Production of high fructose syrup from Asparagus inulin using immobilized exoinulinase from Kluyveromyces marxianus YS-1.
Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial purification by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 degrees C, which was 5 degrees C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 degrees C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a significant improvement in the thermal stability of the biocatalyst after immobilization. Apparent K (m) values for inulin, raffinose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (E (a)) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co(2+) and Mn(2+) enhanced the activity, whereas Hg(2+) and Ag(2+) were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was effectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.
Carbohydrate and Protein Biotechnology Laboratory, Department of Biotechnology, Punjabi University, Patiala, Punjab, India. rssingh11@lycos.com
Ram Sarup
RS
Singh
Rajesh
R
Dhaliwal
Munish
M
Puri
D027761:Asparagus Plant; D019149:Bioreactors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005632:Fructose / Q000096:biosynthesis*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007444:Inulin / Q000378:metabolism*; D007716:Kluyveromyces / Q000201:enzymology* / Q000235:genetics; D018517:Plant Roots; D013395:Sucrose / Q000378:metabolism; D013696:Temperature
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D005632:Fructose; D013395:Sucrose; D007444:Inulin; D006026:Glycoside Hydrolases; C023086:inulinase