St. Xavier’s College, Ahmedabad
Biocatalysis has evolved from a specialty technique for chiral resolution to a developed and ubiquitous technology in contemporary chemical synthesis and production. Facilitated by advances in bioinformatics, enzyme engineering, synthetic biology, and computational modeling, enzymes are now at the heart of sustainable and efficient manufacture of pharmaceuticals, fine chemicals, foodstuffs, and materials. Bench-mark achievements, like the nitrile hydratase-based acrylamide process and Evonik's enzymatic emollient esters, already illustrate the industrial promise of biocatalysis. Yet, wider application remains limited by factors such as stability of the enzyme, cost of production, infrastructure, and scalability. Advances in directed evolution, machine learning, and rational enzyme design in the past few years have also hastened the discovery of stable biocatalysts, while tools in retrosynthetic planning and hybrid chemo-enzymatic approaches widen the synthesis toolbox. Industrial viability more and more depends upon Key Performance Indicators (KPIs) such as yield, enantiomeric excess, product titer, space–time yield (STY), and catalyst loading, by which chemists can assess scalability and economics. Furthermore, developments in enzyme immobilization and the exploitation of non-conventional media have improved stability and expanded process utility. This review gives industrial chemists a working guide by emphasizing enzyme discovery and engineering breakthroughs, successful industrial applications, and the utility of KPIs to inform transformation decisions. In the end, biocatalysis is becoming an unsung pillar of green and industrially feasible chemical production, marrying lab innovation with industrialization.
Biocatalysis, the use of enzymes to catalysed chemical transformations, has undergone a remarkable transition over the past few decades. Once regarded as a niche technique for chiral resolution, it has now emerged as a mainstream and versatile strategy for chemical synthesis and large-scale manufacturing. Enzymes, by virtue of their high catalytic efficiency, stereoselectivity, and ability to operate under mild reaction conditions, provide clear advantages over traditional chemical catalysts. These features have positioned biocatalysis as a powerful driver of sustainable and resource-efficient chemical manufacturing, in line with global efforts toward green chemistry and reduced environmental impact.
The industrial significance of biocatalysis is exemplified by landmark processes such as the use of nitrile hydratase for acrylamide production, which achieves unmatched productivity and selectivity, and Evonik’s enzymatic route to emollient esters, which outcompeted conventional high-temperature chemical methods by reducing energy consumption and purification steps. Beyond specialty applications, enzymes are now deployed across diverse sectors including pharmaceuticals, food and beverages, agriculture, and fine chemicals, with the global industrial enzyme market steadily expanding and dominated by companies such as Novozymes, DuPont, and Roche. Importantly, biocatalysis has become a cornerstone of pharmaceutical manufacturing, enabling the cost-effective and enantioselective synthesis of active pharmaceutical ingredients (APIs) and chiral intermediates.[3] [4]
Despite these advances, the broader industrial adoption of biocatalysis faces challenges. Stability and cost remain critical bottlenecks: while certain enzymes like hydratases are inexpensive and robust, others such as cytochrome P450s can be costly to produce and often lack the durability required under industrial conditions. Moreover, infrastructure and economic barriers—including the need for facility modifications, high enzyme production costs, and tight development timelines in fast-moving industries like pharmaceuticals—can hinder large-scale implementation. Enzyme function outside aqueous environments further complicates their industrial use, though studies have demonstrated that enzymes retain activity, and in some cases gain enhanced properties, in organic solvents and non-conventional media.[5]
The last decade has witnessed significant breakthroughs that have expanded the reach of biocatalysis. Advances in bioinformatics, protein databases, directed evolution, ancestral sequence reconstruction (ASR), and machine learning have revolutionized enzyme discovery and engineering. The Nobel Prize in Chemistry (2018) awarded to Frances H. Arnold for directed evolution highlights the transformative impact of these approaches. Similarly, the advent of cheap synthetic genes, rational enzyme design tools, and retrosynthetic biocatalytic planning frameworks has accelerated route development, making enzymatic processes more predictable and scalable. The integration of biocatalysis with other catalytic strategies—including metal, organo-, photo-, and electro-catalysis—has further matured, providing chemists with hybrid platforms for complex synthesis. To evaluate feasibility and guide industrial adoption, Key Performance Indicators (KPIs) such as yield, enantiomeric excess, product titer, space–time yield (STY), and catalyst loading have emerged as essential benchmarks for decision-making in early-stage development.[6]
Key points
Enzymatic synthesis
1) Alcohol
Chiral alcohols are important components in many pharmaceuticals, fine chemicals, and agrochemicals. In the pharmaceutical industry, molecules that contain chiral hydroxyl (–OH) groups are often used as key building blocks for active drug ingredients. Bio catalysis is a popular method for producing chiral alcohols because it offers several benefits: it provides good control over stereochemistry, operates under mild conditions, avoids the use of metal catalysts, and is more environmentally friendly.
