Potential Of Biocatalysis Research Articles

Harnessing the potential of deep eutectic solvents in biocatalysis: design strategies using CO2 to formate reduction as a case study

IntroductionDeep eutectic solvents (DESs) have emerged as green solvents with versatile applications, demonstrating significant potential in biocatalysis. They often increase the solubility of poorly water-soluble substrates, serve as smart co-substrates, modulate enzyme stereoselectivity, and potentially improve enzyme activity and stability. Despite these advantages, screening for an optimal DES and determining the appropriate water content for a given biocatalytic reaction remains a complex and time-consuming process, posing a significant challenge.MethodsThis paper discusses the rational design of DES tailored to a given biocatalytic system through a combination of experimental screening and computational tools, guided by performance targets defined by solvent properties and process constraints. The efficacy of this approach is demonstrated by the reduction of CO2 to formate catalyzed by NADH-dependent formate dehydrogenase (FDH). By systematically analyzing FDH activity and stability, NADH stability (both long-term and short-term stability after solvent saturation with CO2), and CO2 solubility in initially selected glycerol-based DESs, we were able to skillfully guide the DES screening process.Results and discussionConsidering trade-offs between experimentally determined performance metrics of DESs, 20% solution of choline chloride:glycerol in phosphate buffer (ChCl:Gly80%B) was identified as the most promising solvent system for a given reaction. Using ChCl:Gly as a co-solvent resulted in an almost 15-fold increase in FDH half-life compared to the reference buffer and stabilized the coenzyme after the addition of CO2. Moreover, the 20% addition of ChCl:Gly to the buffer improved the volumetric productivity of FDH-catalyzed CO2 reduction in a batch system compared to the reference buffer. The exceptional stability of the enzyme in this co-solvent system shows great potential for application in continuous operation, which can significantly improve process productivity. Additionally, based on easily measurable physicochemical solvent properties and molecular descriptors derived from COSMO-RS, QSAR models were developed, which successfully predicted enzyme activity and stability, as well as coenzyme stability in selected solvent systems with DESs.

Heme‐Enzymatic Biocatalysis for C−C or C−N Bond Formation

AbstractThe C−C or C−N bond formation is critical in the synthesis of pharmaceuticals and other value‐added products; however, traditional metal‐catalysed synthesis has brought about environmental and resource issues. A plethora of engineered heme‐dependent enzymes, such as cytochrome P450, have exhibited enormous potential in biocatalysis for C−C or C−N bond formation. With the development of computational and spectroscopic methods, the mechanisms underlying heme‐catalysed C−C or C−N bond formation have been extensively investigated. In the presence of carbene or nitrene precursor, an active iron porphyrin carbene (IPC) or iron porphyrin nitrene (IPN) is formed, which subsequently reacts with a second substrate to form new C−C or C−N bonds. Apart from the widely studied IPC/IPN‐facilitated catalytic pathway, halide‐initiated radical cyclization pathway and Cpd‐I‐catalysed diradical pathway have also been proposed. These mechanistic insights have enabled rational engineering and de novo design of heme enzymes. This review summarises recent mechanistic advances in heme enzymatic C−C or C−N bond formation and presents successful applications of mechanism‐based enzyme design. It would shed light on the development of tailored biocatalysts for the synthesis of complex but valuable industrial products.

Glucose-6-phosphate dehydrogenase and its 3D structures from crystallography and electron cryo-microscopy.

Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme in the pentose phosphate pathway. It has been extensively studied by biochemical and structural techniques. 13 X-ray crystal structures and five electron cryo-microscopy structures in the PDB are focused on in this topical review. Two F420-dependent glucose-6-phosphate dehydrogenase (FGD) structures are also reported. The significant differences between human and parasite G6PDs can be exploited tofind selective drugs against infections such as malaria and leishmaniasis. Furthermore, G6PD is a prognostic marker in several cancer types and is also considered to be a tumour target. On the other hand, FGD is considered to be a target against Mycobacterium tuberculosis and possesses a high biotechnological potential in biocatalysis and bioremediation.

Hydrogen-bonded supramolecular biohybrid frameworks for protein biomineralization constructed from natural phenolic building blocks.

