IUPAC classification of enzymes based on reactions they catalyze.
Biocatalysis has been continuously evolving as an essential tool which is playing a significant role in the industrial synthesis of chemicals, active pharmaceuticals, pharmaceutical intermediates, etc. where the high-yielding chemo-, regio-, and enantioselective reactions are needed. Despite its vital importance, industrial biocatalysis is facing certain limitations such as operational stability, economic viability, efficient recovery, and reusability. The limitations mentioned can be overcome by the isolation of specific enzyme producers from extreme environment by protein engineering, bioinformatics, and recombinant DNA technologies. Recently, chemoenzymatic pathway and biological cascade reactions have also been developed and designed to perform the synthesis of pharmaceuticals. In this chapter, we compile the broad applications of biocatalysts in the synthesis of pharmaceuticals.
- substrate specificity
Biocatalysis is appropriately defined as the enzyme-based applications for the transformation of molecular substrate into several natural as well as synthetic chemicals [1, 2]. The enzymes used in the process are in the form of cell lysate, whole cells, or purified enzyme and are prepared either as recombinant expressed proteins in different host cells or expressed in their native cells itself . The key players of biocatalysis are biocatalysts or enzymes that have been divided into six classes by the IUPAC nomenclature system based on the reactions they catalyze , as shown in Table 1.
|Enzyme class||IUPAC code||Catalyzed reactions||Important subclasses|
|Hydrolases||EC3||Hydrolytic reactions and their reversal||Esterases, glycosidases, lipases, proteases, peptidases, amidases|
|Oxidoreductases||EC1||Redox reactions||Dehydrogenases, oxidases, oxygenases, peroxidases, reductases|
|Transferases||EC2||Functional group transformation, addition/elimination involving C-C, C-N, and C-C bond formation or breakage||C1-transferases, glycosyltransferases, aminotransferases, phosphotransferases|
|Lyases||EC4||Elimination reactions||Aldolases, decarboxylases, dehydratase, few pectinases|
|Isomerases||EC5||Molecular isomerizations||Epimerases, racemases intramolecular transferases|
|Ligases/synthetases||EC6||Formation of a covalent bond joining two molecules together, coupled to hydrolysis of an ATP or analog||C-C, C-N, C-O, C-S ligases|
Enzymes as biocatalysts are incredibly proficient and are always preferred to conventional chemical processes. It is due to the fact that enzyme-based biocatalysis has distinct advantages over chemical reactions such as (1) significant specificity towards catalyzed reactions and recognized substrates, (2) simplified synthetic route, (3) high yields with exceptional regio-, chemo-, and stereoselectivities, (4) minimum energy requirements, and (5) generation of less by-products and wastes [5, 6, 7, 8]. Another preferred advantage includes whole bioprocess and bulk operations being carried out under mild conditions at elevated rates and with extreme specificity and with minimum environmental and physiological toxicity, thus making them an ideal candidate in the development and improvement of sustainable chemical processes [9, 10, 11, 12].
However, despite holding tremendous potential, biocatalysis has an inevitable pitfall associated with it when extreme conditions of industrial processes are to be considered. An efficient biocatalyst needs to be compatible enough with specific properties such as thermostability, catalytic ability, substrate specificity, and operational stability in turbulent flow regimes, toxic, hazardous solvents, and substrate inhibition [13, 14, 15, 16, 17, 18, 19, 20, 21].
Thus, there is a need for the identification and production of stable biocatalysts with broad industrial applicability by exploring and screening novel microbes or identification of new genes with desired properties through the analysis of genes responsible for enzyme production and stability. Further enhancement of the enzyme properties can be done by applying protein engineering tools such as molecular docking, directed evolution, molecular modeling, and process engineering [22, 23, 24, 25].
2. Scenario of biocatalysis in pharmaceuticals industries and its pertinent applications
In 1992, Roger Sheldon estimated environmental impact factor (E factor) (kg waste/kg product) for several chemical industries, and an E factor of 25–>100 was noted in the pharmaceutical industries . Thus, to reduce the harmful impact of pharmaceutical manufacturing processes and making it more sustainable, “green chemistry” has been increasingly adopted. An efficient biocatalytic process encompasses the “12 principles of green chemistry” to an extent which give it an edge over other technologies , as shown in Figure 1.
In Europe, a project CHEM21 was launched by the collaboration of both government and industries for the implementation of green technology in the chemical and pharmaceutical sectors [28, 29, 30]. The project was launched because of the replacement of biocatalysis over chemical in the synthesis of pharmaceuticals involving several redox reactions, chiral amine synthesis, and regio- and stereospecific hydroxylation of abundant compounds [18, 28, 31]. Since then biocatalysis has been profitably used for the production of pharmaceutically active chemicals and several blockbuster drugs at the industrial level and some of which are mentioned below:
Sitagliptin—Sitagliptin, an antidiabetic compound, was successfully produced via biocatalytic approach. It finds application in the treatment of type II diabetes and is sold under the trade name “Januvia” by Merck [32, 33]. This work was accomplished by engineering R-selective transaminase (R-ATA, ATA-117) from Arthrobacter species by researchers at Codexis and Merck. The drug produced was having 99.95% enantiopurity even in the presence of 1 M i-PrNH2 with 50% DMSO and at a temperature >40°C . Conventionally, it was prepared using rhodium, a heavy metal as a catalyst. However, on comparing both processes, the biocatalytic method showed a massive reduction in waste as well as the use of heavy metal. Besides this, the overall yield and productivity were increased by 10 and 53% . The R- and S-selective ATA was also used in the production of a variety of drugs such as niraparib and the production of an antagonist of orexin receptor with the formation of inhibitor of JAK kinase pathway [35, 36, 37, 38].
