Competitive Product Inhibition Research Articles

Approximation of intra-particle reaction/diffusion effects of immobilized enzyme system following reverse Michaelis–Menten (rMM) mechanism: third degree polynomial and Akbari–Ganji methods

Two approximate analytical expressions based on third degree polynomial and Akbari–Ganji’s method (AGM) were derived for the reaction/diffusion controlled kinetics of an immobilized enzyme (IE) systems. The approximation methods predict substrate concentration profile and effectiveness factor (eta) in a porous spherical particle. The reaction is assumed to follow reverse Michaelis–Menten (rMM) kinetics. The approximate methods predictions were comparable to that of numerical solution (using the Matlab finite difference function, bvp4c) at wide range of {phi }^{2} and {y}_{o} especially at low {phi }^{2} and high {y}_{o} (polynomial equation) and low {phi }^{2} and low {y}_{o} (AGM equation). Although the approximate solution was derived for rMM kinetics, the results can be used to describe other important enzymatic reaction kinetics such as simple Michaelis–Menten (MM) kinetics and MM with competitive product inhibition kinetics. A necessary design equation should be satisfied when using polynomial or AGM approximation for different enzyme kinetic equations. In this work, two examples of enzymatic reactions of industrial interest were studied, namely glucose-fructose isomerization follows rMM kinetics and hydrolysis of lactose follows Michaelis–Menten (MM) equation with competitive product (galactose) inhibition. Predictions of the developed third degree polynomial and AGM approximation equations agree with that of numerical solution, the percentage relative error for the effectiveness factor was less than 11 in comparison with the numerical solution. Good agreement between approximate and numerical estimations demonstrates the validity of these approximation methods.

Characterization of tyrosine ammonia lyases from Flavobacterium johnsonian and Herpetosiphon aurantiacus.

p-Coumaric acid (pCA) can be produced via bioprocessing and is a promising chemical precursor to making organic thin film transistors. However, the required tyrosine ammonia lyase (TAL) enzyme generally has a low specific activity and suffers from competitive product inhibition. Here we characterized the purified TAL variants from Flavobacterium johnsoniae and Herpetosiphon aurantiacus in terms of their susceptibility to product inhibition and their activity and stability across pH and temperature via initial rate experiments. FjTAL was found to be more active than previously described and to have a relatively weak affinity for pCA, but modeling revealed that product inhibition would still be problematic at industrially relevant product concentrations, due to the low solubility of the substrate tyrosine. The activity of both variants increased with temperature when tested up to 45°C, but HaTAL1 was more stable at elevated temperature. FjTAL is a promising biocatalyst for pCA production, but enzyme or bioprocess engineering are required to stabilize FjTAL and reduce product inhibition.

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed invivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Purification and characterization of a fungal aspartic peptidase from Trichoderma reesei and its application for food and animal feed protein hydrolyses.

Various neutral and alkaline peptidases are commercially available for use in protein hydrolysis under neutral to alkaline conditions. However, the hydrolysis of proteins under acidic conditions by applying fungal aspartic peptidases (FAPs) has not been investigated in depth so far. The aim of this study, thus, was to purify a FAP from the commercial enzyme preparation, ROHALASE® BXL, determine its biochemical characteristics, and investigate its application for the hydrolysis of food and animal feed proteins under acidic conditions. A Trichoderma reesei derived FAP, with an apparent molecular mass of 45.8kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SDS-PAGE) was purified 13.8-fold with a yield of 37% from ROHALASE® BXL. The FAP was identified as an aspartate protease (UniProt ID: G0R8T0) by inhibition and nano-LC-ESI-MS/MS studies. The FAP showed the highest activity at 50°C and pH4.0. Monovalent cations, organic solvents, and reducing agents were tolerated well by the FAP. The FAP underwent an apparent competitive product inhibition by soy protein hydrolysate and whey protein hydrolysate with apparent Ki -values of 1.75 and 30.2mg*mL-1 , respectively. The FAP showed promising results in food (soy protein isolate and whey protein isolate) and animal feed protein hydrolyses. For the latter, an increase in the soluble protein content of 109% was noted after 30 min. Our results demonstrate the applicability of fungal aspartic endopeptidases in the food and animal feed industry. Efficient protein hydrolysis of industrially relevant substrates such as acidic whey or animal feed proteins could be conducted by applying fungal aspartic peptidases. © 2022 Society of Chemical Industry.