Hydrogen bond-mediated supramolecular crystalline materials, such as hydrogen-bonded organic frameworks, offer a promising strategy for protein biomineralization, yet the intricate design and multi-step synthesis of specific orthogonal units in molecular building blocks pose a significant synthetic challenge. Identifying new classes of natural building blocks capable of facilitating supramolecular framework construction while enabling stable protein binding has remained an elusive goal. Here, we introduce a versatile assembly strategy enabling the organization of diverse proteins and phenolic building blocks into highly crystalline hydrogen-bonded supramolecular phenolic frameworks (ProteinX@SPF). The natural ellagic acid (EA) exhibits a centrosymmetric structure with catechol groups on each molecular side, facilitating hydrogen bonding with protein amino acid residues for primary nucleation. Subsequently, EA self-assembles into ProteinX@SPF through hydrogen bonding and π-π interactions. The multiple hydrogen-bonding interactions impart structural rigidity and directional integrity, conferring ProteinX@SPF biohybrids with remarkable resistance to harsh conditions while preserving protein bioactivity. Additionally, the supramolecular stacking induced by π-π interactions endows ProteinX@SPF with long-range ordered nanochannels, which can serve as the gating to sieve the catalytic substrate and thus enhance the biocatalytic specificity. This work sheds light on biomineralization with natural building blocks for functional biohybrids, showing enormous potential in biocatalysis, sensing, and nanomedicine.

Characterization of Phenylalanine Ammonia Lyases from Lettuce (Lactuca sativa L.) as Robust Biocatalysts for the Production of d- and l-Amino Acids.

Phenylalanine ammonia lyase (PAL) catalyzes the reversible conversion of l-phenylalanine into the corresponding trans-cinnamic acid, providing a route to optically pure α-amino acids. We explored the catalytic function of all five PALs encoded in the genome of lettuce (Lactuca sativa L.) that are previously known to be involved in wound browning. All LsPALs were active toward l-phenylalanine in the ammonia elimination reaction and displayed maximum activity at 55-60 °C and pH 9.0-9.5. However, four of them, LsPAL1-LsPAL4, showed significantly higher activity and thermal stability than LsPAL5, as well as a broader substrate spectrum including some challenging substrates with steric demanding or electron-donating substituents. The best one LsPAL3 was subjected to the kinetic resolution of a panel of 21 rac-phenylalanine derivatives, as well as the ammonia addition of 21 cinnamic acid derivatives. It showed excellent enantioselectivity in most cases and significantly better activity than previously described PALs for a number of challenging non-natural substrates, demonstrating its great potential in biocatalysis.

Challenges and Opportunities for the Large-Scale Chemoenzymatic Glycoengineering of Therapeutic N-Glycosylated Monoclonal Antibodies

Variability in the glycosylation profile of therapeutic monoclonal antibodies (mAbs), due to recombinant production technologies, leads to inconsistencies in effector functions and pharmacokinetic properties, both batch-to-batch and within single batches. It also poses regulatory concerns over the effectiveness of commercially available formulations. In vitro chemoenzymatic glycoengineering of variants displaying a homogeneous glycan profile is a trending strategy for ensuring consistent, controlled, and enhanced therapeutic performance, but reported successes are largely limited to small-scale applications. The major challenges for the industrial-scale introduction of the technique stem from the need for activated sugar donors, which can participate in undesired side reactions, and from the economic cost of the additional enzymatic steps and purification stages. While recent developments within the area address some of these obstacles, it appears that more effort is required in order to access the untapped potential of biocatalysis to enable the robust production of therapeutically superior constructs.

The ornithine cyclodeaminase/µ-crystallin superfamily of proteins: A novel family of oxidoreductases for the biocatalytic synthesis of chiral amines

