Suzana Ferreira-Dias

Contribution of response surface design to the development of glycerolysis systems catalyzed by commercial immobilized lipases

Abstract Two commercial immobilized lipases (“Lipozyme® IM” and “Novozym® 435”) were tested as biocatalysts for the glycerolysis of olive residue oil in n -hexane aimed at the production of monoglycerides (MG) and diglycerides (DG). A central composite rotatable design (CCRD) was followed to model and optimize glycerolysis as a function of both the amount of biocatalyst ( L ) and of the molar ratio glycerol/triglycerides (Gly/TG). For both biocatalysts, the production of free fatty acids (FFA) was described by second order models. In terms of MG and DG production, as well as of TG conversion, the best fits were obtained with first-order models. The highest MG productions were in the range 43–45% (w/w, on the basis of total fat) for both biocatalysts tested at a (Gly/TG) ratio of one. In the case of “Novozym 435”, the lowest load used (12%, w/w) gave the best results, in contrast with “Lipozyme IM” with which a concentration of about 26% (w/w) was necessary to obtain the highest production. Under these conditions, the amount of FFA produced was about 2% and 10% (w/w), respectively, for “Novozym 435” and “Lipozyme IM” catalyzed systems. Considering both FFA production and lipase loading, “Novozym 435” was shown to be a better biocatalyst for the glycerolysis of olive residue oil in n -hexane, aimed at the production of MG, than “Lipozyme IM”.

Response surface modelling of the production of ω-3 polyunsaturated fatty acids-enriched fats by a commercial immobilized lipase

Abstract The aim of this study was to model the production of fats, enriched with ω-3 polyunsaturated fatty acids (ω-3 PUFA) for nutraceutical purposes, via the response surface methodology. These fats were obtained by transesterification of palm oil stearin (POS) with a concentrate (EPAX 2050TG) of triglycerides enriched with ω-3 PUFA and soybean oil, catalysed by a commercial immobilized Candida antarctica lipase (“Novozym 435”). The initial water activity ( a w ) of the biocatalyst, POS and EPAX 2050TG concentrations, time and temperature showed a significant effect on the transesterification reaction, as well as on the competing reactions of hydrolysis and lipid oxidation. Depending on the factors included, the transesterification reaction was described either by first- or second-order models. The production of free fatty acids, which is ascribed both to the hydrolytic reaction and the mechanism of lipase-catalysed transesterification, showed a second-order dependence on the initial a w of the biocatalyst.

Production of monoglycerides by glycerolysis of olive oil with immobilized lipases: effect of the water activity

The production of monoglycerides by glycerolysis of olive oil catalyzed by lipases from Candida rugosa, Chromobacterium viscosum and Rhizopus sp. immobilized in a hydrophylic polyurethane foam was investigated. The effect of the amount of aqueous phase used for foam polymerization on the competing reactions of glycerolysis and hydrolysis was studied. The highest monoglyceride production was achieved with the C. rugosa lipase which was thus selected for subsequent studies.The extent to which hydrolysis and glycerolysis occur and the influence of the initial aw of the system on both reactions were also investigated. In glycerolytic reaction systems, initial rates of fatty acid release were always higher than in hydrolytic systems. At aw values lower than 0.43, hydrolysis was completely repressed, although glycerolysis still occurred. This suggests that hydrolysis of the ester bond in the glyceride, promoted by glycerol, is the first reaction step.In glycerolysis, initial rates of FFA and DG production increased exponentially with the initial aw of the system.The lowest total conversion (in terms of % TG consumed) at 48 hours was obtained at intermediate aw values; higher conversions at extreme aw indicated an increase in hydrolytic and glycerolytic rates, at high and low aw respectively.The yield of MG increased with decreasing aw. The highest yield of MG (∼70%, w/w) was obtained at the lowest aw used (0.23). The initial aw of the reaction system is an important parameter in glycerolysis.

Production of olive oil enriched with medium chain fatty acids catalysed by commercial immobilised lipases.

Structured triacylglycerols, containing medium chain fatty acids, were produced by acidolysis of virgin olive oil with caprylic or capric acid, at a molar ratio of olive oil:fatty acid of 1:2, at 45°C for 24h, in solvent-free media or in n-hexane, catalysed by Thermomyces lanuginosa (Lipozyme TL IM), Rhizomucor miehei (Lipozyme RM IM) and Candida antarctica (Novozym 435) immobilised lipases. Incorporations were always greater for capric than for caprylic acid. For both acids, higher incorporations were always attained in solvent-free media: the highest caprylic acid incorporations were obtained with Novozym 435 (25.5mol%) and Lipozyme RM IM (25.7mol%), while similar capric acid incorporations were obtained with all biocatalysts (27.1-30.4mol%). After 10 repeated uses of Lipozyme RM IM, the same incorporation level of capric acid was obtained at the end of each 23h batch. However, with caprylic acid, a first-order deactivation was observed (half-life time, t1/2=299h). During acidolysis with both acids, Novozym 435 (t1/2=217h, for caprylic, t1/2=225h, for capric acid) and Lipozyme TL IM (t1/2=50.4h, for caprylic; t1/2=47.2h, for capric acid) presented first-order deactivation. All biocatalysts presented 1,3-regioselectivity.

A comprehensive study of reaction parameters in the enzymatic production of novel biofuels integrating glycerol into their composition

A comprehensive study of critical parameters in the pig pancreatic lipase (PPL) catalysed transesterification of sunflower oil to novel biofuels integrating glycerol into their composition is reported. The influence of oil/alcohol ratio, temperature, quantity of enzyme and water added and pH have been investigated. The enzymatic activity of PPL was found to be greatly influenced by the pH, reaching notable activities at high pH values (10-12), in contrast to other lipases. The addition of small quantities of NaOH (up to 0.1 mL) as coadjuvant in the transesterification reaction enhances the activity of the enzymes. This remarkable behaviour, reported for the first time, may pave the way for the utilisation of these relatively cheap enzymes in large scale commercial biodiesel production. Besides, a novel biofuels containing glycerol into their composition as mono- and diglycerides using PPL as biocatalyst has been developed.

