The Study In Expression And Application Of Enoate Reductases

In the field of biocatalysis, efficient source of catalytic reaction of enzyme isvery important. enoate reductases is a kind of new enzyme can highly selectivereduction of carbon carbon double bond, its efficient catalytic enzyme source isbecoming a hot spot of research.Saturated compound with electron-withdrawing groups play an important rolein medicine, hormone, spices, and pesticide production.α,β-unsaturatedcompounds with electron-withdrawing groups can make olefin passivationby withdrawing groups, carries on the selective reduction is a challenging task.Research suggested that biological reduction of carbon carbon double bond by enoatereductases with high selectivity, good yield, mild conditions is an environmentalfriendly synthetic way It is very few for the research of highly efficient catalyticenoate reductases, almost no application can be used for industrial production.In thispaper, expression and optimization the enoate reductase gene from bacillus subtilis,the soluble protein after Ni-affinity chromatography were used to the catalyticreaction. The catalytic conditions were also optimized.Due to the catalytic process ofERs require expensive coenzyme NAD(P)H, coenzyme regeneration system wasstudied, and related conditions optimized.At the same time, this is the first study ofimmobilized enoate reductases.ERs gene BS1K from bacillus subtilis successfully expressed in escherichia coliBLP.Induced condition is: the IPTG concentration is0.2mM,25℃,120rpm and12h.Expressed soluble protein was purfied using Ni-affinity chromatography,optimalconcentration of imidazole elution is80mM.Purification of enoate reductase for the catalytic reaction depend on NADH, thesubstrate types include: α,β-unsaturated carbonyl compound, acid, ester, nitro andnitro compounds.The enzyme have good catalytic effect for unsaturated carbonylcompounds.4-Methoxy benzalacetone as model to determine the best catalyticconditions:7.5mg/mL enzyme concentration,11mg/mL of NADH, Na2HPO4-citralas buffer,10mM substrate concentration.In this condition, the reaction conversionrate can reach more than99%.The recombinant of enoate reductase has the reducingpower for α,β-unsaturated nitro and nitro alcohols with reducing power.Among them, 1-bromo-4-(2-nitro ethenyl) benzene in PBS buffer conversion rate higher than theTris-HCl, up to37%.This enzyme have not catalytic ability for open chain unsaturatedacid and ester compounds with an electron-withdrawing groups.enoate reductase BS1K success on the catalytic on former chiral compounds of α，β-unsaturated nitro and nitro alcohols,enoate reductase success on the catalytic,generated by the product, the chiral carbon in the positionof α is racemic form, Cβproduct has good enantioselectivity (81%).This article used the NAD+/GDH coenzyme regeneration system to catalyticreaction, through the comparison, coenzyme regeneration system of catalyticconversion rate is greater than the NADH to participate in, the latter is greater than thebacteria catalytic reaction.In addition, coenzyme regeneration system can catalytic (1-nitroprop-1-en-2-yl) benzene as coenzyme NADH alone cannot catalytic.Using the technology of free carrier immobilized-crosslinked enzymeaggregates(CLEAs) technology for immobilization BS1K.The experiment is dividedinto two parts: single and double enzyme immobilized.Single immobilized enzyme is immobilized experiments of the recombinantenoate reductase.After precipitation and crosslinking conditions optimization, finallyyield the39%activity recovery.Optimization of the immobilized conditions asfollows:3.5M ammonium sulfate as precipitantan slow shocked45min,4%(v/v)glutaraldehyde as crosslinking agent crosslinking3h.The repeated useof immobilized enzyme was investigated the residual activuty of immobilizedenzyme is81.4%after seven times.Optimization of the immobilized conditions on ER-GDH as follows:7Mammonium sulfate as precipitantan slow shocked2h,3%(v/v) glutaraldehyde ascrosslinking agent crosslinking3h. In the end yield the45.7%activity recovery ofBS1K and4.25%of GDH.After seven times using,the residual activuty is76%。The experiment successfully cloned the olefinic bond reductase and applied to aseries of substrate for enzyme catalytic reaction,coupled with the immobilizedexperiments would provide new research ideas for the synthesis of chiral compoundsand industrial large-scale applications.