PRODUCTION, CHARACTERIZATION AND OPTIMAL PERFORMANCE STUDIES OF GLUCOSE ISOMERASE BYACHROMOBACTER XYLOSOXIDANS MCK-4 ISOLATED FROM STARCH MILLING WASTES.
ABSTRACT
D-glucose/xylose isomerase (D-xylose ketol isome rase; EC 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. It is used essentially for the production of high fructose corn syrup (HFCS); the sweetener of choice in the beverage industry. Seventy five (75) bacteria were isolated from starch milling wastes in Nsukka town, Enugu state, Nigeria. The isolates were screened for their ability to produce glucose isomerase (GI) and the isolate Achromobacter xylosoxidans mck-4( accession number -JQ724537) gave the highest activity of intracellular GI, in our production medium after 48hr. incubation, pH 7 and temperature of 30˚C. Seven crude organic carbon sources were evaluated for their ability to induce GI production and corn cob (3.088 ± 0.075 U/ml), wheat bran (3.206 ± 0.107 U/ml) and Rice bran (3.185 ± 0.158 U/ml) gave good enzyme activities at 20 g/l, 15 g/l and 15 g/l respectively. Three crude nitrogen sources were evaluated and Soy flour (3.451 ± 0.029 U/ml), Soy chaff (3.49 ± 0.079 U/ml) gave good enzyme activities at 15 g/l respectively. Three pure carbon sources were evaluated and Sucrose gave best optimal enzyme activity (4.044 ± 0.03 U/ml) at 17.5 g/l followed by xylose at 17.5 g/l (3.088 ± 0.0595 U/ml) and glucose at 15 g/l (2.257 ± 0.08 U/ml) and for the pure nitrogen sources, Yeast extract (4.224 ± 0.079 U/ml) and NH₄NO₃ (2.202 ± 0.051 U/ml) gave the best activity. The sodium dodecyl sulphate polyacrylamider gel electrophoresis (SDS PAGE) showed that the enzyme was partially purified and the band was near the protein marker of 43,000 Daltons. It was observed that the enzyme was purified 4.754 fold with specific activity of 6.461 U/mg of protein and 30.9% enzyme recovery in the final desalted sample. The Km value calculated for glucose by Lineweaver Burk plot is 0.89 moles and Vmax was 6.435 U/mg. The highest activity of the enzyme was observed at temperature of 70˚C and pH 7. The enzyme activity was maximally activated by the cations; magnesium and cobalt with magnesium having the highest effect followed by manganese and cobalt. The observed parameters for the production of GI by Achromobacter xylosoxidans mck-4 are well in the range of economic feasibility, but the pathogenic/ no GRAS status of the organism limits its commercial industrial exploitation. These results indicate that these agro-residues (wheat bran, corn cob, Rice bran, Soy flour, Soy chaff) can be used for industrial production process of glucose isomerase (GI) and subsequently high fructose corn syrup (HFCS). This will reduce the production cost (expensive xylose inducer) as well as solve the problem of disposal of tonnes of agro- residue wastes produced every year in an agriculture dominated country like Nigeria. This is the first report to our knowledge stating the production and characterization of glucose isomerase from Achromobacter xylosoxidans mck-4 utilizing crude agro-residues in Nigeria and beyond.