Biocatalysis Guide

Introduction

The increasing complexity of active pharmaceutical ingredients (APIs) demands efficient and sustainable synthesis routes, prompting the pharmaceutical industry to adopt innovative methodologies. Biocatalysis has emerged as a particularly attractive solution, as it enables streamlined syntheses under mild reaction conditions with intrinsically safer reaction profiles compared with conventional chemistry.

The ACS Green Chemistry Institute Pharmaceutical Roundtable (GCIPR) has developed a Biocatalysis Guide to provide a quick reference to the most common enzyme classes used in the pharmaceutical industry for retrosynthetic analysis. This high-level visual guide gives the enzyme description, substrate scope rating, and a generic reaction scheme. Updated in 2026, enzymes and reactions are now ordered based on the number of peer reviewed publications at various scales.

Name

[generic; specific examples]

Scheme
Key info

Cofactor:
Bold = cofactor or sub-stoichiometric additive/enzyme
Bold + Italics = cofactor that requires a recycling system

Substrate scope:
Broad to Medium to Limited

Most commonly used biocatalytical transformations

≥2 Peer reviewed examples of reactions scaled to ≥ 1 kg, or multiple double digit gram. Enzymes available at > 100 g scale

Hydrolase

[lipases, esterases, PGA]

R, R’, R” can contain asymm centers, often used for kinetic resolutions and desymmetrizations. When immobilized can tolerate organic solvents.

Broad Substrate Scope

Ketoreductase

[KRED, carbonyl-reductase, alcohol dehydrogenase]

R- and S-selectivities available. Dynamic kinetic resolutions possible within R’ and R” groups. Eqm usually favors alcohol product. Can run in oxidative direction.

Broad Substrate Scope

Transaminase

[aminotransferase, ATA, TA, w-TA]

R- and S-selectivities available. Dynamic kinetic resolutions possible. Eqm usually favors ketone, requires driving towards amine product.

Broad Substrate Scope

Iminereductase

[IRED, reductive aminase, amine dehydrogenase]

Asymmetric intermolecular reductive amination with IRED and RedAm. Some IRED only acitive on preformed imines.

Medium Substrate Scope

Enereductase

[enoate reducatase, ERED]

Trans reduction of the alkene. Selectivity can be engineered, steric crowding generally poorly tolerated. Eqm requires driving

limited substrate scope

Nitrilase

[NIT]

Irreversible conversion of nitrile to acid (enzymes that convert nitrile to amide are nitrile hydratases). Used in kinetic resolutions or chemoselective hydrolysis of one nitrile over another.

medium substrate scope

Aldolase

Several classes of aldolase, e.g. DERA (deoxyribose aldolase), others such as pyruvate and fructose aldolase also known.

limited substrate scope

Amino acid dehydrogenase

[AADH, LAADH, DAADH]

Most commonly used in the ‘reverse’ direction to form novel amino acids. R- and S- selective enzymes available. Deracemization of amines when coupled to compatible chemical reductant.

medium substrate scope

α-Ketoglutarate dependent dioxygenase

An α-ketoglutarate dependent enzyme catalyzes the aerobic, unspecified hydroxylation of the amino acid D-proline. Iron (II) salts, α-ketoglutarate are required reagents, whereas ascorbic acid is not always required.

Non-heme Fe(II)- and α-ketoglutarate dependent enzymes using O2 as oxidant. Ascorbic acid generally required. Enzymes available for regio- and stereoselective hydroxylation of cyclic as well as acyclic amino acids. Non-amino acids can also be substrates.

limited substrate scope

Ammonia lyase

[amino acid ammonia lyase]

PAL phenylalanine ammonia lyase, TAL tyrosine ammonia lyase most commonly used but others available. Used in the amino acid forming direction with very high ammonia concentrations to drive equilibrium.

medium substrate scope

Baeyer-Villiger monooxygenase

[BVMO, cyclohexane monooxygenase]

Asymmetric BV reaction, asymmetric sulfide oxidation to sulfoxide.

limited substrate scope

≥ 2 Peer reviewed examples of reactions scaled to ≥ 10 g

Hydroxynitrile lyase

[HNL]

Catalyzes the formation and hydrolysis of α-hydroxy nitriles from/to aldehydes and cyanide. Used in a commercial approach to mandelic acid.

limited substrate scope

Nitrile hydratase

[HNL]

Irreversible conversion of nitrile to amide (enzymes that convert nitrile to acid are nitrilases). Kinetic or dyamic resolution possible with enolisable proton.

medium substrate scope

Epoxide hydrolase

[EH]

Irreversible conversion of epoxide to diol. Mostly used for kinetic resolution (KR). Some EHs are stereoconvergent (SC), ie convert a racemic epoxide to single enantiomer diol. Different mechanistic classes exist.

limited substrate scope

Monoamine oxidase

[MAO]

Desymmetrization of pyrrolidines, and trap of imine. Primary amine oxidation. Deracemization of amines when coupled to compatible chemical reductant.

medium substrate scope

Alcohol oxidase

[AO]

Galactose oxidase is shown aerobically oxidizing the 6-hydroxl group D-galactose to an aldehyde. Copper (I) salt, horse radish peroxidase and catalase are necessary additives in this reaction.

