Gene Cloning And Expression Of A Phosphomannose Isomerase From Bacillus Cereus CZ And Characterization Of The Recombinant Enzyme

The phosphomannose isomerasesis ametal-dependentmonomeric isomerasewhichcan reversibly catalyze the conversion of fructose-6-phosphate andmannose-6-phosphate in organism and plays an important role in metabolism of mannose and synthesis of GDP-Mannose.In addition,the phosphate isomerasescan catalyze the reversible reaction among phosphatesugars and some of monosaccharides can be catalyzed,which is significant for the synthesis of some rare monosaccharide had not been founded in nature.These rare monosaccharidescan be widely used in medicine,industry and so on.Phosphomannose isomerase gene as the marker gene,is extensively used in transgenic plants such as monocots and dicots.Due to the advantages of harmless to human body and ecosystem and have no impact on growth and development of host plants,PMI/Mannose screening system possess a lot of potential application value in transgenic technology.Thus,there are substantial research value on the isolating and purifing phosphomannose isomerase which has high purity and activity from heterologous gene expression and on the preliminary study of enzymatic properties.In this study,we extracted the genome-wide of Bacillus cereus CZ preserved by our laboratory and founded the 16 S rRNA of Bacillus cereus CZ is highly homologous with Bacillus thuringiensis through blast analysis in the web of the National Center for Biotechnology Information?NCBI?.Specific primerswere designed based on the pmi gene sequence from Bacillus thuringiensis to amplify the full lengthgene of pmiin Bacillus cereus CZ strains,and then heterologous expressed in Escherichia coli.The pmi gene obtained by PCR amplification was gel-purified,ligated into expression vector pET-22b?+?and transformed into Escherichia coli BL21?DE3?.The recombinant strain expressed recombinant phosphomannose isomerasesuccessfully.The results showed that pmi gene?948 bp?was clonedand encoded 316 amino acids.The crude recombinant enzyme was purified by His Trap HP Ni-affinity chromatography and the molecular mass of the purified recombinant enzyme was detected about 40.8 kDa.The enzymatic characteristics analysis showed that the optimal reaction temperature was estimated to be 35?and the enzymatic activity retained relatively stable at 30 40?.Meanwhile,the optimal pH of the recombinant enzyme was around 7.0,and the relative enzymatic activity retained 60% in weak alkaline environment for 12 h.Variousmetal ion with low concentrations,such as Ni²⁺,Ca²⁺,Zn²⁺,Cu²⁺,Mg²⁺,could activate the recombinant enzymatic activity in different degree.1mmol/LMn²⁺ had most obvious activation,whereas Co²⁺ had obviously inhibitory effect on the enzyme.In addition,the recombinant enzyme showed the highest substrate specificity on L-ribulose.