Immobilization of Lipase from Candida Rugosa onto Selected Matrices for Use in Enantioselective Preparation of (-)-Menthyl Butyrate
Products of pure enantiomer are in great demand and have been receiving attentions since the past few decades. Reasons which make these products of interest are their efficacies and specificities compared to the racemic mixture or the other enantiomer, which is often inactive or exhibit toxicity....
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Format: | Thesis |
Language: | English English |
Published: |
2004
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Online Access: | http://psasir.upm.edu.my/id/eprint/296/1/549607_FS_2004_8.pdf |
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Summary: | Products of pure enantiomer are in great demand and have been receiving
attentions since the past few decades. Reasons which make these products
of interest are their efficacies and specificities compared to the racemic
mixture or the other enantiomer, which is often inactive or exhibit toxicity. In
recent years, enzymes are finding increasing acceptance as catalysts in the
synthesis of these enantiomerically pure products due to their high
enantioselectivity, commercial availability and environmental friendly
reactions.
In this study, among seven lipases tested, Candida rugosa lipase exhibited
the best ability to catalyze the resolution of (±)-menthol in organic solvent.
The lipase was immobilized on different carriers including a newly
synthesized Mg/Al-hydrotalcite (HT), a kind of layered double hydroxide
material prepared at molar ratio 4 of Mg: Al. Method of immobilization used was a simple physical adsorption method. Lipase immobilized on Amberlite
XAD7, Eupergit C, Eupergit C 250 L and the newly synthesized hydrotalcite,
exhibited highest enantioselectivity and activity, and was chosen for further
characterization.
Immobilization of lipase onto these supports was confirmed using a Scanning
Electron Microscope (SEM). Native lipase was not easily dispersed and
suffers dehydration of cell as a result of lyophilization. For the immobilized
lipases, direct lipase-support interaction can be clearly viewed.
Experiment was carried out in different organic solvents and at different water
activities (aw). High yield (60 %) and optical purity (> 90 %) of the product
was obtained in hexane with butyric anhydride as the acyl donor at 30 oC
using Candida rugosa lipase immobilized on Amberlite XAD7. Yield of (-)-
menthyl butyrate were found to be dependant on the water activity (aw = 0.33
– 0.53). Highest yield of (-)-menthyl butyrate (66 %) was achieved using
Amberlite XAD7-lipase as catalyst after 60 hours of continuous reaction while
highest production of (-)-menthyl butyrate using other immobilized lipases (35
– 60 %) was achieved after about 24 – 28 hours of incubation.
The effects of various temperatures, storage conditions, stability in organic
solvents and lipase reusability were investigated for their influence on the
enzymatic enantioselective formation of (±)-menthyl butyrates. The
immobilized lipases retained high catalytic activity (53 – 83 %) and show
increased stability (2 – 3 folds) compared to the native lipase in all cases.Although lipase activity decreased as temperature is increased, immobilized
lipase especially Eupergit C-lipase was least affected and showed highest
activity (83 %) even upon incubation at 70 oC.
During storage at different temperatures for 60 days, all lipases retained 100
% of their initial activity when stored at –20 oC. Their activities were found to
decrease gradually as temperature was raised. When storage temperatures
were raised to 0 and 4 oC, immobilized lipases exhibited 1.6 – 2.4 folds
higher catalytic activities (62 – 87 %) than native lipase (35 – 39 %).
Immobilized lipases showed slightly higher activities of 1 – 2 folds to the
native lipase upon storage at room temperature (26.5 oC) for 60 days.
In this study, interestingly, immobilized lipases were still active (relative
activities of 46 – 61 %) even after ten days of incubation in hexane. In
addition, their activities showed low effect, with 24 - 49 % decline in relative
activities even after 5 cycles of washing with 20 mL of hexane. Furthermore,
the immobilized lipases showed possible and efficient reuse with HT-lipase
exhibiting excellent half life (t½) of 16 days. Other immobilized lipases,
however, showed lower half-life, t½ = 3.5 for Amberlite XAD7-lipase and t½ =
9.0 for Eupergit C 250 L-lipase and Eupergit C-lipase. |
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