Two of the most common enzymatic approaches to make chiral alcohols are:
These methods are widely used and will be highlighted in specific case studies Their growing use is also evident from a patent study done between 2014 and 2019.One challenge with using KREDs is their need for expensive cofactors like NADH or NADPH. However, this issue has been largely solved at an industrial level: NADH recycling is now efficiently done using isopropanol as a hydride donor, without needing a second enzyme. NADPH recycling typically uses glucose dehydrogenase (GDH), with glucose as a co- substrate [7]
a) Chiral Alcohols Produced by KREDs
(S)-5Dulox Alcohol, a key intermediate in the synthesis of the antidepressant Duloxetine, can be efficiently produced using biocatalysis.
BASF’s approach uses two highly selective keto reductases (KREDs):
These enzymes can reduce the unstable chloro-ketone 3 to the desired (S)-alcohol (S)-4. An evolved enzyme variant works well in mixed solvent systems, is less affected by product inhibition, and allows NADH recycling using rac-2-butanol or isopropanol. The product (S)-4 is then aminated to yield (S)-5. An alternative route uses a more stable starting material, dimethylammonium-ketone 6, and the KRED RtSCR9 from Rhodosporidium toruloides. This method works at high concentration and delivers excellent enantioselectivity.[8]
[ Scheme 1. Routes to key precursor (S)-5 for the anti-depressant Duloxetine using KREDs]
LNP023, a treatment for kidney disease caused by inflammation (with more uses under clinical review), previously had a synthesis route that involved hazardous chemicals like sodium hydride and dimethylacetamide, raising safety concerns for large-scale production. Additionally, the earlier process had poor enantio- and diastereoselectivity, resulting in unwanted stereoisomers.
To improve the process, an enzymatic ketone reduction step using a KRED was introduced to establish one of the two stereocenters.[9]
This new approach significantly improved the synthesis by offering:
Scheme 2. Synthesis of the alcohol intermediate 10 of LNP023 by a KREDs
Industrial-scale processes using multiple biocatalysts to set several chiral centres have shown the strong potential of enzymatic synthesis.[10]
One notable example is Pfizer’s synthesis of a gamma secretase inhibitor:
Both fragments were produced with high stereopurity.
Pfizer utilized commercially available enzymes for screening, which ensured:
Scheme3. A route to the chiral intermediate 130f a gamma-secretase inhibitor using a KRED and a transamine.
b) Chiral Alcohols Produced by Lipases
Lipase-catalyzed kinetic resolution of racemic alcohols has historically been important in asymmetric synthesis. However, with the advent and commercial availability of keto reductases (KREDs), the synthetic role of lipases has diminished—unless used in dynamic kinetic resolution, desymmetrization, or when both enantiomers are useful. Despite this, lipases remain relevant, particularly for regioselective alcohol synthesis and desymmetrization. A notable recent example is the selective acylation of cyclopentene diol, a key step in prostaglandin synthesis, using lipase QL from Alcaligenes sp., successfully scaled up to 200 kg.[11]
Scheme4. Selective mono-acylation of diol 21 by lipase.
2) Amines
Chiral amines are critically important in the pharmaceutical and agrochemical industries. Over 90% of top-selling or newly approved small-molecule drugs are amines or derived from them, most of which are chiral. Additionally, around 30% of crop protection agents are chiral amines. As a result, the production of optically pure amines is a major focus in biocatalysis.[12]
A) Optically active amine
The transaminase-catalyzed reaction is the most versatile biocatalytic method for producing primary amines, converting carbonyl compounds into amines via reductive amination using a sacrificial amine donor. This approach has been extensively reviewed and recognized for its effectiveness in large-scale applications.
Scheme5. Trans amines-catalyzed reductive amination exemplified (S)-moipa.
