MOLECULAR EXPRESSION OF THERMOSTABLE XYLOSE ISOMERASE (XYLA) ISOLATED FROM BREVIBACILLUS FORMOSUS NF2 AND ITS BIOPHYSICAL CHARACTERISTICS

Abstract

The study aims to produce catalytic active xylose isomerase enzyme for the production of high fructose corn syrup as an alternative source of health hazardous inverted and non-calorific sweeteners. Xylose isomerase gene of Brevibacillus formosus NF2 was cloned in pTZ57R/T vector and expressed in Escherichia coli using pET28a(+) vector. The over-expressed xylose isomerase was purified to homogeneity using UNOsphere Q anion exchange column chromatography and Resource ISO pre-packed hydrophobic interaction column chromatography followed by 70% ammonium sulphate precipitation. Using this optimized approaches, we obtained 39.03% yield and enzyme was 8.13 folds purified. Size of xylose isomerase enzyme was estimated to be ~38 kDa using SDS-PAGE. We further characterised that purified xylose isomerase enzyme showed optimal activity at 55 ℃ and pH 7.0. Xylose isomerase retained 100% activity at 45 ℃ and 55 ℃ for 56 h, retained 90-95% of its activity at pH range of 6.5-7.5 for 40 min, exhibited maximum activity in the presence of 5 mM Mg²⁺ and strictly inhibited by Ca²⁺. The enzyme was capable to produce 54.67% of D-fructose from conversion of D-glucose. The kinetic parameters K_m, V_max, K_cat and K_cat/K_m were determined to be 3.73 mM, 65.36 µmol/min, 19395 sec^-1 and 5200 sec^-1/mM for D-glucose and 2.12 mM, 243.9 µmol/min, 72374 sec^-1 and 34139 sec^-1/mM for D-xylose. Taken together, our finding provide foundational work on the exploitation of xylose isomerase enzyme from Brevibacillus formosus NF2 potential candidate for the production of high fructose corn syrup (HFCS) at industrial scale.