Mattias Persson

Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity

The influence of the reaction conditions on the enantioselectivity of reactions catalysed by lipases or hydroxynitrile leases (HNLs) in organic solvents was investigated. The lipases catalysed kinetic resolution of chiral secondary alcohol, or chiral carboxylic acids and the HNLs catalysed asymmetric addition of hydrogen cyanide to aldehydes. The temperature effects on enantioselectivity, were studied in detail. From measurements of the enantiomeric ratio (C) at different temperatures the activation parameters DeltaDeltaH(#) and DeltaDeltaS(#) were determined. In the lipase-catalysed reactions the enthalpic and entropic effects on E always counteracted, while in a few of the HNL-catalysed reactions, DeltaDeltaH(#) and DeltaDeltaS(#) had opposite sign, and therefore the effects cooperated to give high E values (-RTInE = DeltaDeltaG(#) = DeltaDeltaH(#) - TDeltaDeltaS(#)). In all the HNL-catalysed reactions and most of the lipase-catalysed ones, the enantioselectivity increased with decreasing reaction temperature. However, in one of the lipase-catalysed reactions, the enantioselectivity decreased with decreasing temperature. The theoretical background of these observations wars discussed. In the HNL-catalysed reactions, the enantioselectivity increased with increasing water content up to water saturation, while in the lipase-catalysed reactions the opposite trend was found in one case and in the others no significant effect was observed. Solvent mixtures of diisopropylether and hexane were used to obtain solvents with different log P values. The log P value of the solvent did not influence the enantioselectivity in the HNL-catalysed reactions. while the enantioselectivity increased with increasing log P value in two of the lipase-catalysed reactions. The reaction temperature was shown to be a very useful way to influence enzyme selectivity and the effects obtained could be rationalised. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalise and predict

Water activity dependence of lipase catalysis in organic media explains successful transesterification reactions

The water activity dependence of lipase kinetics in organic media was evaluated using lipases from Rhizopus oryzae and Candida rugosa immobilised on polypropene EP-100. The conversion studied was the transesterification of ethyl decanoate to hexyl decanoate with hydrolysis to decanoic acid as competing reaction. The reactions were carried out at controlled water activity in diisopropyl ether. Substrate inhibition was observed at hexanol concentrations of 100 mM or higher. The Rhizopus lipase expressed the highest activity and the best selectivity for transesterification at the lowest water activity (a(w) = 0.06). The Candida lipase expressed the highest transesterification/hydrolysis ratio at a(w) = 0.11 and the highest total activity at a(w) = 0.53. Several glycosidases previously tested under conditions similar to those used here expressed both maximal total activity and the best selectivity at water activities close to 1.0. The water activity dependence of the lipases is thus fundamentally different from that of glycosidases and it is a major part of the reason why lipases are more suited for transferase-type reactions than the glycosidases

Factors Governing the Activity of Lyophilised and Immobilised Lipase Preparations in Organic Solvents

Active site titration and activity measurements were performed in hexane on lyophilised lipase preparations containing different amounts of phosphate buffer and lipase immobilised on porous polypropylene. Lyophilisation of Thermomyces lanuginosus lipase with large quantities of phosphate salts (200 mM) increased the specific activity fourfold, and the number of rapidly titratable active sites increased to 50 % from the 13 % observed when smaller amounts of phosphate buffer were used (20 mM) during lyophilisation. The phosphate buffer worked as an immobilisation matrix for the lipase, and the increase in specific activity was at least partly due to decreased mass transfer limitations. When lipase was immobilised on porous polypropylene, the specific activity was 770 times higher than that of the best freeze‐dried preparation. At optimal enzyme loading, 93 % of the enzyme molecules were titrated at a high rate; this indicates that this adsorption on a hydrophobic surface was a very efficient means of reducing mass transfer limitations and of immobilising the enzyme in its active conformation for use in organic solvents. The variation in specific activity with water activity was found to correlate very well with the variation in titratable active sites when lipases from Burkholderia cepacia and Thermomyces lanuginosus were immobilised on porous polypropylene. The catalytic activity per competent active site was thus constant over the whole range of water activities.