The success of lipase technology in resolving simple chiral amines is due to the exceptionally high activity of certain lipases, such as Burkholderia plantarii lipase (BPL) and Candida antarctica lipase B (CALB). These enzymes, immobilized on polyester resin, selectively acylate the (R)-enantiomer, as shown with 1-phenylethylamine. A major breakthrough came when researchers at BASF and Bayer discovered that using methoxyacetic acid esters significantly enhanced the reaction rate.[13]
Scheme6. Lipase-catalyzed kinetic resolution of racemic benzylamine exemplified for 1-phenylethylamine 31.
3) Carbonyls, Carboxylic Acids and Derivatives
While aldehydes and ketones are typically produced chemically, biocatalytic methods offer advantages such as "all-natural" appeal in flavors and fragrances, and high regioselectivity. Two main biocatalytic approaches are alcohol oxidation (using dehydrogenases or oxidases) and C–C bond formation (via aldolases or lyases). A notable recent example is from the Hollmann group, which used Pleurotus eryngii aryl alcohol oxidase (PeAAOx) with catalase to efficiently oxidize alcohols like trans-2-hexen-1-ol to aldehydes in high yields and at large scale. The process demonstrated very high catalytic efficiency, with PeAAOx reaching a TTN of 2.2 million. Additionally, the Turner group engineered a choline oxidase variant with broad substrate scope for primary alcohols, offering a promising platform for future industrial development.[14]
Scheme7. Oxidation of trans-2-hexen-1-ol 49 to trans-2-hexen-1-al 50 by an aryl alcohol oxidase.
4) Carboxylic Acids and Esters
Carboxylic acids are essential in various industries, including chemical, food, materials, and pharmaceuticals. Biocatalysis is particularly well-suited for producing high-value chiral carboxylic acids. Thanks to recent progress in directed evolution and enzyme cascade technologies, biocatalytic methods have also shown promise for producing bulk carboxylic acids at the laboratory scale.
A) Acid Production via Hydrolysis of Nitriles
One of the most effective biocatalytic methods for producing carboxylic acids is through hydrolysis of nitriles using nitrilases or a nitrile hydratase–amidase system, which are highly active and don’t require external cofactors. These enzyme-based processes have been used at industrial scale for over two decades—for example, in the production of nicotinic acid (by Lonza) and (R)-mandelic acid (by BASF and Mitsubishi Rayon).
A particularly elegant process involves dynamic kinetic resolution of cyanohydrins, allowing for a theoretical 100% yield of (R)-mandelic acid. The Wei group developed a highly productive method using E. coli expressing a nitrilase (BCJ2315) from Burkholderia cenocepacia, achieving 350 g/L of product in 24 hours with 97.4% enantiomeric excess (ee), 93% isolated yield, and excellent space-time yield (STY) and catalyst efficiency.
However, for sterically hindered substrates like (R)-o-chloromandelic acid, used in making the drug (S)-Clopidogrel, most natural nitrilases are not efficient. A mutant version of was engineered to improve activity and selectivity, producing 93 g/L of (R)-o-chloromandelic acid in just 3 hours with 98.7% ee and a high STY of 31 g/L·h.
Another route to α-hydroxy acids is through hydroxynitrile lyases (HNLs), which catalyze the enantioselective addition of HCN to aldehydes. This method has also been used industrially for over 20 years, notably by DSM. For example, engineered HNLs from almond (PaHNL) have shown high activity and stability under acidic conditions. The A111G variant of PaHNL produced (R)-cyanohydrin with 97% ee and 96% yield at a high STY of 60 g/L·h. [15]
Scheme8. a) A nitrilase process (dynamic kinetic resolution) and b) a related hydroxynitrile lyase process for the synthesis of (R)-o-chloromandelic acid for the production of clopidogrel 64
Production of Pharmaceutical Enzymes
The majority of industrial enzymes, including those used in the pharmaceutical sector, are produced via microbial fermentation, primarily using bacteria and fungi. These microorganisms are favored efficiently scaled up in large fermentation tanks. Widely used species include E. coli, Bacillus subtilis, various lactic acid bacteria, filamentous fungi like Aspergillus spp. and Trichoderma atroviride, and yeasts such as Saccharomyces cerevisiae and Pichia pastoris.[16]
Advancements in genetic engineering have enabled these organisms to produce enzymes in significantly higher quantities. Choosing the right microbial strain is crucial for industrial success, especially strains that naturally secrete enzymes into the fermentation broth, which simplifies purification and reduces costs—though not all strains possess this trait.
