Publications
2009
C. Delattre, M. A. Vijayalakshmi
Journal of Molecular Catalysis B: Enzymatic 60 (2009) 97–105
Recent research in the area of bioactive carbohydrates has shown the efficiency of oligosaccharides as signal molecules in a lot of biological activities. Newly observed functions of oligosaccharides and their abilities to act as specific regulatory molecules on various organisms have been more and more described. A successful development of these bioactive molecules in future needs efficient processes for specific oligosaccharides production. To exploit them for putative industrial scale up processes, two main strategies are currently investigated: the synthesis (chemical or bioconversion processes) and the polysaccharide cleavage (chemical, physical or biological processes). Nevertheless, if new manufacturing biotechnologies have considerably increased the development of these functional molecules, the main drawback limiting their biological applications is the complexity to engender specific glycosidic structures for specific activities. In the recent years, new enzymatic reactors have been developed, allowing the automatic synthesis of oligosaccharide structures. This review focuses on the knowledge in the area of bioactive oligosaccharides and gives the main processes employed to generate them for industrial applications with challenges of monolith microreactors.
2008
C. Delattre, P. Michaud, M. A. Vijayalakshmi
Journal of Chromatography B, 861 (2008) 203–208
Fast production and purification of α-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 °C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.
2007
K. Isobe, Y. Kawakami
Journal of Chromatography A, 1144 (2007) 85-89(2007) 85-89
A convection interaction media (trade name CIM, Sartorius BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.
E. Müller, C. Mann
Journal of Chromatography A, 1144 (2007) 30-39(2007) 30-39
The electro-acoustic effects, namely the ion vibration potential (IVP) and the colloidal vibration current (CVI), colloidal vibration potential (CVP) first described by P. Debye [P. Debye, J. Chem. Phys. 1 (1933) 13], are a result of charge separation of bound or free ions at different degrees by ultrasonic waves. Today commercial instruments are available to investigate liquid homogeneous and heterogeneous systems. In the present paper the application of this technique for the characterization of salts, protein solutions and resins for biochromatography is shown and valuable information about resins can be derived in a short time. Various resins were investigated with the following results: (1) the CVI magnitude is dependent of several parameters (such as particle size distribution, volume fraction, density difference); (2) the CVI is influenced by the surface modification of the resins. Polymeric modifications decrease the value of CVI. The CVI is generally lower for high capacity resins; (3) the measurement of the electro-acoustic effects can be used to detect small changes in resins. The CVI is dependent of the amount of adsorbed protein in “native” and denatured state.
I.Vovk, B. Simonovska
Journal of Chromatography B, 849 (2007) 337-343
The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; Mr 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with Mr 82 kDa) and a basic protein with Mr 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM®) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2 M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5 M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with Mr 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH3CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5 mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.
I. Vovk, B. Simonovska
Journal of Chromatography A, 1144 (2007) 90-96(2007) 90-96
An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, Sartorius BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 μg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 μg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5 mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.
R. Nicoli, N. Gaud, C. Stella, S. Rudaz, J.-L. Veuthey
Journal of Pharmaceutical and Biomedical Analysis 48 (2008) 398–407
The preparation and characterization of three trypsin-based monolithic immobilized enzyme reactors (IMERs) developed to perform rapid on-line protein digestion and peptide mass fingerprinting (PMF) are described. Trypsin (EC 3.4.21.4) was covalently immobilized on epoxy, carboxy imidazole (CDI) and ethylenediamine (EDA) Convective Interaction Media® (CIM) monolithic disks. The amount of immobilized enzyme, determined by spectrophotometric measurements at 280 nm, was comprised between 0.9 and 1.5 mg per disk. Apparent kinetic parameters K*m and V*max, as well as apparent immobilized trypsin BAEE-units, were estimated in flow-through conditions using N-α-benzoyl-l-arginine ethyl ester (BAEE) as a low molecular mass substrate. The on-line digestion of five proteins (cytochrome c, myoglobin, α1-acid glycoprotein, ovalbumin and albumin) was evaluated by inserting the IMERs into a liquid chromatography system coupled to an electrospray ionization ion-trap mass spectrometer (LC-ESI–MS/MS) through a switching valve. Results were compared to the in-solution digestion in terms of obtained scores, number of matched queries and sequence coverages. The most efficient IMER was obtained by immobilizing trypsin on a CIM® EDA disk previously derivatized with glutaraldehyde, as a spacer moiety. The proteins were recognized by the database with satisfactory sequence coverage using a digestion time of only 5 min. The repeatability of the digestion (R.S.D. of 5.4% on consecutive injections of myoglobin 12 μM) and the long-term stability of this IMER were satisfactory since no loss of activity was observed after 250 injections.
