Enzyme Inactivation Process Research Articles

Impact of thermal ultrasound on enzyme inactivation and flavor improvement of sea cucumber hydrolysates

In this study, the effects of the thermal ultrasonic enzyme inactivation process on flavor enhancement in sea cucumber hydrolysates (SCHs) and its impact on the inactivation of neutral proteases (NPs) were investigated. The body wall of the sea cucumber was enzymatically hydrolyzed with NPs. On the one hand, the structure of NPs subjected to different enzyme inactivation methods was analyzed using ζ-potential, particle size, and Fourier transform infrared (FT-IR) spectroscopy. On the other hand, the microstructure and flavor changes of SCHs were examined through scanning electron microscopy, E-nose, and gas chromatography-ion mobility spectrometry (GC-IMS). The results indicated that thermal ultrasound treatment at 60 °C could greatly affect the structure of NPs, thereby achieving enzyme inactivation. Furthermore, this treatment generated more pleasant flavor compounds, such as pentanal and (E)-2-nonenal. Hence, thermal ultrasound treatment could serve as an alternative process to traditional heat inactivation of enzymes for improving the flavor of SCHs.

Stabilization of Rice Bran: A Review.

One of the major problems in food science is meeting the demand of the world's growing population, despite environmental limitations such as climate change, water scarcity, land degradation, marine pollution, and desertification. Preventing food from going to waste and utilizing nutritive by-products as food rather than feed are easy and powerful strategies for overcoming this problem. Rice is an important staple food crop for more than half of the world's population and substantial quantities of rice bran emerge as the main by-product of rice grain milling. Usually, rice bran is used as animal feed or discarded as waste. Although it is highly nutritious and comprises many bioactive compounds with considerable health benefits, the rapid deterioration of bran limits the exploitation of the full potential of rice bran. Hydrolytic rancidity is the main obstacle to using rice bran as food, and the enzyme inactivation process, which is termed stabilization, is the only way to prevent it. This study reviews the methods of stabilizing rice bran and other rice-milling by-products comprising rice bran in the context of the efficiency of the process upon storage. The effect of the process on the components of rice bran is also discussed.

Effects of thermal processing on the flavor molecules of goat by-product hydrolysates

The aim of this study was to obtain flavor molecules from goat by-product hydrolysates, emphasizing the thermal action during processing. A mixture of by-products submitted or not to the inactivation of endogenous enzymes was used, followed by hydrolysis with the proteolytic enzyme Alcalase® (Bacillus licheniformis), and autoclaving after hydrolysis. The production of hydrolysates provided both quantitative and qualitative data on the precursors involved in the aromatic formation of protein hydrolysates. The inactivation process of endogenous enzymes resulted in hydrolysates with a higher degree of hydrolysis and greater protein content. The autoclaving process produced a significant increase in the concentration of free amino acids and maltose and a reduction in the glucose content. Application of the two heat treatments resulted in the production of goat by-product protein hydrolysates with different volatile profiles. The goat by-product protein hydrolysate without heat treatment but with autoclaving (HCA), showing a higher concentration of flavor precursors and the formation of heterocyclic volatiles, is expected to impact the aroma quality of goat hydrolysates.

Application of ultrasound technology in processing of ready-to-eat fresh food: A review.

With the increase in food standardization and the pace of modern life, the demand for ready-to-eat foods is growing. The strong processing conditions of traditional technology often accelerate the rate of deterioration of quality, and microbes are the safety hazard of ready-to-eat foods. Ultrasound technology is an environmentally friendly technology that hardly causes thermal damage to raw materials. In this paper, the ultrasound technology is used in the disinfection, sterilization, enzyme inactivation, desensitization, dehydration, curing, tenderization and cooking process of fresh food from the perspective of microbial safety and quality of fresh food. The cavitation effect of ultrasound can improve the mass transfer rate of infiltration processes such as dehydration and curing, promote the oxidation of lipids and proteins for enrich the flavor of meat products, improve the microbiological safety and reduce the sensitization by destroying the integrity of the microbial cells and the conformation of the protein. In addition, ultrasound as an auxiliary processing technology can reduce the damage of traditional production technology to reserve the quality and nutritional value of food. Ultrasound has proved to be an efficient and green processing technology for ready-to-eat food.

