Biocatalysis | Enzymatic Catalysis

Biocatalysis, or enzymatic catalysis, is the use of biologically active components to catalyze chemical transformations. Biocatalysis facilitates a spectrum of primarily carbon-centric reactions that occur in environments ranging from cell-free, fully in vitro to fermentation-mediated processes in living cell culture.

Biocatalysis represents a useful alternative to traditional chemical catalysis for a number of reasons. Enzymatic biocatalyst reactions:

• Are highly chemo-, regio- and enantiospecific
• Often have rapid kinetics
• Operate under milder conditions than chemical catalysts
• Eliminate the issue of waste, toxicity and cost of metal catalysts
• Reduce energy requirements associated with chemical reactions

Directed engineering of biocatalysts improve stability in solvents at elevated temperatures, enabling broad adoption of biocatalysis in the pharmaceutical, chemical, biofuel and food industries.

Biocatalysis is associated with “green”, sustainable and cost-effective chemical product manufacturing. Relative to chemocatalysis, biocatalysts offer inherent advantages for synthesis including:

• Significant compound diversity, either from natural or genetically engineered sources to tackle a broad range of chemical transformations
• Specificity of biocatalytic reactions that mitigate the need for certain classical chemical synthesis/processing strategies such as the use of blocking/deblocking groups, enantiomeric mixture separation and work-up to remove unwanted by-products
• Simplification of infrastructure and raw material requirements as many biocatalysis processes can be performed in single-pot reactions and eliminate reaction steps required by traditional chemical catalysis  

Enzymes used in biocatalysis have important and frequently leveraged abilities which include:

A) Functional group or site-specific binding and target recognition of other biologics or small molecules

B) Promoted association or displacement of other biologics or small molecules

C) Structural or electrostatic features that facilitate ion transport

D) Ordered to disordered structural transformations and, thus, varied catalytic activities

E) Allosteric or indirect modifications which induce or suppress enzyme activity

Research into improved or modified recombinant biocatalysts continues to expand their utility in harsh chemistry, improve reaction scope, enzyme robustness and reusability. Natural and engineered biocatalysts are now available to perform key chemical transformations such as the formation of C-C bonds, amide bonds, forming chiral amines and C-H functionalization.

The scope of enzymes used for biotransformations is extraordinarily broad and cross virtually all industrial sectors including, food, pharma, textiles, biofuels, paper, chemicals and household products. In the synthesis of fine chemicals, pharmaceuticals and related intermediates, biocatalysis (i.e. enzymatic catalysis or enzyme-mediated catalysis) has rapidly grown as an enabling technology. Enzymes are available for various biotransformations such as oxidations, reductions, additions and eliminations. 

The six major classes of enzymes and application examples: 

1. Oxidoreductases catalyzing molecular oxidations and reductions. Monooxygenase enzymes with molecular oxygen enables C-O bond formation, and specific enzymes have been engineered to enable oxidations of alcohols, ketones, aldehydes and amines. With respect to reductions, ketoreductases (KREDS) and dehydrogenases enable the synthesis of enantiospecific alcohols. Other enzymes can reduce bonds such as C=N, transforming nitriles to amines. 

2. Transferases are useful for transferring functional groups from one molecule to another. Kinase and phosphatases (addition and removal of phosphates) are especially important for molecular signaling cascades, DNA replication, RNA transcription and other functions. There are also many biologically significant transferases which serve functions, such as, conversion of ketones or aldehydes to amines and which can do so stereoselectively.

3. Hydrolases catalyze hydrolysis of various substrates. Nucleases or proteases are prolific and essential in nucleic acid and peptide recycling. Hydrolases such as lipase, protease and acylase act as catalysts for Michael 1,4-additions, widely used to form C-C and carbon-heteroatom bonds.

4. Lyases generally catalyze reactions through formation or elimination of double bonds. Different than a substitution reaction by hydrolases, carboxylase or decarboxylase reactions are common in pharmaceutical biocatalysis for the addition or removal of CO₂, as are C-N lyases which can be used to generate amino acids including substituted aspartic acids and alanines.

5. Isomerases enable rearrangement of atoms in a molecule. Isomerases, such as racemases, invert stereochemistry at the target chiral carbon, and cis-trans isomerases catalyze the isomerization of cis-trans isomers in alkenes or cycloalkanes. The conversion of glucose to fructose via glucose isomerase represents a major industrial enzymatic biotransformation.

