23 Lipases-the most promiscuous enzymes

Prof. M. N. Gupta

1. Objectives

•  To understand what is enzyme promiscuity
•  To learn about different classes of enzyme promiscuity
•  Broad specificity of lipases
•  Promiscuous reactions of lipases

2. Concept Map

3. Description

We recognise that when it comes to specificity, not all enzymes are created equal! We are normally introduced the concept of enzyme specificity with the illustrative example of proteases. Trypsin is highly specific, chymotrypsin somewhat less so. Proteases like papain and ficin are fairly nonspecific.

Enzymes also show surprising amount of nonspecific binding. Textile dyes are known to bind to variety of enzymes. Aptamers, peptides and many polymers bind to the enzymes with varied selectivity. These facts are exploited in protein purification.

In this module we will discuss some consequences of the deviations from the specificity in an extreme way. This behaviour is termed as enzyme promiscuity.

We will show that lipases can act upon substrates which are neither fats or oils or esters. That they can catalyse unexpected reactionS such as aldol condensation.

This phenomenon of enzyme promiscuity is obviously of interest to organic chemists as it enables use of lipase in synthesis of even more wider range of products.

Lipases are not the only class of enzyme to show the phenomenon of promiscuity, hence we start with a general discussion and then move to illustrations which focus on promiscuity of lipases.

Enzyme specificity

While introducing enzyme as biocatalysts, it is normal to talk of their specificity. Specificity in fact could be various kinds: substrate specificity, regiospecificity and stereospecificity. As far as substrate specificity is concerned it is also normal to add that some enzymes are very specific (trypsin which will selectively cleave peptide bonds involving arginine or lysine residues). On the other hand, papain or ficin have somewhat more broad specificity. Actually, our idea of substrate specificity is introduced with EC classification of enzymes. All the three examples cited above are hydrolases (ECX) and belong to the subclass of protease (ECX.Y). (Table 1)

The EC classification is based upon reaction type and not the substrate specificity of the individual enzymes. Appreciating this will help us in understanding this phenomenon of enzyme promiscuity.

Enzyme promiscuity

During last several years, a large number of C-C bond formation reactions catalyzed by lipases have been reported. Such reactions are very useful in organic synthesis, so this promiscuous behaviour has caught the attention of biochemists. Lipases, classified as hydrolases, in such reactions, show a synthetic activity. In fact, such behaviour had been noticed by biochemists for a long time in sporadic cases. Both lipases and proteases have been known to show esterase activity. Thus lipases are assayed by both triglyceride hydrolysis (tributyrin hydrolysis assay) as well as ester hydrolysis (p-Nitrophenyl butyrate hydrolysis assay). This was clearly understood in terms of their reaction mechanism and active site geometry.

We have known instances of enzymes promiscuity much earlier!

It was also rationalized by pointing out that this can be treated as a case of broad specificity and both were hydrolase activities. Perhaps well known oxygenase activity of Rubisco was not considered a reason to rethink our notion of enzyme specificity. It is interesting to note that oxygenase activity of Rubisco has been considered as an outcome of evolutionary history of the enzyme. It is believed that when Rubisco evolved, there was no O2 in the atmosphere. It seems that later on evolution did not correct for this “accidental promiscuity” which leads to photorespiration. It is worth noting that carboxylase and oxygenase activity are two different reaction types and have different EC numbers.

Condition promiscuity

Principle of microscopic reversibility states that are reactions can be reversed. Consider the following ester hydrolysis by a lipase:

RCOOR’ + H2O ↔ RCOOH + R’OH

For this reaction, we start with an ester. We normally use excess of H2O to make sure that adequate hydrolysis occurs.

Suppose, we start with the products on right hand side instead and carry out the reaction in the absence of water. [In module 29, we will learn several ways of doing it]. Say in excess of R’OH itself. In such cases, it has been shown that lipase catalyzes the reverse reactions and the ester is indeed formed. As this requires a different reaction medium it has been suggested that this is simply called condition promiscuity and this be included as a class of enzyme promiscuity.

Condition promiscuity is shown by many enzymes, lipase is an important example. (see module 29).

Substrate promiscuity

Enzymes may use the same chemistry on a different substrate. This kind of behaviour is termed as substrate promiscuity or substrate ambiguity. One estimate shows that, as one would expect, substrate promiscuity is more wide spread than catalytic promiscuity (also called reaction promiscuity). This has been in classical enzymology referred to as broad specificity or relaxed specificity.

One can also include coenzyme promiscuity under substrate promiscuity. Coenzyme promiscuity can be exemplified by a dehydrogenase being able to work with both NAD+ and NADP+ to a varying degree.

Lipases show very high promiscuity as they accept wide range of esters.