Kinetics of peroxidase inactivation, color and temperature changes during pumpkin (Cucurbita moschata) blanching using infrared heating

Fresh Pumpkin is sensitive to microbial or enzymatic spoilage even in refrigeration temperatures, thus; blanching before freezing or canning is a proper way for preservation and commercialization of this product. In this study infrared irradiation was chosen as heating source and peroxidase as an indicator of enzyme inactivation adequacy for pumpkin blanching. Pumpkin purée was treated under 5 levels of temperature and 2 levels of sample thickness. Effect of thermal processing on the kinetic parameters of peroxidase inactivation (k, Ea, D and Z values) and color changes (ΔE) was investigated. Peroxidase inactivation of pumpkin samples followed a first order kinetics model. Calculated activation energies and Z values of enzyme inactivation process for 4 and 8 mm thickness levels were 130.63, 119.46 kJ mol−1 and 18.18, 17.85 °C, respectively. No significant difference was observed between activation energies and Z values of two thickness levels. But, the effect of temperature and thickness on inactivation rate constants (k) and D values was significant (P < 0.05). Investigation on the kinetics of color change was carried out by the calculation of ΔE changes during l log enzyme inactivation period. In this study, sample thickness showed a more significant effect on ΔE variations than temperature.

Detection and Structural Characterization of Nucleophiles Trapped Reactive Metabolites of Limonin Using Liquid Chromatography-Mass Spectrometry.

Limonin (LIM), a furan-containing limonoid, is one of the most abundant components of Dictamnus dasycarpus Turcz. Recent studies demonstrated that LIM has great potential for inhibiting the activity of drug-metabolizing enzymes. However, the mechanisms of LIM-induced enzyme inactivation processes remain unexplored. The main objective of this study was to identify the reactive metabolites of LIM using liquid chromatography-mass spectrometry. Three nucleophiles, glutathione (GSH), N-acetyl cysteine (NAC), and N-acetyl lysine (NAL), were used to trap the reactive metabolites of LIM in in vitro and in vivo models. Two different types of mass spectrometry, a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometry and a LTQ velos Pro ion trap mass spectrometry, were employed to acquire structural information of nucleophile adducts of LIM. In total, six nucleophile adducts of LIM (M1–M6) with their isomers were identified; among them, M1 was a GSH and NAL conjugate of LIM, M2–M4 were glutathione adducts of LIM, M5 was a NAC and NAL conjugate of LIM, and M6 was a NAC adduct of LIM. Additionally, CYP3A4 was found to be the key enzyme responsible for the bioactivation of limonin. This metabolism study largely facilitates the understanding of mechanisms of limonin-induced enzyme inactivation processes.

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 102 M-1 min-1. Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Effect of mutations on the thermostability of Aspergillus aculeatus β-1,4-galactanase

New variants of β-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of β-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions were experimentally validated by measurements of the melting temperature (Tm) by differential scanning calorimetry. We found that the Tm is elevated by 1.1°C for G306A, slightly increased (in the range of 0.34 to 0.65°C) for D182N, D58N, Y295F and unchanged or decreased for S185D/Q188T and G104D/A156R. The Tm changes were in the range predicted by PROPKA. Given the experimental errors, only the D58N and G306A show significant increase in thermodynamic stability.Given the practical importance of kinetic stability, the kinetics of the irreversible enzyme inactivation process were also investigated for the wild-type and three variants and found to be biphasic. The half-lives of thermal inactivation were approximately doubled in G306A, unchanged for D182N and, disappointingly, a lot lower for D58N. In conclusion, this study tests a new method for estimating Tm changes for mutants, adds to the available data on the effect of substitutions on protein thermostability and identifies an interesting thermostabilizing mutation, which may be beneficial also in other galactanases.

Structural stability and Sin a 1 anti-epitope antibody binding ability of yellow mustard (Sinapis alba L.) napin during industrial-scale myrosinase inactivation process.

