The Study Of Immobilized Carboxylation Enzyme Based On Novel Microporous Materials

The enzymatic carbon fixation is a promising approach to deal with greenhouse gas emission.There are two major approaches,one is the CO₂ reduction,which is usually accompanied with energy consumption.And the other is the carboxylation,which is less energy requirement.The carboxylation converts CO₂ to organic carbon without extra requirement of reduction power and is hoped as a greener solution.Furthermore,many products from carboxylation is valuablefine chemical intermediates.Here,an enzyme,2,3-dihydroxybenzoic acid decarboxylase(2,3-DHBD)from Aspergillus Oryzae,was chosen as the model to investigate the enzymatic carboxylation of catechol.Firstly,the expression of 2,3-DHBD in Escherichia coli BL21 was optimized.Then,the purified 2,3-DHBD was used for two aspects:the preparation for immobilized 2,3-DHBD and the build of possible enzymatic reaction route.It all started with heterologous expression 2,3-DHBD in E.coli BL21.The optimal conditions for the expression are as below:After the growth reached to OD600=0.5 at 37?C,the IPTG was added to the final concentration 0.5 m M,and induced at 20?C for 8-12 h.After purified by Ni-NTA and FPLC,the purified 2,3-DHBD was stored at-80?for further use.For measure the purified enzyme activity,carboxylation activity has been tested in standard carboxylation system.The result shown that 200?g enzyme catalyzed producing 3?mol2,3-DHBA in 15 min.For the purpose of immobilizing 2,3-DHBD on a yolk-shell nano particle Co@Si O₂,series Co@Si O₂with different cobalt content has been used for preparing immobilized2,3-DHBD.The immobilization was performed on ice in p H 8.0 phosphate buffer.The immobilization efficiency for series carrier were 34-90?g/mg,the immobilization efficiency has negative correlation with the cobalt content of carrier.The carboxylation activity of immobilized 2,3-DHBD were 0.87-1.42 IU/mg at saturation adsorption condition.And the enzyme activity has positively correlated with the cobalt content of series carrier.The immobilized enzyme preparation by carrier with high cobalt content(20.99%)has approach to freebased enzyme.The other application of 2,3-DHBD was establishment of cascaded enzymatic reaction,which co-production of gluconolactone and 2,3-DHBA from glucose and CO₂ fixation.The demo system used glucose,phenol and ascorbic acid to produce 2,3-DHBD by the combination of two enzymes,glucose oxidase(GOD)and 2,3-DHBA.Step 1 oxidation was catalyzed by GOD to produce hydrogen peroxide.Then,step 2 was spontaneous reaction which phenol hydroxylate to produce catechol.The last step was carboxylation catalyzed by2,3-DHBD.Catechol catalyzed by 2,3-DHBD to produce 2,3-DHBA in potassium bicarbonate system.Through whole cascaded enzymatic reaction,confirmed the optimal initial amount of substrate:10 m M phenol,10 m M glucose and 8 m M ascorbic acid.The hydroxylation produced 0.82 m M catechol through optimal hydroxylation reaction system,the efficiency of hydroxylation was the bottleneck of whole cascaded reaction.Then,0.24m M 2,3-DHBA had compound by 2,3-DHBA through carboxylation reaction.The efficiency of carboxylation in cascaded reaction was showed no difference with individual reaction catalyzed by 2,3-DHBD.This thesis provides some references to bio-carboxylation and C1 resources utilization.