M. Bartolini, V. Cavrini, V. Andrisano
Journal of Chromatography A, 1144 (2007) 102–110
The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized on a monolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For this application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective Interaction Media (CIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, low nonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms of action of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver–Burk plot analysis. Analyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak area. AChE-CIM-IMER kinetic parameters (Kmapp and vmaxapp ) were derived as well as inhibitory constants (Kiapp of selected compounds. Moreover, noteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbamoylation and decarbamoylation steps in pseudo-irreversible binding of carbamate derivatives (physostigmine and rivastigmine). AChE-CIM-IMER appeared to be a valid tool to determine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained with the classical methods with the free enzyme. Furthermore, after inactivation by carbamates, activity could be fully recovered and the AChE-CIM-IMER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the first phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracy and reproducibility and with saving of time and materials.
2005
K. Isobe, Y. Kawakami
Journal of Chromatography A, 1065 (2005) 129-134
Chromatography conditions for two types of convection interaction media (CIM) tube monolithic column, DEAE-8 and C4-8, were investigated using three enzymes from different microorganisms. The enzymes were adsorbed on a CIM DEAE-8 tube column under the same conditions as conventional DEAE columns. The CIM C4-8 tube column required a high concentration of ammonium sulfate compared to the conventional C4 column for adsorbing the enzymes. The separation of enzymes on the CIM tube column chromatography was not affected at flow rates between 0.15 and 1.25 volumes of the column per min. Both columns were successfully applied to the purification of enzymes from crude enzyme solution. Thus, both CIM tube monolithic columns proved useful in greatly reducing the purification time, and could be used at any stage of enzyme purification.
I. Vovk, B. Simonovska, M. Benčina
Journal of Chromatography A, 1065 (2005) 121-128
One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.11) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM®) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.
Y.-P. Lim, D. Josić, H. Callanan, J. Brown, D. C. Hixson
Journal of Chromatography A, 1065 (2005) 39–43(2005) 39–43
Epoxy-activated monolithic CIM disks seem to be excellent supports for immobilization of protein ligands. The potential use of enzymes, immobilized on monolithic disks for rapid preparative cleavage proteins in solution was investigated. Digestion of complex plasma proteins was demonstrated by using inter-alpha inhibitors with elastase, immobilized on epoxy-activated CIM disks. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAb 69.31) was developed. MAb 69.31 blocks the inhibitory activity of inter-alpha inhibitor proteins to serine proteases. These results suggest that the epitope defined by this antibody is located within or proximal to the active site of the inhibitor molecule. This antibody, immobilized on monolithic disk, was used for very rapid isolation of inter-alpha proteins. The isolated complex protein was used for enzymatic digestion and isolation of cleavage products, especially from inter-alpha inhibitor light chain to elucidate precisely the target sequence for MAb 69.31 by N-terminal amino acid sequencing. Bovine pancreatic elastase immobilized on monolithic disk cleaves inter-alpha inhibitor protein complex into small fragments which are still reactive with MAb 69.31. One of these proteolytic fragments was isolated and partially sequenced. It could be shown that this sequence is located at the beginning of two proteinase inhibitor domains of the inter-alpha inhibitor light chain (bikunin). Elastase immobilized on monolithic disk offers a simple and rapid method for preparative isolation of protease cleavage fragments. The immobilized enzyme is stable and still active after repeated runs. A partial or complete digestion can be achieved by varying the flow rate.
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M. Bartolini, V. Cavrini, V. Andrisano
Journal of Chromatography A, 1065 (2005) 135-144
The aim of the present study was to optimize the preparation of an immobilized acetylcholinesterase (AChE)-based micro-immobilized enzyme reactor (IMER) for inhibition studies. For this purpose two polymeric monolithic disks (CIM, 3 mm × 12 mm i.d.) with different reactive groups (epoxy and ethylendiamino) and a packed silica column (3 mm × 5 mm i.d.; Glutaraldehyde-P, 40 μm) were selected as solid chromatographic supports. All these reactors were characterized in terms of rate of immobilization, stability, conditioning time for HPLC analyses, optimum mobile phase and peak shape, aspecific interactions and costs. Advantages and disadvantages were defined for each system. Immobilization through Schiff base linkage gave more stable reactors without any significant change in the enzyme behaviour; monolithic matrices showed very short conditioning time and fast recovery of the enzymatic activity that could represent very important features in high throughput analysis and satisfactory reproducibility of immobilization yield. Unpacked silica material allowed off-line low costs studies for the optimization of the immobilization step.