This study investigated the structural stability of yellow mustard (YM, Sinapis alba L.) napin and the changes of its Sin a 1 anti-epitope antibody-binding ability during myrosinase enzyme inactivation process. The food industry uses myrosinase-inactive non-pungent YM for uses beyond spice applications. Napin was isolated from seeds received from an industrial processor before (YM + M) and after (YM - M) myrosinase inactivation. Secondary and tertiary structural features and surface hydrophobicity parameters of napin were analyzed. The Sin a 1 content in YM seeds and the stability of Sin a 1-containing napin during simulated in vitro gastrointestinal (GI) digestion were determined by a non-competitive indirect enzyme-linked immunosorbent assay using the Sin a 1 anti-epitope antibody (AE-Ab) as the primary Ab. YM napin retained the dominant alpha-helical components of secondary and tertiary structure folds during this process. YM - M napin showed changes in hydrophobicity parameters of the molecules and binding ability of AE-Ab: 2.19 ± 0.48 g per 100 g of YM - M seeds vs. 1.49 ± 0.16 g per 100 g YM + M seeds. YM - M proteins were more susceptible for in vitro GI digestion and also showed a 30% reduction in AE-Ab binding ability upon digestion of napins. This suggests that the myrosinase inactivation process has induced the surface modification of napin, exposing Sin a 1 epitope, leading to an increase in AE-Ab binding. However, the epitope region of YM - M napin showed improved susceptibility for hydrolysis during GI digestion resulting in fewer available epitope regions, suggesting a possible reduction in napin immune reactivity.

Factors impeding enzymatic wheat gluten hydrolysis at high solid concentrations

Enzymatic wheat gluten hydrolysis at high solid concentrations is advantageous from an environmental and economic point of view. However, increased wheat gluten concentrations result in a concentration effect with a decreased hydrolysis rate at constant enzyme-to-substrate ratios and a decreased maximum attainable degree of hydrolysis (DH%). We here identified the underlying factors causing the concentration effect. Wheat gluten was hydrolyzed at solid concentrations from 4.4% to 70%. The decreased hydrolysis rate was present at all solid concentrations and at any time of the reaction. Mass transfer limitations, enzyme inhibition and water activity were shown to not cause this hydrolysis rate limitation up to 50% solids. However, the hydrolysis rate limitation can be, at least partly, explained by a second-order enzyme inactivation process. Furthermore, mass transfer impeded the hydrolysis above 60% solids. Addition of enzyme after 24 h at high solid concentrations scarcely increased the DH%, suggesting that the maximum attainable DH% decreases at high solid concentrations. Reduced enzyme activities caused by low water activities can explain this DH% limitation. Finally, a possible influence of the plastein reaction on the DH% limitation is discussed.

Characterization of unstable enzyme systems which evolve according to a three-exponential equation

The time course of an enzyme catalyzed reaction is normally followed either by monitoring the instantaneous concentration or velocity of an enzyme species or a product. In many enzyme catalyzed reactions these time variations are multi-exponential. The accurate fit of the relevant curves to obtain the kinetic parameters involved can be difficult using conventional methods (Galvez et al. in J Theor Biol 89:37–44, 1981; Garcia-Canovas et al. in Biochim Biophys Acta 912:417–423, 1987; Tudela et al. in Biochim Biophys Acta 912:408–416, 1987; Teruel et al. in Biochim Biophys Acta 911:256–260, 1987; Garrido del Solo et al. in Biochem J 294:459–464, 1993; Varon et al. in Int J Biochem 25:1889–1895, 1993; Garrido del Solo et al. in An Quim 89:319–324, 1993; Varon et al. in J Mol Catal 83:273–285, 1993; Garrido del Solo et al. in Biochem J 303(Pt 2):435–440, 1994; Garrido del Solo and Varon in An Quim 91:13–18, 1995; Garrido del Solo et al. in Biosystems 38:75–86, 1996; Garrido del Solo et al. in Int J Biochem Cell Biol 28:1371–1379, 1996; Garrido del Solo et al. in Int J Biochem Cell Biol 30:735–743, 1998; Varon et al. in J Mol Catal 59:97–118, 1990). In order to circumvent such difficulties Arribas et al. (J Math Chem 44:379–404, 2008) proposed an evaluation method which is applicable regardless of the complexity of the kinetic equation. This procedure is based on the numerical determination of statistical moments from experimental time progress curves. The fitting of these experimentally obtained moments to the corresponding theoretical symbolic expressions allows, in most cases, all the individual rate constants involved to be evaluated. In this paper we perform a general analysis that can be applied to any unstable enzyme system described by a three-exponential equation and apply it to a substrates induced enzyme inactivation process that is described by this type of equation. To verify the goodness of the method we have simulated time progress curves and applied the suggested procedure to these curves, obtaining kinetic parameters values very close to those used to obtain simulated curves. Finally, we compare our results with those obtained in previous contributions in which other procedures were used.

