APPLICATION OF LIPASES

Dairy Industry Lipases are extensively used in the dairy industry for the hydrolysis of milk fat. Current applications include the flavour enhancement of cheeses, the acceleration of cheese ripening , the manufacturing of cheese like products, and the lipolysis of butterfat and cream. The free fatty acids generated by the action of lipases on milk fat endow many diary products, particularly soft cheeses, with their specific flavour characteristics. Thus the addition of lipases that primarily release short chain (mainly C4 and C6) fatty acids lead to the development of a sharp,tangy flavour, while the release of medium chain (C12,C14) fatty acids tend to impart a soapy taste to the product. In addition, the free fatty acids take part in simple chemical reactions, as well as being converted by the microbial population of the cheese. This initiates the synthesis of flavour ingredients such as acetoacetate, beta-keto acids, methyl ketones, flavour esters and lactones. The intensive use of lipases in cheese making started in the U.S.A after the Second World War. It was engendered by the Food and Drug Administration's ban on the import of rennet paste from Europe because of the impurity and unsatisfactory microbiology of the product. This paste was used by the manufacturers of traditional Italian cheeses ( Provolone, Romano, Mozzarela, Parmesan) and the first lipase cocktails were introduced as a substitute to create the typical lipolytic flavour of these varieties.

The traditional sources of lipases for cheese flavour enhancement are animal tissues, especially pancreatic glands(bovine and porcine) and the pre-gastric tissues of young ruminants(kid,lamb,calf). The latter are more commonly used in cheese making. The commercial pregastric lipases are available in the form of liquid extracts, pastes and vaccum or freeze dried powders. Each type of pre-gastric lipase gives rise to its own charecteristic flavour profile : a buttery and slightly peppery flavour(calf) ; a sharp 'piccante' flavour (kid); a strong 'pecerino' also described as 'dirty sock' flavour(lamb). A whole range of microbial lipase preparations has been developed for the cheese manufacturing industry: Mucor meihei(Piccnate, Gist-Brocades; Palatase M, Novo Nordisk), Aspergillus niger and A.oryzae (Palatase A, Novo Nordisk; Lipase AP, Amano; Flavour AGE, Chr. Hansen) and several others. These microbial lipases are used not only for flavour enhancement and the acceleration of the ripening of specific cheeses such as blue, but in some cases they have also successfully replaced pre-gastric lipases . A range of cheeses of good quality was produced by using individual microbial lipases or mixtures of several preparations. Apart from substitution of rennet paste and flavour enhancement, lipases are widely used for imitation of cheeses made from ewe's or goat's milk. Addition of lipases to cow's milk generates a flavour rather similar to that of ewe/goat milk. This is used for producing cheeses like Feta, Manchego and Romano from cow's milk. When added to certain blue cheeses, lipase imitate the taste of Roquefort, which is normally produced from sheep's milk. Similarly, the addition of lipases to

pasteurised milk leads to the development of the normal flavour of Ras or Konpanisti, which traditionally are produced from raw milk. Lipases also play a crucial role in the preparation of socalled enzyme modified cheeses (EMC). EMC is a cheese that is incubated in the presence of enzymes at elevated temparature in order to produce a concentrated flavour for use as an ingredient in other products (dips,sauses,dressings,soups,snacks etc.). Typically the concentration of free fatty acids is ten times higher in EMC than in that of the corresponding young cheese. EMC technology is widely used in the U.S.A.

Detergents Enzymes can be used in the laundry detergents and automatic dish washing machines detergents. Enzymes can reduce the environmental load of detergent products, since they save energy by enabling a lower wash temperature to be used; allow the content of other, often less desirable, chemicals in detergents to be reduced; are biodegradable, leaving no harmful residues; have no negative impact on sewage treatment processes; and, do not present a risk to aquatic life. Other enzymes are currently widely used in household cleaning products. A great deal of research is currently going into developing lipases which will work under alkaline conditions as fat stain removers.

Oleochemical Industry The scope for the application of lipases in the oleochemical industry is enormous. Fats and oils are produced world wide at a level of approximately 60

million tonnes per annum and a substantial part of this(more than 2 million tonnes per annum) is utilised in high energy consuming processes such as hydrolysis, glycerolysis and alcoholysis. The conditions for steam fat splitting and conventional glycerolysis of oils involve high temparatures of 240-260 degree C and high pressures (methanolysis is currently performed under slightly milder conditions). The resulting products are often unstable as obtained and require re-distillation to remove impurities and products of degradation. In addition to this, highly unsaturated heat sensitive oils cannot be used in this process without prior hydrogenation. The saving of energy and minimisation of thermal degradation are probably the major attractions in replacing the current chemical technologies with biological ones. However, in spite of their apparent superiority, enzymic methods have not as yet attained a level of commercial exploitation commensurate with their potential. There have been several communications about relatively small scale enzymic fat splitting processes for the production of some high value polyunsaturated fatty acids and the manufacture of soap. For instance Miyoshi Oil & Fat Co., Japan, reported the commercial use of Candida cylindracea lipase in the production of soaps. The company claimed that the enzymic method yielded a superior product and was cheaper overall than the conventional Colgate-Emery process. There are probably several reasons for the generally disappointing level of commercial applications of lipase in this sector at present. First of all, the oleochemical

industry is very conservative, owing to huge capital investments involved. Therefore one cannot expect rapid changes. Secondly until recently the high cost of lipases remained prohibitive for the manufacturing of bulk products. The introduction of the new generation of cheap and very thermostable enzymes should change the economic balance in favour of lipase use. Thirdly, some concern has been expressed by chemical engineers with regard to running and controlling enzymic processes on the required scale. However the recent commercialisation of several lipase based technologies has proved their feasibility unambiguosly. The future of lipases look rather promising in the context of oleochemistry. Some fats are much more valuable than others beacuse of their structure. Less valuable fats can be converted into more useful species using blending of chemical methods but these tend to give quite random products. Lipase catalysed transesterification of cheaper oils can be used, for example to produce cocoa butter from palm midfraction.

