Soundar Divakar

Enzymatic synthesis of isoamyl acetate using immobilized lipase from Rhizomucor miehei

The effects of important reaction parameters for enhancing isoamyl acetate formation through lipase-catalyzed esterification of isoamyl alcohol were investigated in this study. Increase in substrate (acid) concentration led to decrease in conversions. A critical enzyme concentration of 3 g l(-1) was detected for a substrate concentration of 0.06 M (each of alcohol and acid). Solvents with partition coefficient higher than 1000 (log P>3.0) supported enzyme activity to give high conversions. Acetic acid at higher concentrations could not be esterified easily probably owing to its role in lowering the microaqueous pH of the enzyme. Extraneous water/buffer addition decreased the isoamyl acetate yields slightly ( approximately 10%) at 0.005-0.01% v/v of the reaction mixture and drastically (>40%) at above 0.01% v/v. Buffer saturation of the organic solvent employed improved esterification (upto two-fold), particularly at moderately higher substrate concentrations (>0.18 M). Employing acetic anhydride instead of acetic acid resulted in a two-fold increase in the yields (at 0.25 M substrate). Use of excess nucleophile (alcohol) concentration by increasing the alcohol/acid molar ratio resulted in higher conversions in shorter duration (upto eight-fold even at 1.5 M acetic acid). Yields above 80% were achieved with substrate concentrations as high as 1.5 M and more than 150 g l(-1) isoamyl acetate concentrations were obtained employing a relatively low enzyme concentration of 10 g l(-1). The operational stability of lipase was also observed to be reasonably high enabling ten reuses of the biocatalyst.

Response surface methodological approach for the synthesis of isobutyl isobutyrate

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was used for the synthesis of isobutyl isobutyrate by direct esterification of isobutyric acid and isobutyl alcohol. Response surface methodology based on a five-variable central composite rotatable design was used to determine the effect of solvent (log P ranging from 0.49 to 4.5), acid concentration (0.05–0.25 M), alcohol to acid ratio (1.25:1), enzyme concentration (25–225 mg), incubation period (24–120 h) and incubation temperature (30–70°C) on the esterification reaction. The extent of esterification was good at all acid concentrations employed in the range 0.05–0.25 M with increase in enzyme concentration. Even with a low enzyme concentration of 25 mg, 50% conversion was observed. The enzyme was active at all the temperatures ranging from 30 to 70°C even up to an incubation period of 120 h. The results obtained show that the yields were higher in solvents having log P>2, i.e., when solvents of higher non-polarity were employed. Optimum conditions for predicting maximum ester yield were 200 mM using 0.2 M, isobutyric acid; 0.2 M, isobutanol; enzyme concentration, 225 mg; incubation period, 72 h and hexane (log P=3.5) as reaction media at reaction temperature, 70°C. Under the above mentioned conditions, the experimental yield was 195 mM, which is well matched with the predictive yield.

Optimization of isoamyl acetate production by using immobilized lipase from Mucor miehei by response surface methodology.

Immobilized lipase from Mucor miehei was employed for the esterification of isoamyl alcohol with acetic acid in n-heptane solvent. The important process variables studied were enzyme/substrate (E/S) ratio, alcohol (acid) concentration, and incubation period. Based on Box-Behnken design of experiments, a second order response function was developed. The percentage esterification increased with both E/S ratio and time and decreased with alcohol (acid) concentration. The model indicated optimum conditions for maximum esterification ranging from 20 to 99.6% in the alcohol (acid) concentration range of 0.031 to 0.3 M for a range of E/S ratios 8.33 to 50 g/mol, which were in good agreement with the experimental yields.

An esterification method for determination of lipase activity

The choice of a lipase for an esterification reaction can be determined from the esterification reaction between butyric acid (0.16 M) and butanol (0.33 M) at 50 °C and agitated with 10 mg lipase. The decrease in butyric acid is measured by titration and 1 unit of lipase activity is defined as 1 μmol butyric acid consumed per min per mg lipase.

Applications of surface plots and statistical designs to selected lipase catalysed esterification reactions

Abstract The usefulness of several statistical designs in experimental optimisation including Box-Behnken, Central Composite Rotatable and Plackett–Burman designs in lipase catalysed esterification reactions is presented. Analyses of several response surface plots obtained by employing statistical designs in lipase catalysed esterification reactions have indicated that such plots could be grouped into four types to explain esterification behaviour in the presence of different kinds of substrates and reaction conditions. Similarly, a Plackett–Burman design helps in the selection of the most probable organic acid or alcohol in a mixture, for a facile esterification reaction. Predominant acid binding in preference to alcohol gives rise to dome-shaped response surface plots. Predominant alcohol binding in preference to acid give rise to inverted dome-shaped plots. The competitive and inhibitory nature of substrates are brought out clearly in response surface plots. Rate plots show linear relationships between time and substrate with smooth surfaces within the limits of the variables employed. There are other uneven plots, which reflect the effect of variables like buffer pH, buffer volume and temperature on the activity of lipase employed. The stability of lipases under drastic esterification conditions of temperature and solvents was also studied.

Preparation of stearoyl lactic acid ester catalyzed by lipases from Rhizomucor miehei and porcine pancreas optimization using response surface methodology

Abstract The esterification reaction between stearic acid and lactic acid using Rhizomucor miehei lipase and porcine pancreas lipase was optimized for maximum esterification using response surface methodology. The formation of the ester was found to depend on three parameters namely enzyme/substrate ratio, lactic acid (stearic acid) concentration and incubation period. The maximum esterification predicted by theoretical equations for both lipases matched well with the observed experimental values. In the case of R. miehei lipase, stearoyl lactic acid ester formation was found to increase with incubation period and lactic acid (stearic acid) concentrations with maximum esterification of 26.9% at an enzyme/substrate (E/S) ratio of 125 g mol−1. In the case of porcine pancreas lipase, esterification showed a steady increase with increase in incubation period and lactic acid (stearic acid) concentration independent of the E/S ratios employed. In the case of PPL, a maximum esterification of 18.9% was observed at an E/S ratio of 25 g mol−1 at a lactic acid (stearic acid) concentration of 0.09 M after an incubation period of 72 h.

