Seo Woo Yong
(Department of Environmental Engineering and Biotechnology, Myongji University)
,
Lee Kisay
(Department of Environmental Engineering and Biotechnology, Myongji University)
Optimal conditions for the in situ immobilization of lipase in aldehyde-silica packed columns, via reductive amination, were investigated. A reactant mixture, containing lipase and sodium borohydride (NaCBH), was recirculated through an aldehyde-silica packed column, such that the covalent bonding o...
Optimal conditions for the in situ immobilization of lipase in aldehyde-silica packed columns, via reductive amination, were investigated. A reactant mixture, containing lipase and sodium borohydride (NaCBH), was recirculated through an aldehyde-silica packed column, such that the covalent bonding of the lipase, via amination between the amine group of the enzyme and the aldehyde terminal of the silica, and the reduction of the resulting imine group by NaCBH, could occur inside the bed, in situ. Mobile phase conditions in the ranges of pH $7.0{\~}7.8$, temperatures between $22{\~}28^{circ}C$ and flow rates from $0.8{\~}1.5\;BV/min$ were found to be optimal for the in situ immobilization, which routinely resulted in an immobilization of more than 70 mglipase/g-silica. Also, the optimal ratio and concentration for feed reactants in the in situ immobilization: mass ratio [NaCBH]/[lipase] of 0.3, at NaCBH and lipase concentrations of 0.75 and 2.5 g/L, respectively, were found to display the best immobilization characteristics for concentrations of up to 80 mg-lipase/g-silica, which was more than a 2-fold increase in immobilization compared to that obtained by batch immobilization. For tributyrin hydrolysis, the in situ immobilized lipase displayed lower activity per unit mass of enzyme than the batch-immobilized or free lipase, while allowing more than a $45\%$ increase in lipase activity per unit mass of silica compared to batch immobilization, because the quantity of the immobilization on silica was augmented by the in situ immobilization methodology used in this study.
Optimal conditions for the in situ immobilization of lipase in aldehyde-silica packed columns, via reductive amination, were investigated. A reactant mixture, containing lipase and sodium borohydride (NaCBH), was recirculated through an aldehyde-silica packed column, such that the covalent bonding of the lipase, via amination between the amine group of the enzyme and the aldehyde terminal of the silica, and the reduction of the resulting imine group by NaCBH, could occur inside the bed, in situ. Mobile phase conditions in the ranges of pH $7.0{\~}7.8$, temperatures between $22{\~}28^{circ}C$ and flow rates from $0.8{\~}1.5\;BV/min$ were found to be optimal for the in situ immobilization, which routinely resulted in an immobilization of more than 70 mglipase/g-silica. Also, the optimal ratio and concentration for feed reactants in the in situ immobilization: mass ratio [NaCBH]/[lipase] of 0.3, at NaCBH and lipase concentrations of 0.75 and 2.5 g/L, respectively, were found to display the best immobilization characteristics for concentrations of up to 80 mg-lipase/g-silica, which was more than a 2-fold increase in immobilization compared to that obtained by batch immobilization. For tributyrin hydrolysis, the in situ immobilized lipase displayed lower activity per unit mass of enzyme than the batch-immobilized or free lipase, while allowing more than a $45\%$ increase in lipase activity per unit mass of silica compared to batch immobilization, because the quantity of the immobilization on silica was augmented by the in situ immobilization methodology used in this study.
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제안 방법
In this study, the feasibility of an in situ immobilization method, with the immobilization of lipase taking place on a modified silica support, for the purpose of possible use in a low-pressure preparative system was investigated. Various conditions of the in situ immobilization reaction were optimized in order to increase the quantity of immobilized lipase per unit mass of silica support, These conditions included the reactant composition and concentration, pH of the mobile phase, buffer strength, temperature and flow rate.
In this study, the optimal conditions for in situ immobilization of lipase in an aldehyde-silica packed column were investigated, using sodium borohydride (NaCBH) as a reducing agent. The optimal mobile phase conditions for in situ immobilization were within the following ranges: pH 7.
대상 데이터
Porcine pancreatic lipase (Sigma L3126) which is known to have a hydrolytic activity of 30~90 units/mg for triacetin and 100-400 units/mg for olive oil [11], was used in this study. One unit of this enzyme hydrolyzes 1 μmole equivalent of fatty acids in 1 min.
The Davisil 663XWP (extraowide pore) spherical silica gel (35-75 μm diameter, 500 A pore size) was purchased from Supelco (Bellefonte, PA, USA). The reagents for the silica derivatization and lipase immobilization, such as 3- glycidoxy-propyltrimethoxysilane, toluene, trimethylamine, periodic acid and sodium cyanoborohydride (NaCBH), were all obtained from Aldrich (St. Louis, MO, USA).
이론/모형
Aldehyde-silica, which was used as a support material for the lipase immobilization, was prepared, essentially according to Larsson's method [12]. Twenty grams of dried silica was first converted to epoxy-silica, by epoxidation with 3-glycidoxypropyltrimethoxysilane (10 mL) in the presence of trimethylamine (0.
In situ immobilization, however, allowed for more than 45% more lipase activity per unit mass of silica than the batch immobilization. This enhancement in the total activity was due to an increase in the quantity of immobilization onto the silica by the in situ immobilization methodology used in this study.
성능/효과
The amount of immobilized lipase after 5 days, via batch mode method, was only about 35 mg-lipase/g-silica, even though the contact time was much longer than in the in situ mode of immobilization (data not shown). This comparison demonstrated that the in situ immobilization technique used in this study enhanced the immobilization efficiency through close and repeated contact between the enzyme and silica particles in the packed-bed environment.
참고문헌 (17)
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