Pharmaceutical Industry The vast variety of synthetic pharmaceuticals and agrochemicals containing one or more chiral centres, are stll being sold as racemates. This is despite the fact that the desired biological activity resides in one particular enantiomer. A single isomer is preferable to a racemate, but there are severe technical and/or economic problems with the production of single isomers.

The usefulness of lipases in the preparation of chiral synthons is well recognised. The resolution of 2halopropionic acids, starting materials for the synthesis of phenoxypropionate herbicides is being carried out on a 100-kg scale by Chemie Linz Co.(Austria) under a license from the Massachusetts Institute of Technology. The process is based on the selective esterification of (S)-isomers with butanol catalysed by porcine pancreatic lipase in anhydrous hexane. Typically , > 99% enantiomeric excess(e.e) is obtained at 75% of the theoritical yield and the resolution is complete in several hours. Generally, the lipase mediated resolution of 2-substituted propionic acids, and especially 2-aryl derivatives, have been the subject of intensive investigations. A substantial body of literature exists on the production of both (R) and (S) isomers of alpha(*sub)-phenoxypropionic acids, which are useful synthons for the preparation of enantiomerically pure herbicides and non-steroidal antiinflammatory drugs (naproxen,ibuprofen) respectively. The required optically pure derivative can be obtained directly via the (trans)esterification or hydrolysis of the corresponding ester. These resolutions have been performed on a multi-kilogramme scale by several companies world-wide. Another instance of commercial application of lipases to the resolution of racemic mixtures is the hydrolysis of epoxy alcohol esters. The highly enantioselective hydrolysis of (R,S)-glycidyl butyrate has been developed by DSM-Andeno (the Netherlands). The reaction products, (R)-glycidyl esters and (R)-glycidol, are readily converted to (R)-and (S)-glycidyltosylates, which are

very attractive intermediates for the preparation of optically active beta-blockers and a wide range of other products. A similar technology has been commercialized by Sepracor Inc.(USA). This company has successfully operated a multi-kilogramme scale membrane bioreactor to produce the 2(R),2(S) methyl methoxyphenyl glycidate, the key intermediate in the manufacture of the optically pure cardiovascular drug diltiazem. 2(S),3(R)methoxyphenyl glycidic acid, the product of enzymic hydrolysis, was found to be unstable under the conditions of the reaction, and the resultant aldehyde inhibited the lipase activity and reduced the lifetime of the enzyme. Both problems were overcome by the introduction of a multi-phase membrane reactor where the aldehyde byproduct reacted in situ with bisulphite to form a noninhibitory, water soluble adduct, extracted into the aqueous phase. Lipases have been found useful as industrial catalysts for the resolution of racemic alcohols. Enantiomerically pure endo-2-norborn-2-ol is an important chiral intermediate in the preparation of some prostaglandins, steroids and carbocyclic nucleoside analogues. Bend Research Inc(USA) have developed a two-step resolution process . The process involved acylation of the (R)-alcohol with butyric anhydride, mediated by Candida cylindracea lipase, followed by the hydrolysis of the (R)-ester catalysed by the same enzyme. The first resolution resulted in the enantiomerically pure (S)-alcohol (e.e > 98%) and (R)-ester(e.e-78%) which was further enriched by the back conversion to (R)-alcohol(e.e > 98%). the resolution was performed on a multi-kilogramme scale in

a permselective membrane bioreactor specially designed to facilitate product recovery and to minimise product inhibition. Lipases are currently being used by many pharmaceutical companies world-wide for the preparation of optically active intermediates on a kilo-gramme scale. A number of relatively small biotechnological companies, such as Enzymatix in the U.K, specialise in biotransformations and offer a whole variety of intermediates prepared via lipase mediated resolution. Regioselective modifications of polyfunctional organic compounds is yet another area of expanding lipase application. In may cases, lipases have been shown to acylate or deacylate selectively one or several hydroxyl groups of similar reactivity in carbohydrates, polyhydroxylated alkaloids and steroids.Apart from the synthesis of sugar based surfactants, lipases wer successfully applied in the regioselective modification of castanospermine. a promising drug for the treatment of AIDS. Thus lipases have become a conventional research tool in many organic chemistry laboratories. As a result they are readily incorporated into synthetic routes especially when optical purity of the final product is essential.

Cosmetic Industry Although the cost of lipase catalysed esterification remains too high for the manufacturing of bulk products, the synthesis of several speciality esters has found its way in the market place. Unichem International has launched the production of isopropyl myristate, isopropyl

palmitate and 2-ethylhexylpalmitate for use as an emollient in personal care products such as skin and suntan creams, bath oils etc. Immobilised Rhizomucor meihei lipase was used as a biocatalyst in the solvent free esterification, which was driven to completion by vaccum distillation of the water produced during the reaction. The company claims that the use of the enzyme in place of the conventional acid catalyst gives products of much higher quality, requiring minimum downstream refining. Batches of several tonnes have been successfully produced at Unichem's factory in Spain. Wax esters (esters of fatty acids and fatty alcohols) have similar applications in personal care products and are also being manufactured enzymically (Croda Universal Ltd). The company uses Candida cylindracea lipase in a batch bioreactor. According to the manufaturer, the overall production cost is slightly higher tha that of the conventional method, but the cost is justified by the improved quality of the final product.

Medical applications Possible medical applications of lipases are under consideration, for example inhibition of the human enzyme as a method of reducing fatty acid absorption is being investigated as a possible treatment for obesity.