Lipase-Mediated Biocatalysis as a Greener and Sustainable Choice for Pharmaceutical Processes


Why choose biocatalysis?

The phenomenon of speeding up a chemical or biological reaction in the presence of a catalyst is termed catalysis. The use of chemical catalysis has been the heart of innumerable chemical processes since times immemorial. Many efficient chemical processes have been developed in the past for the synthesis of fine chemicals like active pharmaceutical ingredients and their intermediates. But these processes are less specific, can lead to

Pharmaceutical Biocatalysis: Important Enzymes, Novel Targets, and Therapies Edited by Peter Grunwald

Copyright © 2021 Jenny Stanford Publishing Pte. Ltd.

ISBN 978-981-4877-13-8 (Hardcover), 978-1-003-04539-7 (eBook) the formation of racemic mixtures, are generally more expensive, and can have an overall negative impact on the environment. Therefore, there is an increasing demand for "Biocatalysts" or enzymes for industrial processes and considerable efforts are being made to search for such enzymes (Rozzell, 1999) (Table 1.1).

The umbrella of biocatalysis is vast enough to cover myriad of industrial applications including detergent formulations, feed and food enzymes, production of biodiesel, and most importantly the production of fine chemicals like agro-chemicals and pharmaceuticals. Biocatalytic processes have multiple advantages including better chemical precision, mild operating conditions, use of renewable catalysts, higher turnover numbers, fewer side reactions, increased specificity and selectivity that result in lowering the costs and making the process more sustainable and viable (Rozzell, 1999) (Fig. 1.1). The greener methods of biosynthesis and conversions are thus becoming the backbones of many sustainable technologies in today's era (Tucker and Faul, 2016).

Advantages of biocatalysis

Figure 1.1 Advantages of biocatalysis.

Table 1.1 Important biocatalytic reactions catalyzed by different classes of enzymes

Reaction catalyzed

Enzyme class




lipase, esterase, protease, nitrilase, nitrile hydratase

Oxidation or reduction


Dehydrogenase, oxygenase, oxidase


non-hydrolytic bond


Dehydratase, decarboxylase

Transfer a group from one molecule to another


Transaminase, glycosyl transferase




mutase, racemase

Bond formation requiring triphosphate



Hydrolases as Biocatalysts

Hydrolases are a group of enzymes catalyzing basic reactions such as bond cleavage (hydrolysis), condensation, and alcoholysis. Hydrolases include glycosidases, proteases, nitrilases, esterases, and lipases. These enzymes constitute about 95% of the total production of industrial enzymes. Hydrolases are chemically unique enzymes that can withstand harsh environmental conditions. Lipases and esterases are capable of functioning in the presence of water-miscible as well as immiscible solvents (Bornscheuer and Kazlauskas, 2006). This provides a basis for their utilization in biocatalysis at a larger scale.

Lipases: A General Account

Both lipases (triacylglycerol acylhydrolase EC and esterases (carboxylic-ester hydrolase EC catalyze the hydrolysis of esters, but lipases preferentially act on emulsified substrates (Bornscheuer and Kazlauskas, 2006). While other hydrolases function in one homogenous phase and follow enzyme kinetics that follows Michaelis-Menten equations, lipases are unique in the manner that they catalyze reactions only at a lipid-water interface. This phenomenon called "interfacial activation” (Jaeger and Reetz, 1998) is necessary for the activity of lipases as the substrates for these enzymes i.e., the triglycerides are insoluble in water.

Lipases are one of the most widely employed enzymes in the pharmaceutical sector. Sources of lipases are spread across almost all forms of living entities. They can be derived from plants, animals, and microbes. However, microbial lipases are commercially more important as they can exhibit high stability and activity under diverse experimental conditions. They possess several unique qualities that make them the catalysts of choice for processes to be carried out at higher temperature ranges and in non-aqueous environments. Increasing demand for industrial lipases has been accelerating the search for new lipases with improved properties and diverse potential applications (Kanmani etal., 2015).

Selective screening of microorganisms is one of the most efficient methods for finding novel enzymes viable for the industry. Various simple quantitative methods used for detecting lipase activity in microorganisms include the addition of a lipidic source to the solid culture medium so that hydrolysis can be detected on a plate. Basic indicator dyes such as Phenol red, Nile blue, Victoria blue, Methyl red, Rhodamine B, etc., are used to aid the visibility of clear zones or halos in such methods (Kouker and Jaeger, 1987). For quantitative estimation of the lipolytic activity, para-nitrophenyl esters of fatty acids are majorly used which on hydrolysis, release para-nitrophenol, which is a colored compound that can be estimated spectrophotometrically at 410 nm (Vorderwulbecke et aL, 1992). These methods of lipase estimation can be very cumbersome if multiple samples are to be assessed in one go, where a quantitative high throughput screening method using a Rhodamine B-olive oil emulsion for lipase enzyme has been used (Zottig et al., 2016).

For improving the efficacy of lipase-mediated biocatalysis, a number of modifications in the enzyme are carried out including enzyme immobilization. This increases the number of enzyme molecules per unit area, thus increasing the enzyme stability, easier and enzyme-free product recovery (Sharma et al., 2001). Different lipase immobilization methods include gel-entrapment, encapsulation, covalent bonding, adsorption on hydrophobic supports and the use of nano-materials. Biodegradable natural polyaminosaccharides like chitin and chitosan, and another food-grade polymeric support, Amberlite FPX-66, have also been used to immobilize Candida antarctica lipase В (CAL-B). These natural supporting materials have rendered thermostability and re-usability of the enzyme up to 80 cycles (Kralovac et ah, 2010; Silva et ah, 2012).