Many sub-types with different substrate selectivities e.g. galactose oxidase (GO) acts on primary alcohols in polyols and benzylic alcohols. Kinetic resolutions possible. Oxygen mass transfer limited.

limited substrate scope

Halohydrin dehalogenase

[HHDH]

Two reactions are shown. This first is halohydrin dehalogenasae catalyzing a reaction of generic 1-haloalkan-2-ol with cyanide or azide to give a generic 1-cyano or 1-azido-alkan-2-ol product. The second reaction is the halohydrin dehalogenasae catalyzing the ring opening of a generic terminal epoxide with cyanide or azide to give the same product. No cofactors are required.

Catalyze the conversion of vicinal halohydrins to epoxides, as well as epoxide ring opening. Closely related to some epoxide hydrolases.

limited substrate scope

Unspecific peroxygenase

[UPO]

Fungal heme containing enzymes use hydrogen peroxide as oxidant and require no cofactors. They have varying oxidative capabilities including:

Hydroxylation, epoxidation, N- or S- oxidation, bromination, dealkylation

medium substrate scope

Tryptophan synthase

[TrpB]

Native reaction forms L-tryptophan using PLP cofactor. Many non-canonical amino acids have been produced with engineered variants.

limited substrate scope

≥ 1 Peer reviewed example of reactions scaled to multi-mg

Carboxylic acid reductase

[CAR]

Multidomain enzyme that uses ATP to transform the carboxylic acid to a thioester, and then reduces the thioester to the aldehyde with NADPH.

limited substrate scope

Halogenase

[CAR]

Halogenation of aromatic rings. Halogenation takes place via a halogenated lysine residue. Regiochemistry can be controlled via directed evolution of the enzyme.

limited substrate scope

Cytochrome P450

[P450]

Heme containing enzymes using oxygen as oxidant. Requires electron transfer proteins either as part of the enzyme or added enzymes, often nicotinamide dependent. They have varying oxidative capabilities including:

Hydroxylation, desaturation, epoxidation, N- or S-oxidation, dealkylation

limited substrate scope

Amide ligase

[amide synthetase]

ATP dependent amide formation between acid and amine.

limited substrate scope

Nicotinamide cofactor recycling

The image shows the closely related structures of the reduced (NADPH and NADH) and oxidized (NADP+ and NAD+) of the nicotinamide cofactors. The difference between the two series is that NADP series as a 2’-phosphate on the adenyl nucleotide.

Most commonly used reductive nicotinamide regenerating systems

The image shows the conversion of D-glucose to D-gluconic acid, with concomitant conversion of oxidized to reduced cofactors.

Glucose dehydrogenase

[GDH]

Gluconic acid formation drops reaction pH, and may required the use of a pH stat. Highly active enzyme. Active on both NAD+ and NADP+.

The image shows the conversion of a generic secondary alcohol to a generic ketone, with concomitant conversion of oxidized to reduced cofactors.

Ketoreductase

[KRED, alchol dehydrogenase]

Uses an alcohol, such as isopropanol, to reduce NAD(P)+ For ketone reductions the KRED often is dual purpose, reducing the desired substrate and oxidizing IPA. Reaction is reversible.

The image shows the conversion of formic acid to carbon dioxide, with concomitant conversion of oxidized to reduced cofactors.

Formate dehydrogenase

[FDH]

Irreversible conversion of formic acid salts to CO2. Generally less active than GDH. Often NAD selective.

Less commonly used reductive nicotinamide regenerating systems

For oxidative approaches

Irreversible conversion of reduced co-factor to oxidized cofactor in presence of O2. NADH or NADPH activity available. Sacrificial substrate approach (similar to KRED + IPA). Use unsaturated donor that can aromatize when oxidized.

Non-enzymatic methods

Electrochemical
Potentially the ‘greenest’ approach, still in development.

Photochemical
Still in development.

Non-abundant metal hydrogenation
Still in development, but questionable sustainability.

Adenosine triphosphate (ATP) recycling

The image shows the structures of the adenosine-5’-phosphate series from adenosine (no phosphate) to adenosine triphosphate

Most commonly used ATP regenerating systems

Acetate kinase

[AcK]

Acetylphosphate is relatively easy to make, but hydrolytically unstable. Underused in industry.

