Lipases and Proteases

The application of lipases in synthetic biotransformations encompasses a wide range of solvolytic reactions of the carboxyl group, such as esterification, transesterification (alcoholysis), perhydrolysis, and aminolysis (amide synthesis) [103]. Transesterification and amide synthesis are preferably performed in an anhydrous medium, often in the presence of activated zeolite, to suppress unwanted hydrolytic side reactions. CaLB (which readily tolerates such conditions [104,105]), PsL, and PcL are often used as the biocatalyst [106]. 

Lipase-catalyzed transesterification to prepare polyesters (replacing the traditional chemical polymerization at 200 °C) has received considerable attention in recent years. CaLB was found to mediate polyester synthesis in the ionic liquids [BMIm][BF4], [BMIm][PF6], and [BMIm][ Tf2N] at 60°C [110, 111, 112], but the molecular weight of the product was rather low compared with that in a solventless system [113], perhaps owing to the high viscosity of ionic liquid media. 

The synthesis of long-chain fatty acid esters of carbohydrates is inherently more demanding. It was found that glucose did not react with vinyl laurate in a pure ionic liquid medium, but in biphasic tert-butyl alcohol/[BMIm][PF6], glucose could be acylated by the vinyl esters of O, 2-Cu, fatty acids. The best results were obtained with CaLB, which was twice as active as TIL, and the selectivity for acylation at C-6 was high [114]. The esterification of glucose with palmitic acid, which is, in an industrial context, to be preferred over transesterification, has recently been demonstrated in tert-butyl alcohol/[BMIm][PF6] medium [115]. 

The enzymatic esterification of polysaccharides, e.g., glucomannan, a copolymer of glucose and mannose, has also been reported [118]. 

Acylations of carbohydrate derivatives such as alkyl glucosides and galactosides have also been successfully performed in ionic liquids [63]. Similarly, the flavonoid glycosides naringin and rutin were acylated with vinyl butyrate in ionic liquid media in the presence of a number of lipases, e.g., CaLB (Novozym 435), immobilized TIL, and RmL [119]. The products are of interest for application as strong antioxidants in hydrophobic media. 

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Non-enteric-coated pancreatic enzyme supplements can be used for initial therapy. The relative dose of amylase, lipase, and protease may be increased until control of pain and fatty diarrhea is achieved or the patient experiences intolerable side effects. If pain and diarrhea control are achieved, the patient can be transitioned to an enteric-coated supplement to maximize compliance. A reasonable example starting regimen is Viokase-8, six tablets with each meal and at bedtime, given with famotidine 20 mg at bedtime. 

Pancreatic enzyme supplements should be taken immediately prior to meals to aid in the digestion and absorption of food. Alternately, patients can supplement their diet with medium chain triglycerides (MCTs) or ingest foods rich in MCTs since they do not require pancreatic enzymes for absorption. An appropriate regimen incorporates the successful doses of each enzyme (amylase, lipase, and protease) from the starting non-enteric-coated regimen. As with the previous example, a patient stabilized on Viokase-8, six tablets with each meal, can be transitioned to Pancrease MT-16 three tablets with meals. The famotidine can then be discontinued. 

Figure 3 Typical efficiency of 4-FPBA in liquid detergent containing lipase and protease. In this case the efficiency is around 100 times that of boric acid...

Enzyme and Nonenzyme Catalysts By nature, enzymes themselves are chiral and they catalyze a variety of chemical reactions with stereoselectivity. These reactions include oxidation, reduction, and hydration. Examples of enzymes are oxidases, dehydrogenases, lipases, and proteases. Metoprolol, an adrenoceptor-blocking drug, is produced using an enzyme-catalyzed method. 

A critical consideration in the development of biocatalytic systems is the form in which the enzyme or enzyme system is going to be used. There are two general approaches. One is to use isolated enzymes. If these are inexpensive, they can be used as disposable biocatalysts, as is the case for glucose isomerase, ° which is the key biocatalyst in the production of high-fructose corn syrups from starch, or the lipases and proteases that are present in detergents. Alternatively, if enzymes are expensive to produce, they can be immobilized and used repeatedly by recovering the enzyme particles after each use. 

Most kinetic resolutions of pharmaceutical intermediates that have been reported involve the use of hydrolases, particularly lipases and proteases. This is because many hydrolases are commercially available (in bulk and kit form), do not require cofactors and are active in many organic solvents (see Section 1.4). Processes can therefore, often be developed rapidly, using high substrate concentrations and without specialist knowledge. 

Exocrine pancreatic insufficiency is most commonly caused by cystic fibrosis, chronic pancreatitis, or pancreatic resection. When secretion of pancreatic enzymes falls below 10% of normal, fat and protein digestion is impaired and can lead to steatorrhea, azotorrhea, vitamin malabsorption, and weight loss. Pancreatic enzyme supplements, which contain a mixture of amylase, lipase, and proteases, are the mainstay of treatment for pancreatic enzyme insufficiency. Two major types of preparations in use are pancreatin and pancrelipase. Pancreatin is an alcohol-derived extract of hog pancreas with relatively low concentrations of lipase and proteolytic enzymes, whereas pancrelipase is an enriched preparation. On a per-weight basis, pancrelipase has approximately 12 times the lipolytic activity and more than 4 times the proteolytic activity of pancreatin. Consequently, pancreatin is no longer in common clinical use. Only pancrelipase is discussed here. 