Boceprevir—This is a product of chiral amine synthesis and is marketed by Merck under trade name Victrelis. It is used for the treatment of chronic hepatitis C infections. In the production process, monoamine oxidase (MAO) from fungus Aspergillus niger was used for the asymmetrical amine oxidation of bicyclic proline intermediate . The biocatalytic process increased yield by 150%, with an overall reduction in raw materials and side products as waste. At present, engineered monoamine oxidase (MAO) is also used in the production of another hepatitis C drug, telaprevir [34, 40], and various other synthetic drugs such as solifenacin, levocetirizine along with few natural alkaloid products (confine, harmicine, elegance, and leptaflorine).
Montelukast—Montelukast or Singulair (trade name) is an anti-asthmatic drug marketed by Merck . The engineered keto-reductase (KRED) was used for the production of montelukast, which displayed significant enantioselectivity (99.9%) and was stable in 70% organic solvent and temperature of 45°C . The biocatalytic method was advantageous in the sense that it omitted the use of hazardous chemical catalyst chlorodiisopinocampheylborane (DIP-CI), which was conventionally used. Several other drugs such as atorvastatin, crizotinib, duloxetine, and phenylephrine were also developed by biocatalytic process using KRED from bacterium Lactobacillus kefir .
Atorvastatin—It comes from the statin family and is marketed under the trade name Lipitor by Pfizer. This drug reduces cholesterol levels by inhibiting the synthesis of cholesterol in the liver . Atorvastatin production is also carried out by employing KRED for the production of hydroxy nitrile, an important intermediate. It is a multienzyme process involving glucose dehydrogenase (GDH), KRED, and halohydrindehalogenase (HHDH). Thus, the process is environmentally as well as economically feasible.
Pregabalin—Pregabalin, a lipophilic GABA (γ-aminobutyric acid) analog, finds use in the treatment of various central nervous system ailments including neuropathic pain, fibromyalgia, epilepsy, and anxiety [43, 44]. Its production was carried out by biocatalytic conversion of rac-2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester to 2-carboxyethyl-3-cyano-5-methylhexanoic acid using lipolase. A heat-promoted decarboxylation of 2-carboxyethyl-3-cyano-5-methylhexanoic acid yielded (S)-3-cyano-5-methylhexanoic acid ethyl ester, which is a principal known precursor of pregabalin . The mentioned chemoenzymatic synthesis route not only produced increased yields of pregabalin (40–45%) but also eliminated wastes and usage of organic solvent.
7-ACA (7-aminocephalosporic acid)—Cephalosporin has been extensively used as semisynthetic antibiotics; it acts on bacterial cell wall (peptidoglycan) synthesis. 7-Aminocephalosporanic acid (7-ACA), the critical intermediate or precursor for the production cephalosporins, is biocatalytically produced by enzymatic deacylation of cephalosporin-C (CPC). A two-step enzymatic process utilizes D-amino acid oxidase (DAAO) and 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase (GLA) for two consecutive reactions. Also, a single-step conversion from CPC to 7-ACA has been reported . It has been successfully applied for the conversion of CPC to 7-ACA at industrial level . Similarly, 6-aminopencillanic acid has been reported for the synthesis of semisynthetic penicillins using penicillin acylase .
Some other noteworthy examples and recent progress being made in pharmaceutical synthesis using enzymes from various sources are represented in Table 2.
|Biocatalysts||Microbial sources||Pharmaceutical compounds||References|
|Lipase B||Candida antarctica||Reboxetine|||
|Carbonyl reductase (YlCR2)||Yarrowia lipolytica||Statins|||
|Acyltransferase (LovD)||Whole-cell Escherichia coli strain overexpressing LovD||Simvastatin||[52, 53]|
|Engineered cyclohexanone monooxygenase||—||Armodafinil|||
|(+)-γ-lactamases||Bradyrhizobium japonicum USDA 6||Carbovir, abacavir, melogliptin||[5, 55]|
|Immobilized lipase||Thermomyces lanuginosus||Rasagiline mesylate (active ingredient of AZILECT®)|||
|Expressing tyrosine phenollyase||Erwinia herbicola cells|||
|E. coli cells expressing cellobiose 2-epimerase||Caldicellulosiruptor saccharolyticus||Lactulose|||
Biocatalysis has made a remarkable journey so far and has been successfully applied for the numerous biotransformation processes in several industries. It has benefitted nearly all sectors, particularly chemical and pharmaceuticals. The flourishing development of economically viable and sustainable chemoenzymatic processes highly depends on the broader availability and applicability of enzymes with robust performance irrespective of extreme conditions. Recent surveys have shown that most of the biocatalysts are being used in the synthesis of pharmaceuticals or drugs or intermediates replacing some of the chemical processes, but their stability, selectivity, and specificity are of prime concern.
4. Future prospects
Based on the literature available on the role of biocatalysts in the drug/pharmaceutical synthesis, biocatalysts with improved desired characteristics can be achieved by a multifaceted approach, as shown in Figure 2.
Several tools and techniques represented above will enhance the biocatalytic stability, activity, enzyme-substrate affinity, and thermostability and will lead to higher yield. Also, the incorporation of artificial metabolic pathways, cell factory design, and nanotechnology approaches will further aid towards a suitable biocatalytic process. It will also ensure the quality and productivity of the drugs manufactured by optimizing safe process development. Thus, we envision that biocatalysis will be a more radical approach that is going to feat the arena of pharmaceutical manufacturing as well as other sectors such as bioenergy and waste treatment that are far more challenging at present.
Conflict of interest
The authors declare that there are no conflicts of interests whatsoever.