Polyphosphate kinase

[PPK]

Type III PPK can convert AMP to ATP, other classes convert AMP to ADP. Polyphosphate is very cheap, and hydrolytically stable. Not all phosphate units are transferred. Underused in industry.

Phosphoenolpyruvate kinase

[PK]

Phosphoenolpyruvate (PEP) is expensive, used mostly in academic settings.

Adenylate kinase

[AK]

Used in combination with another enzymes that convert AMP –> ADP (e/g. PPT).

Less commonly used ADP/ATP regenerating systems

Creatine kinase

Stable creatine phosphate and thiophosphate is made chemically. Enzyme can transfer either phosphate or thiophosphate.

Polyphosphate transferase

[PPT]

Polyphosphate is very cheap, and hydrolytically stable. Not all phosphate units are transferred. Underused in industry.

The image shows the reversible action of adenylate kinase on ADP and ATP converting to two equivalents of ADP, whereupon acetate kinase converts them both to ATP via transfer of phosphate from acetyl phosphate.

Combinations for recycling AMP to ATP

AK forms ADP, which is acted upon by AcK in presence of acetylphosphate to give ATP. PPK and polyphosphate could be used in place of AK and acetylpphosphate.

Download the Biocatalysis Guide

Biocatalysis Guide in A4 format

References

Hydrolase

Development of a Chemoenzymatic Manufacturing Process for Pregabalin. C. A. Martinez et al., Org. Process Res. Dev. 2008, 12, 3, 392–398. 3500 kg scale.
The Development of a Manufacturing Route for the GPIIb/IIIa Receptor Antagonist SB-214857-A. Part 2: Conversion of the Key Intermediate SB-235349 to SB-214857-A. R. J. Atkins et al., Org. Proc. Res. Dev. 2003, 7, 5, 663–675. 250 kg scale/14kg scale.
A Practical and Robust Process to Produce SB-214857, Lotrafiban, ((2S)-7-(4,4‘-Bipiperidinylcarbonyl)-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1, 4-Benzodiazepine-2-acetic Acid) Utilising an Enzymic Resolution as the Final Step. T. C. Walsgrove et al., Org. Proc. Res. Dev. 2002, 6, 4, 488–491. 80 kg scale.

Hydrolase Reagent Guide

Ketoreductase

Development of a Biocatalytic Process as an Alternative to the (−)-DIP-Cl-Mediated Asymmetric Reduction of a Key Intermediate of Montelukast. J. Liang et al., Org. Process Res. Dev. 2010, 14, 1, 193–198. 230 kg scale.
Development of a First-Generation Stereocontrolled Manufacturing Process of TRPA1 Inhibitor GDC-6599. A. Y. Hong et al., Org. Process Res. Dev. 2024, 28, 6, 2325–2333. 120 kg scale.
A green-by-design biocatalytic process for atorvastatin intermediate. S. K. Ma et al., Green Chem., 2010, 12, 81-86. >100 kg scale (unpublished)
Bio- and Chemocatalysis for the Synthesis of Late Stage SAR-Enabling Intermediates for ROMK Inhibitors and MK-7145 for the Treatment of Hypertension and Heart Failure. R. T. Ruck et al., Org. Process Res. Dev. 2021, 25, 3, 405–410. 97 kg scale.
Chemical and Biochemical Approaches to an Enantiomerically Pure 3,4-Disubstituted Tetrahydrofuran Derivative at a Multikilogram Scale: The Power of KRED. A. Bigot et al., Org. Process Res. Dev. 2024, 28, 12, 4467–4476. 40 kg scale.
A Chemoenzymatic Route to Chiral Intermediates Used in the Multikilogram Synthesis of a Gamma Secretase Inhibitor. M. Burns et al., Org. Process Res. Dev. 2017, 21, 6, 871–877. 35 kg scale.
Merging Biocatalysis, Flow, and Surfactant Chemistry: Innovative Synthesis of an FXI (Factor XI) Inhibitor. B. Wu et al., Org. Process Res. Dev. 2020, 24, 11, 2780–2788. 3.5 kg scale.
Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 5─Chemoenzymatic Diastereoselective Fluorination/DKR. S. D. McCann et al., Org. Process Res. Dev. 2024, 28, 2, 441–450. 1 kg scale.

