Rapeseed lipases

Production of TAG enriched with polyunsaturated fatty acids (PUFA) via lipase catalysis in codliver oil has also been described (Haraldsson et ah, 1989). Preparation of y-linolenic acid enriched TAG via rapeseed lipases (Syed Rahmatullah et aL, 1994 Jachmanian and Mukerjee, 1996) and microbial lipases (Akoh et aL, 1995, 1996) has also been reported. Incorporation of eicosapentaenoic and docosahexaenoic acids into peanut oil via transesterification catalysed by microbial lipases has been reported by Sridhar and Lakshminarayana (1992). According to Wada and Koizumi (1986) and Kimoto et aL (1994) interesterification of fish oils to form TAG containing PUFA in the 2-position rather than the 1,3-positions increases the oxidative stability of the modified oil as compared to its original counterpart. 

Rapeseed lipase (Brassica nap us L.) Enrichment of GLA in evening primrose oil and of DH A in cod Ever oil by esterification with butanol Hills ei a/. (1990)... 

Both maize and rapeseed lipases were more active on tri-, than on di-or monollnoleln. Castor bean lipase was equally active on tri-, di-, and monollnoleln, as has been reported realier (9). These relative activities are different from those of pancreatic lipase which is more active on tri-than dlllnoleln, and Is Inactive on monollnoleln. 

Lipase from Aspergillus niger Transesterification 2-ethyl-1- hexanol and rapeseed oil Oil/water (97/3) 41.1 103... 

D-glycerol has been isolated from various red algae,377,378 rubber latex,284 turnips and rapeseed,179,294 and wheat flour.358 It is likely that this compound is a product of enzymic hydrolysis (by D-galacto-lipase)352 of DGDG. 

Kalo, P., Vaara, K., Antila, M. 1986b. Changes in triglyceride composition and melting properties of butter fat fraction/rapeseed oil mixtures induced by lipase catalysed interesterification. Fette, Seifen, Anstrichmittm. 88, 362-365. 

Figure 2.5. Stabilization of immobilized C. antarctica lipase in the second-step reaction by addition of vegetable oil and glycerol. The reaction was repeated at 30 °C with 6 wt% immobilized lipase by transferring the lipase to a fresh substrate mixture every 24 h. A, A reaction mixture was composed of dehydrated first-step product, rapeseed oil, and MeOH. Rapeseed oil was added to give the acylglycerol content of 50%, and the amount of MeOH was an equimolar amount to unreacted FAs. B, A reaction mixture was prepared by adding 10wt% glycerol to the mixture used in Figure A. O, The content of FAMEs at 2h , at 4h , at 24h. Dotted lines indicate the content of FAMEs before the reaction (44.1 wt%).

Li, L., Du, W., Liu, D., Wang, L., and Li, Z. 2006. Lipase-catalyzed transesterification of rapeseed oils for biodiesel production with a novel organic solvent as the reaction medium. /. Mol. Catal. B Enzym., 43, 58-62. 

A new industrial application for lipases has been developed in the field of renewable energy. Lipases are used for methylation of plant oils such as rapeseed oil. The resulting methylated oils are used as a replacement or supplement for fossil fuel-derived diesel. As of today, the costs of the biocatalyst, however, prevent its commercial application and chemical processes such as saponification of fats with sodium methoxide are currently preferred. 

The reaction is catalyzed by a variety of both acids and bases but simple bases such as NaOH and KOH are generally used for the industrial production of biodiesel [200, 201]. The vegetable oil feedstock, usually soybean or rapeseed oil, needs to be free of water (<0.05%) and fatty acids (<0.5%) in order to avoid catalyst consumption. This presents a possible opportunity for the application of enzymatic transesterification. For example, lipases such as Candida antarctica B lipase have been shown to be effective catalysts for the methanolysis of triglycerides. When the immobilized form, Novozyme 435, was used it could be recycled 50 times without loss of activity [201, 202]. The presence of free fatty acids in the triglyceride did not affect the enzymes performance. The methanolysis of triglycerides catalyzed by Novozyme 435 has also been successfully performed in scC02 as solvent [203]. 

Recent studies have attempted to improve the efficiency of epoxidation under milder conditions that minimize the formation of byproducts. Chemo-enzymatic epoxidation uses the immobilized lipase from Candida antartica (Novozym 435) (56) to catalyze conversion of fatty acids to peracids with 60% hydrogen peroxide. The fatty acid is then self-epoxidized in an intermolecular reaction. The lipase is remarkably stable under the reaction conditions and can be recovered and reused 15 times without loss of activity. Competitive lipolysis of triacylglycerols is inhibited by small amounts of fatty acid, allowing the reaction to be carried out on intact oils (57). Rapeseed oil with 5% of rapeseed fatty acids was converted to epoxidized rapeseed oil in 91% yield with no hydroxy byproducts. Linseed oil was epoxidized in 80% yield. Methyl esters are also epoxidized without hydrolysis under these conditions. 

One of the largest uses for lipases174 may be in the hydrolysis and transesterification of oils and fats.175 The reactions with enzymes are much gentler than the ones used today, such as hydrolysis of a fat or oil in water at 150 260 C for 3-24 h. They are also less capital-intensive. Lipases can be used for the hydrolysis and subsequent reesterification with butyl alcohol of olive and rapeseed oils in 100% conversion. An Aspergillus lipase hydrolyzed various fats and oils in the presence of an aqueous buffer in 90-99% yields in 2-24 h.176 Much of the lipase remained in the emulsion at... 