Pharmaceutical enzymes are typically manufactured using GRAS (Generally Recognized As Safe) microbes. The production relies on either submerged fermentation (SmF) or solid-state fermentation (SSF). While SmF is more commonly used across industries, SSF is gaining popularity in certain specialized applications due to its specific advantages.[17]
Enzyme Production Through SmF
Submerged fermentation (SmF) takes place in a liquid medium where nutrients are dissolved or suspended, allowing microorganisms to grow in suspension. It is often used to produce extracellular and intracellular enzymes, especially with aerobic microbes in stirred-tank reactors.
SmF is popular because it can scale from small pilot fermenters (around 100 litres) to large industrial vessels (millions of litres). It also benefits from advanced process controls for temperature, pH, dissolved oxygen, and foam. While it allows for efficient mass and heat transfer, it does consume more energy.
There are four main types of SmF processes:
Enzyme Production Through Solid-State Fermentation
Solid-state fermentation (SSF) is an effective method for enzyme production. It offers several advantages over submerged fermentation (SmF), such as higher product yields, lower wastewater generation, simpler equipment, and fewer labor requirements. However, SSF is generally better suited for cases where the fermented product is used directly, rather than when pure enzymes are needed.
SSF commonly uses plant-based waste as substrates, like wheat bran, rice bran, sugarcane bagasse, wheat and rice straw, sawdust, corncobs, banana and cassava waste, oil cakes, and palm oil mill waste. These low-cost materials effectively support the growth of microorganisms that produce enzymes.[19]
Applications of Enzymes in Pharmaceutical Manufacturing
(A) Enzymes for the synthesis of antimicrobials
(I) Synthesis of 6-amino penicillanic acid by penicillin acylases
Penicillin acylases are enzymes that break the acyl side chain of penicillin to create 6-aminopenicillanic acid (6-APA) and an organic acid. Various microorganisms, including bacteria, actinomycetes, yeasts, and fungi, produce these enzymes.
They are categorized into three types based on substrate specificity:
Initially, 6-APA was made chemically with toxic substances, but this method was replaced by enzyme-based techniques, particularly using penicillin G acylase, due to better yields and safer conditions.[20]
Later, penicillin V acylase became a more effective option because:
Scheme 9. Penicillin acylase-catalyzed synthesis of some important semi-synthetic beta-lactam antibiotics.
(II) Synthesis of semisynthetic penicillins by penicillin acylases
Semisynthetic penicillins provide benefits over natural penicillin G and V. They offer greater stability, better absorption, fewer side effects, and improved effectiveness against antibiotic-resistant bacteria. [22]
Their large-scale production involves combining the beta-lactam core with suitable D-amino acids, a reaction that penicillin acylases catalyze. [23]
Additional key uses of penicillin G acylase include:
(III) Synthesis of the beta-lactam antibiotic key intermediate, 7-aminocephalosporanic acid
Cephalosporin-derived antibiotics are important and effective medications for treating bacterial infections. Many semisynthetic beta-lactam antibiotics, such as cefotaxime, ceftriaxone, cefuroxime, and cefdinir, are commercially available. Newer drugs like ceftobiprol and ceftaroline fosamil effectively target MRSA (methicillin-resistant Staphylococcus aureus).
Cephalosporin C is a commonly used and affordable starting material for producing 7-aminocephalosporanic acid (7-ACA), an essential intermediate for semisynthetic cephalosporins. It is obtained through microbial fermentation and can be turned into 7-ACA through amide bond cleavage. [24]
The enzymatic synthesis of 7-ACA involves several steps:
Scheme 10. Penicillin G acylase catalyzed enantioselective acylation of the L-enantiomers of methyl esters of phenylglycine and 4-hydroxyphenylglycine.