A probabilistic model for risk assessment of residual host cell DNA in biological products

Biological products such as viral vaccines manufactured in cells contain residual DNA derived from host cell substrates used in production. It is theoretically possible that the residual DNA could transmit activated oncogenes and/or latent infectious viral genomes to subjects receiving the product, and induce oncogenic or infective events. A probabilistic model to estimate the risks due to residual DNA is proposed. The model takes account of enzyme inactivation process. It allows for more accurate risk assessment when compared to methods currently in use. An application of the method to determine safety factor of a vaccine product is provided.

Microwave and Radio Frequency Heating

This paper brings to perspective issues related to research initiatives for the application of microwave (MW) and radiofrequency (RF) applications in foods. Both MW (300 MHz and 300 GHz) and RF waves (3 kHz — 300 MHz) are part of the electromagnetic spectrum that result in heating of dielectric materials by induced molecular vibration as a result of dipole rotation or ionic polarization. They have been credited with volumetric heat generation resulting in rapid heating of foodstuffs. Due to their lower frequency levels, RF waves have a larger penetration depth than MW and hence could find better application in larger size foods. Besides the popular domestic use of MW ovens, commercialized applications of MW/RF heating include blanching, tempering, pasteurization, sterilization, drying, rapid extraction, enhanced reaction kinetics, selective heating, disinfestations, etc. This paper reviews the current status and research needs for in-packaged sterilization technologies for commercial applications. Technological challenges include process equipment design, microbial destruction and enzyme inactivation kinetics, temperature and process monitoring, and achieving of temperature uniformity. Other issues also relate to the use of packaging material in in-package sterilization applications, package/container concerns in domestic MW ovens, receptor technology for creating dry-oven conditions, modeling and time-temperature process integrators. There is also the issue of non-thermal and enhanced thermal effects of microwave heating on destruction kinetics.

Palmito de pupunha (Bactris gasipaes Kunth.) composição mineral e cinética de enzimas oxidativas

A análise da presença de enzimas oxidativas como a peroxidase (POD) e a polifenoloxidase (PPO) e o controle da atividade destas enzimas são importantes na preservação e no processamento de alimentos. Este trabalho teve por objetivo determinar a atividade enzimática da polifenoloxidase (PPO) e da peroxidase (POD) do palmito de pupunha, bem como avaliar o comportamento destas enzimas frente ao tratamento térmico e assim calcular a cinética de inativação térmica das mesmas para suas porções termorresistente e termolábil. Para a extração de peroxidase (POD) e polifenoloxidase (PPO) de palmito, utilizou-se solução tampão fosfato de sódio 100 mM com diferentes pHs (5,5; 6,0; 6,5 e 7,0). O melhor pH de extração da POD foi 5,5 e da PPO, 6,5. Estes extratos foram tratados em diferentes temperaturas (65, 70, 75 e 80 °C) por períodos de 1 a 10 minutos. A POD e a PPO sofreram um decréscimo de 70 e 80%, respectivamente, em relação às suas atividades iniciais. As energias de ativação, nas temperaturas estudadas, para a porção termolábil e termorresistente da peroxidase foram 154,0 e 153,0 kJ.mol-1, respectivamente, enquanto que para a polifenoloxidase foram 26,3 e 27,0 kJ.mol-1, respectivamente. Resultados apresentaram valores que estão dentro da faixa de energia de ativação reportada para o processo de inativação térmica de enzimas.