KRED Reagent Guide

Transaminase

A Chemoenzymatic Route to Chiral Intermediates Used in the Multikilogram Synthesis of a Gamma Secretase Inhibitor. M. Burns et al., Org. Process Res. Dev. 2017, 21, 6, 871–877. 25 kg scale.
Evolution of a Green and Sustainable Manufacturing Process for Belzutifan: Part 5─Chemoenzymatic Diastereoselective Fluorination/DKR. S. D. McCann et al., Org. Process Res. Dev. 2024, 28, 2, 441–450. 12 kg scale.
Application of Transition-Metal Catalysis, Biocatalysis, and Flow Chemistry as State-of-the-Art Technologies in the Synthesis of LCZ696. X. Gu et al., J. Org. Chem. 2020, 85, 11, 6844–6853. 4.4 kg scale.
Preclinical Toxicology Supply for a Complex API Enabled by Asymmetric Catalysis and Rapid Chemical Development: IL-17A Inhibitor LY3509754. T. Beauchamp et al., Org. Process Res. Dev. 2025, 29, 3, 889–908. 1.2 kg scale.
From Chiral Resolution to Diastereoselective Ellman Chemistry to Biocatalysis: Route Evolution for the Efficient Synthesis of the Tetrahydrobenzoazepine Core of BTK Inhibitor BIIB091. C. Li et al., Org. Process Res. Dev. 2023, 27, 8, 1463–1473. 1 kg scale.
Biocatalytic Asymmetric Synthesis of Chiral Amines from Ketones Applied to Sitagliptin Manufacture. C. K. Saville et al., Science 2010, 329 (5989), 305-309. 1 kg in Sl.

Transaminase Reagent Guide

Enereductase

Rapid Development of a Scalable Reduction of R-Carvone Utilizing Commercially Available Biocatalysts. S. M. McKenna et al., Org. Process Res. Dev. 2025, 29, 6, 1571–1576. 100 kg scale.
Identification and Implementation of Biocatalytic Transformations in Route Discovery: Synthesis of Chiral 1,3-Substituted Cyclohexanone Building Blocks. T. Hadi et al., Org. Process Res. Dev. 2018, 22, 7, 871–879. 40 kg scale.
Enantioselective Enzymatic Reduction of Acrylic Acids. C. An et al., Org. Lett. 2020, 22, 21, 8320–8325. 1.6 kg scale.

Ene Reductases Reagent Guide

Nitrilase

Efficient Chemoenzymatic Synthesis of Optically Active Pregabalin from Racemic Isobutylsuccinonitrile. Q. Zhang et al., Org. Process Res. Dev. 2019, 23, 9, 2042–2049. 300 kg scale.
Asymmetric Synthesis of Akt Kinase Inhibitor Ipatasertib. C. Han et al., Org. Lett. 2017, 19, 18, 4806–4809. 200 kg scale.
Enzyme Optimization and Process Development for a Scalable Synthesis of (R)-2-Methoxymandelic Acid. M. E. Scott et al., Org. Process Res. Dev. 2022, 26, 3, 849–858. 40 kg scale.
Identification and Implementation of Biocatalytic Transformations in Route Discovery: Synthesis of Chiral 1,3-Substituted Cyclohexanone Building Blocks. T. Hadi et al., Org. Process Res. Dev. 2018, 22, 7, 871–879. 20 kg scale.
Process Development for the Production of (R)-(−)-Mandelic Acid by Recombinant Escherichia coli Cells Harboring Nitrilase from Burkholderia cenocepacia J2315. H. Wang. Et al., Org. Process Res. Dev. 2015, 19, 12, 2012–2016. 3.5 kg scale.

Epoxide Hydrolase

Development of an Enzymatic Process for the Production of (R)-2-Butyl-2-ethyloxirane. G.-D. Roiban et al., Org. Process Res. Dev. 2017, 21, 9, 1302–1310. 20 g scale.
Enantioselective Hydrolysis of Racemic and Meso-Epoxides with Recombinant Escherichia coli Expressing Epoxide Hydrolase from Sphingomonas sp. HXN-200: Preparation of Epoxides and Vicinal Diols in High ee and High Concentration. S. Wu et al., ACS Catal. 2013, 3, 4, 752–759. 10 g scale.
Cascade Biocatalysis for Sustainable Asymmetric Synthesis: From Biobased l-Phenylalanine to High-Value Chiral Chemicals. Y. Zhou et al., Angew. Chem. Int Ed., 2016, 55 (38),11647-11650. 1.7 g scale.
Enantioselective Bio-Hydrolysis of Various Racemic and meso Aromatic Epoxides Using the Recombinant Epoxide Hydrolase Kau2. W. Zhao et al., Adv. Synth. Catal., 2015, 357 (8), 1895-1908. 1 g scale.

