Lipases interfacial activation

Hydrolysis of substrates is performed in water, buffered aqueous solutions or biphasic mixtures of water and an organic solvent. Hydrolases tolerate low levels of polar organic solvents such as DMSO, DMF, and acetone in aqueous media. These cosolvents help to dissolve hydrophobic substrates. Although most hydrolases require soluble substrates, lipases display weak activity on soluble compounds in aqueous solutions. Their activity markedly increases when the substrate reaches the critical micellar concentration where it forms a second phase. This interfacial activation at the lipid-water interface has been explained by the presence of a... 

Cutinase is a hydrolytic enzyme that degrades cutin, the cuticular polymer of higher plants [4], Unlike the oflier lipolytic enzymes, such lipases and esterases, cutinase does not require interfacial activation for substrate binding and activity. Cutinases have been largely exploited for esterification and transesterification in chemical synthesis [5] and have also been applied in laundry or dishwashing detergent [6]. 

In 1958 Sarda and Desnuelle [79] discovered the lipase activation at the interfaces. They observed that porcine pancreatic lipase in aqueous solution was activated some 10-fold at hydrophobic interfaces which were created by poorly water-soluble substrates. An artificial interface created in the presence of organic solvent can also increase the activity of the lipase. This interfacial activation was hypothesized to be due to a dehydration of the ester substrate at the interface [80], or enzyme conformational change resulting from the adsorption of the lipase onto a hydrophobic interface [42,81,82]. 

In case of lipases, one of the simplest methods to combine an enzyme with an organic solvent is to coat the lipase with a lipid or surfactant layer before lyophilisation. It is estimated that about 150 surfactant molecules are sufficient for encapsulating one lipase molecule. Following this route the surfactant coated lipase forms reverse micelles with a minimum of water concentration. The modified lipases are soluble in most organic solvents, and the reaction rates are increased compared to the suspended hpases due to the interfacial activation [59,60]. 

The influence of surfactant on the catalytic activity of lipases in water is well known. The addition of surfactants can enhance the activity and enantioselectivity of these enzymes in aqueous solutions [94] due to the interfacial activation and due to the emulsification of hydrophobic substrates. 

Finally, the purity-related performance of the enzyme preparation would appear to be a constant throughout a series of measurements. This, however, may not be the case when several enzyme species are present and different activities are expressed as a function of the medium composition [100]. Although it would appear that this source of error is abolished by the present-day availability of highly pure enzyme preparations (but see also [101, 102]), the intrinsic properties of i.e. Upases may lead to different E-values as a result of interfacial activation [103] and the conformation of the lid structure of lipases [56]. 

Although many biochemical reactions take place in the bulk aqueous phase, there are several, catalyzed by hydroxynitrile lyases, where only the enzyme molecules close to the interface are involved in the reaction, unlike those enzyme molecules that remain idly suspended in the bulk aqueous phase [6, 50, 51]. This mechanism has no relation to the interfacial activation mechanism typical of lipases and phospholipases. Promoting biocatalysis in the interface may prove fruitful, particularly if substrates are dissolved in both aqueous phases, provided that interfacial stress is minimized. This approach was put into practice recently for the enzymatic epoxidation of styrene [52]. By binding the enzyme to the interface through conjugation of chloroperoxidase with polystyrene, a platform that protected the enzyme from interfacial stress and minimized product hydrolysis was obtained. It also allowed a significant increase in productivity, as compared to the use of free enzyme, and simultaneously allowed continuous feeding, which further enhanced productivity. 

The active site of serine proteases is characterized by a catalytic triad of serine, histidine, and aspartate. The mechanism of lipase action can be broken down into (i) adsorption of the lipase to the interface, responsible for the observed interfacial activation (ii) binding of substrate to enzyme (iii) chemical reaction and (iv) release of product(s). 

The location of the acyl chain is of primary importance in the binding process because of its size. Due to the movement of lid during interfacial activation, a hydrophobic trench is created between the lid and enzyme surface. The trench size is ideal to accommodate the acyl chain. Interactions between the non-polar residues of the trench and the non-polar acyl chain stabilize the coupling. It has been postulated that the configuration of the trench is responsible for substrate specificity. This hypothesis seems plausible since lipases usually discriminate against certain acyl chain lengths, degrees of unsaturation, and location of double bonds in the chain. Any of these factors could affect the interaction between the acyl chain and the trench. 

True lipases show the interfacial activation phenomenon in their catalytic activity pattern. At low concentration of water-insoluble substrates, lipases are almost inactive, and the hydrolytic activity does not increase linearly. At a certain substrate concentration, however, the hydrolytic activity of lipases increases rapidly and the lipase kinetics resembles normal enzyme kinetics. This boost in activity is related to the formation of water-insoluble substrate aggregates such as micelles or another second phase. Only when this second phase is present, do lipases become fully active. This interfacial activation is caused by a large conformational change in the 3D structure of the lipases. In their water-soluble form, the active site is covered by a lid, which prevents the substrates from reaching it. At the lipidAvater interface, the lid is opened and the active site is accessible to the substrates. In addition, the now accessible area is mainly hydrophobic, which gives the open-form lipase the shape and behavior of conventional surfactant molecules with a hydrophilic and a hydrophobic moiety in one single molecule. 