Accidental vs Induced promiscuity

Enzymes in their wild form (without any mutation) show all the three kinds of promiscuity. The three kinds of promiscuity shown by many lipases are further illustrated below.

Protein engineering has been used to generate or improve enzyme promiscuity.

Ser105Ala mutant of CAL B showed 100x higher specific rate for some C-C bond reactions.

This is generally referred to as induced promiscuity.

At one time it was believed that catalytic rates of promiscuous reactions are slower than those observed during normal catalysis. Later data showed that rate acceleration upto 1018 have been observed even for promiscuous reactions.

Conformational flexibility is believed to be an important parameter which decides the probabilityof the enzyme involved in catalysing a promiscuous reaction. The conformational flexibility around active site is especially believed to play an important role.

Hydrophobic bonds are dependent upon desolvation and are entropically driven. These are not so much dependent upon stereochemical fit between two entities. Hence, substrate hydrophobicity facilitates catalytic promiscuity.

Optimum pH is a well known characteristic of the enzymes. In some cases, the optimum pH of an enzyme in a normal reaction and promiscuous reaction can be different. This is because the two reactions may be facilitated by different protonation states of the same catalytic residues.

Substituting different metal ions is known to induce promiscuous activities. Of course, since lipases are not metalloenzymes, this is not of direct relevance to lipases.

It is believed that primordial enzymes were highly promiscuous as fewer enzymes were expected to help the organism survive. Divergent evolution via gene duplication and mutations was driven by selective pressure to evolve more specific enzymes.

Specificity of lipases

Before we discuss promiscuous reactions of lipases let us familiarize ourselves with specificity of lipases. All lipases are part of α/β hydrolase fold family. The core of parallel β- strands is surrounded by α-helices.

The active site of lipase consists of a catalytic triad of Ser, His and Asp/Glu. The active site nucleophilic serine is at a hairpin turn between a β-strand and α-helix and is a part of highly conserved pentapeptide sequence Gly-X-Ser-X-Gly. The only exception is CAL B which does not have this pentapeptide around the active site.

As fats/oils are their natural substrates, it is common to talk of regiospecificity of lipases vis-à-vis acyl chain on the 3 carbons of glycerol. Many nonspecific lipases hydrolyze ester bonds at all the 3 C-atoms.

In such cases, diglycerides and monoglycerides are intermediates but these do not accumulate.

The reason is that these are hydrolyzed quite often even more rapidly than triglyceride.

There are other lipases which are 1,3 specific. These lipases release fatty acids from 1 and 3 position. These also give monoglycerides and diglycerides. Obviously, 2-Monoglyceride accumulates in such cases.

Burkholderia cepacia (previously called Pseudomonas cepacia) lipase is a nonspecific lipase. So is the lipase from Candida rugosa. The lipase from Thermomyces lanuginosus is another important nonspecific lipase.

The catalytic promiscuity of lipases consists of formation of C-C bond, C-heteroatom bonds, heteroatom-heteroatom bonds and even oxidative processes. C-C bond formations by aldolases are very useful synthetic reactions in organic chemistry.

Aldol condensation has been reported to be catalyzed by several groups. Both CAL B and porcine pancreatic lipase has been found to given excellent conversions albeit with long reaction times.

Limited solubility of many aldehydes in aqueous buffers results in most of the reactions being carried out either with very small concentration of aldehydes or adding a small % of water soluble organic solvents.

If one of the substrates is a water miscible organic solvent this problem does not arise.

However, a large amount of water miscible organic solvents results in lipases being inactivated.

Hence, optimization by considering various trade-offs is very necessary.

The medium also plays an important role in controlling enantioselectivity even for these catalytically promiscuous reactions. The reaction between p- nitrobenzaldehyde and acetone catalysed by lipases illustrates this.

With 20% water (v/v), 96% conversion and 15 % ee was observed. In nearly anhydrous medium % ee improved to a value of 44% but significant drop in % conversions was reported.

CAL B has also been shown to catalyze a Michel addition of an α/β unsaturated aldehydes or ketone to a 1,3-dicarbonyl compound. The mutant Ser105Ala was found to be better than wild type CAL B for michael addition between acetyl acetone and acrolein by a factor of 36X in terms of reaction rates.

This result as well as quantum molecular modelling in the context of aldol reaction catalyzed by CAL B indicate that the active site Ser is not involved in the catalysis of these C-C bond formations. Presumably, Histidine acts as the base. It is not clear why replacing Ser accelerates the catalytic rates.

The lipase from Mucor miehei has been shown to catalyze one pot Mannich reaction between acetone, aniline and various aromatic aldehydes. The reaction medium was an aqueous buffer.

Michael addition between secondary amines such as pyrrolidine, piperidine and diethylamine and acrylonitrile illustrates the formation of other types of bonds catalyzed by lipases.

