Klebsiella Pneumoniae 2e Expression Of Glycerol Dehydratase Activating Factor And Its Catalytic Properties

With the large-scale development of the biodiesel industry,the production of crude glycerin,a low-value by-product,has also increased rapidly.The production and accumulation of a large amount of crude glycerin has become one of the important factors affecting the sustainable development of the biodiesel industry.Direct conversion of crude glycerol into high-value chemical 1,3-propanediol through microbial fermentation is currently a promising way to utilize crude glycerol.Glycerol dehydratase is an important rate-limiting enzyme in the synthesis of 1,3-PDO by microorganisms.This enzyme plays a crucial role in the synthesis rate and yield of 1,3-PDO.Therefore,it is extremely important to study glycerol dehydratase for fermentation.Of.In this study,A screened crude glycerol-tolerant strains Klebsiella sp.2e was used as the research object.By analyzing the genome of Klebsiella pneumoniae 2e,it was found that there are two genes of the Klebsiella pneumoniae 2e genome relating to the glycerol dehydratase activating factor.Further bioinformatics analysis indicates that the two genes are likely to encode other glycerol dehydratase activating factors.In this paper,the two genes were first analyzed by bioinformatics,and the two glycerol dehydratase activator genes were cloned and expressed.The enzymatic properties of the two genes were studied and the following results were obtained:1.Glycerol dehydratase activating factor genes orfW and orfY were analyzed using molecular bioinformatics.The sequence similarity of orfW and was 65.47%.The orfW gene belongs to the Cob-adeno-trans superfamily and has the function of activity factor.The protein domain of orfY protein is the same as that of dhaG.The three-dimensional structure information of these two genes and the construction of evolutionary tree were obtained by homology modeling.It was found that the orfW gene is similar to the reactivating enzyme of Lactobacillus deliverance diol dehydratase.Meanwhile,orfY gene is similar to the reactivating enzyme of Klebsiella pneumoniae glycerol dehydratase in the position of evolutionary tree.The results showed that the two genes had the function of glycerol dehydratase reactivating enzyme.2.Using Klebsiella pneumoniae 2e genomic DNA as a template,the glycerol dehydratase activating factor genes orfW and orfY were obtained by PCR amplification and linked to the expression vector pcold-TF,respectively.Then the recombinant plasmids pcold-orfW and pcold-orfY were obtained.After sequencing,the plasmids were transformed into Escherchia.coli Rosseta(DE3)respectively.Than the objective product was induced to express,and the soluble protein was obtained and purified.SDS-PAGE analysis of the purified protein showed that there were specific protein at 70 kDa and 67 kDa,and the result was consistent with the expectation.Klebsiella pneumoniae 2e glycerol dehydratase activation experiments were performed in the presence of coenzymes B12,ATP,and Mg2+.The results showed that the expression products of the recombinant plasmids pcold-orfW and pcold-orfY had glycerol dehydratase activating factor activity.3.In the enzyme activity analysis,the inactivated GDHt was activated by orfW and orfY respectively,and the activation degree gradually increased with the increase of time.The optimal temperature and pH of orfW were 37? and 8.0,respectively.The optimal temperature and pH of orfY are 30? and 9.0,respectively.The activation effect of orfW is stronger than orfY.When orfW and orfY jointly activate the inactivated GDHt,the results of their interaction are lower than the content of propionaldehyde produced by orfW alone,but the enzyme activity is higher than orfY.The results showed that orfW and orfY were both activators of GDHt,which could activate the inactivated GDHt.4.The transcription levels of the genes orfW,orfY,dhaF and dhaG in Klebsiella pneumatiae 2e in different concentrations of crude glycerol impurities were analyzed.The results showed that the glycerol dehydratase in Klebsiella pneumatiae 2e was not affected by crude glycerol because of the interaction of four GDHt activating factors.