| With the continuous development of science and technology,the functionality of sugar has been more and more in-depth research and development.In the traditional mode of production,a variety of functional sugar is mainly chemical synthesis.However,the chemical synthesis process is complex,the production cost is high and it is also easy to cause environmental pollution.These factors restrict the further development of functional sugar industry.Using modern biotechnology to produce functional sugar has the advantage of easy operation,low cost,good specificity,mild reaction conditions,pure product,high yield and so on.So it has great potential for development.In the process of using modern biotechnology to produce functional sugar,nucleotide sugar is an essential sugar donor substrate.The production of nucleotide sugar depends on the high performance of the enzyme.UDP-galactose(UDP-Gal)and UDP-xylose(UDP-Xyl)are two important nucleotide sugars,but they are difficult to obtain and expensive.UDP-glucose(UDP-Glc)is readily available and inexpensive,and UDP-Gal and UDP-Xyl can be prepared using UDP-Glc as substrate through related enzymes.UDP-glucose 4-epimerase(UGE)can isomerize UDP-Glc to UDP-Gal.UDP-glucose dehydrogenase(UGD)can catalyze UDP-Glc to UDP-GlcA,then UDP-xylose synthase(UXS)can catalyze UDP-GlcA to UDP-Xyl.In the dissertation,the three enzymes were taken as the research objects,and the enzyme genes(CGIUGE,CGIUGD and CGIUXS)were discovered from the genome of Crassostrea gigas.The three genes were cloned,then the enzymes from the three genes were expressed and studied.The results showed that the catalytic efficiency of the three enzymes was relatively high,and the reaction conditions were mild,which was suitable for the production of UDP-Gal and UDP-Xyl.In particular,the CGIUGE has strong catalytic ability,good stability,so it has great potentiality for development.At the same time,the synthesis of UDP-Xyl by one pot reaction was also studied in this dissertation.The product was successfully obtained and the conversion rate was high.Details of the results are showed below:1.Cloning,sequence analysis and protein expression of the genes CGIUGE,CGIUGD and CGIUXSAccording to the known the Crassostrea gigas genome sequence,preliminary identified a UGE gene,a UGD gene and a UXS gene in the genomes,were named CGIUGE,CGIUGD and CGIUXS.Extraction of RNA from fresh oysters,and reverse transcription to cDNA,then cDNA was taken as a template to clone the three genes.Recombinant expression vectors were constructed,then recombinant expression vectors were put into E.coli BL21(DE3).Amino acid sequence analysis showed that the CGIUGE and CGIUGD genes were highly conserved with other eukaryotes,and the similarity of some sequences were more than 60%,but the conservatism were not high compared with Prokaryotes.Compared with the first two enzymes,the conservatism was significantly low between CGIUXS and the Isoenzymes that have been found,suggesting that the UXS enzyme obtained from this study may have unique enzymatic properties.After expression and purification,SDS-PAGE assay showed clear bands for CGIUGE,CGIUGD and CGIUXS,with each having the same molecular weight as estimated in theory.2.Activity test and enzymology characterization of recombinant CGIUGEA new method for the determination of the enzyme activity of UGE isomerase was established.Based on this,the detection method for the enzymatic properties of UGE isomerase was studied.The results of the determination of the activity showed that CGIUGE was active and the activity was very high.The detection results of the enzyme characterization showed that the optimal pH value of the CGIUGE was 8.5,the optimal reaction temperature was 25?,16 ? and 37 ? also had strong activity,did not require metal ions as coenzyme.Ni2+had a strong effect on the enzymatic activity of CGIUGE,Cu2+,Mn2+and Zn2+could inhibit the enzyme activity of CGIUGE,while Ca2+,Co2+,Fe3+and