• The Ornithine Cyclodeaminase/μ-Crystallin (OCD/CRYM) superfamily, a ubiquitously distributed but relatively unexplored family of proteins catalyzing imine reduction, is reviewed. • The physiological role and biochemical properties of the known members are summarized. • The structure and enzymatic mechanism in the superfamily are critically analyzed. • The current and prospective applications are discussed in detail. Biocatalysis, one of the most ecologically friendly methods for synthesising chiral synthons, has emerged as a desirable process for manufacturing active pharmaceutical ingredients and agrochemicals, most of which contain one or more chiral amine moieties. Compared with the traditional biocatalytic processes for the synthesis of chiral amines involving lipases or transaminases, enzymatic imine reduction is a more promising approach. The single-step enzymatic reduction of prochiral imines to the corresponding amines can yield 100% of the required enantiomer without any by-products. Furthermore, the reduction of imines generated in situ through the condensation of amines and carbonyl compounds can be used to synthesise almost any primary, secondary, or tertiary amine. In the past decade, several imine-reducing enzyme families, such as Streptomyces imine reductases (IReds), native amine dehydrogenases and engineered leucine/phenylalanine dehydrogenases and opine dehydrogenases, have been explored. The ornithine cyclodeaminase/μ-crystallin (OCD/CRYM) superfamily consists of proteins capable of imine reduction, which have been relatively unexplored regarding the synthesis of chiral amines. The proteins in this family are ubiquitously distributed in all three domains of life and catalyse diverse and unique reactions. This review is aimed at summarising current knowledge on this superfamily and exploring its potential in biocatalysis. After a brief discussion of their discovery, the known members of the OCD/CRYM superfamily are broadly classified on the basis of the reactions that they catalyse, and their biochemical characteristics and biological roles are described in detail. This is followed by a discussion of the overall structure, active sites and proposed reaction mechanisms, with common themes drawn among members. Finally, the applications of these enzymes, particularly in the synthesis of various chiral synthons, are summarised.

Integration of natural deep-eutectic solvent and surfactant for efficient synthesis of chiral aromatic alcohol mediated by Cyberlindnera saturnus whole cells

Efficiency of whole-cell biocatalysis in aqueous buffer solution is mainly limited by the low cellular membrane permeability and poor solubility of hydrophobic non-natural substrate. In this study, highly efficient bioreduction of 3,5-bis(trifluoromethyl)acetophenone (BTAP) to (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol ((S)-BTPE) with excellent enantioselectivity (> 99.9 % ee) by a novel yeast isolate Cyberlindnera saturnus ZJPH1807 was achieved via a integrated strategy of natural deep-eutectic solvent (NADES) and surfactant. The introduction of L-carnitine: lysine (C:Lys, molar ratio 1:2) in the reaction medium improved cell membrane permeability effectively and relieved BTAP toxicity to the cells. The Tween-80 addition further accelerated catalytic yield by increasing solubility of BTAP for 7.6-fold compared with neat aqueous buffer solution. After the optimization of key reaction parameters, a 81.0 % yield within 24 h under 500 mM substrate loading was achieved in the established C:Lys/Tween-80-containing system, compared with a 76.4 % yield for 30 h in aqueous buffer system under 200 mM BTAP concentration. This biocatalytic process was also feasible at 500 mL preparation scale with a 79.7 % (S)-BTPE yield under 500 mM BTAP. The developed combinational strategy of NADES with surfactant is an effective approach for enhancing bioreductive efficiency of hydrophobic substrates mediated by whole-cell catalyst, and has great potential in biocatalysis.

Function‐Oriented Natural Product Synthesis

AbstractNatural products and their derivatives have long been used as medicinal agents, and they still make up a significant fraction of clinically approved drugs. Natural product synthesis provides a rich and unparalleled opportunity to develop new synthetic transformations, conceive novel and general strategies to access complex structures, and study the mechanism of action of bioactive targets. The combination of the tools and principles of chemistry, together with the tools of modern biology, allows us to create complex synthetic and natural molecules, comprising processes with novel biological, chemical and physical properties. This account will illustrate the opportunities that lie at this interface between synthetic organic chemistry and chemical biology by describing a series of examples that we are actively working on in our laboratory at Peking University. We take the inspiration from mother nature to develop new synthetic strategies to achieve the efficient synthesis of complex natural products. In addition, we also conduct chemical biology studies for these bioactive natural products to elucidate their cellular targets and mode of action. Moreover, we further use bioactive natural products to explore new biology and develop novel drug candidates for human diseases, such as cancers and infectious diseases. What is the most favorite and original chemistry developed in your research group?By far the most original and ground‐breaking work we have done is the discovery of the first naturally occurring TRUE Diels‐Alderase which shows tremendous synthetic potential in biocatalysis for efficiently making structurally complex and bioactive molecules.How do you get into this specific field? Could you please share some experiences with our readers?I have a great passion in synthetic chemistry, and I am always fascinated by making organic molecules that may help us explore new biology and develop medicine for human diseases. Therefore, my research programs are relatively broad, spanning from natural product synthesis, chemical biology, synthetic biology, biocatalysis to drug discovery.What is the most important personality for scientific research?In my personal opinion, curiosity, ambition, and dedication are the most important personalities for scientific research. What is your favorite journal(s)?My favorite journals are: 1) Cell, when you read a Cell paper, you can get a full story of the original discovery and understand the detailed logic on how the research is planned and conducted; 2) Accounts of Chemical Research, this journal provides a short review on how a chemist establishes a specific new research field, very inspiring!What's your hobbies?I love many sports, soccer, American football, basketball, etc. I am also good at Seal Cutting, one of the four major traditional Chinese arts.If you have anything else to tell our readers, please feel free to do so.I would like to share one advice with the young scientists who just start their independent research career: BE DIFFERENT from your Ph.D. and Postdoc advisors, try to establish your own research direction! It's always easy to quickly get paper published when you take advantage of your advisor's research system, but it's very challenging to establish your own reputation and prove your originality in this way.