Continuous production of a chiral amine in a packed bed reactor with co-immobilized amine dehydrogenase and formate dehydrogenase

A continuous flow packed bed reactor system was developed for the asymmetric reductive amination of 5-methyl-2-hexanone with a chimeric amine dehydrogenase (AmDH) and the formate dehydrogenase (FDH) from Candida boidinii. Both enzymes were co-immobilized onto the Nuvia® IMAC resin from Bio-Rad which employs the well-established binding chemistry between 6xHis-tagged proteins and immobilized Ni2+. Binding was robust under reaction conditions and continuous operation in a packed-bed flow column for more than five days was achieved. At 40 °C, conversion of 20 mM 5-methyl-2-hexanone ranged between 19 and 48%, with volumetric productivities between 443 and 107 g/L·day. The apparent biocatalyst half-life was 159 h and the absence of both internal and external mass transfer limitations was confirmed. Conversion was limited by modest competitive product inhibition of (R)-amine on ketone and an excess of enzyme over cofactor concentration, which precludes the use of Michaelis-Menten kinetics and results in a rate decrease of 80% compared to conditions where [S] ≫ [E]. The relationship between flow rate, conversion, and productivity was examined, with important lessons for reaction engineering. The reported work represents the first stable application of AmDHs in a continuous flow reactor for the production of chiral amines.

Variants at position 603 of the CGTase from Bacillus circulans STB01 for reducing product inhibition

Inhibition of cyclodextrin glycosyltransferases (CGTases) by their product cyclodextrins limits the efficiency of cyclodextrin production. In an effort to produce variants with good activity but reduced product inhibition, six mutants were constructed at position 603 of the CGTase from Bacillus circulans STB01, which exhibits mixed-type product inhibition. In a kinetic analysis, N603I showed reduced noncompetitive inhibition while N603K, N603H and N603R showed increased noncompetitive inhibition. Unexpectedly, N603E and N603D exhibited reductions in both competitive and noncompetitive product inhibition. Noncompetitive product inhibition is closely related to the interaction between the cyclodextrin and the enzyme in maltose binding site 2 (MBS2). Structural models led to the suggestion that there is increased interaction between maltose binding sites 1 and 2 in mutants N603E and N603D, which may have led to the unexpected results. N603D exhibited a 23.9% greater cyclodextrin yield per gram of enzyme than the wild-type, suggesting it has potential for industrial use. Further reductions in product inhibition may be gained through studies of maltose binding site interactions.

Assessing the impact of product inhibition in a chromogenic assay

Chromogenic substrates (CS) are synthetic substrates used to monitor the activity of a target enzyme. It has been reported that some CSs display competitive product inhibition with their target enzyme. Thus, in assays where enzyme activity is continuously monitored over long periods of time, the product inhibition may significantly interfere with the reactions being monitored. Despite this knowledge, it is rare for CSs to be directly incorporated into mathematical models that simulate these assays. This devalues the predictive power of the models. In this study, we examined the interactions between a single enzyme, coagulation factor Xa, and its chromogenic substrate. We developed, and experimentally validated, a mathematical model of a chromogenic assay for factor Xa that explicitly included product inhibition from the CS. We employed Bayesian inference, in the form of Markov-Chain Monte Carlo, to estimate the strength of the product inhibition and other sources of uncertainty such as pipetting error and kinetic rate constants. Our model, together with carefully calibrated biochemistry experiments, allowed for full characterization of the strength and impact of product inhibition in the assay. The effect of CS product inhibition in more complex reaction mixtures was further explored using mathematical models.

Ammonolysis of (5S)-N-(tert-butoxycarbonyl)-5-(methoxycarbonyl)-2-pyrroline with immobilized Candida antarctica lipase B (CAL B) in a packed bed reactor

Abstract Conversion of (5S)-N-(tert-butoxycarbonyl)-5-(methoxycarbonyl)-2-pyrroline to its corresponding amide, an important intermediate in the synthesis of the dipeptidyl peptidase IV (DPP4) inhibitor Saxagliptin, was carried out by ammonolysis reaction catalyzed by immobilized Candida antarctica lipase B (Novozyme 435) in a packed bed reactor. The reaction proceeds smoothly at 50 °C in anhydrous tert-butanol containing 1.25 M ammonia and follows typical Michaelis-Menten kinetics with competitive product inhibition. The estimated kinetic parameters were Vmax 40 ± 4.4 mM h−1g−1, Km (216 ± 22 mM) and Ki 303 ± 31 mM. At substrate concentration of 30 mg/mL (132 mM) and flow rate of 0.1 mL/min, the product is obtained in >98% yield and 98.5% purity at steady state in a column (100 cm × 1.2 cm) packed with 40 g immobilized CAL B.