6. Ligases form new chemical bonds by joining two molecules to form a larger molecule. One of the most important applications is the use of DNA ligase in the formation of recombinant DNA molecules and are the complement to endo- or exonucleases.

Producing enzyme catalysts. Generally, natural or recombinant enzymes for biocatalysis are first synthesized through fermentations most often utilizing bacteria, but are occasionally derived from yeast cultures as well. Although enzymes may be produced in more complex mammalian cell cultures, bacteria and yeast usually contain sufficient and naturally occurring molecular pathways to achieve the folding and moderate post-translational modifications needed for recombinant biocatalytic enzymes. Enzymes produced through such fermentations are usually harvested by lysis or other disruption of the cell structures to release the recombinant enzymes and are further purified in a process similar to other biologic pharmaceuticals. Fermentation, harvest and downstream processing present their own challenges which are addressed by leveraging Process Analytical Technology (PAT).

Stabilization and immobilization. Enzyme immobilization is used to achieve more stable, active and reusable enzymes. Generally, cross-linked enzyme aggregates (CLEAs) can be formed with a number of different solid substrates such as silica, resins or polymers such as PEG and even other complexes such as lipid-nanoparticles (LNPs). For some flow chemistry applications, CLEAs can also be formed on surfaces and sensors such as those used in surface-enhanced measurements for both analytical as well as catalytic purposes. The challenges of related component synthesis, adsorptions, conjugations, or other associations, as well as product stability and formulation are addressed by similar solutions for vaccine formulation and adjuvant processes.

Product capture and isolation. After biocatalysis, CLEAs are separated from product and residual reactants and may then be recycled or otherwise retained. Small molecule products may then be isolated through filtrations or chromatography processes, although many processes continue to favor methods of crystallization. Large molecule biocatalysis products would generally continue with traditional downstream processing workflows.

Understanding the practical half-life and stability of any free-, substrate-bound or surface-immobilized enzyme can be critical to the design of proper biocatalysis protocols. Considerations include:

• Solvents - Many natural enzymes struggle to cope with organic solvent reactions. A constantly evolving profile of engineered or recombinant enzymes are improving compatibility with organic solvent conditions. Recombinant enzymes are now capable of catalyzing reactions in organic solvent environments, for example, enabling the use of modified phosphoramidites in oligonucleotide synthesis.
• pH - Enzymes generally operate best within narrow pH ranges and tolerate pH variation <0.5 pH units. Thus, pH control is often a critical parameter for consideration for many biocatalytic reactions and may be difficult or tedious to control manually. 
• Temperature - Natural enzymes and their recombinant counterparts function efficiently at specific temperatures with fairly tight ranges. Temperature deviations may cause changes in reaction kinetics, product yield or other critical process outcomes.
• Dissolved Oxygen - This critical process parameter, especially important for oxygenation and reduction reactions, is usually controlled by sparge or headspace dosing of oxygen or inert gas. In order to ensure that oxidoreductase enzymes can operate at peak efficiency, optimizing the quantity and mass transfer of dissolved oxygen (including bubble characteristics) into the reaction mixture is critical.
• Mixing, Fluid Dynamics and Gases - Proper mixing apparatus can have a great effect on the life cycle of biocatalytic enzymes, especially over long duration, flow chemistry or perfusion style runs. Scale-up of biocatalytic reactions may be suboptimal if mixing is insufficiently characterized. Mixing is also critical for proper gas distribution and mass transfer.
• Reaction Stoichiometry - Correct reagents-to-enzymes ratios are critical and can be can be determined by Design of Experiments (DoE) studies, automated by chemical reactors and aided by PAT integrated in process development.

Development and scale-up of biocatalytic reactions is dependent on the nature of the specific biocatalysts used, substrates, co-factors and reaction conditions applied. As in chemical catalysis, this complex, interdependent matrix of variables is best understood by careful control of reaction variables when combined with information from real-time analytical measurements. However, much of the process development, scale-up and data density limitations for modern processes are tied to the continued manual orientation of biocatalysis reactions, which have significant time and resource costs. Manual operation implies personnel present at the bioreactor for near-constant tending, adjustment and sampling in order to meet increasing regulatory and institutional demand for process characterization and technology transfer.

