Alain Marty

Continuous enzymatic transesterification of high oleic sunflower oil in a packed bed reactor: influence of the glycerol production

Abstract The transesterification of high oleic sunflower oil with butanol by the immobilized Lipozyme® in n -hexane was carried out in a continuous packed bed reactor, oleic acid, butyl ester, and glycerol being formed as the main products. It was found that glycerol, insoluble in n -hexane, remained in the reactor adsorbed onto the enzymatic support, leading to a drastic decrease in enzymatic activity. The phenomenon involved in this loss of activity was attributed to the formation of an hydrophilic hindrance around the enzyme resulting in diffusion limitations of the hydrophobic substrate from the organic phase to the enzyme. To recover enzymatic activity, several solutions are proposed. The addition of silica gel into the enzymatic bed to adsorb the produced glycerol did not enable this loss of activity to be avoided. In order to enhance the solubility of glycerol in the reaction medium as soon as it was produced, n -hexane amended acetone was used as solvent, but high conversion of sunflower oil was not restored. Finally, by intermittent rinsing of the catalyst bed with a solution of tertiary alcohol amended with water to obtain the optimal thermodynamic water activity of 0.54, glycerol was eliminated from the reactor, and high conversion was maintained. This semi-continuous process allowed not only the synthesis of oleic acid butyl ester but also the two products to be recovered separately.

The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications.

Lipases are serine hydrolases that catalyze in nature the hydrolysis of ester bonds of long chain triacylglycerol into fatty acid and glycerol. However, in favorable thermodynamic conditions, they are also able to catalyze reactions of synthesis such as esterification or amidation. The non-conventional yeast Yarrowia lipolytica possesses 16 paralogs of genes coding for lipase. However, little information on all those paralogs has been yet obtained and only three isoenzymes, namely Lip2p, Lip7p and Lip8p have been partly characterized so far. Microarray data suggest that only a few of them could be expressed and that lipase synthesis seems to be dependent on the fatty acid or oil used as carbon source confirming the high adaptation of Y. lipolytica to hydrophobic substrate utilization. This review focuses on the biochemical characterization of those enzymes with special emphasis on the Lip2p lipase which is the isoenzyme mainly synthesized by Y. lipolytica. Crystallographic data highlight that this latter is a lipase sensu stricto with a lid covering the active site of the enzyme in its closed conformation. Recent findings on enzyme conditioning in dehydrated or liquid formulation, in enzyme immobilization by entrapment in natural polymers from either organic or mineral origins are also discussed together with long-term storage strategies. The development of various biotechnological applications in different fields such as cheese ripening, waste treatment, drug synthesis or human therapeutics is also presented.

Comparison of lipase-catalysed esterification in supercritical carbon dioxide and in n-hexane

SummaryOleic acid esterification by ethanol has been performed by an immobilized lipase fromMucor miehei in supercritical carbon dioxide and in n-hexane as solvents. In both media, determination of apparent kinetic constants has been achieved and influence of water content has been shown to be different due to various rates of water solubilities. Stability of the lipase has been proved to be correct and similar in both solvents. Inhibition by ethanol excess has been found but is greater in n-hexane. That can explain the higher initial velocities obtained in supercritical carbon dioxide for the highest ethanol concentrations.

Enzymes in non-conventional phases

The use of enzymes for technical applications has become increasingly important in different areas of biotechnology such as the food or pharmaceutical industries. Various processes have been developed using soluble or immobilized enzymes mainly in aqueous reaction phases. However, the catalytic activity of enzymes not only in water, but also in other solvents was first investigated at the beginning of the century (Bourquelot, E. and Bridel, M., 1911). Apart from water as the exclusive solvent and reaction phase, many kinds of solvent systems for enzymatic reactions became the subject of intensive research over the last 10-15 years (Buckland, Dunnill and Lilly, 1975; Klibanov, Samokhin, Martinek and Berezin, 1977; Nakanishi and Matsuno, 1986; Chen and Sih, 1989; Klibanov, 1990; Blanch, 1992; Carrea et al., 1992).

Selection of CalB immobilization method to be used in continuous oil transesterification: analysis of the economical impact.