Aldolase

Preparation of β-hydroxy-α-amino Acid Using Recombinant ᴅ-Threonine Aldolase. S. L. Goldberg et al., Org. Process Res. Dev. 2015, 19, 9, 1308–1316. ~4 kg scale.
An efficient process for production of ɴ-acetylneuraminic acid using ɴ-acetylneuraminic acid aldolase. M. Mahmoudian et al., Enzyme and Microbial Technology 1997, 20 (5), 393-400. Scale: 817 g limiting reagent.
Development of an efficient, scalable, aldolase-catalyzed process for enantioselective synthesis of statin intermediates. W. A. Greenberg et al., PNAS, 2004, 101 (16), 5788-5793. 11.1 g scale.
Design of an in vitro biocatalytic cascade for the manufacture of islatravir. M. A. Huffman et al., Science 2019, 366 (6470), 1255-1259. 8.75 mmole,~ 1.5 g scale (>1 kg unpublished).
Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase. S. Jennewein et al., Biotechnology Journal 2006, 1 (5), 537-548. Scale: 0.4 g limiting substrate ~ 1.3 g product
Application of Threonine Aldolases for the Asymmetric Synthesis of α-Quaternary α-Amino Acids. J. Blesl et al., ChemCatChem., 2028, 10 (6), 3453-3458. 19 g scale, but low yield of ~800 mg.
ᴅ-Serine as a Key Building Block: Enzymatic Process Development and Smart Applications within the Cascade Enzymatic Concept. N. Ocal et al., Org. Process Res. Dev. 2020, 24, 5, 769–775. ~100 mg scale.

Adolase Reagent Guide

Monoamine Oxidase

Efficient, Chemoenzymatic Process for Manufacture of the Boceprevir Bicyclic [3.1.0]Proline Intermediate Based on Amine Oxidase-Catalyzed Desymmetrization. T. Li et al., J. Am. Chem. Soc. 2012, 134, 14, 6467–6472. 40 g scale (> 1 kg unpublished).
Enantioselective oxidation of O-methyl-N-hydroxylamines using monoamine oxidase N as catalyst. T.S. C. Eve et al., Chem. Commun., 2007, 1530-1531. 1.2 g scale.
A Chemo-Enzymatic Route to Enantiomerically Pure Cyclic Tertiary Amines. C. J. Dunsmore et al., J. Am. Chem. Soc. 2006, 128, 7, 2224–2225. 0.4 g scale.

Baeyer-Villiger Monooxygenase

Development and Scale-Up of an Improved Manufacturing Route to the ATR Inhibitor Ceralasertib. M. A. Graham et al., Org. Process Res. Dev. 2021, 25, 1, 43–56. 64 kg scale.
Development and Scale-up of a Route to ATR Inhibitor AZD6738. W. R. F. Goundry et al., Org. Process Res. Dev. 2019, 23, 7, 1333–1342. 62 kg scale.
Baeyer–Villiger Monooxygenase-Mediated Synthesis of Esomeprazole As an Alternative for Kagan Sulfoxidation. Y. K. Bong et al., J. Org. Chem. 2018, 83, 14, 7453–7458. 30 g scale.
The First 200-L Scale Asymmetric Baeyer−Villiger Oxidation Using a Whole-Cell Biocatalyst. C. V. F. Baldwin et al., Org. Process Res. Dev. 2008, 12, 4, 660–665. 200 L scale.

BVMO Reagent Guide

Amino Acid Deydrogenase

Preparation of an Amino Acid Intermediate for the Dipeptidyl Peptidase IV Inhibitor, Saxagliptin, using a Modified Phenylalanine Dehydrogenase. R. L. Hanson et al., Adv. Synth. Catal., 2007, 349 (8-9), 1369-1378. 40 kg scale.
A one-pot system for production of l-2-aminobutyric acid from l-threonine by l-threonine deaminase and a NADH-regeneration system based on l-leucine dehydrogenase and formate dehydrogenase. R. Tao et al., Biotechnol. Lett., 2014, 36, 835–841. 4.6 kg scale.
Preparation of (S)-1-Cyclopropyl-2-methoxyethanamine by a Chemoenzymatic Route Using Leucine Dehydrogenase. W. L. Parker et al., Org. Process Res. Dev. 2012, 16, 3, 464–469. 50 g scale.
Asymmetric Synthesis of iso-Boc (S)-2-Amino-8-nonenoic Acid in One Through-Process. J. Park et al., Org. Process Res. Dev. 2016, 20, 1, 76–80. ~15 g scale.
Biocatalytic synthesis of (2S)-5,5,5-trifluoroleucine and improved resolution into (2S,4S) and (2S,4R) diastereoisomers. H. Biava et al., Tet. Lett., 2013, 54 (28), 3662-3665. 1.5 g scale.