Unattended operation of biocatalysis is possible through lab automation platforms that respond to dynamic reaction conditions continuously measured though integrated sensors and process analytical technologies (PAT). Automated synthesis reactor platforms facilitate faster experimentation with minimized human error or intervention. Paired with modular, automated reactor sampling for offline chromatographic analysis, all critical process parameters (CPP) can be overlaid and trended. Parameter control and process analytic data is automatically recorded in real time and structured for data-management and archive. Reduced experimental variability and increased reproducibility across different reactor scales further streamlines evidence-based scale-up.

• EasyMax and OptiMax automated reactors have important roles ensuring that the effect of key variables of biotransformations, such as temperature, pH, mixing rates and dosing, are precisely controlled, effectively modelled and recorded.
• ReactIR and ReactRaman in-situ spectroscopic technologies are useful for tracking and measuring key reaction species in real time. ReactIR tracks key analytes in solution without interference from suspended particles such as whole cells.
• ParticleTrack with FBRM technology measures particle size and distribution in heterogeneous suspensions. EasyViewer captures in-situ images of particles and performs real-time image analysis on system turbidity, shape, size, morphology, count and distributions.
• EasySampler enables fully automated and unattended sampling of chemical reactions, including quenching and dilution of samples in preparation for offline analysis.

EasyMax automated reactor is a complete workstation for parameter optimization in biocatalysis to support development, scale-up and execution. This fully automated system enables precise control over process parameters.

• Manage CPPs including source material identification, dosing rates, temperature, agitation, headspace, sparging, pH measurement and titration
• Readily integrates with in-situ analysis for biocatalysis, including ReactIR (FTIR), ReactRaman (Raman), ParticleTrack (FBRM), EasyViewer (in-situ image analysis) and EasySampler (automated, in-situ reactor sampling)
• Run experiments when unattended; execute intelligent automated sampling when offline analysis is required
• iC Software enables automated execution and control of all experiments and peripheral equipment as well as full data capture and record keeping

ReactIR uses FTIR spectroscopy to measure both aqueous and organic-soluble components in biocatalysis studies. ReactIR measurements are not impacted by suspended solids, bubbles or other particulate mass.

ReactRaman uses Raman spectroscopy to provide molecular analysis of particles and reaction analysis.

• Monitors multiple product quality attributes in situ and in real time in a vessel or flowing process stream
• Eliminates delays associated with grab samples
• Quantifies the impact of processing steps on product yield and process efficiency
• Provides actionable data analytics for real-time decision making and control
• Supports ICH regulatory guidelines through data-rich experimentation and digitalization
• Aids in scale-up from R&D to manufacturing
• Measures analyte concentrations as low as 500 ppm in volumes from few milliliters to thousands of liters

ParticleTrack uses Focused Beam Reflectance Measurement (FBRM) technology and is an optical probe-based instrument that is inserted directly into a vessel to track changing particle size and count in real time. ParticleTrack uses Chord Length Distribution (CLD) measurements to obtain Particle Size Distribution (PSD).

• Particles and particle structures are monitored continuously, as experimental conditions or parameters vary
• Characterize solid-phase particle synthesis, enzyme immobilization, cleavage and related process morphologies
• Track batch or continuous growth of biocatalysis products during biocatalysis in real time with no sample removal required
• Characterize particle system conditions which affect nucleation, precipitations, polymorphism and other system attributes

EasyViewer is a probe-based particle size analyzer that is inserted directly into a vessel to capture images of the particle system. EasyViewer performs image analysis and measures particle count, size distribution and morphology from time-resolved inline images of the process allowing populations to be trended over time.

• Particles and particle structures are monitored continuously, as experimental conditions or parameters vary
• Characterize solid-phase particle synthesis, enzyme immobilization, cleavage and related process morphologies
• Measure mass transfer (K_La) through bubble characterization
• Track batch or continuous growth of biocatalysis products during crystallization in real time with no sample removal required
• Characterize particle systems conditions which affect nucleation, precipitation, polymorphism and other system attributes

ReactIR Provides Insight into Optimizing the Synthesis of the Ester

Allsop, G. L., Carey, J. S., Joshi, S., Leong, P., & Mirata, M. A. (2021). Process Development toward a Pro-Drug of R-Baclofen. Organic Process Research & Development, 25(1), 136–147

In the development of complex molecules, chemists search for ways to simplify multi-step syntheses. The use of enzymes either as catalysts and in the resolution of racemic compounds is well-established. In-situ analytical techniques are complementary and highly useful for aiding in the optimization of chemical and biochemical transformations in the individual synthesis steps.

