Carboxyl ester lipase substrates

M. Lindstrom, B. Stemby, and B. BorgstrOm. Concerted action of human carboxyl ester lipase and pancreatic lipase during lipid digestion in vitro importance of the physicochemical state of the substrate. Biochim. Biophys. Acta 959 178 (1988). 

M. Lindstrom, J. Persson, L. Thum, and B. BorgstrOm. Effect of pancreatic phospholipase A2 and gastric lipase on the action of pancreatic carboxyl ester lipase against lipid substrates in vitro. Biochim. Biophys. Acta 1084 194 (1991). 

The bile-salt-dependent lipase of pancreatic juice has many names such as cholesterol esterase, nonspecific lipase, the most rational being carboxyl ester lipase [27], In the case of water-insoluble substrates this enzyme has an absolute requirement for bile salts specifically having hydroxyl groups in the 3a and la positions [28.29]. The best documented role for this enzyme is to allow the absorption of dietary cholesterol, through hydrolysis of cholesterol esters in the lumen. The enzyme also catalyzes the esterification of cholesterol and a role for it has been proposed in cholesterol absorption [30]. In addition, a wide range of primary and secondary fatty acyl esters including glycerides, vitamin A and E esters are hydrolyzed by this enzyme. 

It has been suggested that carboxyl ester lipase has two different bile-salt-binding sites [34-36], one nonspecific for the bile salt structure and one specific for primary bile salts causing the di(poly)merization. An interesting question is whether the bile salts regulate substrate specificity not only by bile salt-enzyme interactions but also by forming the appropriate substrate surface structure [27]. 

In addition to the now well-documented lipase system, cows milk contains several other carboxyl ester hydrolases, collectively referred to as esterases. These are distinguished from lipases by their ability to act on ester substrates in solution rather than in an emulsified form (Jaeger et al., 1994) and/or by their preference for hydrolysing esters of short-chain rather than long-chain acids (Okuda and Fujii, 1968). 

Lipases are of remarkable practical interest since they have been used in numerous biocatalytic applications, such as kinetic resolution of alcohols and carboxyl esters (both in water and in non-aqueous media) [1], regioselective acylations of poly-hydroxylated compounds, and the preparation of enantiopure amino acids and amides [2, 3]. Moreover, lipases are stable in organic solvents, do not require cofactors, possess broad substrate specificity, and exhibit, in general, a high enantioselectivity. All these features have contributed to make hpases the class of enzyme with the highest number of biocatalytic applications carried out in neat organic solvents. 

Pancreatic lipase can be considered a model for all other lipases. Most if not all of these enzymes seem to be nonspecific carboxyl ester hydrolases of the serine histidine type. Their specificity consists, by definition, in their ability to hydrolyze insoluble substrates, but apart... 

Enzymes that hydrolyze lysophospholipids are found in nearly all tissues and organisms. They seem to be non-specific esterases of the serine-histidine type (25) and hardly deserve the name lysophospholipase because they also hydrolyze esters other than phospholipids. They should probably be considered together with such enzymes as cholesterol esterases and monoglyceride lipases as amphiphilic carboxyl ester hydrolases. These non-specific esterases have a preference for amphiphilic (hydrophilic-lipophilic) substrates. Such an enzyme may perhaps hydrolyze lysophospholipis, monoglycerides, diglycerides, and cholesterol esters. 

In addition to hydrolyzing carboxylic ester bonds, lipases can catalyze a variety of esterification reactions in non-aqueous media. Industrial applications of lipases have focused on their enantioselectivity, regioselectively and substrate... 

To the secondary aliphatic alcohols, which have been resolved into their enantiomers, belong a variety of hydroxy carboxylic esters and acids (35,100-102,125,126, 131-140, 150, 151, 166, 168-172, 174, 182, 183), some hydroxy ketones (128-130, 141) and a crown ether derivative (203) (Table 11.1-20). Even the tetraphenylpor-phyrin derivatives 204 and 205 were substrates for different lipases. 

The in situ racemization can be achieved by different means either spontaneously or catalytically. Due to their chemical properties certain substrates may racemize spontaneously under the reaction conditions. Useful catalysts could be ordinary chemicals such as bases, transition metal complexes and in theory another type of biocatalyst. Having identified a suitable enzyme promoting the enantiomer-differentiating process by hydrolysis or alcoholysis of a carboxylic ester or by acylation of an alcohol one has to find the appropriate racemizing catalyst. Lipase and catalyst must tolerate each other they must work under identical conditions. The product must be chemically and configurationally stable in the presence of the catalyst. 

Lipases catalyze the hydrolytic cleavage of triglycerides into fatty acids and glycerol, or into fatty acids and mono or di-glycerides, at oil interfaces in nature. However, this hydrolytic reaction can be reversed and transformed into reactions of esterification (inter or transesterification), alcoholysis or aminolysis by engineering the medium polarity or the water content of the medium. Therefore, substrates for lipases can be esters, like the natural triglyceride substrates in hydrolytic reactions or, if the reaction is reversed, carboxylic acids, alcohols, amines or esters. The reaction medium not only determines the direction of the reaction (hydrolytic or synthetic), but also determines the solubility and stability of lipase substrates. Therefore, lipase activity and selectivity are strongly influenced by reaction medium. 

A representative selection of ester substrates, which have been hydrolyzed in a highly selective fashion is depicted in Scheme 2.62 [234, 438-441]. The wide substrate tolerance of this enzyme is demonstrated by a variety of carboxyl esters bearing a chiral center in the alcohol- or the acid-moiety. In addition, desymme-trization of meso-iorms was also achieved. In general, good substrates for CALB are somewhat smaller than those for Candida rugosa lipase and typically comprise (acetate or butyrate) esters of sec-alcohols in the (co-1)- or (co-2)-position with a straight-chain or monocyclic framework. 

Most such carboxylic ester hydrolases (Upases) contain a Ser-His-Aspartate/ Glutamate catalytic triad (Serl05-His224—Aspl87) in the active site, and share (at least in part) the common structural framework of the a,P-hydrolase fold. This fold is composed mainly of parallel P-sheet, flanked on both sides by a-helices [51]. A unique structural feature common to all lipases is a lid or flap composed of an amphiphilic a-helix peptide sequence which, in its closed conformation, prevents access of the substrate to the catalytic site [52]. When the lid is opened. 

Williams and coworkers have reported a DKR of ot-bromo [56a] and a-chloro esters [56b]. In the latter case, the KR is catalyzed by commerdally available cross-linked enzyme crystals derived from Candida cylindracea lipase. The racemization takes place through halide 5 2 displacement. The DKR is possible because the racemization of the substrate is faster than that of the produd (carboxylate). For the ester, the empty ii (C=0) orbital is able to stabilize the Sn2 transition state by accepting... 

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