2,4-Dichlorophenol Enzymatic Removal and Its Kinetic Study Using Horseradish Peroxidase Crosslinked to Nano Spray-Dried Poly(Lactic-Co-Glycolic Acid) Fine Particles.

Horseradish peroxidase (HRP) catalyzes the oxidation of aromatic compounds by hydrogen peroxide via insoluble polymer formation, which can be precipitated from the wastewater. For HRP immobilization, poly(lactic-co-glycolic acid) (PLGA) fine carrier supports were produced by using the Nano Spray Dryer B-90. Immobilized HRP was used to remove the persistent 2,4-dichlorophenol from model wastewater. Both extracted (9-16 U/g) and purified HRP (11-25 U/g) retained their activity to a high extent after crosslinking to the PLGA particles. The immobilized enzyme activity was substantially higher in both the acidic and the alkaline pH regions compared with the free enzyme. Optimally, 98% of the 2,4-dichlorophenol could be eliminated using immobilized HRP due to catalytic removal and partly to adsorption on the carrier supports. Immobilized enzyme kinetics for 2,4-dichlorophenol elimination was studied for the first time, and it could be concluded that competitive product inhibition took place.

Diffusion, adsorption and reaction of glucose in glucose oxidase enzyme immobilized mesoporous silica (SBA-15) particles: Experiments and modeling

Glucose oxidase (GOD) enzyme is widely used as a sensing molecule for glucose and is thus immobilized in different porous hosts for sensing applications. We have synthesized different morphologies of mesoporous silica (SBA-15) host particles and impregnated GOD in it. Rod and cuboid shaped SBA-15 particles having internal pore diameters of 6.8 and 11.4nm respectively, were synthesized, in order to measure enzyme activity. The latter primarily determines the performance of a glucose sensor. In this quest, we therefore developed a mathematical model to predict variation of glucose concentration with time, by considering diffusion, adsorption and reaction rates of glucose by immobilized GOD. The model was validated with our own experiments, by fitting diffusion coefficient of glucose inside mesopores and the competitive product inhibition constant of GOD. The validated model indicates that, specific activity of GOD immobilized in SBA-15 is lower at higher GOD loading. Thus, this provides insight into the effect of pore-confinement on diffusion and reaction kinetics of the substrate (glucose) by an immobilized enzyme (GOD). Understanding the interplay of chemical transport and reaction kinetics issues in a porous solid nanoparticle host (SBA-15) can lead to superior detection and measurement of glucose concentration.

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase: PRODUCT INHIBITION, COOPERATIVITY, AND MAGNESIUM ACTIVATION

In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mm tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 ± 4.9 min(-1), the Km for ATP by 0.104 ± 0.024 mm, and the Ki for ADP by 0.037 ± 0.007 mm. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 ± 0.1, indicating noncooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 ± 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, although larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 ± 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, and empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (24:1, L8S8/Rca6) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg(2+)] in response to changes in available light.

Description of the diffusive–convective mass transport in a hollow-fiber biphasic biocatalytic membrane reactor

The substrate transport in a biphasic, biocatalytic, capillary membrane layer has been investigated. The measured data of oleuropein hydrolysis, in olive mill wastewater, have been evaluated in both a well-mixed tank reactor and a polysulphone, biocatalytic, capillary membrane reactor. The β-glucosidase enzyme was immobilized in the sponge layer of the asymmetric, hydrophilic membrane layer. Strong, competitive product inhibition, applying Michaelis–Menten kinetics with product inhibition for evaluation of the measured data, has been obtained in the mixed tank reactor while the reaction did not show inhibition in the biocatalytic membrane layer. Applying the kinetic data for the oleuropein hydrolysis, the performance of a biocatalytic membrane reactor has been discussed under different operating modes. The effect of the lumen radius, membrane thickness, location of the inlet of the substrate, the inlet concentration and Peclet number as well as the effect of the external mass transfer resistances have been discussed and illustrated. It has been shown that all parameters mentioned above can have a strong effect on membrane performance. The model and its presented, so-called forward sweep numerical solution method, where concentration of the first sublayer is given by an explicit expression of closed form, can essentially help the reader estimate the effect of the operating parameters on the performance of a biocatalytic, capillary membrane reactor. The simulation results enable the user to select the right choice between operating conditions, providing a high efficiency membrane reactor.

