Cloning,Expression And Characterization Of Recombinant Alcohol Dehydrogenase 4 Of Kluyveromyces Marxianus GX-15

Kluyveromyces marxianus has recently been thought to be the optimal strain for utilization in ethanol production because of its good feature of producting ethanol at higher temperature and great suitability for substrates.After efforts,one strain of high-temperature K.marxianus yeast which has a high yield of alcohol was screened in our laboratory.This strain could grow and produce ethanol even at elevated temperatures up to 40 ?.The mechanisms why this strain could produce alcohol at high temperature were studied in our laboratory.These mechanisms included the heat shock proteins,trehalose and the saturation degree of membrane fatty acid.However,the relationship of alcohol dehydrogenase of K.marxianus which is an important enzyme in ethanol metabolism to alcohol production at high temperature has not been reported so far.Our topic will carry out the heterologous expression of 4 isozymes of alcohol dehydrogenase in K.marxianus GX-15,and try to compare their biochemical properties in order to analysis the mechanism of the alcohol production at high-temperature.The heterologous expressions of ADH1 and ADH2 of K.marxianus GX-15 have been successfully conducted in our laboratory.This experiment will study the ADH4 gene.The gene(ADH4)of K.marxianus GX-15 was amplified by PCR,and then recombined with plasmid pET-32a(+)to produce the recombinant plasmid pET32-ADH4.The pET32-ADH4 was transformed into E.coli Rosseta(DE3)expression host which were then induced at 20? for 16 h.After ultrasonic lysing and centrifugation,the recombinant protein showed the molecular mass of 60 kDa by SDS-PAGE,which was consistent with the predicted size.The recombinant protein was purified by Ni-NTA affinity chromatography.The results of the study of enzymatic properties showed that the optimal temperature of the recombinant protein was 50 ? and the optimal pH was 7.0.The optimal substrate for ADH4 is ethanol and the Km and Vmax of ADH4 for ethanol were 204.5 mM and 62.5 ?mol min-1 mg-1,respectively.The recombinant protein had no catalytic activity to acetaldehyde.