Abir B. Majumder

Designing cross-linked lipase aggregates for optimum performance as biocatalysts

Cross-linked enzyme aggregates (CLEAs) are prepared by precipitation of an enzyme and then chemical cross-linking the precipitate. Three CLEAs of lipase with glutaraldehyde concentrations of 10 mM (CLEA A), 40 mM (CLEA B) and 60 mM (CLEA C) were prepared. Studies show that there is a trade-off between thermal stability vs transesterification/hydrolysis rate vs enantioselectivity. The initial rates for transesterification of β-citronellol for the uncross-linked enzyme and CLEAs A, B and C were 243, 167, 102 and 40 µmol mg−1 h−1, respectively. Their thermal stabilities in aqueous media, as reflected by their half-life values at 55°C, were 6, 9, 13 and 16 h, respectively. The enantioselectivity, E values (for kinetic resolution of β-citronellol by transesterification) were 19, 74, 11 and 6, respectively. These results show that CLEA C was the most thermostable; the uncross-linked enzyme was best at obtaining the highest transesterification rate; and CLEA A was best suited for the enantioselective synthesis. Scanning electron microscopy (SEM) showed that the morphology of CLEA was dependent upon the extent of cross-linking.

Stabilization of Candida rugosa lipase during transacetylation with vinyl acetate

An optimally prepared Candida rugosa lipase aggregate cross-linked with bovine serum albumin, was found to overcome acetaldehyde deactivation during transacetylation of a series of benzyl alcohols with vinyl acetate. The formulation, under the same reaction conditions, exhibited 4-30x enhancement in the reaction rate as compared to the celite immobilized lyophilized formulation and 25-133x enhancement as compared to the free lyophilized enzyme depending upon the alcohol chosen. The racemic 1-phenylethanol, taken as one of the alcohols, underwent a more efficient enantioselective transacetylation giving 80% enantiomeric excess of the product, (R)-1-phenylethyl acetate, at 38% conversion (E = 15) within 24h while the enzyme immobilized on celite gave 83% enantiomeric excess at 18% conversion (E = 13) during the same period of time.

Increasing the catalytic efficiency of Candida rugosa lipase for the synthesis of tert-alkyl butyrates in low-water media

Abstract Reactions involving tert-alcohols and their esters are generally not catalyzed by lipases. Candida rugosa lipase is one of the few lipases which shows at least limited catalytic activity towards tert-alcohols and their esters. Using transesterification of tributyrin with tertiary butyl and amyl alcohols as a model reaction, it is shown that precipitation of lipase by a tertiary alcohol in the presence of a buffer with optimum concentration enhances the catalytic activity 7 fold as compared to rates obtained with lyophilized powders. Optimization of the ratio of triglyceride to tert-alcohols and medium engineering gave an initial rate which was 41 times higher than that obtained with lyophilized powders. Hence, use of a simple enzyme formulation, coupled with optimization of reaction conditions led to Candida rugosa lipase becoming a useful catalyst for catalyzing transesterification involving tertiary alcohols.

Enantioselective transacetylation of (R,S)-β-citronellol by propanol rinsed immobilized Rhizomucor miehei lipase

BackgroundUse of enzymes in low water media is now widely used for synthesis and kinetic resolution of organic compounds. The frequently used enzyme form is the freeze-dried powders. It has been shown earlier that removal of water molecules from enzyme by rinsing with n-propanol gives preparation (PREP) which show higher activity in low water media. The present work evaluates PREP of the lipase (from Rhizomucor miehei) for kinetic resolution of (R,S)-β-citronellol. The acylating agent was vinyl acetate and the reaction was carried out in solvent free media.ResultsThe PREP, with 0.75% (v/v, reaction media) water, was indeed found to be more efficient and gave 95% conversion to the ester. Using this PREP, with no added water, 90% ee for (R)-(+)-β-citronellyl acetate at 45% conversion (E = 42) was obtained in 4 h. The control with freeze-dried enzyme, with zero water content, gave 78% ee at 30% conversion (E = 13). FT-IR analysis showed that PREP had retained the α-helical content of the enzyme. On the other hand, freeze-dried enzyme showed considerable loss in the α-helical content.ConclusionThe results show that PREP may be a superior biocatalyst for enantioselective conversion by enzymes in low-water media.

Decarboxylative aldol reaction catalysed by lipases and a protease in organic co-solvent mixtures and nearly anhydrous organic solvent media

Abstract The effects of the choice of lipase, reaction medium, immobilization, presence of additives and temperature on conversion and stereoselectivity during a lipase catalysed decarboxylative aldol reaction were examined. It was shown that Candida antarctica lipase B (CALB) catalysed a decarboxylative aldol reaction between 4-nitrobenzaldehyde and ethyl acetoacetate in a 60% acetonitrile–40% aqueous buffer co-solvent mixture. Interestingly, free and immobilized forms of CALB showed opposite enantioselectivity in this media. The addition of 30 mol% imidazole increased the reaction rate from 8.5 to 55.7 μM min− 1 mg− 1. A 98% conversion could be achieved in 14 h (instead of 168 h) by adding imidazole. Other lipases also catalysed this reaction in different reaction media to a varying extent. With Mucor javanicus lipase in 30% DMSO, 20% enantiomeric excess (ee) of the (R)-product was observed. CALB also catalysed this reaction in nearly anhydrous acetonitrile. In the presence of cross-linked protein coated microcrystals of CALB, 90% conversion was obtained in this media in 24 h. A commercially available protease, alcalase, was also found to catalyse this reaction. While low water media gave poor conversion, the reaction in aqueous–60% acetonitrile co-solvent mixture gave 99% conversion in 72 h, provided imidazole was used as an additive.

Lipase-Catalyzed Condensation Reaction of 4-Nitrobenzaldehyde with Acetyl Acetone in Aqueous–Organic Cosolvent Mixtures and in Nearly Anhydrous Media

Abstract The nature of the product(s) in lipase-catalyzed reaction of acetyl acetone with 4-nitrobenzaldehyde was found to depend upon the source of lipase and the reaction medium. Mucor javanicus lipase was found to give 70% aldol with 80% enantiomeric excess in anhydrous t-amyl alcohol. A 2:2 adduct was formed by the dimerization of the aldol along with an unsaturated cyclic ether as the side products in varying proportions depending upon the reaction medium and the lipase used. [Supplementary materials are available for this article. Go to the publishers online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.] GRAPHICAL ABSTRACT