Activity and batch operational stability of Candida rugosa lipase immobilized in different hydrophilic polyurethane foams during hydrolysis in a biphasic medium

Abstract The aim of this study was to investigate eventual relationships between some physico-chemical properties (e.g. porosity, aquaphilicity, partition coefficient for oleic acid and drying curves) of relatively hydrophilic polyurethane foams and the activity and batch operational stabiliy of Candida rugosa lipase immobilized within these foams. Two biocompatible polyurethane pre-polymers (“HYPOL FHP 2002TM” and “Hypol FHP X4300TM” from Hampshire Chemical GmbH, Germany) were tested as immobilization supports. The model reaction was the hydrolysis of crude olive residue oil in a biphasic aqueous/n-hexane medium.Drying curves under normal and reduced pressures suggested that water molecules are more strongly bound to the “FHP 2002” than to “FHP X4300” foams. This is in agreement with the higher aquaphilicity value estimated for the “FHP 2002” foam (3.7 vs 2.8).For every enzyme loading tested, hydrolysis efficiency was considerably higher for the lipase in “FHP X4300” foam when compared to the other counterpart. However, internal mass transfer limitations seem to be more severe with “FHP X4300” foams.Operational stability was evaluated in 10 consecutive batches (1 batch = 23 hours) for both immobilized preparations. A fast deactivation was observed for both biocatalysts. However, a slightly higher operational stability was observed for the lipase in “FHP 2002” foam. For the lipase in “FHP X4300” foam, the activity decay can be explained by a dramatic lipase leakage from the foam observed along successive batches. For the lipase in “FHP 2002” foam, no significant enzyme loss was observed along the reutilizations probably due to a higher number of multi-point attachment between the lipase and its support.In fact, activity and operational stability of Candida rugosa lipase in “FHP 2002” and “FHP X4300” foams appear to be related with the strength and/or the number of covalent binding between the enzyme and the support rather than to the physico-chemical properties evaluated in this work.

Biodiesel production from crude Jatropha oil catalyzed by non-commercial immobilized heterologous Rhizopus oryzae and Carica papaya lipases.

The aim of this study was to evaluate the feasibility of biodiesel production by transesterification of Jatropha oil with methanol, catalyzed by non-commercial sn-1,3-regioselective lipases. Using these lipases, fatty acid methyl esters (FAME) and monoacylglycerols are produced, avoiding the formation of glycerol as byproduct. Heterologous Rhizopus oryzae lipase (rROL) immobilized on different synthetic resins and Carica papaya lipase (rCPL) immobilized on Lewatit VP OC 1600 were tested. Reactions were performed at 30°C, with seven stepwise methanol additions. For all biocatalysts, 51-65% FAME (theoretical maximum=67%, w/w) was obtained after 4h transesterification. Stability tests were performed in 8 or 10 successive 4h-batches, either with or without rehydration of the biocatalyst between each two consecutive batches. Activity loss was much faster when biocatalysts were rehydrated. For rROL, half-life times varied from 16 to 579h. rROL on Lewatit VPOC 1600 was more stable than for rCPL on the same support.

Genetic characterization of Listeria monocytogenes food isolates and pathogenic potential within serovars 1/2a and 1/2b.

A total of 39 Listeria monocytogenes strains isolated from raw milk, smoked meat, chicken carcass and reference strains, belonging to serovars 1/2a, 4a, 1/2b, 3b and 4b, were analysed by RAPD and by polymorphisms of the virulent genes inlAB and iap. Ten isolates, belonging to serovars 1/2a and 1/2b and, collected from raw milk and smoked meat, were further tested for pathogenicity by IP injection into mice. The clustering of the 39 L. monocytogenes strains in 3 groups at 0.45 similarity level, based on molecular typing, was observed. Distribution of serovars in these clusters was in agreement with the proposed three Listeria monocytogenes lineages. Within serovar 1/2b, the 50% lethal dose (LD50) ranged from 8.4 x 10(4) to 1.7 x 10(6) cfu.ml(-1). One of the serovar 1/2b strains, isolated from smoked meat, exhibited the lowest virulence potential evaluated by LD50 and by mean time to death (MTD) and, from this point of view, was completely different from the other strains. Our results suggest the existence of heterogeneity in virulence levels within serovars 1/2a and 1/2b. However, when comparing the isolates based on genotyping, virulence indicators and food origin, no relation could be assessed.

Enzymatic glycerolysis of olive oil: A reactional system with major analytical problems

Major analytical problems were encountered while carrying out the lipase catalyzed glycerolysis of olive oil in n-hexane. Since direct quantification of monoglycerides could not be achieved, an alternative methodology is proposed: the estimation of monoglyceride production from a mass balance after having assayed for the unconverted triolein, as well as for the diolein and free fatty acids formed.

Glycerolysis of olive oil: batch operational stability of Candida rugosa lipase immobilized in hydrophilic polyurethane foams

The operational stability of the Candida rugosa lipase immobilized in a hydrophilic polyurethane foam was evaluated in consecutive batches for the glycerolysis of olive oil in n-hexane, aimed at the production of monoglycerides.Glycerol controlled the glycerolysis in the system under study, since it is both a substrate and a powerful water binder that reduces the water activity of the reaction medium and of the microenvironment. Two sets of experiments were carried out under different glycerol/triglyceride ratios. After 345 hours of consecutive 23 hours batches no lipase inactivation was observed.