In this work, the researchers were involved in developing a multi-kilogram synthesis for the drug arbaclofen placarbil, used for alcohol abuse disorders. One step involved the synthesis of a key succinate ester intermediate compound and subsequent resolution of the ester to a single (S)-enantiomer. To obtain this single enantiomer in sufficient quantities, they explored two different approaches, preparative chiral chromatography and enzymatic resolution. The latter approach was deemed advantageous, and they found that C. antartica A lipase immobilized on polymethacrylate (IMMCALA-T2-150 lipase) provided the viable route to the resolved ester succinate intermediate.

To initially synthesize the succinate ester, they found that the analogous thiocarbonate compound reacted with sulfuryl chloride, to yield the desired ester. In order to optimize the yield of the ester, and minimize a by-product, ReactIR was employed. In-situ FTIR showed that when the thiocarbonate compound and sulfuryl chloride reacted, an intermediate chloroformate compound formed that had limited thermal stability. Once this intermediate formed and prior to decomposition, N-hydroxysuccinimide was added, followed by the addition of triethylamine. The addition of triethylamine was highly exothermic and led to the chloroformate intermediate transforming into the desired succinate ester.

ReactIR Tracks Key Biocarbonate Concentration

Pesci, L., Gurikov, P., Liese, A., & Kara, S. (2017). Amine-Mediated Enzymatic Carboxylation of Phenols Using CO₂ as Substrate Increases Equilibrium Conversions and Reaction Rates. Biotechnology Journal, 12(12), 1700332

The authors found that dihydroxybenzoic acid (de)carboxylases enzymes catalyze the reversible regio-selective ortho-(de)carboxylation of phenolics and are of significant interest since they perform carboxylations at much lower temperatures and pressures than respective chemocatalysts. In this current work, they report that 2,3-dihydroxybenzoic acid (de)carboxylase from Aspergillus oryzae acts as a catalyst to enable the ortho-carboxylation of phenolic molecules, and this is associated with the simultaneous amine moderated transformation of the co-substrate CO₂ to bicarbonate in situ.

The authors state that the nature of the co-substrate CO₂, or the hydrated form bicarbonate, is important in enzymatic carboxylations since the mechanism of substrate binding and activation determines the actual carboxylation agent. In their work, a ReactIR equipped with a diamond ATR insertion probe was used to track the formation of bicarbonate. As a result of this work, they report the development of reaction conditions that significantly improve both conversion and reaction rate for the biocatalytic carboxylation of catechol.

ReactIR Investigates Oxidative Coupling With Various Substrates and Aids in Optimizing Reaction Conditions

Engelmann, C., Illner, S., & Kragl, U. (2015). Laccase initiated C-C couplings: Various techniques for reaction monitoring. Process Biochemistry, 50(10), 1591–1599.

The authors report investigating the use of the fungal enzyme laccase (Novozym 51003) for oxidative C-C coupling in phenolic compounds. Laccase has shown applicability to oxidize phenols, anilines, etc into quinones with reduction of O₂ to water. Laccasse coupling is typically considered not selective, and therefore the authors initiated a study of laccase oxidation in various phenolic substrates to determine the effectiveness/selectivity of the C-C coupling. They determined that oxidative dimerizations of 2,6 disubstituted phenols were not only highly selective but also capable of being significantly scaled up. They demonstrated that the resultant 2,6 diisopropyl phenol oxidative product was easily reduced to the analogous biphenol compound.

In this study, the researchers initially selected target substrates via oxygen measurement during laccase initiated oxidations. They then used ReactIR (in-situ FTIR spectroscopy) to investigate the effectiveness and selectivity of the laccase initiated coupling reaction with the specific phenolic substrates and to define the optimum conditions to maximize the product. In-situ FTIR studies demonstrated that regioselective, symmetrical diquinone synthesis occurred rather rapidly. Furthermore, in-situ FTIR permitted monitoring of polymerizations that occurred via the laccase oxidations, and since the FTIR-ATR method tracks solute analytes only, polymeric precipitates did not interfere with the measurement.  

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