobilized enzymes also had excellent storage stability and reusability,and both SBA-15-R and PEGDGE-SBA-15-R were used for the synthesis of isoprene at room temperature.SBA-15-R retained more than 50% and 80% of their activities after 30 days of storage at room temperature,and more than 37% and 83% of their activities after five consecutive operational uses,respectively.This experiment realized the efficient in vitro cascade reaction and recycling of these five enzymes by the adsorption-crosslinking coupled immobilized enzyme method,and provided a technical reserve for the construction of an immobilized enzyme-fermentation synergistic reaction system to solve the problem of low yield in the production of isoprene by biofermentation alone.