Product inhibition in native-state proteolysis.

The proteolysis kinetics of intact proteins by nonspecific proteases provides valuable information on transient partial unfolding of proteins under native conditions. Native-state proteolysis is an approach to utilize the proteolysis kinetics to assess the energetics of partial unfolding in a quantitative manner. In native-state proteolysis, folded proteins are incubated with nonspecific proteases, and the rate of proteolysis is determined from the disappearance of the intact protein. We report here that proteolysis of intact proteins by nonspecific proteases, thermolysin and subtilisin deviates from first-order kinetics. First-order kinetics has been assumed for the analysis of native-state proteolysis. By analyzing the kinetics of proteolysis with varying concentrations of substrate proteins and also with cleavage products, we found that the deviation from first-order kinetics results from product inhibition. A kinetic model including competitive product inhibition agrees well with the proteolysis time course and allows us to determine the uninhibited rate constant for proteolysis as well as the apparent inhibition constant. Our finding suggests that the likelihood of product inhibition must be considered for quantitative assessment of proteolysis kinetics.

Theoretical analysis of intrinsic reaction kinetics and the behavior of immobilized enzymes system for steady-state conditions

Mathematical modeling of immobilized enzymes under different kinetics mechanism viz. simple Michaelis–Menten, uncompetitive substrate inhibition, total competitive product inhibition, total non-competitive product inhibition and reversible Michaelis–Menten reaction are discussed. These five kinetic models are based on reaction diffusion equations containing non-linear terms related to Michaelis–Menten kinetics of the enzymatic reaction. Modified Adomian decomposition method is employed to derive the general analytical expressions of substrate and product concentration for all these five mechanisms for all possible values of the parameters ΦS (Thiele modulus for substrate), ΦP (Thiele modulus for product) and α (dimensionless inhibition degree). Also we have presented the general analytical expressions for the mean integrated effectiveness factor for all values of parameters. Analytical results are compared with the numerical results and also with the limiting case results, which are found to be good in agreement.

Mechanistic Study of Manganese-Substituted Glycerol Dehydrogenase Using a Kinetic and Thermodynamic Analysis

Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

Ester ammoniolysis in an enzymatic membrane reactor

• A gas–liquid enzymatic membrane reactor was implemented for direct ammoniolysis. • Ethyl octanoate was converted to octanamide by immobilized C. antarctica lipase B. • High enzyme stability and selectivity toward the amide (95% at 40 °C) was achieved. A form of generating amides is by reaction of esters with ammonia (ammoniolysis) or with amines (aminolysis). This reaction can be catalyzed by free or immobilized lipases. The aim of this work is to develop a gas–liquid enzymatic membrane reactor in which the ammoniolysis of ethyl octanoate, catalyzed by Candida antarctica lipase B (EC 3.1.1.3), is improved through immobilization of the biocatalyst onto a membrane surface which also serves as gas–liquid interphase. After the selection of a suitable membrane for the process, data show that the higher the initial substrate concentration, the lower the reaction rate. A Michaelis–Menten type model with competitive product inhibition correlated well with the kinetic data. In addition, the temperature dependence of the reaction rate goes through a maximum. The yield of the amide was always above 92% in the range 25–55 °C, with a maximum of 95% at 40 °C. At 40 °C, and after 24 h of reaction, values comparable to those reported in the literature on the free enzyme-catalyzed ammoniolysis were achieved, but with much lower enzyme/substrate ratio. Moreover, the immobilized enzyme kept its activity for a long period of time (three weeks).

Precise Supramolecular Control of Selectivity in the Rh-Catalyzed Hydroformylation of Terminal and Internal Alkenes