Mg2+ had no obvious effect on CGIUGE.With the increase of concentration,P-ME,Urea,Imidazole and SDS could decrease the enzyme activity of CGIUGE,and SDS decreased the activity of CGIUGE obviously.CGIUGE was stable at 37? and 42? but at 50? and 60? easy deactivation.Km value of CGIUGE for UDP-Gal was 21?mol/L,Vmax value was 19?mol/L/min,and Kcat value was 0.39 min-1.3.Activity test and enzymology characterization of recombinant CGIUGD and recombinant CGIUXS.The results of enzyme catalyzed reaction products were analyzed by UPLC.The results showed that CGIUGD catalyzed the formation of UDP-GlcA by UDP-Glc and CGIUXS catalyzed the formation of UDP-Xyl by UDP-GlcA.These show that both CGIUGD and CGIUXS are active.CGIUGD and CGIUXS reaction products UDP-GlcA and UDP-Xyl were further verified by MALDI-TOF.The detection results of the enzyme characterization showed that the optimal pH value of CGIUGD was 9.0,the optimal reaction temperature was 37? did not require metal ions as coenzyme.Co2+,Cu2+,Fe3+and Zn2+could inhibit the enzyme activity of CGIUGD,but Ca2+,Mg2+,Mn2+and Ni2+had no obvious effect on CGIUGD.In a certain range of concentration,the activity of CGIUGD was enhanced by ?-ME,and the activity was strongest at the concentration of 10 mM ?-ME.When the concentration of Urea was lower,the activity of CGIUGD could be enhanced,but with the increase of Urea concentration,the activity of CGIUGD was inhibited.The effect of Imidazole on CGIUGD was similar to Urea,but not as significant as Urea.Different concentrations of Triton-X100 had little effect on the enzyme activity of CGIUGD.SDS could significantly inhibit the enzyme activity of CGIUGD,and the higher the concentration,the stronger the inhibition.CGIUGD was stable at 37 0C,but at 42 0C,50?and 60? easy deactivation.UDP-Xyl could produce feedback inhibition on CGIUGD.Km value of CGIUGD for UDP-Glc was 20 ?mol/L,Vmax value was 34 ?nol/L/min,Kcat value was 0.505 min-1.Km value of CGIUGD for NAD+was 54 ?mol/L,Vmax value was 22 ?mol/L/min,and Kcat value was 0.653 min-1.The detection results of the enzyme characterization showed that the optimal pH value of CGIUXS pH was 7.5,the optimal reaction temperature was 37? did not require metal ions as coenzyme.Co2+,Cu2+,Fe3+,Mg2+,Mn2+,Zn2+could enhance the enzyme activity of CGIUXS,especially the Mn2+enhancement effect is the most significant.However,Ca2+,Fe2+and Ni2+had no significant effect on enzyme activity.In a certain range of concentration,the activity of CGIUXS was enhanced by ?-ME,and the activity was strongest at the concentration of 10 Mm ?-ME.Urea could inhibit the enzyme activity of CGIUXS,and the higher the concentration,the stronger the inhibition ability.Imidazole had little effect on the enzyme activity of CGIUXS.In a certain concentration range,Triton-X100 could enhance the enzyme activity of CGIUXS,and the higher the concentration,the stronger the enhancement effect.CGIUGE was stable at 37? and 42?,but at 50? and 60? easy deactivation.Km value of CGIUXS for UDP-GlcA was 28?mol/L,Vmax was 16 ?molL/min,and Kcat value was 0.014 min-1.4.UDP-Xyl synthesis in one-pot reactionOne-pot reaction with both CGIUGD and CGIUXS successfully converted UDP-Glc directly into UDP-Xyl.The best ratio between CGIUGD1 and CGIUXS was 2:5,the optimal pH was 8.0,the optimal temperature was 37? in the Single factor experiments,Orthogonal test indicated the best combination for these factors were pH7.5,37?,2:5 between CGIUGD and CGIUXS,and the productivity of UDP-Xyl was 72.681%under these conditions.Temperature had the greatest effect on the reaction in the three factors,and the ratio of enzyme was second.The reaction system was expanded from 20?L to lmL on the basis of this orthogonal experiment,and the productivity of UDP-Xyl changed from 72.681%to 69.721%,and the change was little.It showed that this method had the latent capacity to be applied in actual production. |
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