Figure 2.1 Most lipases are interfacially activated. When immobilized on hydrophobic carriers they are assumed to be in their active conformation.

Another novel approach to prepare catalytically active bio-imprinted enzymes has been reported by Braco and co-workers [20]. This approach, known as interfacial activation , involves lyophilisation of lipolytic enzymes in the presence of phospholipid-based liposomes as the template. The efficacy of this imprinting process is controlled by the interface between the liposomes and the enzymes. This strategy to prepare bio-imprinted enzymes is illustrated in Fig. 10.2. It was found that the amphiphiles do not provide mere lyoprotection, rather they are responsible for generating imprinting-induced conformationally rigid active sites. This technique was tested with a number of lipases and the resulting imprinted enzymes showed enhanced catalytic activity in anhydrous media [21]. 

As stated earlier, lipases act at the interface between hydrophobic and hydrophilic regions, a characteristic that distinguishes lipases from esterases. Similar to serine proteases, lipases share the nucleophile-histidine-acidic residue catalytic triad that manifests itself as either a Ser-His-Asp triad or a Ser-His-Glu triad. The enzyme s catalytic site often is buried within the protein structure, surrounded by relatively hydrophobic residues. An a-helical polypeptide structure acts as a cover, making the site inaccessible to solvents and substrates. For the lipase to be active, the a-helical lid structure has to open so that the active site is accessible to the substrate. The phenomenon of interfacial activation is often associated with reorientation of the lid, increasing the hydrophobicity of the surface in the vicinity of the active site and exposing it. The opening of the lid structure may be initiated on interaction with an oiFwater interface. 

Despite their different physical and biochemical properties, most lipases and phospholipases share a common structural element an a-helical loop ( lid ) that covers the active site. Since the opening of the lid exposes a large hydrophobic patch, the resulting open conformation is thermodynamically unfavorable in solution. In contrast, in the presence of a lipid interface the open conformation is stabilized by the interaction with lipids. Many lipases and phospholipases show higher activity on interfaces than with free lipids (interfacial activation). It has long been considered that interfacial activation and lid opening are correlated. However, a number of enz3unes, such as CalB, possess a lid structure but do not show interfacial activation [18-20]. 

Conflicting hypotheses were also put forward with respect to the mechanism of interfacial activation. Desnuelle et al. (1960) were the first to suggest that a conformational change in the enzyme could be responsible for the enhancement of activity at the oil-water interface. There were also other hypotheses. For example. Wells (1974) suggested that the apparent activation of lipases is due to the orientation of the scissile ester bond on the surface of micelles Brockerhoff (1968), on the other hand, pointed to the possibility of differences in solvation of the ester bond in solution versus a lipid phase, whereas Brockman et al. (1973) postulated that a steep substrate concentration gradient at the interface may provide an explanation. 

In lipases the existing database regarding the oxyanion holes is still limited. In RmL two amide groups (residues 145 and 146) were originally proposed as likely candidates for this function (Brady et al., 1990). However, structural analyses of the two RmL-inhibitor complexes (Brzozowski et al., 1991 U. Derewenda et al., 1992) revealed that the oxyanion hole is likely to be fully formed only after the conformational change associated with interfacial activation, and that it is made up of both the amide and the side-chain hydroxyl of Ser-82 (Fig. 6). A hy-... 

In GcL Schrag et al. (1991) postulate that Ala-218 (again structurally equivalent to 145 in RmL) and Ala-132 play the same role in the stabilization of the intermediates. However, assuming that conformational rearrangements will also occur in this lipase during interfacial activation, the confirmation of this proposal will have to await a structural description of an enzyme-inhibitor complex. 

Since the pioneering studies of the phenomenon by Sarda and Des-nuelle (1958), many authors have hypothesized about the molecular basis of interfacial activation (see Section I). The present structural evidence supports the original proposal put forward by Desnuelle et al. (1960), who postulated a conformational change in the enzyme, fixing itself at the interface. All crystal structures of lipases clearly show that a change is necessary to expose the catalytic centers, which in the native enzymes are buried under various surface loops, or lids. 

It is, however, the role of colipase, a cofactor unique to the pancreatic enzyme, that is of particular significance. CLP was originally believed to function by anchoring the lipase molecule to the bile salt-covered surface of the lipid micelle (Canioni et al., 1977 Verger et al., 1977), thereby playing a key role in the interfacial activation. The enhancement of activity of hPL in the presence of CLP is approximately 10-fold. Abousalham et al. (1992) used limited proteolytic degradation of pancreatic lipases by chymotrypsin as a tool to obtain information re-... 

Brzozowski, A. M., Derewenda, U., Derewenda, Z. S., Dodson, G. G., Lawson, D. M., Turkenburg, j. P., Bjorkling, F., Huge-jensen, B., Patkar,S. A.,andThim, L. (1991). A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature (London) 351, 491-494. 

Van Tilbeurgh, H., Egloff, M.-P., Martinez, C., Rugani, N., Verger, R. and Cambiliau, C. (1993) Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography. Nature 362, 814-820... 

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