Enzymatic transesterification of triglycerides in a continuous way is always a great challenge with a large field of applications for biodiesel, bio-lubricant, bio-surfactant, etc. productions. The lipase B from Candida antarctica (CalB) is the most appreciated enzyme because of its high activity and its non-regio-selectivity toward positions of fatty acid residues on glycerol backbone of triglycerides. Nevertheless, in the field of heterogeneous catalysis, we demonstrated that the medium hydrophilic nature of the support used for its commercial form (Lewatit VPOC1600) is a limitation. Glycerol is adsorbed onto support inducing drastic decrease in enzyme activity. Glycerol would form a hydrophilic layer around the enzyme resulting in diffusional limitations during triglyceride transfer to the enzyme. Accurel MP, a very hydrophobic macroporous polymer of propylene, was found not to adsorb glycerol. Immobilization conditions using this support were optimized. The best support was Accurel MP1001 (particle size<1000 μm) and a pre-treatment of the support with acetone instead of ethanol enables the adsorption rate and the immobilized enzyme quantity to be maximized. An economical approach (maximization of the process net present value) was expanded in order to explore the impact of immobilization on development of an industrial packed bed reactor. The crucial ratio between the quantity of lipase and the quantity of support, taking into account enzyme, support and equipped packed bed reactor costs was optimized in this sense. The biocatalyst cost was found as largely the main cost centre (2-10 times higher than the investments for the reactor vessel). In consequence, optimal conditions for immobilization were a compromise between this immobilization yield (90% of lipase immobilized), biocatalyst activity, reactor volume and total investments.

Lipases: An Overview

Lipases are ubiquitous enzymes, widespread in nature. They were first isolated from bacteria in the early nineteenth century and the associated research continuously increased due to the particular characteristics of these enzymes. This chapter reviews the main sources, structural properties, and industrial applications of these highly studied enzymes.

Use of supercritical CO2 for bone delipidation

Supercritical carbon dioxide was used for bone delipidation. It appeared that this technology is very efficient since supercritical CO2 is able to diffuse into microporous solids much better than liquids and that it has a good solvent capacity for lipids. This extraction is the ideal first step of any bone processing because microporosity of bone tissue becomes much more accessible, which may enhance osteoconduction once implanted. Moreover, it is safe since it involves no toxic chemical and is potentially usable with allografts as well as xenografts.

Efficient lipase catalysed production of a lubricant and surfactant formulation using a continuous solvent-free process.

The transesterification of sunflower oil with a high oleic acid residue content (typically 83.5%) with butanol-1 by immobilised Lipozyme was carried out in a solvent free system and in a continuous way. During the first 6 h of reactor operation, a transition phase was observed, in which the main products were butyl ester and glycerol. This latter being insoluble in the reaction mixture, it is adsorbed onto the enzyme support thus leading to a decrease in enzyme performance. Step by step, less and less glycerol is produced and finally when glycerol is no longer produced a steady state is attained. The product composition is a mixture of butyl ester (65 molar%), monoglyceride (26 molar%), diglyceride (6 molar%) and residual triglyceride (3 molar%). This mixture has interesting lubricant and surfactant properties. The reactor was maintained without any loss in activity for a period of 3 months. This result is very different to that obtained using an organic solvent (n-hexane) which leads to a total loss of enzyme activity within a few hours.

Improvement of Yarrowia lipolytica Lipase Enantioselectivity by Using Mutagenesis Targeted to the Substrate Binding Site

Enhanced enantioselectivity: The resolution of 2‐bromo‐arylacetic acid esters by Lip2p lipase from Yarrowia lipolytica was improved through mutagenesis of the substrate binding site. Position 232 was identified as crucial for the discrimination. Saturation of this position led to the identification of variant V232S, which has a tremendously increased activity and E value as compared to the parental enzyme.

Continuous lipase-catalyzed production of esters from crude high-oleic sunflower oil

The objective of this work was to develop an economically relevant enzymatic process of butyl ester production using crude high-oleic sunflower oil. Novozym 435, a non-regiospecific biocatalyst, provided the best compromise between activity and butyl-ester yield. The inhibition caused by the presence of phopholipids in crude oil was eliminated by using tert-butanol. It demonstrates the key role of the medium polarity in order to insure the stability of a process. Initial substrate concentrations and their molar ratio were optimized in a continuous packed-bed reactor to maximize product yield and productivity. The best compromise was obtained for an initial oil concentration of 500 mM and a molar ratio of 5. It enabled a high productivity of 13.8 tons year(-1)kg Novozym 435(-1) with a butyl-ester purity of 96.5% to be obtained. Experiments with the continuous reactor were performed over 50 days without any loss of enzyme activity.