2004
H. Podgornik, A. Podgornik
Journal of Chromatography B, 799 (2004) 343–347
Different chromatographic methods including chromatofocusing are used for separation of manganese peroxidase (MnP) isoforms and their isolation from the fungal growth medium. We tested strong anion exchange methacrylate based monolithic columns as a stationary phase for fast separation of MnP’s. Sodium acetate buffers of two different pH values (6 and 4) were used for formation of reproducible pH gradient. The entire cycle, involving analysis and column regeneration, was completed in 3 min. Use of pH gradient showed better MnP isoform separation comparing to the salt gradient, while application of combined pH–salt gradient, resulted in further improvement.
E. Vlakh, A. Novikov, G. Vlasov, T. Tennikova
Journal of Peptide Science, 10: 719–730 (2004)
Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing β-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods.
The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.
E. G. Vlakh, A. Tappe, C. Kasper, T. B. Tennikova
Journal of Chromatography B, 810 (2004) 15–23
Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.
E. G. Vlakh, A. Tappe, C. Kasper, T. B. Tennikova
Journal of Chromatography B, 810 (2004) 15–23
Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.
M. Bartolini, V. Cavrini, V. Andrisano
Journal of Chromatography A, 1031 (2004) 27–34(2004) 27–34
The development and characterization of a human recombinant acetylcholinesterase (hrAChE) micro-immobilized-enzyme reactor (IMER), prepared by using an in situ immobilization procedure is reported. hrAChE was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12mm x 3mm i.d.), previously derivatized with glutaraldehyde. The optimal conditions for the immobilization were: 12 μg of enzyme dissolved in 800 μl of phosphate buffer (50 mM, pH 6.0). The mixture was gently agitated overnight at 4 °C. The resulting Schiff bases were reduced by cyanoborohydride and the remaining aldehydic groups were condensed with monoethanolamine. Under these conditions, 0.22 U of hrAChE were immobilized with retention of 3.0% of the initial enzymatic activity. The activity of the immobilized hrAChE was stable for over 60 days. The activity and kinetic parameters of the hrAChE micro-IMER were investigated by inserting the micro-IMER in a HPLC system and it was demonstrated that the enzyme retained its activity. The micro-IMER was characterized in terms of units of immobilized enzyme and best conditions for immobilization yield. IMERs were compared for their relative enzyme stability, immobilized units, yield and aspecific matrix interactions. The effect of AChE inhibitors was evaluated by the simultaneous injection of each inhibitor with the substrate. The relative IC50 values were found in agreement with those derived by the conventional kinetic spectrophotometric method. In comparison with previously developed AChE-based IMERs, AChE monolithic micro-IMER showed advantages in terms of reduction of analysis time (2 min), lower aspecific matrix interactions and lower backpressure. Included in a HPLC system, it can be used for the rapid screening of new compounds’ inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analyzed in continuous.
K. Benčina, A. Podgornik, A. Štrancar, M. Benčina
Journal of Separation Science 2004, 27, 811-818
Monolithic Convective Interaction Media (CIM) have been activated with epoxide and imidazole carbamate functionalities and used as supports for covalent immobilization of protein A, deoxyribonuclease I, and trypsin. The efficiency of immobilization for these proteins was determined from the amount of bound IgG, degradation of DNA, and hydrolysis of Nα-benzoyl-L-arginine ethyl ester, respectively. The respective biological activities of trypsin and the binding capacity of protein A immobilized via imidazole carbamate groups were 11.45 and 2.25 times higher than those obtained for epoxide matrix while they were practically equal for deoxyribonuclease I. The kinetics of immobilization was studied in detail for trypsin under dynamic conditions and revealed that the enzyme immobilized via imidazole carbamate groups already reached its highest activity in 5 min. In contrast, a much longer time was required for immobilization via epoxy groups.
P. N. Nesterenko, M. A. Rybalko
Mendeleev Commun. 2004
The continuous flow gradient and its effect on chromatographic parameters were investigated for the separations of inorganic anions on a monolithic porous disk with bonded hydroxyproline residues.
2003
E. G. Vlakh, G. A. Platonova, G. P. Vlasov, C. Kasper, A. Tappe, G. Kretzmer, T. B. Tennikova
Journal of Chromatography A, 992 (2003) 109–119
The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-l-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.