Preparation Of NADH Regeneration System And Preparation Of Petroleum Degradation

Petroleum leaking into the environmental during the process of extraction,storage and refining contaminated the soil and ground water and poison thebiology. The most effective pathway to degraded the petroleum inenvironmental was micro-biology degradation during their metabolism with thepetroleum became to harmless CO2and H2O. Intracellular enzymes ofmicrobiology play main role during the petroleum biodegradation. It wasconsidered that the petroleum-degrading enzymes extracting from the microbialcell to deal with the petroleum molecule directly could enhance the degradingeffective.Formate dehydrogenase (FDH) from the C.boidinii was expressed inE.coli and extract from intracellular and then co-immobilized with thepetroleum-degrading enzymes on diatomite in this paper. The FDH couldprovide reduced state constantly for the petroleum-degrading enzymes and thepetroleum-degrading process may operate continuously, which could reduce thecost of petroleum-contamination treatment. The major content and conclusionof this paper as follow:(1) Firstly, we extract the genome of C.boidinii and gain the fdh gene byPCR, and then the fdh gene was analyzed.(2) Then, after connecting with the plasmid pET28a(+), the fdh gene wastransformed into the strain E.coli BL21(DE3) and strain E.coli E.coli BL21(pET28a(+)-fdh) was constructed.(3) The strain E.coli BL21（pET28a(+)-fdh）was cultured and then inducedwith IPTG for4hours after transfer into new medium. The E.coli cell wascollected and ultrasonication, duration of radiation each time2s (with a2sinterval), ultrasonic power320W, and total irradiation time10minutes, torelease the intracellular enzymes and it is showed that the enzyme activity inevery gram cell was1.19U.(4)2mg of petroleum-degrading enzyme was immobilized on1gramdiatomite, and then co-immobilized with FDH, it was showed that theimmobilized-enzyme has the highest activity when FDH added0.06U.(5) Petroleum-degrading experiment was carried out with1gramimmobilized-enzyme when temperature was30°C and it is showed that it could degrade83.3%of100mg petroleum in10hours.Petroleum-degrading efficiency of enzyme was enhanced obviously afteradding FDH. It is considered that the petroleum-degrading enzyme agentproduced in this paper could be use in the recovery project of the petroleumcontamination.