Recombinant Expression Of D-arabinose Isomerase From Geobacillus Pallidus And Its Application

D-arabinose isomerase （referred to D-AI, EC5.3.1.3）, which can catalyze D-arabinose（aldose） isomerized into D-ribulose （ketose）.In this paper, we resrarched into the recombinantexpression of D-AI which also has a high activity in an acidic condition and studied theconversion of D-tagatose. D-tagatose is a kind of natural ketone sugar and relatively rare inthe nature. It has a very special application value in terms of food, medical and health care. Inrecent years, studies have found that D-tagatose is not just a rare sugar but a new type offunctional sweetener, and it has the effect of reducing calories, falling blood sugar, preventingtooth decay and protecting health.In this paper, the screening of D-AI has carried on the catalytic reaction and pro-duct analysis, finaly we determined that D-AI have the ability to catalyze D-galactoseisomerized into D-tagatose. The product of reaction has been identified as D-tagatoseby HPLC detection and no other byproducts. On this basis, we researched on the clo-ning and expression D-AI gene of G.pallidus, and induction the production of enzymeof expression optimization, separation and purification of enzyme, and research the en-zyme properties and the biotransformation of D-tagatose, including the following parts:1. We found the coding sequence of D-AI gene of G.pallidus in the GenBank,and its registration number in the database is AB429010. Then we designed primersand got the D-AI gene sequences through PCR amplification. The D-AI gene sequen-ces contains1788bp, encoding595amino acid residues, and its translation obtainedof protein molecular weight is66,237Da through software calculation.2. The D-AI gene was inserted into the expression vector pET-28a（+）, and transformedinto the expression host E.coli BL21（DE3） for expression, and the recombinant strain withD-AI insert was successfully constructed the target gene with the C-terminal fusion His-Tag.The induction of expression of the recombinant engineered bacteria conditions are optimizedto determine the optimal conditions is: the recombinant bacteria were cultured for4hours at37℃and then added to a final concentration of1.0mM of IPTG, and then incubated at30℃induced6hours, and the highest enzyme activity can reached10.0U/mL.3. The recombinant D-AI with His-Tag was purified by Ni affintychromatography.The D-AI is intracellular enzyme, before the column purification, it has to ultrasonicdisruption, and then obtained the crushed crude enzyme by centrifuged. Finally, weobtain a single strip by polyacrylamide gel electrophoresis.4. We studied the enzymatic properties of recombinant D-AI and that under theconditions of60℃and pH7.0, the catalysis to D-tagatose showed the highest activi-ty.And it has good stability at the range of pH6.0to8.0and a range of30℃to7 0℃. Mn²⁺,Zn²⁺,Mg²⁺,Ca²⁺,Fe²⁺,Cu²⁺,Ba²⁺and other divalentmetal ions had activationeffecton the D-AI, especially Mn²⁺, while Mg²⁺and Cu²⁺has inhibitory effect on theD-AI,and the inhibitory effect of Cu²⁺was more significant. The Km and Vmax for D-galactose were296.85mmol/L and151μg/min/L.5. A study was carried out on the conversion of D-AI biosynthesizing into D-tagatose.Using crude enzyme solution of D-AI catalyze D-galactose to D-tagatose, we can concludethat the optimum concentration of galactose is18g/L, and the conversion can reached41.6%after36hours’ reaction.