Structural features of lipases

The mystery related to the unique catalytic activity of lipases could not get solved without the knowledge of their structural features. The structural features of lipases started to get disclosed with the early elucidations of the popular 2.4 A resolution structure of the human pancreatic lipase (Winkler et ah, 1990) which was found to be a 449 amino acid residue chain, with Serl52 as the nucleophilic residue vital for the catalytic activity. Another study revealed the 1.9 A resolution structure of the Rhizomucor meihei lipase (Derewenda et ah, 1992) reflecting a single polypeptide chain of 269 residues folded in a unique manner with Serl44 as the nucleophilic residue vital for the catalytic activity. Both of these reports highlighted the special features of the catalytic triad existing at the enzyme active site similar to that of the serine proteases (Dodson and Wlodawer, 1998).

The crystallography data of different lipases shows that even on having different amino acid sequences, different chain lengths, and architectures, these enzymes fold in a similar manner and have similar catalytic sites. A special fold that exists at the active site of these enzymes has a core of parallel (3-sheets which are surrounded by multiple a-helices along with the catalytic triad of Ser, His and Glu/Asp present there (Ollis et al., 1992). This fold is called the cc/p hydrolase fold which makes the lipases to be called as a/p hydrolases.

The catalytic triad in almost all the ot/p hydrolases studied so far has been observed to possess a nucleophilic center residue, a catalytic acid residue, and a hystidine residue in the same order as this. The nucleophilic center residue is a serine, cystiene or aspartate residue (in case of lipases, it is always a serine residue) while the catalytic acid residue can be a glutamate or an aspartate residue (Jaeger et al., 1999). These structural characteristics also explain the basis of the basic mechanism responsible for increased catalytic efficiency of lipases existing at heterogeneous surfaces or the "interfacial activation."

Reaction mechanism of lipases

As mentioned earlier, lipases belong to the class of serine hydrolases. The catalytic triad serine-histidine-aspartate is commonly present on the surface of their active sites. At first, the covalent acyl-enzyme intermediate is formed as the OH" group of nucleophilic Ser residue reacts with the lipidic substrate with the help of neighboring His and Asp residues (Fig. 1.2). Multiple types of nucleophiles can react with this intermediate and this gives rise to the broad range of functionality of lipases (Kazlauskas, 1994; Jaeger et al., 1999).

Properties of lipases

Microbial lipases are the most versatile enzymes that can bring about a huge range of bioconversions (Vulfson, 1994) such as hydrolysis, esterification, transesterification, interesterification, alcoholysis, aminolysis, and acidolysis (Jaeger et al., 1994; Pandey et al., 1999; Kim et al., 2002a,b). Various unique qualities of lipases and esterases are responsible for imparting such high applicability/potential to these enzymes, which include organic solvent tolerance, thermostability, and their uniquely selective nature to transform different substrates to specific products. These enzymes are also known to exhibit substrate specificity, regiospecificity, stereospecificity and functional group specificity (Brockman and Borgstorm, 1984; Jaeger and Reetz, 1998).

Solvent tolerance

Water is known to be a poor solvent in preparative organic chemistry. The asymmetric hydrolyzing ability of lipases has been known for years which is based on the fact that lipases prefer to carry out hydrolysis in aqueous environments while in the

Reaction mechanism of a Rhizomucor meihei lipase

Figure 1.2 Reaction mechanism of a Rhizomucor meihei lipase.

non-aqueous environment, esterification, and transesterification reactions are favored (Klibanov, 1990). The benefits of reactions occurring in non-aqueous environments can be counted as the shift from hydrolysis to synthesis by esterification or transesterification, lower risks of contamination, and reduced formation of undesirable side reactions (Priyanka et al., 2019). Therefore, the choice of non-aqueous solvent systems/reaction environment is of utmost importance in biocatalytic processes.


Lipases generally exhibit this unique property of sustaining and maintaining the catalytic activity even under higher temperatures. A large number of lipases functioning in the range of 30-70°C have been reported. These thermozymes are attractive troubleshooters for processes such as those of organic synthesis. They offer additional advantages including the lower chances of microbial contamination, decreased viscosity of the reaction media, zero costs of maintaining setups at room temperature and increased enzyme activity. Talaromyces thermophilus lipase has been used in the synthesis of the popular anti-convulsant drug Pregabalin (Ding et ah, 2018). The thermostable lipase from Bacillus cereus C71 showed a preference for (R)-enantiomer of 2-arylpropanoate esters (Chen et al., 2007). A number of thermoalkaline lipases from Candida albicans as well as Candida rugosa have been used for the synthesis of chiral drugs and their intermediates as discussed in the text below.


The chemical selectivity of enzymes can be divided into four discrete categories:

i. Substrate selectivity (the ability to distinguish and act on a subset of compounds within a larger group of chemically related compounds)

ii. Stereoselectivity (the ability to act on a single enantiomer or diastereomer selectively)

iii. Regioselectivity (the ability to act on one location in a molecule selectively)

iv. Functional group selectivity (the ability to act on a single functional group selectively in the presence of other equally reactive or more reactive functional groups).

Due to their selective nature, lipases are known to aid the dynamic and kinetic resolution of various molecules, selective acylation and decylation of various drug moieties and resolution of sterically hindered compounds. Lipases are also used for the generation of enantiomerically enriched primary and secondary alcohols, chiral carboxylic acids and secondary amines (Isaksson et al., 2006). Lipases are also used for the synthesis of biopolymers such as polyesters and polyphenols, in the kinetic resolution of racemic mixtures, in secondary hydrolysis reactions, in transesterification and alcohol etherification reactions (Jaeger and Eggert, 2002).

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