Ammonia Lyase

Calcium Alginate Bead Immobilization of Cells Containing Tyrosine Ammonia Lyase Activity for Use in the Production of p-Hydroxycinnamic Acid. R. J. Trotman et al., Biotechnol. Progress 2008, 23 (3), 638-644. >4 kg scale.
Toward a Scalable Synthesis and Process for EMA401, Part III: Using an Engineered Phenylalanine Ammonia Lyase Enzyme to Synthesize a Non-natural Phenylalanine Derivative. L. A. Hardegger et al., Org. Process Res. Dev. 2020, 24, 9, 1763–1771. 2 kg scale.
Asymmetric Synthesis of (S)-2-Indolinecarboxylic Acid by Combining Biocatalysis and Homogeneous Catalysis. B. de Lange et al., ChemCatChem., 2011, 3 (2), 289-292. 18.1 g scale.
Cascade Biocatalysis for Sustainable Asymmetric Synthesis: From Biobased ʟ-Phenylalanine to High-Value Chiral Chemicals. Y. Zhou et al., Angew. Chem. Int Ed., 2016, 55 (38),11647-11650. 1.7 g scale.
Bacterial Anabaena variabilis phenylalanine ammonia lyase: A biocatalyst with broad substrate specificity. S. Lovelock et al., Bioorg. & Med. Chem., 2014, 22 (20), 5555-5557. 1 g scale.

Alcohol Oxidase

Development of a Biocatalytic Aerobic Oxidation for the Manufacturing Route to Islatravir. M. H. Shaw et al., Org. Process Res. Dev. 2024, 28, 9, 3545–3559. 7.6%w/w of 350 kg soln, ~ 26.6 kg scale.
Biocatalytic Oxidation in Continuous Flow for the Generation of Carbohydrate Dialdehydes. S. C. Cosgrove et al., ACS Catal. 2019, 9, 12, 11658–11662. 2.1 g scale.
Design of an in vitro biocatalytic cascade for the manufacture of islatravir. M. A. Huffman et al., Science 2019, 366 (6470), 1255-1259. 8.75 mmole,~ 1.5 g scale.
Scope and Synthetic Applications of the Aryl-Alcohol Oxidase from Streptomyces hiroshimensis (ShAAO). C. Ascasco-Alegre et al., Org. Lett. 2025, 27, 43, 12086–12091. Low scale but lots of examples.
Microsome-bound alcohol oxidase catalyzed production of carbonyl compounds from alcohol substrates. A. Kakoti et al., J. Mol. Cat. B: Enzymatic 2012, 78, 98– 104. Low scale but lots of examples.

Iminereductase

Biocatalytic reductive amination from discovery to commercial manufacturing applied to abrocitinib JAK1 inhibitor. R. Kumar et al., Nat. Catal., 2021, 4, 775–782. 230 kg scale.
Engineered Imine Reductase for Larotrectinib Intermediate Manufacture. Q. Chen et al., ACS Catal. 2022, 12, 23, 14795–14803. 1.6 kg scale.
Imine Reductase-Catalyzed Synthesis of a Key Intermediate of Avacopan: Enzymatic Oxidative Kinetic Resolution with Ex Situ Recovery and Dynamic Kinetic Reduction Strategies toward 2,3-Disubstituted Piperidine. Z. J. Pótáriné et al., Org. Process Res. Dev. 2025, 29, 4, 1093–1102. 1 kg scale.
Chiral synthesis of LSD1 inhibitor GSK2879552 enabled by directed evolution of an imine reductase. N. Schober et al., Nat. Catal., 2019, 2, 909–915. 392 g scale.
Technical Considerations for Scale-Up of Imine-Reductase-Catalyzed Reductive Amination: A Case Study. A. Bornadel et al., Org. Process Res. Dev. 2019, 23, 6, 1262–1268. 1 g scale.

Imine Reductase Reagent Guide

Nitrile Hydratase

Nitrile hydratase-mediated conversion of acrylonitrile by Rhodococcus rhodochrous (RS-6). R. Sahu et al., J. Chem. Tech. & Biotech., 2021, 96 (4), 1080-1085. 400 g scale.
Enzymatic hydrolysis of cyanohydrins with recombinant nitrile hydratase and amidase from hodococcus erythropolis. Ch. Reisinger et al., Biotechnol. Lett., 2004, 26, 1675–1680. 1 g scale.
High-yield continuous production of nicotinic acid via nitrile hydratase–amidase cascade reactions using cascade CSMRs. L. Cantarella et al., Enz. & Microbial Tech., 2011, 48 (4–5), 345-350. Scale: flow, isolated amount not stated.

