Cloning And Identification Of Xylitol Dehydrogenase Gene From Three Yeast Strains And Study On The Difference Of Protein Properties

At present,the most excellent strain for the production of fuel ethanol from lignocellulose.However,Saccharomyces cerevisiae could not ferment xylose to produce ethanol,and it could only be fermented.Currently the most widely used strategy is introduced to the xylose metabolic pathway in the Saccharomyces cerevisiae by xylose reductase and xylitol dehydrogenase to convert xylose to xylulose and xylose.However,this kind of metabolic method often showed the accumulation of xylitol and ethanol production is not ideal,there are related experiments show that the efficient expression of XYL2 is a necessary condition to improve the efficiency of xylose fermentation strains.Therefore,this paper selects the laboratory has isolated yeast strains for ethanol production and more efficient xylose three,namely Meyerozyma guilliermondii,Torulaspora delbrueckii and Pichia kudriavzevii/Issatchenkia orientalis,amplified the encoding xylitol dehydrogenase gene,construct recombinant Saccharomyces cerevisiae strains can utilize xylitol,study the properties of protease expression of xylitol dehydrogenase gene,for the separation of Candida tropicalis SHC-3 by Pichia stipitis CBS6054 and reported as control.The results of the study are as follows:(1)The three were not reported in the xylitol dehydrogenase gene and pYES2/NT-B plasmid to construct expression recombinant plasmid into xylitol dehydrogenase of Saccharomyces cerevisiae INVscl,and gene fragments were sequenced and analyzed.The experimental results show that three kinds of recombinant strains and compare two kinds of recombinant strains can successfully expression of xylitol dehydrogenase,and has the ability to metabolize xylitol;through the comparison of protein sequences,we found that the five enzymes belong to the intermediate chain dehydrogenase family(MDR),and there is a similar binding region in the amino acid sequence of the target protein and zinc binding region,while other regions is different,also has obvious specificity.(2)The expression of three xylitol dehydrogenase gene and fluorescent protein into Saccharomyces cerevisiae,inducing the expression of green fluorescent protein and observe the position of its expression,and two kinds of xylitol dehydrogenase expression sites with known green fluorescent protein were compared.The results show that the three xylitol dehydrogenase gene in this study,the expression and location of Candida tropzicalis and Pichia stipitis xylitol dehydrogenase in the cytoplasm,nucleus and cell wall were unable to see the fluorescence reaction,shows five kinds of experiments using xylitol dehydrogenase are cytosolic protein.(3)Study on properties of three kinds of xylitol dehydrogenase,by Pichia stipitis and Candida tropicalis were also added some in-depth study of the enzymatic property.The results show that five kinds of xylitol dehydrogenase have strict dependence on coenzyme NAD+,the enzyme activity was greatly inhibited when EDTA was added,which verified the binding structure of zinc and coenzyme.Pichia kudriavzevii xylitol dehydrogenase specific activity up to 14.66 U/mg,Torulaspora delbrueckii xylitol dehydrogenase activity for a minimum of 10.71 U/mg,the other three were at about 13 U/mg,and there are significant differences in different sources of xylitol dehydrogenase and itsenzymatic properties,the best in the pH8.5 and temperature of 35? activity from Torulaspora delbrueckii xylitol dehydrogenase,the value of Km is 20.96 mM and the maximum reaction rate is the highest,a small amount of Mg2+ can promote the activity of Ca2+ has obvious inhibitory effect on the Meyerozyma guilliermondii;the xylitol dehydrogenase in pH8.0 and temperature of 35? activity was best,in addition has effect of low concentration of mercaptoethanol;The best is the utilization efficiency of xylitol is Issatchenkia orientalis xylitol dehydrogenase,because of its highest affinity,the best catalytic effect,pH8.0 and temperature of 45? under the condition of optimum activity,Mg2+ can significantly promote the activity of the enzyme,xylitol dehydrogenase significantly inhibited effect on other Ni2+ on the basic no,in the presence of Na+ There will be a significant promoting effect on the enzyme activity,and has high tolerance for K+,the xylitol dehydrogenase can be highly tolerant to salt ions,but the addition of beta mercaptoethanol has obvious inhibitory activity;the optimum temperature is 35? of Pichia stipitis xylitol dehydrogenase,was significantly lower than that of other enzymes.The high temperature fermentation;the optimum temperature is 50? of Candida tropicalis xylitol dehydrogenase,suitable for use in high temperature fermentation,the enzyme activity by inhibition of Cu2+ than the other xylitol dehydrogenase is weak,Mg2+ can greatly promote the enzyme activity,adding beta mercaptoethanol with little effect.The research results make us related mechanism of Sacchaomyces erevisiae xylose metabolism are more in-depth understanding and Research on different protein properties of xylitol dehydrogenase can lay the foundation for the construction of a high efficient xylose fermenting yeast and subsequent in-depth study,has important scientific and practical significance to realize the efficient production of cellulose fuel ethanol.