1,3-specific lipases

Catalysts and enzymes Chiral synthesis BINAP" Novozym 388 2,2 -Bis (diphenylphosphino) l,l -binaphthyl 1,3-Specific lipase Rhodia, France Solvias, Switz. Novozymes, Denmark... 

A number of specific lipases are used for ester synthesis (eq. 4) transesterification, eg, acidolysis with 1,3 specific lipase (eq. 5) and hydrolysis reactions, eg, with nonspecific lipase (eq. 6). 

Lipases can be classified into groups that reflect their specificity. The common lipases include non-specific lipases that do not discriminate between the position or the type of the fatty acid on the triacylglycerol (e.g., lipase from Candida cylindracea) and 1,3-specific lipases that act only at the sn-l and sn-3 positions of the triacylglycerol (e.g., lipases from Aspergillus niger and Rhizopus species). In addition, some lipases are specific for a specific fatty acid type (e.g., lipase from Geotrichum candidum). 

When a 1,3-specific lipase is used for interesterification, the enzymatically-modified product has some different properties compared to those of a chemically-interesterified product. For example, the dropping point of butterfat was increased slightly by chemical interesterification whereas interesterification by a 1,3-lipase from Rhizopus arrhizus led to a 2-4°C decrease in dropping point. Although both methods of interesterification reduced hardness, the magnitude of the effect was greater for the enzymatically-interesterified fat (Marangoni and Rousseau, 1998). 

Interesterification can also be catalysed by enzymes, many of which show useful specificities. The 1,3-specific lipases, such as those derived from Aspergillus niger, Mucor javanicus, M. miehei, Rhizopus arrhizus, R. delemar, and R. niveus, are particularly useful for interesterification. They are used to effect acyl exchange at the sn- and 3 positions while leaving acyl groups at the sn-2 position unchanged. 

Stmctured lipids possessing n-3 PUFAs, such as EPA and DHA, located at mid position, with MCFAs at the end positions, have gained considerable attention as nutritional and health supplements. These TAGs provide rapid delivery of energy via oxidation of the MCFAs and, at the same time, supply metabolically functional fatty acids in the same molecule. Senanayake and Shahidi (51) used an immobilized in-1,3-specific lipase from Mucor miehei to incorporate capric acid into seal blubber oil containing EPA and DHA. On enzyme-catalyzed acidolysis, a stmctured lipid containing 27.1% capric acid, 2.3% EPA, and 7.6% DHA was achieved. Lipase from Mucor miehei incorporated capric acid predominantly at the in-1,3 positions of the stmctured lipid. 

FIGURE 6.1 Structured lipids produced from an MCT and an LCT catalyzed by sn-1,3-specific lipase. M = medium chain FA L = long chain FA. 

FAs liberated from food during absorption are metabolized more easily if they are short or medium chain, i.e., C10 or below. The sn-2 monoacylglycerols can be absorbed directly. Therefore, essential or desired FAs are most efficiently utilized from the sn-2 position in acylglycerols. In accordance with this, TAGs with short-chain FAs (SCFAs) or MCFAs at the sn-1 and sn-3 positions and PEFAs at the sn-2 position are rapidly hydrolyzed with pancreatic lipase (sn-1,3-specific lipase) and absorbed efficiently into mucosal cells. SCFAs or MCFAs are used as a source of rapid energy for infants and patients with fat malabsorption-related diseases. LCFAs, especially DHA and arachidonic acid, are important in both the growth and development of an infant, while n-3 PEFAs have been associated with reduced risk of cardiovascular disease in adults (Christensen et al., 1995 Jensen et al., 1995). 

Modified fats and oils 1,3-Specific lipases (migration of acyl groups) Japan, U.K. n. a. 1100... 

High-performance health beneficial emulsifier (i.e., the DHA-bound lysophos-pholipid) can be prepared from squid meal phospholipid because squid meal phospholipid contains DHA exclusively in position sn-2 (Hosokawa 1996). For this reason, if we eliminate the fatty acid moiety in the sn-1 position by applying position 1,3-specific lipase, or phospholipase Aj, we can obtain the sn-2 DHA-bound lyso-phospholipid. As a substrate for the desired reaction, squid meal phosphatidylcholine (PC) is the most useful so far. 

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). 

Availability of 1,3-specific lipases capable of modifying the 1,3 fatty acid composition of oils via interesterification has made it possible to produce cocoa butter-like products resembling cocoa butter in both composition and functional characteristics. Butter substitutes produced by this approach are granted GRAS (generally recognized as safe) status (Anon, 1988). 

Figure 3.87 Transesterification of triacylglycerols catalysed by sn-1,3 specific lipase A and B = acyls bound at sn-l,3-positions, X and Y = acyls bound at sn-2 positions.

Lipolase Gene technology 7-12 20-70 1,3-Specific lipase with broad substrate specificity... 

Fuji Oil (Matsuo et al, 1981) filed the first patents for the lipase-catalysed synthesis of a cocoa butter equivalent from palm oil and stearic acid. Both companies currently manufacture it using 1,3-selective lipases to replace palmitic acid with stearic acid at the 5n-l and sn-2> positions. The reactions usually performed are transesterification or acidolysis of cheap oils using tristearin or stearic acid as the acyl donors and a 1,3-specific lipase (Figure 3). Suitable starting oils besides palm oil mid-fractions are sunflower, rapeseed (Adlercreutz, 1994) and olive (Chang et al, 1990) oils. 

Reaction temperature is well known for its effect on enzyme activity. Most lipases are active in the temperature range 40-75 °C. Within this temperature range, higher temperature usually increases the reaction rate. Nevertheless, lipases are deactivated faster at higher reaction temperature due to protein denaturation. Cheong and co-workers (2007) found that 1,3-specific lipase from RML was quite stable at 65 °C and could be reused in at least ten continuous batches of enzymatic partial hydrolysis (120h) to produce DAG without significant loss of catalytic activity. 

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