CapiPy: python-based GUI-application to assist in protein immobilization.

Protein immobilization, while widespread to unlock enzyme potential in biocatalysis, remains tied to a trial an error approach. Nonetheless, several databases and computational methods have been developed for protein characterization and their study. CapiPy is a user-friendly application for protein model creation and subsequent analysis with a special focus on the ease of use and interpretation of the results to help the users to make an informed decision on the immobilization approach which should be ideal for a protein of interest. The package has been tested with three separate random sets of 150 protein sequences from Uniprot with more than a 70% overall success rate (see Supplementary information and Supplementary Dataset). The package is free to use under the GNU General Public License v3.0. All necessary files can be downloaded from https://github.com/drou0302/CapiPy or https://pypi.org/project/CapiPy/. All external requirements are also freely available, with some restrictions for non-academic users. Supplementary data are available at Bioinformatics online.

Purification and immobilization of His-tagged organophosphohydrolase on yolk−shell Co/C@SiO2@Ni/C nanoparticles for cascade degradation and detection of organophosphates

The synthesis or decomposition of complex substances through the combination of metals and enzymes is desirable but still a huge challenge. A kind of yolk-shell structured Co/C@SiO2@Ni/C nanocomposites were prepared by the polyhedral ZIF-67@SiO2 composite as a precursor through the extended stöber method and subsequent carbonization treatment. The SiO2 as an intermediate protective layer stabilized the morphology and structure of the carbonized products. The metallic nickel nanoparticles were uniformly distributed on the surface of Co/C@SiO2@Ni/C for highly specific absorption of His-tagged organophosphohydrolase (OpdA) from cell lysates via affinity, and therefore an integrated nanocatalyst (INC) named OpdA@Co/C@SiO2@Ni/C was successfully prepared. The prepared INC was applied to achieve a one-pot chemoenzymatic cascade degradation of organophosphates (OPs) to p-aminophenol (PAP) within 30 min. In addition, the INC proved to be a simple and high-sensitivity nanocatalyst for the detection of methyl-parathion with a low limit of detection (0.30 μM). The INC exhibited improved pH stability, thermal stability, SDS resistance, and storage stability compared to that of free enzymes. When OpdA@Co/C@SiO2@Ni/C was reused for 7 cycles, the cascade degradation efficiency of methyl-parathion could retain more than 60 %. This study provides a strategy for constructing a multifunctional nanocatalyst, and further demonstrated its potential in biocatalysis, accurate detection of OPs and remediation of environmental pollution.

