Immobilization of cyclodextrin glycosyltransferase for the production of ascorbic acid glucoside

Abstract

This study aims to immobilize cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. A11 and characterize the immobilized CGTase for the production of 2-O-∝-glucopyranosyl-L-ascorbic acid (AA-2G). CGTase was immobilized on various carriers including alumina, silica, activated carbon and chitosan by covalent linkage using bifunctional agent glutaraldehyde. Alumina was selected among tested carriers, because of the highest immobilized activity. The immobilization parameters such as concentration of coupling agent, enzyme/support ratio and coupling time were optimized. It was determined that the optimum conditions for enzyme immobilization were to activate alumina with 2% γ-aminopropyltriethoxysilane and 1% glutaraldehyde. The activated alumina was then incubated with the enzyme solution for 6 hours at ℃, using an enzyme to the support ratio of 14 units per gram alumina. The best preparation of immobilized CGTase retained 31.2% of its original activity (4.36 units/g alumina). After immobilization, the optimum pH of immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60℃). The pH stability of both free and immobilized enzymes were in the range of 5.0-9.0, whereas the thermal stability of immobilized CGTase was slightly higher than that of the free enzyme. The Michaelis-Meten constant for immobilized CGTase (Km = 5.62 ± 0.20 mg/ml) was higher than the free enzyme (Km= 0.59 ± 0.25 mg/ml). The Vmax value of immobilized CGTase (5.82 ± 0.13 U/mg protein) was lower than the free form (9.69 ± 0.38 U/mg protein). The immobilized CGTase exhibited higher stability than the free enzyme when stored at both 4℃ and 25℃ for months. When this immobilized enzyme was used in batch production of AA-2G, the optimal conditions were to incubate 350 units/g β-cyclodextrin of immobilized CGTasw with 4% (w/v) β-cyclodextrin and 2% (w/v) ascorbic acid in the reaction pH of 5.0, for 24 hours at 40℃. Under these conditions, the immobilized CGTase produced AA-2G with 2.92% yield and retained its activity up to 74.4% after being used for 3 cycles.