Glucose isomerase (EC 5.3.1.5), is the third highest value enzyme after amylase and protease. It catalyzes the reversible isomerization of D-glucose to D-fructose. Isomerization of glucose to fructose is of commercial importance in the production of High-Fructose Corn Syrup (HFCS). Glucose has 70-75% the sweetening strength of beet sugar (sucrose), but fructose is twice as sweet as sucrose, so high fructose syrup can be used to replace sucrose as a sweetener. As enzyme itself is very expensive, immobilization of this enzyme on a suitable carrier is necessary for commercial use. The immobilization offers advantages like reusability, greater stability and better process control. Traditional methods of enzyme immobilization can conveniently divided…show more content… Glutaraldehyde (25% v/v), Casamino acid, cysteine Hydrochloride, carbazole and xylose were obtained from Himedia. All other reagents were used of analytical grade.
2.2 Microorganism and Growth
The thermophilic strain of Streptomyces therrnonitrificans (NCIM 2007) was routinely maintained on MGYP slants (malt extract, 0.3%; yeast extract, 0.3%; peptone, 0.5%; glucose, 1%; and agar, 2%; adjusted to pH 7.0 with NaOH) at 50oC. The vegetative growth was colorless to pale yellow turning slightly darker in 2-3 days. Greyish white aerial mycelium developed after 36-48 hr and turned dark grey in 72 hr.
2.3 Production of Glucose isomerase.
The inoculum was prepared in 50ml Callens’ Medium[9]( sorbitol, 2%; Casamino acid, 1%; Yeast extract, 0.5%; KH2PO4, 0.27%; K2HPO4, .52%; MgSO4.7H2O, 0.2%; NH4NO3, 0.3%; Xylose, 1% ) by inoculating with a well sporulated slant of Streptomyces culture, followed by incubating at 500C with shaking (200rpm) for 24hr.
Enzyme production was carried out by transferring inoculums (10%) to 50 ml of the same medium, in each of several 250-ml Erlenmeyer flasks, and then incubated at 50°C for 16 h with shaking…show more content… The process consisted of suspending magnetic particles (200mg) in [3-(2-aminoethylamino)propyl]trimethoxysilane (1ml), deionized water (250µL) and methanol (25mL). The mixture was sonicated for 30 min. After that, glycerol (15 mL) was added, and the solution was heated at 900C for 6 hr with continuous mechanical agitation. The obtained precipitate was washed with water and methanol for four times in each case and dried, yielding a fine powder.
2.8 Protein immobilization
Magnetic CLEAs of Glucose isomerase were prepared as described by Talekar et al (6) - The amino functionalized magnetic particles (50mg) were mixed with 15mL of crude enzyme extract (50 mM potassium phosphate buffer, pH 7.0, containing 1 mM Co2+ and 5 mM Mg2+) and shaken for 15 min at room temperature. Then ammonium sulphate (70%) was added with stirring at 40C for 30 min. After precipitation, 15ml glutaraldyhyde (1%v/v) was added into the suspension and stirred for 3 h at room temperature. After cross-linking, magnetic CLEAs were separated using magnet, washed for three times by sodium phosphate buffer and stored in 0.1 M phosphate buffer (pH 7.0) at 40C.