Improved stability of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by replacement of cysteine residues

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 is a homo-tetrameric protein containing three cysteines per 28 kDa subunit. Under oxidizing conditions the enzyme was found to be susceptible to inactivation which could be prevented by the addition of β-mercaptoethanol or glycerol. Gel filtration experiments and SDS-PAGE analysis revealed that inactivation coincided with monomerization and intramolecular disulfide bond formation. To identify the cysteine residues involved in the inactivation process, a set of cysteine mutant enzymes was constructed. All the purified mutants (C30A, C153S, C229A and C153S/C229A) showed a similar activity as wild-type enzyme, indicating that no cysteine is directly involved in catalysis. The C153S and C30A mutants displayed a higher stability than wild-type enzyme, whereas mutating Cys229 resulted in decreased enzyme stability. SDS-PAGE analysis showed that in wild-type enzyme Cys30-Cys229 and Cys153-Cys229 disulfide bonds were readily formed while almost no formation of the Cys30-Cys153 disulfide bond could be observed. From this, it was concluded that all three cysteine residues are involved in the enzyme inactivation process. The importance of the improved stability of the C153S and C30A mutant enzymes was demonstrated by performing kinetic resolution experiments with racemic 2-chloro-1-phenylethanol, which resulted in higher enantiomeric excess values of the remaining halohydrin when compared to conversions catalyzed by wild-type enzyme.

撹はん型ビーズミルによるパン酵母内酵素の失活を伴う放出過程

Release process of enzymes from baker's yeast accompanied by inactivation was experimentally investigated by using an agitating bead mill. The enzymes examined were Alkaline phosphatase and alcohol dehydrogenase. As a result, the disruption rate of yeast was directly proportional to the product of the concentration of undisrupted yeast and agitating speed of impeller, and the disruption process of yeast was clarified by this analysis. In order to investigate the inactivation process of enzymes, the activity of enzymes within the yeast was measured using a lytic method, the results of which are not influenced by the mechanical force. In the case where there is no inactivation, the release rate of enzymes was directly proportional to the product of the activity of unreleased enzymes and agitating speed of impeller. In the case of inactivation, the release rate of enzymes was directly proportional to the product of the activity of uninactivated enzymes and agitating speed of impeller. The experimentally analyzed models based on the kinetics were found to describe well the release process of enzymes accompanied by the inactivation from baker's yeast, disrupted by the agitating bead mill.

L-Malate Dehydrogenase fromPseudomonas stutzeri:Purification and Characterization

L-Malate dehydrogenase (MDH) fromPseudomonas stutzeriwas purified to homogeneity by a two-step procedure comprising anion-exchange chromatography and affinity chromatography on immobilized anthraquinone α-ketocarboxyl biomimetic dye. The enzyme has molecular mass of 66,500 Da and consists of two identical subunits of molecular mass of approximately 34,000 Da. Initial velocity, product inhibition, and binding studies were consistent with an ordered Bi-Bi mechanism for the enzyme action and the formation of a ternary complex. The enzyme is susceptible to activation and inhibition by its substrates. Thermodynamic analysis and kinetic inhibition studies were performed for determining basic equilibrium and kinetic constants. Malate dehydrogenase was covalently inactivated by a dichlorotriazine dye, Vilmafix Blue A-R (VBAR). The inactivation process follows first-order kinetics, and the results from kinetic analysis suggested the formation of a noncovalent enzyme–dye complex prior to the covalent reaction, withKd84.6 μMand a maximum rate constant 0.16 min−1. The enzyme inactivation process was partially inhibited by substrates and inhibitors. Quantitatively inactivated MDH contained approximately 1 mole of dye per mole of enzyme subunit. The denatured enzyme contains 10 sulfhydryl groups per subunit, as shown after reaction with 5,5′-dithio-bis-(2-nitrobenzoic acid), of which 5 can be titrated also in the native enzyme, exhibiting time-dependent reactivity. One sulfhydryl group is located in the coenzyme binding site. This study shows that the physical and catalytic properties ofP. stutzeriMDH strongly resemble those of the mitochondrial eukariotic enzyme. This finding strengthens the existing view that, in the evolution process, the mitochondrial MDH might have appeared before the cytoplasmic.