(2) Enzymes for the DKR of drugs
Enzymes are very selective and usually act on just one enantiomer (R or S), leaving the other one unchanged. In traditional enzymatic resolution, only half of the racemic mixture can convert to the desired enantiomer, which limits yield. Dynamic Kinetic Resolution (DKR) solves this problem by combining the racemization of the unreacted enantiomer with enantioselective transformation, allowing for up to 100% conversion to the desired enantiomer. [25]
Key Examples of Enzymatic DKR:
1) Yasukawa et al. – DKR of α-aminonitriles:
2) Burkholderia cepacia lipase:
3) Mandelic acid DKR:
(3) Enzymes for the synthesis of amino acids
Amino acids are crucial building blocks for life, important for health and nutrition in humans and animals. Their chirality makes them biochemically significant and useful in chemical synthesis. The nine essential amino acids—including L-leucine, L-isoleucine, L-valine, and others—cannot be produced by the human body and must come from diet. Enzymes and whole-cell biocatalysts are key in producing both proteinogenic and nonproteinogenic D- and L-amino acids, as well as pure amino acid derivatives, widely used in pharmaceuticals, cosmetics, and agrochemicals. The amino acid market has rapidly grown since the 1980s, driven by cost-effective production methods. Among these, fermentation and enzymatic catalysis are the most common due to their efficiency, low cost, and environmental friendliness.[26]
i) Enzymatic production of proteinogenic amino acids
Enzymes have been used for over 40 years in Japan to industrially produce L-amino acids, including key examples like L-methionine and L-valine, produced in large amounts using enzyme membrane reactor technology to minimize enzyme loss. L-methionine, used in special diets, is obtained through enzymatic resolution with acylase from Aspergillus oryzae. L-aspartic acid is made industrially by aspartase, which adds an amino group from ammonia to fumaric acid—this L-aspartate is further used to synthesize the artificial sweetener L-aspartame.[27] L-alanine is made from L-aspartate using aspartate β-decarboxylase. L-cysteine, primarily produced through electrochemical reduction of L-cystine in the past, is now synthesized more efficiently using a three-enzyme system (L-ATC hydrolase, S-carbamoyl-L-cysteine hydrolase, and ATC racemase) acting on DL-2-amino-2-thiazoline-4-carboxylic acid (ATC). Additionally, fermentation using genetically modified E. coli strains, like those with the yciW gene disrupted, has led to significant L-cysteine production. [28]
Scheme 11. Enzymatic transformation of cephalosporin C into 7-ACA catalyzed by D-amino acid oxidase and glutaryl-7-ACA-acylase.
(ii) Enzymatic production of nonproteinogenic amino acids
The enzymatic production of D-amino acids and nonproteinogenic L-amino acids is gaining attention as a green and efficient alternative. D-amino acids often appear as byproducts during the enzymatic resolution of racemic DL-amino acid mixtures for L-amino acids. However, they can also be produced directly—for example, by using D-specific acylases on racemic acetyl amino acids. A key industrial use involves producing D-phenylglycine and p-hydroxy-D-phenylglycine, important for semisynthetic antibiotics like ampicillin and amoxicillin, using a hydantoinase/carbamoylase enzyme system. Recent advances in molecular biology, especially directed evolution, have allowed modification of hydantoinases to switch from D- to L-specificity. Moreover, recombinant whole-cell systems can now coexpress racemases with selective hydantoinases and carbamoylases, facilitating the efficient production of various D- and L-amino acids. A newer, more cost-effective method for D-amino acid production involves dynamic kinetic resolution of N-succinyl amino acids, using D-succinylase and N-succinyl amino acid racemase to selectively hydrolyze the D-form, producing high-purity D-amino acids. [29-30]
(3) Enzymatic synthesis of pregabalin intermediate, (S)-3-cyano-5-methylhexanoic acid
Pregabalin, a strong anticonvulsant for treating seizures, neuropathic pain, and fibromyalgia, works exclusively through its (S)-enantiomer. Because of this, the asymmetric synthesis of the key intermediate, (S)-3-cyano-5-methylhexanoic acid, is vital for its pharmaceutical production. Both chemocatalytic (like bisphosphine rhodium catalysts) and biocatalytic (like nitrilase enzymes) methods have achieved high enantiomeric excess (98% and 97% ee, respectively), but are seen as environmentally and economically unsustainable. To tackle this, a more efficient and eco-friendlier biocatalytic method has been developed using an esterase from Arthrobacter sp. ZJB-09277 via whole-cell catalysis. This approach enables kinetic resolution of racemic esters, yielding (S)-3-cyano-5-methylhexanoic acid with 95.1% ee at a concentration of 44.6 mM, presenting a promising alternative for sustainable pregabalin synthesis. [31-32]
Scheme12. Dynamic kinetic resolution of alpha-amino acids
Scheme 13. Kinetic resolution of alpha-aminonitriles catalyzed by lipase
Enzyme Therapy
1) Proteolytic and glycolytic enzymes for treating damaged tissue
Proteolytic enzymes from bacterial and plant sources have been studied for burn debridement (removing dead skin), but their inconsistent performance and limited effectiveness have kept them from commercial use. The emergence of recombinant DNA technology has led to better enzyme formulations, such as Debrase gel dressing, a mixture of pineapple-derived enzymes. Approved by the US FDA in 2002, Debrase has shown consistent results and is now in Phase IIb clinical trials in the U.S. and Europe for treating deep partial-thickness and full-thickness thermal burns. [33]
2) Enzymes for the treatment of infectious diseases
Lysozyme, a naturally occurring bactericidal enzyme in the human body, is often added to food products and has shown promising activity against HIV by selectively degrading viral RNA, similar to RNase A and urinary RNase U, making it a potential candidate for HIV treatment. Another group of antimicrobial enzymes, chitinases, target chitin, an important structural component of the cell walls of various pathogens like fungi, helminths, and protozoa, making them useful in antimicrobial strategies. Furthermore, lytic enzymes from bacteriophages effectively kill bacteria such as Streptococcus pneumoniae, Clostridium perfringens, and Bacillus anthracis. The use of lytic bacteriophages is being developed as a treatment for bacterial infections, especially those caused by antibiotic-resistant strains, showing potential in fighting drug-resistant pathogens. [34]
Scheme 14. Acylation of racemic amine using penicillin G amidohydrolase.