A central composite rotatable design analysis of lipase catalyzed synthesis of lauroyl lactic acid at bench-scale level

Abstract Porcine Pancreas lipase (PPL) was employed for the reaction between lactic acid and lauric acid at bench-scale level. A Response Surface Methodological (RSM) study was conducted by employing a five-level, five variable, central composite rotatable design (CCRD) in order to understand the esterification behavior of PPL in the lauroyl lactic acid synthesis. Five important variables were considered, namely, enzyme/substrate (E/S) ratio (0.09–1.14 Activity Units/mmole - AU/mmol), lactic acid concentration (5–25 mmol), incubation period (6–54 h), buffer volume (0–0.2 ml) and buffer pH Values (4.0–8.0). Highest ester yield of 6.8 mmol was predicted at the lowest E/S ratio of 0.09 AU/mmol. Lower E/S ratios gave higher yields and higher E/S ratios gave lesser yields. This behavior clearly explained the competitive nature of binding between lauric and lactic acids for the same binding site on the enzyme. Addition of buffer in terms of both volume and pH did not have a profound effect on increase in ester yield. Predicted yields showed good validation with experimental yields when experiments corresponding to selected points on the contour plots were carried out.

Structural Studies on Inclusion Compounds of β-Cyclodextrin with Some Substituted Phenols

A detailed structural study of the inclusion compounds of some substituted phenols such as catechol, guaiacol, protocatechuic aldehyde, vanillin, caffeic acid, ferulic acid and eugenol with β-cyclodextrin (βCD) was carried out by using UV-visible, fluorescence, 1H and solid-state 13C NMR spectroscopic and potentiometric investigations. Based on these studies guaiacol, catechol and eugenol were found to exhibit identical orientations - with the phenyl ring within the cavity and the hydroxyl and methoxyl groups projecting outside; protocatechuic aldehyde, caffeic acid, ferulic acid and vanillin display a different orientation - with the phenol part within the cavity and the aldehyde or carboxyl part projecting outside.

Inclusion of Ring A of Cholesterol Inside the β-Cyclodextrin Cavity: Evidence from Oxidation Reactions and Structural Studies

The disposition of cholesterol inside the β-cyclodextrin cavity(β-CD) was deduced from oxidation of cholesterol secondary alcoholgroups by Ca(OCl)2 and H2O2 in thepyridine–acetic acid system. The amount of cholest-4-ene-3-one formedwas found to be proportional to the concentration of β-cyclodextrin,resulting in 56.1% of ketone. The oxidation rate was enhanced byβ-cyclodextrin and its methyl, polymer and 1 : 1copper(II)–β-cyclodextrin derivatives. Detailed investigationsinvolving UV-visible, 13C- and 1H-NMR(T1, 1D NOE and ROESY) spectroscopic studies were carried out.A binding constant value of 15,385 ± 1500 M-2 wasobtained for the 2 : 1heptakis-2,6-di-O-methyl-β-cyclodextrin(DMβ-CD) : cholesterolcomplex in chloroform from UV studies. Proton and solid state13C-CP MAS spectra of the β-CD–cholesterol mixtureshowed large magnitude shifts for the protons from the wider end of theβ-CD cavity as well as those of ring A and ring B of cholesterol. Both1D NOE and ROESY measurements indicated the proximity between ring A andring B protons of cholesterol and the wider end protons of β-CD andDMβ-CD. Besides, analysis of τc,τi and tau;m from T1measurements showed not only a lowering of rotational motions but a ξvalue of 0.016–0.048 for some of the cholesterol protons, typical of aweak complex. Based on these studies, a probable structure for the 2 : 1complex involving two molecules of β-CD/DMβ-CD was proposed withportions of ring A and ring B being present inside the wider end of theβ-CD/DMβ-CD cavity and ring D and the side chain attached atposition 17, projecting into the wider end of the secondβCD/DMβ-CD molecule.

Thermostability of porcine pancreas lipase in non-aqueous media

Abstract Thermostability of Porcine pancreas lipase (PPL) was studied by monitoring esterification activity and A 290 values by ultra-violet (UV) spectroscopy, in the temperature range 40–80°C for incubation periods up to 10 days through Response Surface Analysis. This novel approach to the problem generated information on the behaviour of PPL under different temperatures for longer periods of incubations, which is essential for employment of lipase in esterification reactions which are time consuming and requires higher temperatures and non-polar solvents. Native PPL, PPL in buffer saturated methylisobutyl ketone (MIBK) and PPL in 0.2 M lactic acid in MIBK were subjected to response surface methodological analyses. Native enzyme showed loss of activity at 60°C probably due to a conformation with a greater unfolding at 60°C than at 40 and 80°C. Longer periods of incubation of PPL at especially 80°C did not affect the active conformation of PPL even after incubation for a period upto 10 days. However, the presence of small amounts of buffer stabilizes the enzyme at 60°C contrary to what was observed with the native enzyme. In the presence of 0.2 M lactic acid, there was a general loss in active conformation and hence activity at all temperatures and periods studied. The results from activity measurements were supported by UV spectroscopic data of the same in every respect indicating that variation in conformational changes is responsible for loss in activity.