Biodiesel fuel can be made by the lipase-catalyzed alcoholysis of oils such as rapeseed oil in over 90% conversion.179 (The reaction is shown in 9.16 with triolein and ethanol for convenience. Rapeseed oil also contains some other fatty acids.)... 

Methanol is often used, but ethanol would be better because it can be made by fermentation. There is a problem with the small amounts of stearates present crystallizing out in cold winters. The use of isopropyl alcohol reduces this problem. The other possibility is to crystallize it out at the plant before it reaches the market. A better solution might be to use a desaturase to introduce a double bond into the stearate. Transesterification of rapeseed oil with 14.6% lipase from C. rugosa (with no added solvent) took place in 1 h.180 When the level of enzyme was dropped to 0.3% the... 

Lipase-Catalyzed Transesteriiication of Rapeseed Oil for Biodiesel Production with tert-Butanol... 

In our study, we conducted the enzyme-catalyzed methanolysis of rapeseed oil using Novozym 435, a well-known nonspecific lipase. Novozym 435 facilitates reactions between a wide variety of alcohols and is also a remarkably heat-tolerant enzyme [6, 8], Watanabe et al. [9] previously reported that immobilized Candida antarctica lipase was inactivated in the presence of more than half the stoichiometric amount of methanol against total fatty acids in the oil. This disadvantage was surmounted by the utilization of three-step methanolysis, in which only one third of the total amount of methanol was added in each stage [7, 9]. 

Methanolysis was conducted in a 20mL reaction bottle, maintained at 40 °C in a rotary shaker at 260rpm. The initial weight of the rapeseed oil was 5g. To prevent direct contact between the lipases and the methanol drops, the methanol was mixed with 4mL tert-butanol and oil followed by the addition of lipases to the mixture. In all experiments without initial water content experiment, there was no set initial amount of water in reactant, with the exception of the water contained within the enzymes themselves. 

In the enzymatic process utilized herein for the production of fatty acid methyl ester fixrm rapeseed oil, several factors are known to influence conversion. The initial step of this study involved the identification of factors likely to influence conversion, fri this study, tert-butanol was applied in lipase-catalyzed methanolysis. fert-Butanol has been utilized previously in several enzymatic process, including sorbitan ester synthesis [6, 15]. It has also been confirmed that terf-butanol is inert in the Novozym 435-catalyzed methanolysis of rapeseed oil for the production of biodiesel [12]. 

Experimental Oils. DAG was prepared by esterifying glycerol with fatty acids from soy bean oil using 1,3-specific lipase and purified by silicic acid chromatography (12). Of the total fatty acids in DAG oil emulsion used in this study, 90% existed as the 1,3-DAG and 1,2-DAG isomers in a ratio of 7 3, whereas <10% of total fatty acids were TAG. The TAG oil emulsion was prepared by mixing rapeseed and safflower oils to make the fatty acid composition almost the same as that of the DAG oil (Table 1). The combustion energies of DAG and TAG measured using a bomb calorimeter did not differ (9.1 kcal/g). 

Test Articles and Dosing. The DAG oil (Econa Oil, Kao Corporation, Biological Science Laboratories, Tochigi, JAPAN) used in this study was prepared from rapeseed oil in the presence of lipase, and was 90% DAG (w/w). The ratio of 1,2-DAG to 1,3-DAG was 3 7. Triacylglycerol oil was prepared from a mixmre of rapeseed, soybean, and safflower oils. This mixture was used to match the major fatty acid composition of the DAG and TAG oils as closely as possible. The intended use of the DAG oil product is as a component in cooking, as a substitute for other oils. 

The analysis of the stereospecific structure of rapeseed oil triglycerides with pancreatic lipase is often accepted as quite straightforward (cf. Breck-enridge, 1979), but in studies with lipases of plant (Rosnitschek and Theimer, 1980), microbial (Kroll et al., 1973) and animal (Myher et al., 1979 Vajreswari and Tulpule, 1980) origins, oils rich in erucic acid tended... 

Li, L., W. Du, D. Liu, L. Wang, and Z. Li. 2006. Lipase-Catalyzed Transesterification of Rapeseed Oils for Biodiesel Production with a Novel Organic Solvent as the Reaction Medium. Journal of Molecular Catalysis B Enzymatic 43 (l-4) 58-62. 

Vegetable oils (such as rapeseed, soybean and palm oils) or animal fats (beef tallow) are used for the production of so-called biodiesel, which is typically made by alcoholysis of oils and fats with methanol, ethanol or propan-l-ol. With the growth in the hydrocarbon chain length of the alcohol, the lipophilicity increases so that the two-phase system does not form in the reaction with propan-l-ol, and glycerol does not separate. Therefore, the industrially important esters, such as propyl or butyl esters as well as esters of secondary alcohols, can be only prepared by direct esterification of fatty acids with the appropriate alcohol. Methyl and especially ethyl esters can also be obtained by enzyme-catalysed alcoholysis using non-specific lipases. 

The data in Table 2 show that lipase activity was only present in germinating seeds and was absent from all other parts of the rapeseed plant. These data are consistent with our ELISA results which show that lipase protein is likewise confined to germinating seeds. 

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