Fate of a terminal olefin with Drosophila microsomes and its inhibitory effects on some P-450 dependent activities.

In vitro bioassays were used to analyze the metabolism of the 11-dodecenoic acid (11-DDNA) by microsomes prepared from Drosophila melanogaster RalDDTR strain. 11-DDNA is metabolized to 11,12-epoxylauric acid (epoxyLA) in a NADPH-dependent way. The microsomal production of epoxyLA reaches a plateau very quickly, suggesting the occurrence of an enzyme inactivation process. After incubation of microsomes with (1-14C)11-DDNA, three proteins of Mr approximately 50 kDa were labeled. 11-DDNA inhibits the microsomal metabolism of lauric acid and 7-ethoxycoumarin in a time and NADPH-dependent process. An inhibition of metabolites generated from DDT and testosterone was also obtained but at higher concentrations. These results are discussed according to the fact that RalDDTR is an insecticide resistant strain characterized as a high metabolizer of the insecticide DDT and also of lauric acid, testosterone, and ethoxycoumarin.

Tritium release from [19-3H]-19,19-difluoroandrost-4-ene-3,17-dione during inactivation of aromatase.

Aromatase is a cytochrome P-450 enzyme involved in the conversion of androst-4-ene-3,17-dione to estrogen via sequential oxidations at the 19-methyl group. Previous studies from this laboratory showed that 19,19-difluoroandrost-4-ene-3,17-dione (5) is a mechanism-based inactivator of aromatase. The mechanism of inactivation was postulated to involve enzymic oxidation at, and hydrogen loss from, the 19-carbon. The deuteriated analogue 5b has now been synthesized and shown to inactivate aromatase at the same rate as the nondeuteriated parent (5). We conclude that C19-H bond cleavage is not the rate-limiting step in the overall inactivation process caused by 5. [19-3H]-19,19-Difluoroandrost-4-ene-3,17-dione (5b) with specific activity of 31 mCi/mmol was also synthesized to study the release of tritium into solution during the enzyme inactivation process. Incubation of [19-3H]19,19-difluoroandrost-4-ene-3,17-dione with human placental microsomal aromatase at differing protein concentrations resulted in time-dependent NADPH-dependent, and protein-dependent release of tritium. This tritium release is not observed in the presence of (19R)-10 beta-oxiranylestr-4-ene-3,17-dione, a powerful competitive inhibitor of aromatase. We conclude that aromatase attacks the 19-carbon of 19,19-difluoroandrost-4-ene-3,17-dione, as originally postulated.

Protecting effect of competitive inhibitors during very intense insolubilized enzyme-activated support multipoint attachments: trypsin (amine)-agarose (aldehyde) system

The effect of the presence of benzamidine, a competitive inhibitor of trypsin, during two different enzyme inactivation processes was studied. The inactivation processes were: thermal inactivation of soluble and insolubilized trypsin and inactivation of trypsin by multipoint covalent attachment to highly activated agarose (aldehyde) gels. In both cases, the presence of benzamidine, strongly adsorbed on the active site of the enzyme, greatly reduces the inactivation of trypsin. In the insolubilization-stabilization of trypsin by multipoint covalent attachment, the presence of benzamidine preserved the enzyme fully active during the insolubilized enzyme-activated support multiinteraction processes. This result was observed even during the most intense process in which eight lysine residues per trypsin molecule remained attached to the support. As a consequence of this result, the insolubilized derivative becomes 10 000-fold more stable than soluble trypsin. On the other hand, the presence of the inhibitor reduces the overall possibilities of enzyme-support multiinteraction; as a result, derivatives prepared in the presence of benzamidine are more active but less stable than derivatives prepared in absence of the inhibitor.