At this point it became logical to raise the question how much water do enzymes really need . In trying to answer to this question it was found that some enzymes-particularly lipases and proteases-were still active even in neat organic solvents. This happened in the early eighties, and was the beginning of so-called non-aqueous enzymology . 

Thus the use and practice of biocatalysis at full scale has waxed and waned over the years. In the past, one factor limiting the use of biocatalysis has been the availability of a variety of enzymes and the time taken to refine/evolve enzymes for specific industrial apphcations. Hydrolytic enzymes such as lipases and proteases designed for other industrial uses such as detergents and food processing have always been available in bulk, and indeed used by process chemists. 

Heat-Resistant Lipases. The heat-resistant lipases and proteinases and their effects on the quality of dairy products have been reviewed (Cogan 1977, 1980). Several reports have linked the lipases from bacteria with the off-flavor development of market milk (Richter 1981 Shipe et al. 1980A Barnard 1979B). The microflora developing in holding tanks at 4°C [and presumably in market milk stored at 40°F (Richter 1981)] may produce exocellular lipases and proteases that may survive ordinary pasteurization and sterilization temperatures. Rancidity of the cheese and gelation of UHT milk appear to be the major defects caused by the heat-resistant enzymes. 

Kishonti, E. 1975. Influence of heat resistant lipases and proteases in psychortrophic bacteria on product quality. Int. Dairy Fed. Doc. 86, pp. 121-124,... 

Optically Active Acids and Esters. Enantioselective hydrolysis of esters of simple alcohols is a common method for the production of pure enantiomers of esters or the corresponding acids. Several representative examples are summarized in Table 4. Lipases, esterases, and proteases accept a wide variety of esters and convert them to the corresponding acids, often in a highly enantioselective manner. For example, the hydrolysis of (R)-methyl hydratropate [34083-55-1] (40) catalyzed by lipase P from Amano results in the corresponding acid in 50% yield and 95% ee (56). Various substituents on the a-carbon (41—44) are readily tolerated by both lipases and proteases without reduction in selectivity (57—60). The enantioselectivity of many lipases is not significandy affected by changes in the alcohol component. As a result, activated esters may be used as a means of enhancing the reaction rate. 

The esterases, lipases, and proteases are often used to prepare chiral intermediates when the reactions are carried out in the synthetic mode. Selected examples of these enzymatic biotransformations will be discussed in the respective sections later in this chapter. The reader is directed to the following reviews and textbooks for additional information, especially regarding resolutions 5,19,26,72,156-159... 

Christen, G.L., Wang, W.-C., Ren, T.-J. 1986. Comparison of the heat resistance of bacterial lipases and proteases and the effect on ultra-high temperature milk quality. J. Dairy Sci. 69, 2769-2778. 

Vercet, A., Lopez, P., Burgos, J. 1997. Inactivation of heat-resistant lipase and protease from Pseudomonas fluorescens by manothermosonication. J. Dairy Sci. 80, 29-36. 

Hydrolytic enzymes such as lipases and proteases catalyze readily reversible reactions and will often promote reverse hydrolysis at reduced water activities. Water can be removed with desiccants, as an azeotrope with a solvent or through application of a vacuum. Lipases have proven particularly useful in this regard, allowing the formation of esters from alcohols and either free carboxylic acids or esters (see Figure 31.12). 

Q14 Pancreatic enzyme preparations contain amylase, lipase and protease enzymes. These supplements are given by mouth and compensate for the reduced or absent pancreatic secretions they assist the digestion of starch, fat and protein. Since the enzymes may be inactivated by gastric acid, they are usually presented in a protected, enteric-coated form which is sprinkled directly on the food. 

Since cystic fibrosis patients lack digestive enzymes, enzyme preparations containing amylase, lipase and proteases are prescribed in order to improve intestinal absorption of nutrients. 

Esterases, lipases, and proteases are widely used in enzymatic resolution processes of racemic carboxy acid ester substrates. Industrially relevant examples are the manufacture of D-)ff-acetylmercaptoisobutyric acid, the optically active side-chain of the ACE inhibitor captopril (43) [104] (Scheme 26), and synthesis... 

Catabolism of chylomicron remnants may be viewed as the second step in the processing of chylomicrons. After the loss of apo C-II and other C and A apoproteins, LPL no longer acts upon the remnants, and they leave the capillary surface. Chylomicron remnants are rapidly removed by uptake into liver parenchymal cells via receptor-mediated endocytosis. Apo E is important in this uptake process. The chylomicron receptors in liver are distinct from the B-E receptor that mediates uptake of LDL. The hepatic receptor for chylomicrons binds with apo E, but not apo B-48. Another receptor, known as the LDL receptor-related protein (LRP), may also function in chylomicron uptake. Chylomicron remnants are transported into the lysosomal compartment where acid lipases and proteases complete their degradation. In the liver, fatty acids so released are oxidized or are reconverted to triacylglycerol, which is stored or secreted as VLDL. The cholesterol may be used in membrane synthesis, stored as cholesteryl ester, or excreted in the bile unchanged or as bile acids. 

Malabsorption requires treatment when steatorrhea is documented (>7 g of fat in the feces per 24 hours while on a diet of 100 g/day of fat) and persistent weight loss occurs despite efforts to correct it. The combination of pancreatic enzymes (lipase, amylase, and protease) and a reduction in dietary fat (to <25 g/meal) enhances the patient s nutritional status and reduces (but does not totally correct) steatorrhea. The success of a pancreatic enzyme preparation requires that it contain a high concentration of lipase and proteases, be enteric-coated to avoid destruction by gastric acid, and be the... 

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