Time Diversification and Estimation Risk

The recommendation that investors with long investment horizons tilt their portfolios toward stocks is commonplace. We used a nonparametric bootstrap approach to investigate whether in a mean-variance-efficient portfolio, the weights for U.S. stocks and U.S. T-bills vary in a systematic manner with investment horizon. This approach allowed us to analyze the impact of estimation risk on the optimal weights of stocks and fixed-income securities. The results show that an investor can gain from time diversification: The weights for stocks in an efficient portfolio were significantly larger for long investment horizons than a one-year horizon. Practitioners commonly recommend that investors with long investment horizons tilt their portfolios toward stocks and away from fixed-income securities. This behavior is an important example of putting into practice the concept of time diversification, which implies that a systematic relationship exists between the portfolio weights for a particular asset class and investment horizon. We analyzed whether mean-variance-efficient portfolio weights for stocks and bills vary significantly with the investment horizon for a buy-and-hold strategy. In this analysis, we kept the risk price, the slope of the efficient frontier, constant while varying the investment horizon from 1 year to 5 years to 10 years. The data were real U.S. return data from 1900 to 1997 for a well-diversified stock portfolio and a short-term, nominally risk-free rate. We presupposed that investors form optimal investment strategies based only on historical estimates of the following parameters or inputs to the optimization problem—means, variances, and covariances. The model we used is an unconditional model in the sense that agents do not explicitly try to model any possible time-series relationships among the assets. We implicitly accounted for any possible time dependencies in the observed return-generating processes, however, by resampling a great number of return series from the original data through the use of a computer-intensive method called “bootstrapping.” In particular, we used a nonparametric moving block bootstrap with a block length of 60 months in which serial dependence and cross-sectional correlation were preserved within the blocks. The real bonus of the bootstrap approach is the possibility of generating empirical distributions of optimal weights. Thus, we could not only analyze the existence of time diversification but could also test whether time diversification is significant in a statistical sense (i.e., if significant statistical differences exist between the optimal weights for different investment horizons). With the bootstrap approach, we could also study the impact of estimation risk (meaning that the true parameters of the return distributions are unknown) on the optimal weights of stocks and bills. In a mean-variance context, estimation risk implies that the inputs to the mean-variance model are only sample estimates, not the true parameters. The results show that estimation errors increase with the risk price and with the investment horizon. The first effect is a result of error maximization, which implies that the optimization framework chooses assets with overestimated expected returns and underestimated risks. The second effect is partly a result of fewer nonoverlapping observations existing at longer investment horizons than at shorter horizons. We provide strong evidence that for all risk prices, the weights of stock in an efficient portfolio are significantly larger for the longer horizons. A tentative explanation is that for certain investment horizons, the return-generating process for stocks is mean reverting and/or the process for bills is positively autocorrelated. Because the return spread between stocks and bills is almost constant over the investment horizons, the change in portfolio weights might stem from the fact that with longer investment horizons, the standard deviation for stocks falls whereas the standard deviation for bills increases. Our evidence supports the existence of time diversification: The weights for stock in efficient portfolios are significantly higher for long investment horizons than for a one-year horizon.

Lipase-catalyzed reactions in organic and supercritical solvents: application to fat-soluble vitamin determination in milk powder and infant formula

The aim of this study was to thoroughly investigate the possibility of using enzyme catalyzed hydrolysis and alcoholysis of ester bonds in vitamin A and E esters to facilitate their determination in different food formulas. Two vitamin esters, retinyl palmitate and α-tocopheryl acetate were used as model compounds and two food formulas, milk powder and infant formula, were used as model matrices. Six lipase preparations and one esterase preparation were investigated in the solvents di-isopropyl ether, hexane/ethanol and supercritical carbon dioxide containing ethanol. Three of the enzyme preparations, lipases from Candida antarctica (Novozyme 435), Rhizomucor miehei (Lipozyme IM) and Pseudomonas cepacia, showed considerably higher activity toward retinyl palmitate than the other four enzyme preparations. There was no observed activity with α-tocopheryl acetate using any of the enzyme preparations. Novozyme 435 showed highest activity in supercritical fluid and generally larger tolerance to variations of the investigated parameters. Using this enzyme preparation in supercritical carbon dioxide containing 3 vol% ethanol and 0.03 vol% water at 366 bar and 80°C, quantitative conversion of retinyl palmitate to retinol was obtained. These conditions were then used for simultaneous lipase-catalyzed reaction and extraction of vitamin A and E from milk powder and infant formula. The developed supercritical fluid extraction method using immobilized Candida antarctica preparation seems to be more beneficial to the oxidation prone vitamins A and E compared to extraction methodologies based on alkaline saponification, resulting in comparatively higher recoveries.

