Construction Of D-Psicose-3-Epimerase Gene Engineered Kluyveromyces Marxianus

D-allulose is a kind of rare sugar,which rarely exists in nature.D-allulose has good processing characteristics,which sweetness is similar to sucrose but low calories.D-allulose possesses great application value in food,health care,and medicine due to the physiological functions of prevention of obesity,hypoglycemic,anti-oxidation,neuroprotective and inhibition for cancer cells,etc.Whole-cell reaction avoids multi-step and time-consuming processes to obtain enzymes.However,the reaction is executed by D-psicose-3-epimerase catalyzed at high temperature,at which the conventional host,such as Escherichia coli and Saccharomyces cerevisiae are easily inactivated and killed after the catalysis.Therefore,it is hardly utilized repeatedly.To realize the repeated utilization of the engineering strains after in-situ catalysis,the high efficient expression vector was constructed by cloning D-psicose-3-epimerase gene,and then transferred into thermotolerant Kluyveromyces marxianus for the high level of expression.Through the cell reaction of engineered K.marxianus,the yield and the conversion rate of D-allulose were improved by optimization of in-situ catalytic process of producing D-allulose from D-fructose.The biological removal of D-fructose from the mixture,reduced the production cost,improved utilization of engineered strains and production efficiency of D-allulose production efficiency.Accordingly,the conclusion was that it was feasible to produce D-allulose from D-fructose by engineered K.marxianus regeneration and cyclic catalytic.The main results were as follows:(1)Cloning and analysis of D-psicose-3-epimerase gene.D-psicose-3-epimerase gene was amplified through PCR from Agrobacterium tumefaciens and cloned into T-vector plasmid,constructed the recombinant plasmid of p UCm-T-dpe and transferred into Escherichia coli DH5? for sequencing and analysis.Homology analysis showed that the nucleotide sequence of dpe gene has 99.45% similarity to A.tumefaciens and 99.65% similarity to the amino acid sequence of A.tumefaciens encoded D-psicose-3-epimerase NCIM:2942(No.KX098480.1).The DNA sequences and structural characteristics of DPEase were analyzed by bioinformatics.The results showed the gene possessed 870 bp open reading frame and encoded 289 amino acids.The protein with a relative molecular of 31493.62 was stable hydrophilic.The protein located at the cell membrane belonged to a hydrophilic protein without signal peptide and transmembrane region.Alpha helix,beta folding,and random coil respectively accounted for 38.41%,47.06% and 14.53% in the secondary structure of the protein.Three-dimensional structure showed that DPEase protein was a tetramer and each monomer had a typical TIM barrel,composed of 8 repeat(?/?)8 structure.(2)Construction of engineered K.marxianus and enzymology characteristics of recombinant DPEase.Using enzyme digestion and T4 ligase connection,the expression vector of p RS42H-dpe was constructed and transferred into thermotolerant K.marxianus by Lithium acetate transformation method.Recombinant K.marxianus No.4 with the highest DPEase enzyme activity was obtained by screening of hygromycin resistance,reverse transcriptional PCR and enzyme activity.The expression of recombinant DPEase reached a maximum of 0.35 mg/m L and the enzyme activity is 6.5 U/m L for 48 h.The purity of recombinant DPEase was up to 95% after purification by Ni2+-Chelating Sepharose Fast Flow affinity chromatography column.Enzymatic properties studies showed that Mn2+ significantly improved the enzyme activity and the optimum temperature and p H value of the enzyme respectively were 55°C and 8.0.(3)Study on the biological removal of D-fructose from the catalytic reaction liquid.By the whole-cell reaction,the concentration of 10 g/L recombinant cell catalyzed 750 g/L D-fructose at 55°C and p H 8.0 to produce 190 g/L D-allulose.Moreover,the results show that it was feasible to produce D-allulose from D-fructose by engineered K.marxianus regeneration and cyclic catalytic.Approximately 100 g residual D-fructose were converted to 34 g ethanol,15 g engineered K.marxianus and the purity of D-allulose above 92% were obtained.Thermotolerant K.marxianus integrated with DPEase gene successfully expressed recombinant DPEase.Using the cell reaction to study the properties of in-situ catalytic by engineered K.marxianus,which can reduce production costs and improve utilization of engineered K.marxianus.This study provide a reference for the industrial production of D-allulose from the repeated utilization of thermotolerant engineered K.marxianus in-situ catalysis and biological removal of D-fructose to purify D-allulose.