Acidothermus Cellulolytics L-Arabinose Isomerase: Cloning, Characterization And Molecular Modification

Dietary structure is attracting more and more attention as the chronic diseases breaking out all over the world. The exploitation of novel sugar substitute has been an important part in the sweetener field. D-tagtatose is a low-calorie functional sweetener and is one of the rare sugars. It has been shown to have numerous health and medical benefits, including treatment of obesity, prevention of cariogenic, adjustment the balance of intestinal microorganisms, and reduction of symptoms of type 2 diabetes. Based on these properties, D-tagatose has attracted a great deal of attention in recent years as a low calorie sugar-substituting sweetener, an intermediate for synthesis of other optically active compounds, and an additive in detergent, cosmetic, as well as in pharmaceutical formulations.Much emphasis has been put on the biotrsformation of D-tagatose in recent years, and L-arabinose isomerase is considered to have the most potential use for D-tagatose production, since it can catalyze the isomerization of D-galactose to D-tagatose and convert L-arabinose to L-ribulose.In this study, an acido-philic stain (Acidothermus cellulolytics)ATCC 43068 with predicted L-arabinose isomerase gene (araA) was previously chosen out for D-tagatose production. The strain was identified to have the ability of producing L-arabinose isomerase and biotransformation of D-tagatose. In the present paper, the L-arabinose isomerase gene from Acidothermus cellulolytics was cloned, sequenced, and expressed in E. coli. The recombinant enzyme was been purified and characterized. And the molecular modifications were carried out to reveal the relationship between the structure and functionality. The main results are as follows:1. The araA gene was obtained trough PCR amplification. DNA sequence analysis revealed an open reading frame of 1,503 bp, encoding a polypeptide of 501 amino acid residues with a calculated molecular mass of 55 k Da. The sequence data of the araA gene from Acidothermus cellulolytics was submitted to the GenBank databases under accession no. GU188440. A homology search revealed that the deduced araA gene product showed 42-62% amino acid identity with L- arabinose isomerases from various microorganisms.2. The araA gene was inserted into the pET-22b(+) vector, and transformed into E. coli BL21(DE3). And two recombinant strains with araA insert, E. coli/pET-araA and E. coli/pET-araA-his with fused C-terminal His-Tag, were constructed. The inducing conditions of the recombinant strain were investigated. Instead of IPTG, L-arabinose was proved to be the best inducer. Maximal expression was observed at 37 oC after 8 h incubation. The acitivity of the recombinant L-arabinose isomerase was detected through whole-cell transformation, and the product was identified to be D-tagatose by HPLC and 13C-NMR. No by-product was detected.3. The recombinant L-arabinose isomerase without His-Tag was purified to electrophoretical homogeneity by heat treatment, ion exchange and gel filtration chromatography. Based on SDS-PAGE and gel filtration analysis, the molecular mass was determined to be 55 kDa, and the L-arabinose isomerase was a tetra-polymer. The recombinant L-arabinose isomerase with His-Tag was purified by Ni affinity chromatography and exhibited a special activity of 4.16 U/mg, while the special activity of L-arabinose isomerase without His-Tag was 20.88 U/mg. According to the great activity difference between the two recombinant enzymes, the L-arabinose isomerase was predicted to be metalloproteinase.4. The properties of this L- arabinose isomerase were investigated. The enzyme showed maximal activity to D-galactose at 75 oC, pH 7.5. It exhibited good thermostability and wide range pH stability. It was highly stable below 65°C and maintained more than 90% activity in the pH range from 6.0 to 8.0. It required divalent metal ions, either Mn2+or Co2+, for both enzymatic activity and thermostability improvement at higher temperatures, while Cu2+ and Hg2+ caused caused activity inhibition. Study on substrate affinity revealed L-arabinse was the best catalytic substrate; D-galactose was the second. The kinetic parameters Km, Vm and catalytic efficient kcat/Km were 15.2 mM, 6.8 U/mg and 24.6 min-1 mM-1 , respectively, and those for D-galactose were28.9 mM, 4.9 U/mg and 9.3 min-1 mM-1, respectively.5. Based on the revealed crystal structure of E. coli L-arabinose isomerase, the structure of Acidothermus cellulolytics L-arabinose isomerase monomer was simulated, and the putative active centre consisting of L20, Y21, Y336, N123, N125, H129 and Mn2+, was pedicted. Through structure comparison and analysis, mutant enzymes L20A, Y21A, Y336A, N123A, N125A and H129A were constructed, of which L20A showed higher activity (2.2-fold) and catalytic efficient for D-galactose than the wild-type enzyme. Y21A, Y336A, N123A, N125A and H129A caused activity decrease for D-galactose, which indirectly proved the important roles of these sites in active site region. The functions of these sites were predicted.