The Study Of The Changed Reaction And Substrate Specificities For Enzymes Expressed As Active Inclusion Bodies

Before 1980's,it was generally accepted that protein in the inclusion body is loss-of functional,and the protein activity was restored after in vitro refolding.In recent years,more and more researches identify that the certain proteins or enzymes,even expressed as inclusion bodies,retain activities.The referred as active inclusion bodies.Active inclusion bodies are superior to the soluble enzymes such as increased expressivity,enhanced protein stability,easier extraction and purification,and repetitive use.In active inclusion bodies,the protein is insolubly aggregated,and the conformational changed is accompanied.However,no information is available on the change of the reaction and substrate specificities for the enzymes expressed in active inclusion bodies.To address this question,we selected three enzymes applied in biotechnology and industry.Uroporphyrinogen III methyltransferase(UMT)as a red fluorescent reporter catalyzes the multistep methylation reactions.The eukaryotic serine racemase(SR)is responsible for racemation of D-serine and its enantiomer L-serine,and dehydration of D-and L-serine.Human D-amino acid oxidase(DAAO)is involved in efficient oxidation of several D-amino acids.It is documented that the yeast DAAO is active in inclusion bodies,but the preferential reactions catalyzed by the other two enzymes in active inclusion bodies are not known.Here,we constructed the three enzymes expressed in soluble and insoluble forms,overexpressed them in E.coli and analyzed the activities.The results are as follows:1.Construction of the enzymes.The tag-free barley UMT variant UMT8 for soluble expression,or that with the C-terminal hydrophobic tag ELK16 to decrease protein solubility was generated,since the fusion partner barely affects the catalysis of the fluorescent reporter.The untagged Zm SR was yielded to prevent the potential inference of fusion partner at N-or C-terminus.The SUMO tagged h DAAO was designed to facilitate part of the tagged protein produced in soluble forms.All of the genes were expressed under the control of the T7 promoter.2.Overexpression of the constructs in E.coli.The constructed expression plasmids were transformed into E.coli BL21(DE3).The target proteins in soluble and insoluble fractions were separated and detected by SDS-PAGE.Fusion of the ELK16 tag rendered the production of the insoluble aggregated UMT8.In the clear lysates and pellets,the maize SR without tag and the h DAAO fused with the SUMO protein samples were examined.3.Monitoring the fluorescence of the soluble and insoluble versions of the UMT8 constructs.The UV-visible spectra showed that two peaks in 354 nm and 375 nm appeared in the soluble fractions containing the recombinant UMT8,which represents di-methylated and tri-methylated fluorescent products.In contrast,the correspondently minor two peaks for soluble fraction containing the expressed UMT8-ELK16 were assayed,suggesting that the insoluble aggregation decreased catalytic efficiency.The fluorescence spectra analysis also displayed that attachment of the ELK16 decreased the cellular accumulation of tri-methylated fluorescent product,denoting that the decrease in protein solubility inhibited the over-methylation reaction by the expressed UMT8.4.Detection of the soluble and insoluble ZmSR for catalyzing racemization and dehydration reactions.The activities of racemase and dehydratase for the solubly expressed Zm SR were detected.In comparison,the insoluble Zm SR lost dehydratase activity.Addition of glycerol,or the reducing reagent dithiothreitol had differential impacts on the activities of racemase and dehydratase for soluble and insoluble Zm SR proteins.The results showed that Zm SR in active inclusion bodies changed the catalytic reaction specificities.5.Analysis of the yield and substrate specificity of the constructed h DAAO in active inclusion bodies.The ratio of SUMO-h DAAO inclusion body to the cells wet weight was optimized.It was about 17.71% after induction at 28 °C for 12 h,but increased up to about 37.99% after induction at 37 °C for 4 h.The insoluble h DAAO displayed the correspondent substrate specificity for oxidizing the three D-amino acids,but the differential activities between the soluble and insoluble h DAAO proteins were slightly different.,suggesting slight conformational change of the active center was occurred in the insoluble h DAAO.In summary,the chosen enzymes are efficiently used as probes for investigating the changed catalytic reactions,or the substrate specificities.The loss of dehydratase activities of the insoluble maize SR makes the enzyme as the special immobilization for potential use in production of D-serine.The tagged insoluble h DAAO retained the correspondent substrate specificity.This character afforded the enzyme in active inclusion bodies easily extracted for screening the specific inhibitor possibly used for therapy of Parkinson's disease.