Whole-Cells of Yarrowia lipolytica Applied in “One Pot” Indolizine Biosynthesis

A series of yeast strains was tested in order to evaluate their catalytic potential in biocatalysis of one-pot indolizine’s synthesis. Yeast cultivation was performed in a submerged system at 28 °C for 72 h at 180 rpm. An assessment of the reagents’ toxicity on yeast viability and metabolic functionality concluded that the growth potential of three Yarrowia lipolytica strains were least affected by the reactants compared to the other yeast strains. Further, crude fermentation products (biomass and cell-free supernatant)—obtained by submerged cultivation of these yeasts—were used in multistep cascade reactions for the production of fluorescent indolizine compounds with important biologic activities. A whole–cell catalyzed multicomponent reaction of activated alkynes, α-bromo-carbonyl reagents and 4,4′-bipyridine, at room temperature in buffer solution led to the efficient synthesis of bis-indolizines 4a, 4b and 4c, in good-to-excellent yields (47%–77%). The metabolites of the selected Y. lipolytica strains can be considered effective biocatalysts in cycloaddition reactions and the high purity and bioconversion yields of the synthesized indolizines indicates a great potential of this type of “green” catalysts. Seeds of Triticum estivum L. were used to investigate the impact of the final products on the germination and seedling growth. The most sensitive physiological parameters suggest that indolizines, at the concentrations tested, have non-toxic effect on germination and seedling growth of wheat, fact also confirmed by confocal laser scanning microscopy images.

Protein‐Structure‐Directed Metal–Organic Zeolite‐like Networks as Biomacromolecule Carriers

Fabrication of zeolite-like metal-organic frameworks (ZMOFs) for advanced applications, such as enzyme immobilization, is of great interest but is a great synthetic challenge. Herein, we have developed a new strategy using proteins as structure-directed agents to direct the formation of new ZMOFs that can act as versatile platforms for the in situ encapsulation of proteins under ambient conditions. Notably, protein incorporation directs the formation of a ZMOF with a sodalite (sod) topology instead of a non-porous diamondoid (dia) topology under analogous synthetic conditions. Histidines in proteins play a crucial role in the observed templating effect. Modulating histidine content thereby influenced the resultant MOF product (from dia to dia + sod mixture and, ultimately, to sod MOF). Moreover, the resulting ZMOF-incorporated proteins preserved their activity even after exposure to high temperatures and organic solvents, demonstrating their potential for biocatalysis and biopharmaceutical applications.

Protein‐Structure‐Directed Metal–Organic Zeolite‐like Networks as Biomacromolecule Carriers

AbstractFabrication of zeolite‐like metal–organic frameworks (ZMOFs) for advanced applications, such as enzyme immobilization, is of great interest but is a great synthetic challenge. Herein, we have developed a new strategy using proteins as structure‐directed agents to direct the formation of new ZMOFs that can act as versatile platforms for the in situ encapsulation of proteins under ambient conditions. Notably, protein incorporation directs the formation of a ZMOF with a sodalite (sod) topology instead of a non‐porous diamondoid (dia) topology under analogous synthetic conditions. Histidines in proteins play a crucial role in the observed templating effect. Modulating histidine content thereby influenced the resultant MOF product (from dia to dia + sod mixture and, ultimately, to sod MOF). Moreover, the resulting ZMOF‐incorporated proteins preserved their activity even after exposure to high temperatures and organic solvents, demonstrating their potential for biocatalysis and biopharmaceutical applications.

Lösungsansätze entlang der chemischen Wertschöpfungskette

In this article, the high potential of biocatalysis as a versatile tool to address industrial challenges is illustrated by three examples from our own research, which was done, in part, jointly with companies from the chemical industry. In these examples, biocatalytic processes were deve -loped for products from different industrial segments. In detail, the pro -ducts being obtained by the presented bioprocesses are from the areas of polymer building blocks, lubricant components and pharmaceuticals.

Deciphering the Structural Basis of High Thermostability of Dehalogenase from Psychrophilic Bacterium Marinobacter sp. ELB17.

Haloalkane dehalogenases are enzymes with a broad application potential in biocatalysis, bioremediation, biosensing and cell imaging. The new haloalkane dehalogenase DmxA originating from the psychrophilic bacterium Marinobacter sp. ELB17 surprisingly possesses the highest thermal stability (apparent melting temperature Tm,app = 65.9 °C) of all biochemically characterized wild type haloalkane dehalogenases belonging to subfamily II. The enzyme was successfully expressed and its crystal structure was solved at 1.45 Å resolution. DmxA structure contains several features distinct from known members of haloalkane dehalogenase family: (i) a unique composition of catalytic residues; (ii) a dimeric state mediated by a disulfide bridge; and (iii) narrow tunnels connecting the enzyme active site with the surrounding solvent. The importance of narrow tunnels in such paradoxically high stability of DmxA enzyme was confirmed by computational protein design and mutagenesis experiments.