Hydroxynitrile Lyase

‘Large-Scale Synthesis of (R)-2-Amino-1-(2-furyl)ethanol via a Chemoenzymatic Approach. T.Purkarthofer et al., Org. Process Res. Dev. 2006, 10, 3, 618–621. 1 kg scale.
Probing o-Diphenylphosphanyl Benzoate (o-DPPB)-Directed C—C Bond Formation: Total Synthesis of Dictyostatin. S. Wünsch et al., Chem. Eur. J., 2015, 21 (6), 2358-2363. 36 g scale.
A Unified Strategy for the Stereospecific Construction of Propionates and Acetate–Propionates Relying on a Directed Allylic Substitution. T. Reiss et al., Chem. Eur. J., 2009, 15 (26), 6345-6348. 14 g scale.
Stereoselective Biocatalytic Synthesis of (S)-2-Hydroxy-2-Methylbutyric Acid via Substrate Engineering by Using “Thio-Disguised” Precursors and Oxynitrilase Catalysis. M. H. Fecter et al., Chem. Eur. J., 2007, 13 (12), 3369-3376. 10.8 g scale.
Chemo-enzymatic synthesis of new ferrocenyl-oxazolidinones and their application as chiral auxiliaries. B. J. Ueberbacher et al., Tet. Asymm., 2008, 19 (7), 838-846. 7 g scale.
Enzyme and Gold Catalysis: A New Enantioselective Entry into Functionalized 4-Hydroxy-2-pyrrolines. B. Ritzen et al., Synlett 2014, 25(2),270-274. 2.4 g scale.
Synthesis of Aromatic 1,2-Amino Alcohols Utilizing a Bienzymatic Dynamic Kinetic Asymmetric Transformation. J. Steinreiber et al., Adv. Synth. Catal., 2007, 349 (8-9), 1379-1386. 1.8 g scale.

HCN Lyase Reagent Guide

Amino Acid Oxidase

Enzymatic Preparation of an (S)-Amino Acid from a Racemic Amino Acid. Y. Chen et al., Org. Process Res. Dev. 2011, 15, 1, 241–248. 15 g substrate (60% potency due to salt and hydrate form) scale.

Halohydrin Dehalogenase

A green-by-design biocatalytic process for atorvastatin intermediate. S. K. Ma et al., Green Chem., 2010, 12, 81-86. 70 g scale (>100 kg unpublished).
HHDH-Catalyzed Synthesis of Enantioenriched Fluorinated β-Hydroxy Nitrile─Process Advances through a Reaction Engineering Approach. N. Milčić et al., Ind. Eng. Chem. Res. 2024, 63, 16, 7051–7063. 0.69 g scale.
A One-Step Biocatalytic Process for (S)-4-Chloro-3-hydroxybutyronitrile using Halohydrin Dehalogenase: A Chiral Building Block for Atorvastatin. N. W. Wan et al., ChemCatChem., 2015, 7 (16), 2446-2450.

Amino Acid Hydroxylase

Biocatalytic Aerobic Oxidation for Large-Scale Production of trans-3-Hydroxy-l-Proline. K.-J. Xioa et al., Org. Process Res. Dev. 2025, 29, 8, 2076–2085. 465 kg scale.
Engineering Hydroxylase Activity, Selectivity, and Stability for a Scalable Concise Synthesis of a Key Intermediate to Belzutifan. W. L. Cheung-Lee et al., Angew. Chem. Int. Ed., 2024, 63 (13), e202316133. 1.5 kg scale.

Tryptophan Synthase (TrpB)

The β-subunit of tryptophan synthase is a latent tyrosine synthase. P. J. Almhjell et al., Nat. Chem. Biol. 2024, 20, 1086–1093. 5 g scale.
Unlocking Reactivity of TrpB: A General Biocatalytic Platform for Synthesis of Tryptophan Analogues. D. K. Romney et al., J. Am. Chem. Soc. 2017, 139, 10769-10776. 1 g scale.
Improved Synthesis of 4-Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima. C. E. Boville et al., J. Org. Chem. 2018, 83, 7447-7452. 0.8 g scale.

Halogenase

Extended Biocatalytic Halogenation Cascades Involving a Single‐Polypeptide Regeneration System for Diffusible FADH2. N. Montua et al, ChemBioChem 2023, 24 (22), e202300478. 800 mg of product scale.

P450

Enabling Selective and Sustainable P450 Oxygenation Technology. Production of 4-Hydroxy-α-isophorone on Kilogram Scale. I. Kaluzna et al., Org. Process Res. Dev. 2016, 20, 4, 814–819. 897 g scale.

Unspecific Peroxygenase

Toward Kilogram-Scale Peroxygenase-Catalyzed Oxyfunctionalization of Cyclohexane. T. Hilberath et al., Org. Process Res. Dev. 2023, 27, 7, 1384–1389. 500 g scale.
Divergent Oxidation Reactions of E– and Z-Allylic Primary Alcohols by an Unspecific Peroxygenase. J. Li et al., Angew. Chem., Int. Ed., 2025, 64 (12), e202422241. 1.25 g scale.