Lipase have been show to catalyze preparations of peroxycarboxylic acids by perhydrolysis of medium chain carboxylic acids by using H2O2 as the perhydrolytic reagent.

The peroxyacids could be used in situ as an oxidising agent to obtain epoxides of alkenes. This epoxidation of alkenes, as a synthetic strategy is of great utility!

The direct epoxidation of α/β- unsaturated aldehydes but-2-enal and 3-phenyl prop-2-enal by using H2O 2 has also been possible. Again Ser105Ala mutant also was able to catalyse this promiscuous reaction.

Synthesis of heterocycles is another useful type of reaction which has been catalyzed by lipases. The reaction between fatty acid esters and 1,2- arylenediamines led to the synthesis of 2-alkyl benzimidazoles.

Similarly synthesis of dihydrofuran by using lipase as a catalyst has been possible. Again such instances are not limited to use of a single lipase. For example, Mucor miehei lipase and porcine pancreatic lipases have been used in synthesis of heterocycles.

Three component Hantzscle reaction has been catalyzed by CAL B to obtain 1,4-dihydropyridines. With 4-nitrobenzaldehyde, acetylacetone and acetamide as the reactants, 90% yield was possible in 72 hrs.

Lipases do not prefer ω-fatty acids as substrates. ω-3 poluunsaturated fatty acids (PUFAs) eicosapentaenoic acid and docosahexaenoic acid (DHA) are important fatty acids. Their presence as fatty acids in oils is considered desirable from nutritional perspective.

DHA for example occurs in fish oils. The DHA concentrates have been obtained by hydrolysing other oils by lipases in aqueous medium. Extensive work on enrichment of ω-3 PUFAs in fish oil by lipases has been carried out.

The synthetic utility of lipases has been exploited at the industrial scale as well. BASF process for obtaining chiral amines involves selective acylation of one enantiomer present in the racemic mixture of anions by using alkyl methoxyacetate as the acyl donor.

The ring opening of an oxazolin with butanol leads to the synthesis of L-tert-leucine. The reaction medium was toluene and lipase from Mucor miehei was used as the catalyst.

Lipases can substitute for phospholipase A2 and A1 in conversion of phospholipids. Such conversions have been carried out in toluene, hexane or even solvent free media.

The starting material is a naturally occurring lipid to obtain a specific glycerophospholipid. Many 1, 3 specific lipases show specificity towards and Sn-1 position of glycerophospholipid, a type of reaction which would normally require phospholipase A1.

As polysaccharides like starch are renewable materials, there has been considerable interest in industry in acylation of starch. An interesting approach has been to use Novozym 435 as the biocatalyst to acylate starch nanoparticle with vinyl acetate. Novozym 435 is immobilized form of CAL B.

Modification of natural glycosides by lipases has been used to enhance their lipophilicity. This enhances their bioavailability by increasing their capacity to get transported across membranes.

Another interesting type of application of lipases is to prepare esters from alcohols and acids, each of which have some useful property. Rutin and vanillyl alcohols are antioxidants. These were esterified with ω-3 fatty acids from fish.

Naringin was converted to narangin 6”- ricinoleate using castor oil itself as acyl donor. L-Ascorbic acid was used to esterify cinnamic acid to create a more powerful antioxidant. CAL B was the enzyme and vinyl cinnamate was found to be best acyl donor among several which were tried.

In many of these applications, lipases have been used in immobilized form. Immobilized enzyme have a long history of applications in industrial enzymology. Most of those applications are in aqueous media.

Immobilization in such cases offer the twin advantage of stabilization and reusability. The former feature is generally exploited for carrying out reactions at higher temperatures. The latter arises from use of solid supports for linking enzymes.

In case of low water media even free enzymes are insoluble. Hence, reusability is not an issue.

The major advantage of immobilization in such media is that mass transfer is improved.

Free enzyme will be in the form of a precipitate or powder and will act as a heterogenous catalyst. After immobilization, enzyme is spread over the surface of the support and offers large catalytic surface.

Table : Specific Activity of TLL in different preparations

In the case of many lipases immobilization also offers another advantage. Many hydrophobic (& in some cases even hydrophilic) surfaces when used as supports result in opening up of the active site molecular lid (a peptide). This also happens when lipase encounters an interface. Hence, the phenomenon is known as interfacial activation of lipases. This enhances lipase activity several times.

Lipases top the growing list of enzymes which are now used in organic synthesis even at the industrial scale. Both their broad specificity and catalytic promiscuity continue to power their synthetic applications.

Summary

•  Enzyme promiscuity
•  Kinds of enzyme promiscuity
•  Example of promiscuous reactions catalyzed by lipases.