In this study, we report a series of DIMPhos ligands L1-L3, bidentate phosphorus ligands equipped with an integral anion binding site (the DIM pocket). Coordination studies show that these ligands bind to a rhodium center in a bidentate fashion. Experiments under hydroformylation conditions confirm the formation of the mononuclear hydridobiscarbonyl rhodium complexes that are generally assumed to be active in hydroformylation. The metal complexes formed still strongly bind the anionic species in the binding site of the ligand, without affecting the metal coordination sphere. These bifunctional properties of DIMPhos are further demonstrated by the crystal structure of the rhodium complex with acetate anion bound in the binding site of the ligand. The catalytic studies demonstrate that substrate preorganization by binding in the DIM pocket of the ligand results in unprecedented selectivities in hydroformylation of terminal and internal alkenes functionalized with an anionic group. Remarkably, the selectivity controlling anionic group can be even 10 bonds away from the reactive double bond, demonstrating the potential of this supramolecular approach. Control experiments confirm the crucial role of the anion binding for the selectivity. DFT studies on the decisive intermediates reveal that the anion binding in the DIM pocket restricts the rotational freedom of the reactive double bound. As a consequence, the pathway to the undesired product is strongly hindered, whereas that for the desired product is lowered in energy. Detailed kinetic studies, together with the in situ spectroscopic measurements and isotope-labeling studies, support this mode of operation and reveal that these supramolecular systems follow enzymatic-type Michaelis-Menten kinetics, with competitive product inhibition.

A Combined Kinetic and Thermodynamic Approach for the Interpretation of Continuous‐Flow Heterogeneous Catalytic Processes

The heterogeneous proline-catalyzed aldol reaction was investigated under continuous-flow conditions by means of a packed-bed microreactor. Reaction-progress kinetic analysis (RPKA) was used in combination with nonlinear chromatography for the interpretation, under synthetically relevant conditions, of important mechanistic aspects of the heterogeneous catalytic process at a molecular level. The information gathered by RPKA and nonlinear chromatography proved to be highly complementary and allowed for the assessment of optimal operating variables. In particular, the determination of the rate-determining step was pivotal for optimizing the feed composition. On the other hand, the competitive product inhibition was responsible for the unexpected decrease in the reaction yield following an apparently obvious variation in the feed composition. The study was facilitated by a suitable 2D instrumental arrangement for simultaneous flow reaction and online flow-injection analysis.

Reassessing the type I dehydroquinate dehydratase catalytic triad: Kinetic and structural studies of Glu86 mutants

Dehydroquinate dehydratase (DHQD) catalyzes the third reaction in the biosynthetic shikimate pathway. Type I DHQDs are members of the greater aldolase superfamily, a group of enzymes that contain an active site lysine that forms a Schiff base intermediate. Three residues (Glu86, His143, and Lys170 in the Salmonella enterica DHQD) have previously been proposed to form a triad vital for catalysis. While the roles of Lys170 and His143 are well defined-Lys170 forms the Schiff base with the substrate and His143 shuttles protons in multiple steps in the reaction-the role of Glu86 remains poorly characterized. To probe Glu86's role, Glu86 mutants were generated and subjected to biochemical and structural study. The studies presented here demonstrate that mutant enzymes retain catalytic proficiency, calling into question the previously attributed role of Glu86 in catalysis and suggesting that His143 and Lys170 function as a catalytic dyad. Structures of the Glu86Ala (E86A) mutant in complex with covalently bound reaction intermediate reveal a conformational change of the His143 side chain. This indicates a predominant steric role for Glu86, to maintain the His143 side chain in position consistent with catalysis. The structures also explain why the E86A mutant is optimally active at more acidic conditions than the wild-type enzyme. In addition, a complex with the reaction product reveals a novel, likely nonproductive, binding mode that suggests a mechanism of competitive product inhibition and a potential strategy for the design of therapeutics.

Kinetics of Alcalase-catalyzed dipeptide synthesis in near-anhydrous organic media

The coupling kinetics of phenylalanine amide and the carbamoylmethyl ester of N-protected phenylalanine in near-anhydrous tetrahydrofuran were investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads; these immobilized enzymes were hydrated prior to use. Near-anhydrous conditions (i.e. extremely low water activity) were maintained by a carefully chosen amount of molecular sieve powder. Kinetic characteristics were determined from reaction time courses up to full conversion at various initial concentrations of substrate and product. These progress curve data were fitted with different kinetic models to determine which of these models best approximates the kinetic properties of the immobilized Alcalase with respect to the coupling under study. An appropriate model of the coupling reaction is necessary in order to design a reactor and predict its performance adequately leading to the practical implementation of biocatalyic peptide synthesis. The kinetics of the coupling were found to be complex and to obey a two-substrate kinetic model with two competitive product inhibition terms. To reduce the effect of the product inhibition on immobilized Alcalase, a reactor should be designed in which at least glycolamide is selectively removed as this was found to be the strongest inhibitor.