| D-allose, one of the rare aldohexoses, has attracted much attention because of its numerous health and medical benefits especially on the anti-tumor activity. Based on these properties, much emphasis has been put on the bioproduction of D-allose in recent years. Interconversion between D-allose and D-psicose by isomerization has been focused on as a commercially attractive enzymatic reaction for the production of D-allose. Ribose-5-phosphate isomerase B is suggested as a practical solution for D-allose production.In this study, a ribose-5-phosphate isomerase gene from Thermotoga lettingae TMO was cloned and expressed in E. coli. The recombinant T. lettingae ribose-5-phosphate isomerase was purified and characterized. And the molecular modifications were carried out to reveal the relationship between the structure and functionality. Then, a co-expression system was constructed to perform a dual-enzyme coupled reaction to biotransform D-fructose to D-allose.The main results are as follows:The ribose-5-phosphate isomerase gene from Thermotoga lettingae TMO was amplified by PCR. A438bp ribose-5-phosphate isomerase gene, encoding a polypeptide of145amino acid residues was obatained and then inserted into the pET-22b (+) vector. The resulting pET-rpiB was transformed into E. coli BL21(DE3).The inducing conditions of the recombinant strain E. coli BL21/pET-rpiB was investigated. Maximal expression was observed at28℃after6h incubation with1mM IPTG. The acitivity of the recombinant ribose-5-phosphate isomerase was detected through whole cell transformation. The product was identified to be D-allose by HPLC, LC-MS-MS, FTIR and’H-NMR.The purification of D-allose using cation exchange resin DTF-01was investigated. The effects of column temperature, sample volume and elution flow rate were discussed. It showed that, when8ml D-allose was loaded with elution flow rate of1ml/min at60℃, collected D-allose products with the purity above90%.The recombinant ribose-5-phosphate isomerase with His-Tag was purified to electrophoretical homogeneity by Ni affinity chromatography. The molecular mass of the recombianant enzyme was estimated to be17kDa by SDS-PAGE and35kDa by size exclusion chromatography. From these results, the recombinant ribose-5-phosphate isomerase from T. lettingae was considered to be a homodimer with two identical subunits. The isoelectric point of the recombinant enzyme was determined to be6.3by isoelectric focusing electro-phoresis.The properties of this ribose-5-phosphate isomerase were investigated. The enzyme showed maximal activity of0.2Umg^D-allose producing at75℃, pH8.0. It exhibited good stability at pH8.0and considerable thermostability with the half life of3.3h at75℃. The kinetic parameters Km, kcat and catalytic efficient kcat/Km were64mM,7.73min-1and0.12mM-1min-1, respectively. The equilibrium ratio value between D-psicose and D-allose was68:32.Based on homology modeling, molecular docking and site-directed mutagenesis methods, T. lettingae ribose-5-phosphate isomerase was modified successfully and13mutants was obtained, including G1y66Ser, G1y66Ala, His133Asp, His133Ala, Tyr42Phe, Tyr42Ala, His9Tyr, His9Ala, Arg132Glu, Arg132Ala, Arg136Glu, Arg136Asp and Arg136Ala. Among these mutants, G1y66Ala, H1s9Ala, Arg132Glu and Arg132Ala showed higher activity of2.08,1.12,2.13and1.32-fold than the wild-type enzyme, respectively. The functions of these sites were predicted.Co-expression plasmid pETDuet-Tlrpi-Ccdpe co-expressing the genes encoding D-psicose3-epimerase from Clostridium cellulolyticum H10and the R132E mutant was constructed successfully. The pETDuet-Tlrpi-Ccdpe plasmid was then transfered into E. coli cells and induced. And then the resulting whole cells harboured two enzymes were used in D-allose production at low cost. D-Allose was obtained from D-fructose with10.7%yield by the dual-enzyme coupled reaction. The dual-enzyme coupled biotransformation was observed to change the reaction balance to the tendency of increasing the first product D-psicose forming. |
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