Carboxylic Acid Reductase

Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity. K. Shi et al., Sci. Adv. 2024, 10 (48). 2.1 g scale.
Cell-free in vitro reduction of carboxylates to aldehydes: With crude enzyme preparations to a key pharmaceutical building block. A. Swartz et al., Biotechnol. J., 2021, 16 (4), 2000315. 1.1 g scale.
Engineering the activity and thermostability of a carboxylic acid reductase in the conversion of vanillic acid to vanillin. Y. Ren et al., Biotechnol. J., 2024, 386, 19-27. 0.5 g scale.

Amide Ligase/Synthetase

Discovery, characterization and engineering of ligases for amide synthesis. M. Winn et al., Nature 2021, 593, 391–398. Scale: 0.9 g limiting acid.
Broad Spectrum Enantioselective Amide Bond Synthetase from Streptoalloteichus hindustanus. Q. Tang et al., ACS Catal., 2024, 14, 2, 1021–1029. Scale: 50 mg limiting acid.

Contributors

David Entwistle¹Francesco Falcioni²Richard Lloyd³Katharina Neufeld⁴Teresa O’Neill⁵Tay Rosenthal⁶Martin Schürmann⁷Zara Seibel¹Juan Velasquez⁸Hao Wu⁹Christiana Briddell¹⁰Isamir Martinez¹⁰

¹Codexis Inc., 200 Penobscot Drive, Redwood City, CA 94063, USA

²AstraZeneca, 1 Francis Crick Ave, Cambridge, CB2 0AA, UK

³GSK, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK

⁴Johnson & Johnson Innovative Medicine, Turnhoutseweg 30, 2340 Beerse, Belgium at the time of contribution, now at Lesaffre, 101 Rue de Menin, 59700 Marcq-en-Barœul, France

⁵Zoetis, 333 Portage St. Kalamazoo, MI 49007, USA

⁶Corteva Agriscience, 9330 Zionsville Road, Indianapolis, IN 46268, USA

⁷InnoSyn B.V., Urmonderbaan 22, 6167 RD Geleen, The Netherlands

⁸Merck & Co., Inc. 126 East Lincoln Avenue, Rahway, NJ 07065, USA

⁹Bohringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA at the time of contribution, now at Pfizer Inc., 22515 – 29th Drive Southeast, Bothell, WA 98021, USA

¹⁰American Chemical Society, 1155 Sixteenth St. NW, Washington, DC, USA 20036

Biocatalysis

The Evolving Landscape of Industrial Biocatalysis in Perspective from the ACS Green Chemistry Institute Pharmaceutical Roundtable

The Evolving Landscape of Industrial Biocatalysis in Perspective from the ACS Green Chemistry Institute Pharmaceutical Roundtable. Francesco Falcioni*, Luke Humphreys*, Richard C. Lloyd*, Hao Wu, Isamir Martinez, Jonathan Jones, Shane McKenna, Katharina Neufeld, Ryan M. Phelan, Tay Rosenthal, Christophe J. Szczepaniak, Kumiko Yamamoto, Scott P. France, and Anna Fryszkowska. ACS Catal. 2025, 15, 12, 10780–10794. https://doi.org/10.1021/acscatal.5c01646.

Biocatalysis Guide

Introduction

Name

Most commonly used biocatalytical transformations

≥2 Peer reviewed examples of reactions scaled to ≥ 1 kg, or multiple double digit gram. Enzymes available at > 100 g scale

Hydrolase

Ketoreductase

Transaminase

Iminereductase

Enereductase

Nitrilase

Aldolase

Amino acid dehydrogenase

α-Ketoglutarate dependent dioxygenase

Ammonia lyase

Baeyer-Villiger monooxygenase

≥ 2 Peer reviewed examples of reactions scaled to ≥ 10 g

Hydroxynitrile lyase

Nitrile hydratase

Epoxide hydrolase

Monoamine oxidase

Alcohol oxidase

Halohydrin dehalogenase

Unspecific peroxygenase

Tryptophan synthase

≥ 1 Peer reviewed example of reactions scaled to multi-mg

Carboxylic acid reductase

Halogenase

Cytochrome P450

Amide ligase

Nicotinamide cofactor recycling

Most commonly used reductive nicotinamide regenerating systems

Glucose dehydrogenase

Ketoreductase

Formate dehydrogenase

Less commonly used reductive nicotinamide regenerating systems

Phosphite dehydrogenase

Enereductase

NAD(P)H oxidase

Non-enzymatic methods

Adenosine triphosphate (ATP) recycling

Most commonly used ATP regenerating systems

Acetate kinase

Polyphosphate kinase

Phosphoenolpyruvate kinase

Adenylate kinase

Less commonly used ADP/ATP regenerating systems

Creatine kinase

Polyphosphate transferase

Combinations for recycling AMP to ATP

References

Contributors

Related Articles

The Evolving Landscape of Industrial Biocatalysis in Perspective from the ACS Green Chemistry Institute Pharmaceutical Roundtable