Immobilisation of lipases by adsorption and deposition: high protein loading gives lower water activity optimum

Two different immobilisation techniques for lipases were investigated: adsorption on to Accurel EP-100 and deposition on to Celite. The specific activities were in the same order of magnitude, 2.9 (μmol min−1 mg protein) when Celite was used as support and 2.3 (μmol min−1 mg−1 protein) when Accurel EP-100 was used as support, even if the amount of lipase loaded differed by 2 orders of magnitude. Immobilisation on Accurel EP-100 was the preferred technique since 40–100 times more protein can be loaded/per g carrier, thus yielding a more active catalyst. The water activity profiles in lipase catalysed esterification were influenced by the amount of protein adsorbed to Accurel EP-100. Higher protein loading (40 mg g−1) resulted in a bell-shaped water activity profile with highest specific activity (6.1 μmol min−1 mg−1 protein) at aw=0.11, while an enzyme preparation with low protein loading (4 mg g−1) showed highest specific activity at aw=0.75.

Enzymatic fatty acid exchange in digalactosyldiacylglycerol.

Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) and heptadecanoic acid in toluene. Lipases from Geotrichum candidum, Alcaligenes sp. and Penicillium camembertii did not catalyse the acidolysis reaction. Rhizopus arrhizus and Rhizomucor miehei (Lipozyme) catalysed the acidolysis but produced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DGDG. The extra acyl group is bound to the primary hydroxyl of the digalactosyl moiety. Candida antarctica also catalysed the acidolysis but the TLC analysis showed bands with higher Rf values than acyl-DGDG, these probably being different tetra and higher esters. R. arrhizus lipase was the most promising enzyme under the conditions used, with no tetra esters being formed and giving the highest reaction rate of the enzymes investigated. Low water activity (0.06 or 0.11) and high fatty acid concentration (400 mM) increased the formation of acyl-DGDG whilst higher water activities (0.33 and 0.54) increased the amount of DGMG when R. arrhizus lipase was used as catalyst. At a water activity of 0.11 and a fatty acid concentration of 400 mM a yield of 24% modified DGDG was obtained. In this product the fatty acid originally present in the sn-1 position had been exchanged by heptadecanoic acid.

Enzymatic conversions of polar lipids. Principles, problems and solutions

This text provides a brief overview of the principles of enzymatic lipid conversion and some recent advances in the enzymatic conversion of glycerophospholipids and galactolipids. Lipases and phospholipases are used to exchange fatty acids or the polar group in the lipids. The reactions can be carried out either as hydrolysis-esterification sequences or as one-step transferase reactions. The scope and limitations of the different methods are discussed.

Lipase-Catalyzed Fatty Acid Exchange In Digalactosyldiacylglycerol. Improvement Of Yield Due To The Addition Of Phenylboronic Acid

Rhizopus arrhizus lipase immobilized on porous polypropylene was used in an acidolysis reaction in toluene at low water activity (0.11) to exchange the fatty acid in the sn-1-position of digalactosyldiacylglycerol (DGDG). Without extra precautions. an yield of only about 20% was obtained, mainly due to side reactions; incorporation of an extra acyl group on the primary hydroxyl of the digalactosyl moiety and acyl migration in partially deacylated reaction intermediates, followed by further hydrolysis. By the addition of phenylboronic acid (equimolar amount with respect to DGDG) both the side reactions were suppressed and the yield of DGDG containing the new fatty acid (heptadccanoic acid) in the sn-1-position increased to about 60%.