Crotonases: Nature’s Exceedingly Convertible Catalysts

The crotonases comprise a widely distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole-mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) both with nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by α-helices, which typically oligomerizes to form a trimer or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis...

Structural, kinetic and operational characterization of an immobilized l -aminoacid dehydrogenase

Abstract l -Alanine dehydrogenase from Bacillus subtillis is a NADH-dependent enzyme that catalyzes the reversible reductive amination of pyruvate using ammonia as amine source. This enzyme has been intensively exploited in biocatalysis but its immobilized form is rarely used. In this work, we have immobilized this enzyme on agarose microbeads activated with glyoxyl groups (aliphatic aldehydes). 85% of the offered enzyme was immobilized on these microbeads and the enzyme recovered 45% of its initial reduction amination activity upon the immobilization. The resulting immobilized preparation was 13 times more thermostable than the soluble enzyme because the immobilization attenuated the negative conformational changes induced by the temperature. The optimally immobilized enzyme was also stabilized against acidic pH. Finally, we have applied this heterogeneous biocatalyst to the synthesis of L-[13N]alanine using pyruvate and [13N]NH4OH obtaining a radiochemical yield of >95% in 20 min. This immobilized enzyme was re-used for up to 5 cycles keeping the maximum yield. Herein, we have fabricated a highly stable and active heterogeneous biocatalyst for reductive aminations of α-ketoacids that has an enormous potential in biocatalysis.

Cold and Hot Extremozymes: Industrial Relevance and Current Trends.

The development of enzymes for industrial applications relies heavily on the use of microorganisms. The intrinsic properties of microbial enzymes, e.g., consistency, reproducibility, and high yields along with many others, have pushed their introduction into a wide range of products and industrial processes. Extremophilic microorganisms represent an underutilized and innovative source of novel enzymes. These microorganisms have developed unique mechanisms and molecular means to cope with extreme temperatures, acidic and basic pH, high salinity, high radiation, low water activity, and high metal concentrations among other environmental conditions. Extremophile-derived enzymes, or extremozymes, are able to catalyze chemical reactions under harsh conditions, like those found in industrial processes, which were previously not thought to be conducive for enzymatic activity. Due to their optimal activity and stability under extreme conditions, extremozymes offer new catalytic alternatives for current industrial applications. These extremozymes also represent the cornerstone for the development of environmentally friendly, efficient, and sustainable industrial technologies. Many advances in industrial biocatalysis have been achieved in recent years; however, the potential of biocatalysis through the use of extremozymes is far from being fully realized. In this article, the adaptations and significance of psychrophilic, thermophilic, and hyperthermophilic enzymes, and their applications in selected industrial markets will be reviewed. Also, the current challenges in the development and mass production of extremozymes as well as future prospects and trends for their biotechnological application will be discussed.

The specificity and kinetic mechanism of branched‐chain amino acid aminotransferase from Escherichia coli studied with a new improved coupled assay procedure and the enzyme's potential for biocatalysis

Branched-chain amino acid aminotransferase (BCAT) plays a key role in the biosynthesis of hydrophobic amino acids (such as leucine, isoleucine and valine), and its substrate spectrum has not been fully explored or exploited owing to the inescapable restrictions of previous assays, which were mainly based on following the formation/consumption of the specific branched-chain substrates rather than the common amino group donor/acceptor. In our study, detailed measurements were made using a novel coupled assay, employing (R)-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans as an auxiliary enzyme, to provide accurate and reliable kinetic constants. We show that Escherichia coli BCAT can be used for asymmetric synthesis of a range of non-natural amino acids such as l-norleucine, l-norvaline and l-neopentylglycine and compare the kinetic results with the results of molecular modelling. A full two-substrate steady-state kinetic study for several substrates yields results consistent with a bi-bi ping-pong mechanism, and detailed analysis of the kinetic constants indicates that, for good 2-oxoacid substrates, release of 2-oxoglutarate is much slower than release of the product amino acid during the transamination reaction. The latter is in fact rate-limiting under conditions of substrate saturation. Branched-chain amino acid aminotransferase EC 2.6.1.42; (R)-2-hydroxyglutarate dehydrogenase EC 1.1.99.2.