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.

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Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.

An analytical procedure was developed for regiodistribution analysis of TAG using alpha-MAG prepared by an ethyl magnesium bromide deacylation. In the present communication, the deacylation procedure is shown to lead to representative alpha-MAG, allowing the composition of the native TAG in the alpha-position to be determined directly. The composition in the beta-position can then be estimated from the composition of the alpha-MAG and TAG according to the formula 3 x TAG - 2 x alpha-MAG. The estimates are superior to those obtained using the alpha,beta-DAG and Brockerhoff calculations as they come closer to the theoretical value and have smaller SD. The present procedure, first demonstrated on a synthetic TAG, was then successfully applied to the analysis of borage oil, milkfat, and tuna oil.

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The unripe fruit of babaco (Vasconcellea x heilbornii; syn. Carica pentagona) contains a latex, similar to that in Carica papaya, which exhibits lipolytic activity. Herein, the regioselectivity, stereoselectivity and typoselectivity in both hydrolysis and acyltransfer reactions of babaco latex lipases were studied and compared to those of Carica papaya latex. In hydrolysis, both biocatalysts are 1,3-regioselective with ratios for 1,2-2,3-diacylglycerols/1,3-diacylglycerol of 6.5 and 21 for babaco and papaya, respectively. In contrast, papaya latex had a slight sn-3 stereopreference. Babaco latex displayed a higher activity on triacylglycerols with short chain and unsaturated fatty acids.

We have examined the possibility of producing analogs of medium-chain triglycerides (MCT) from copra oil, i.e. a triacylglycerol mixture with a high content of medium-chain fatty acid moieties (C 6 -C 10 ). A two-step enzymatic process was used in which copra triacylglycerols were first split with papain lipase by alcoholysis with an alkyl alcohol and then subjected to interesterification with the alkyl esters recovered using papain lipase. Effects of temperature, water activity/content, substrate ratio, biocatalyst amount, and alcohol chain length were also investigated. On the one hand, the sn-3 stereoselectivity of the lipase in the alcoholysis of copra oil with butanol has permitted a direct enrichment of caproic, caprylic and capric moieties in the synthesized butyl esters. Thus, in the batch reactor, the reaction led to about 31% conversion of the oil after 24 h, and the content of C 6 -C 10 acids in the synthesized esters increased from about 16% in the starting oil to almost 42%. A similar enzymatic alcoholysis in a packed-bed column bioreactor gave 31% conversion of the oil after 120 min of reactor residence time. The reaction was also very selective because the C 6 -C 10 fatty acyl groups represented about half of the newly formed butyl esters, whereas they accounted for only 16% of total fatty acids in the starting oil. On the other hand, the transesterification of the alkyl esters recovered (highly enriched in C 6 -C 10 fatty acyl groups) with native copra oil directly led to an increase in the content of MCT in the oil, from 18 mol-% at the beginning of the reaction to 61 mol-% of MCT after a time period of 72 h in the batch reactor.