Jean Graille

Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches

Lipases (triacylglycerol ester hydrolases, EC 3.1.1.3) are ubiquitous enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol. Besides this, they are also efficient in various reactions such as esterification, transesterification and aminolysis in organic solvents. Therefore, those enzymes are nowadays extensively studied for their potential industrial applications. Examples in the literature are numerous concerning their use in different fields such as resolution of racemic mixtures, synthesis of new surfactants and pharmaceuticals, oils and fats bioconversion and detergency applications. However, the drawbacks of the extensive use of lipases (and biocatalysts in general) compared to classical chemical catalysts can be found in the relatively low stability of enzyme in their native state as well as their prohibitive cost. Consequently, there is a great interest in methods trying to develop competitive biocatalysts for industrial applications by improvement of their catalytic properties such as activity, stability (pH or temperature range) or recycling capacity. Such improvement can be carried out by chemical, physical or genetical modifications of the native enzyme. The present review will survey the different procedures that have been developed to enhance the properties of lipases. It will first focus on the physical modifications of the biocatalysts by adsorption on a carrier material, entrapment or microencapsulation. Chemical modifications and methods such as modification of amino acids residues, covalent coupling to a water-insoluble material, or formation of cross-linked lipase matrix, will also be reviewed. Finally, new and promising methods of lipases modifications by genetic engineering will be discussed.

Esterification of phenolic acids from green coffee with an immobilized lipase from Candida antarctica in solvent-free medium

Phenolic acid esters were synthesized by direct esterification catalysed by an immobilized lipase obtained from Candida antarctica. Esterification yields ranged from 3 to 98%; yields seem to be linked to the electronic distribution of phenolic acids which affects the reactivity of the carboxylic function. The carbon chain length of the alcohols also plays an important role. It was observed that even for high yields, reaction times were two days or more.

Fructose oleate synthesis in a fixed catalyst bed reactor

SummaryFructose oleate undergoes continuous synthesis in a fixed catalyst bed reactor using an industrial fixed lipase. The effects of the time spent in the reactor, substrate concentration and effluent recycling are studied. A yield of 83% is obtained by recycling the effluent 3 times.

Acylation over cation-exchanged montmorillonite

The Friedel-Crafts acylation of aromatic compounds (benzene, toluene, xylene) with carboxylic acids (CH3(CH2)n COOH, n = 0–14) was performed over cation-exchanged montmorillonites (H+, Al3+, Ni2+, Zr4+, Ce3+, Cu2+, La3+). Yields in ketones were found to be dependent upon the nature of the interlayer cation and on the acid chain length.

Effect of water activity on enzymatic synthesis of alkylglycosides

Water activity (aw) was studied during biocatalyzed alkylglycoside synthesis using defated raw almond meal displaying a good β glucosidasic activity. Optimum aw were determined for several alcohols used for the alkylation of the sugar. Above and below optimum aw, yield decreased rapidly.

Carica papaya latex lipase : sn-3 stereoselectivity or short-chain selectivity ? Model chiral triglycerides are removing the ambiguity

Short-chain fatty acids are usually located at positionsn-3 in natural triglycerides, particulary in dairy fats. As a result, it is extremely difficult to differentiate betweensn-3 stereospecificity and short-chain typoselectivity in many lipases and acyltransferases that perform in this way. This ambiguity can be removed through successive use of a chiral triglyceride with a short fatty acid in positionsn-1 and of its racemic in controlled hydrolysis reactions. After checking that the proposed method effectively confirmed the type of activity of control biocatalysts (Candida cylindracea nonspecific lipase andMucor miehei 1–3 regiospecific lipase), we confirmed thatCarica papaya latex has a strictsn-3 stereospecificity.

Purification and properties of the lipase fromCandida deformans (zach) langeron and guerra

Palm oil being solid at room temperature could be converted into a fluid oil by substitution of about 40–50% of its palmitic acid. This could be achieved by a fermentation process or using yeast lipase.Candida deformans CBS 2071 seemed suitable for this purpose: therefore, its lipase was isolated and studied. This enzyme was purified by acetone precipitation followed by chromatographies on Sephadex C 50 and Sephadex G 150. The purification factor achieved was ×70, and the protein and activity yields were 0.25% and 18%, respectively. The homogeneity of the purified enzyme was verified by polyacrylamide gel electrophoresis. The enzyme molecular weight was estimated at 207,000. Its activity is optimal between 40 C and 50 C and its optimum pH is 7.0. This enzyme is thermo-resistant and loses only 14% and 18% of its activity, respectively, when heated to 45 C and 55 C for 30 min. Its activation energy was 2.75 kcal.mole−1 and its inactivation energy was around 21 kcal.mole−1. This enzyme is activated by Ca++, Mg++ and Co++ and inhibited by Cu++, Zn++, and p-chloromercuribenzoate (pCMB) and EDTA.The synthesis of this lipase is induced by lipid substrates in the culture medium and inhibited by glucose. This enzyme attacks primarily the 1- (or 3-) position of all triglycerides tested. Hydrolysis was preferential for triglycerides containing short chain fatty acids. The triglycerides with monounsaturated monoacids were more quickly hydrolyzed, than those with saturated monoacids. The presence of two and especially three double bonds in the fatty acid chain seemed to slow down the rate of hydrolysis.

Towards an efficient membrane based vegetable oils refining

Abstract The diversification of the non food uses of vegetable oils and the need for softer processing demands new oil refining processes. Taking advantage of the membrane technology for vegetable oil refining, the preliminary results reported here concern a single dead-end or crossflow microfiltration step. The influence of experimental parameters (temperature, pore size, pressure …) on the flow rate of permeate and on the free fatty acids (FFA) (as soap) and phosphorus retention ratios is discussed. After addition of sodium hydroxide followed by dead-end filtration the quality of the oil meets the quality standard but the main drawback is the very poor flow rate as a consequence of the rapid fouling of membranes. Cross-flow filtration allows a higher flow rate of permeate which shows a slow decrease after operating for several hours, with only a slight loss of quality of the processed oil compared to dead-end filtration. From this preliminary study, the cross-flow microfiltration gives promising results for vegetable oils refining. This is the first report of simultaneous reduction of phosphorus, free fatty acids and water concentrations after a solvent free single microfiltration step.

A study of the influence of the growth media on the fatty acid composition in Candida lipolytica Diddens and Lodder

Candida lipolytica YB 423-12 is able to incorporate fatty acid from the culture medium when lipids are used as carbon substrate. The composition of cell lipids is largely dependent on that of the culture medium. An important Δ 9 desaturase activity acts on incorporated palmitic and stearic acids; and Δ 11-eicosenoic and erucic acids are shortened to oleic acid.

Specificity of #Carica papaya# latex in lipase-catalyzed interesterification reactions

Enzymatic interesterification of the chiral triacylglycerol, 1-butyroyl-2-stearoyl-3-palmitoyl-sn-glycerol (sn-BSP) with trimyristin indicated that the lipase present in Carica papaya latex exhibits an sn3 stereoselectivity. Other interesterification experiments with homogeneous triacylglycerols of varying chain length with tricaprylin showed that this enzyme also has a typoselectivity for short chain fatty acids.