3) Enzymes for the treatment of cancer
Using therapeutic enzymes in cancer treatment is a rapidly growing area. Notably, PEG-immobilized arginine deaminase, which reduces arginine levels, has been shown to inhibit certain cancers like hepatocellular carcinoma and skin cancers that lack argininosuccinate synthetase and are thus arginine-deficient. Another successful enzyme therapy involves PEGylated L-asparaginase (Oncaspar or pegaspargase), which has demonstrated better outcomes in treating acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin’s lymphoma. This works because normal cells can produce asparagine, but cancer
cells cannot, making them vulnerable to depletion of asparagine by the enzyme. While PEG-asparaginase formulations are more expensive than native enzymes, the overall treatment cost is similar, and both asparaginase and PEG-asparaginase are better alternatives to standard chemotherapy due to their effectiveness and targeted action. [35]
Future Prospects of Enzymes in Pharmaceuticals
Enzymes are increasingly replacing traditional chemical catalysts in pharmaceutical manufacturing, suggesting a promising future for their use in this field. Some enzymes, like superoxide dismutase (SOD) and catalase, naturally detoxify the body by working together—SOD transforms the highly toxic superoxide anion into moderately toxic hydrogen peroxide, which catalase then breaks down into harmless water and oxygen. However, despite PEGylation, these enzymes have not yet been effectively used for therapeutic detoxification in humans. Interestingly, studies show they can extend the lifespan of the nematode Caenorhabditis elegans, hinting at potential for human applications in longevity. Improving their structural and functional properties could help reduce organ damage during hemorrhagic shock. Another example is human butyrylcholinesterase, which detoxifies cocaine overdose; structural engineering and directed evolution have enhanced its activity. Overall, directed evolution and protein engineering are expected to create more efficient therapeutic enzymes and biocatalysts for pharmaceutical uses, improving their effectiveness and expanding their potential applications. [36]
Enzymatic activity in organic solvents
Enzyme catalysis in organic solvents is a valuable technique in biotechnology that widens the scope of biocatalysis beyond its natural aqueous environment. The process involves suspending enzymes, which need a small layer of water to maintain their structure, in an organic solvent to catalyze reactions. This method offers unique advantages for industrial chemistry, especially for modifying hydrophobic compounds. [37]
The lack of water can enable enzymatic reactions that are hindered in aqueous environments, such as transesterification, aminolysis, and ester synthesis. However, enzymes usually show much lower activity in organic solvents than in water, even though this drop is not unavoidable. Hydrophobic solvents are generally preferred because they keep essential bound water, unlike hydrophilic ones that strip it away. Since enzymes do not dissolve in organic solvents, they are used as lyophilized powders that must be stirred vigorously to overcome mass-transfer limitations. Lyophilization itself can cause enzyme denaturation, even more than exposure to organic solvent, but this can be reduced by adding lyoprotectants (such as sugars, PEG, salts, ligands, or crown ethers) or using crystalline enzymes or lipid-enzyme complexes. Also, enzymes keep a "pH memory," reflecting the ionization state from their last aqueous environment, and their activity can be boosted by lyophilizing them at optimal pH or adding the right buffer pairs. Structural rigidity from the absence of hydrogen bonding and lower dielectric constants in organic solvents also decreases enzymatic efficiency.[38] This can be improved by adding small amounts of water, hydrogen bond-forming solvents like glycerol, or mild denaturing co-solvents that increase enzyme flexibility without causing complete denaturation. By understanding and addressing these factors, enzymatic activity in organic solvents can be significantly enhanced, approaching or even matching the levels seen in aqueous solutions.[39]
Stability of enzymes in organic solvents
Enzyme Thermal Instability: Two Types
Water plays a key role in both:
Melting Temperature Increases Without Water Bovine ribonuclease melts at:
The same stability is observed when the enzyme is simply exposed to air or argon, indicating that the solvent does not interact directly with the enzyme. [40]
Other features of enzymatic catalysis in organic solvents
Enzymes in organic solvents show a remarkable "molecular memory" effect. Their catalytic behaviour depends on their preparation history due to the conformational rigidity caused by the dry environment. For instance, lyophilized enzymes that were previously exposed to water or ligands before being moved to an organic solvent often display significantly improved activity, changed substrate specificity, and increased stability. These effects disappear when the enzymes are redissolved in water. This phenomenon is due to conformational changes induced by ligands that remain in the dry state. Moreover, organic solvents can provide unique benefits over water, such as reducing unwanted side reactions. For example, mandelonitrile lyase achieves higher enantiomeric purity in organic solvents by preventing non-stereoselective cyanohydrin formation, which occurs in water. Additionally, enantioselectivity can be improved in organic solvents by temporarily enlarging a substrate through salt formation with bulky counter-ions. These would break apart in water but stay intact in non-aqueous media. This approach has significantly enhanced selectivity in lipase-catalyzed reactions, highlighting the unique capabilities of enzymatic catalysis outside of aqueous systems. [41-42]
Practical applications of enzymes in organic solvents
Using organic solvents in enzymatic reactions greatly increases the possibilities for bio trans formations. This approach enables processes that would be difficult or inefficient in water. For instance, direct asymmetric (trans)esterification and acylation allow for the effective production of optically pure acids, alcohols, and amines without needing an initial esterification step. Companies like Chemie Linz, Schering-Plough, and BASF have successfully scaled these methods for pharmaceutical production. Enzymes in organic solvents are also useful for making specialty polymers, such as enantioselective polyesters and polyphenols. These polymers can overcome solubility issues encountered in water. Supercritical fluids, which are even further removed from water, offer better control over polymer properties through pressure tuning. Interestingly, enzymes can also work in gas-phase reactions. This capability supports applications like breath and air analysis, where immobilized bi-enzymatic systems detect volatile compounds such as ethanol or formaldehyde through colour changes.[43-44]
Applications
Enzyme catalysis in organic solvents has found successful applications in several industries:
CONCLUSION
Enzymatic synthesis has become essential in modern pharmaceutical manufacturing. It offers a sustainable, selective, and scalable alternative to traditional chemical methods. Enzymes like KREDs and lipases enable efficient production of chiral alcohols and amines, which are important drug intermediates, under mild and environmentally friendly conditions. Their high regio- and stereoselectivity has been demonstrated in successful drugs like Duloxetine, LNP023, and gamma-secretase inhibitors, showing their value in industry, especially with integrated cofactor recycling. Beyond alcohols and amines, enzymes play a vital role in producing carbonyl compounds, carboxylic acids, esters, and amino acids, including complex molecules like (R)-mandelic acid and pregabalin intermediates. Enzyme therapy has also expanded their medical use to include cancer treatment, infectious diseases, detoxification, and burn care. Improvements in enzyme stabilization, organic solvent systems, and molecular memory have enhanced enzyme function in non-aqueous environments, allowing for various transformations like transesterification and aminolysis. Methods such as directed evolution, protein engineering, and computational design continue to improve enzyme performance and accessibility. In essence, enzymes have evolved from niche biocatalysts to crucial tools in chemical synthesis and therapeutics. Their increasing significance will be key to meeting future needs for greener, more efficient, and innovative pharmaceutical solutions.
REFERENCES
Ishwaki Patel, Enzyme Catalysis in The Synthesis of Pharmaceuticals & Industrial Application and Processes in Organic Solvents, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 10, 2020-2038. https://doi.org/10.5281/zenodo.17385865
10.5281/zenodo.17385865
