A study on enzymatic reaction using a liquid emulsion membrane technique

An enzymatic reaction using a liquid emulsion membrane technique was studied to investigate the effects of some experimental variables on the stability of liquid membrane, enzyme deactivation, and transport of substrates and products. The hydrolysis of L-phenylalanine methyl ester by alpha-chymotrypsin was selected as a model reaction system. First, a transport mechanism for the substrates and products across the membrane was qualitatively identified. Second, it was found that the pH of the internal phase was one of the most important variables to determine the enzyme activity in a liquid membrane. Third, the effect of membrane phase which consists of surfactant, carrier, and organic solvent on the emulsion stability was investigated. It was found that the properties of the organic solvents greatly affect the emulsion stability. For an optimum condition, it was possible to reuse the emulsion which consists of membrane phase and internal phase without further separation. It was finally concluded that the enzyme in a liquid membrane retained 60% of its native activity in spite of vigorous mixing during the emulsification step.     

Integration of bioconversion and downstream processing: starch hydrolysis in an aqueous two-phase system

Integration of bioconversion and the first step(s) of down stream processing can be used as a means to increase the productivity of bioprocesses. This integration also gives the possibility to run the bioconversion in a continuous mode. We demonstrate the use of an aqueous two-phase system in combination with ultrafiltration to accomplish this. Conversion of native starch to glucose by alpha-amylase and glucoamylase was carried out in an aqueous two-phase system in connection with a membrane filtration unit. In this way, a continuous stream of glucose in buffer solution was obtained; the phase-forming polymers as well as the starch-degrading enzymes were recycled, and clogging of the ultrafiltration membrane was avoided. The process was carried out continuously in a mixer-settler reactor for a period of 8 days. The enzyme activities in the top and bottom phases and in the mixing chamber were monitored intermittently throughout the experiment. The optimum pH, temperature, and ionic strength for the activity of the enzyme mixture were determined. The settling time of phase systems containing varying amounts of PEG, crude dextran, and solid starch was studied. The activity and stability of enzyme mixtures was studied both in buffer medium and in the medium containing the polymers. The enzymes were found to be more active and stable in medium containing polymers than in the buffer solutions.     

Detection of native peptides as potent inhibitors of enzymes. Crystal structure of the complex formed between treated bovine alpha-chymotrypsin and an autocatalytically produced fragment, IIe-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp, at 2.2 angstroms resolution

Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in alpha-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 +/- 0.3 x 10(-11) M and thus works as a highly potent competitive inhibitor. The commercially available alpha-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated alpha-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mM sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 M ammonium sulfate. The rectangular crystals of small dimensions of 0.24 x 0.15 x 0.10 mm(3) were obtained. The X-ray intensity data were collected at 2.2 angstroms resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of alpha-chymotrypsin, which was readily interpreted as a 14 residue fragment of alpha-chymotrypsin corresponding to Ile-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 sigma between the active site Ser195 Ogamma and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 angstroms between Ser195 Ogamma and Trp29 C indicating the presence of a covalent linkage between the enzyme and the native fragment. This meant that the enzyme formed an acyl intermediate with the autodigested fragment Ile16-Trp29. In addition to the O-C covalent bond, there were several hydrogen bonds and hydrophobic interactions between the enzyme and the native fragment. The fragment showed a high complementarity with the binding site of alpha-chymotrypsin and the buried part of the fragment matched excellently with the corresponding buried part of Turkey ovomucoid inhibitor of alpha-chymotrypsin.     

Self-assembling of polymer-enzyme conjugates at oil/water interfaces

Interface-binding enzymes are desirable for biphasic reactions in that they offer simultaneous access to substrates dissolved in both phases across the interface. It has been shown that conjugating water-soluble enzymes with hydrophobic polymers facilitated the assembling of enzymes at oil/water interfaces. In this work, the interfacial assembling of alpha-chymotrypsin conjugated with polystyrene, poly(methyl methacrylate), and poly(l-lactic acid) was examined using the pendant drop method. The interface-assembling process of the conjugates from the organic phase followed a similar pattern of that of native alpha-chymotrypsin from the aqueous buffer phase, i.e., the interfacial tension decreased gradually with time. However, when the conjugates were dispersed in the form of particulates in the aqueous phase, in which the conjugate was insoluble, the assembling occurred faster and the interfacial tension quickly approached zero. It was suspected that the assembling in this case involved two steps, i.e., the adsorption of the particulates and the subsequent rearrangement, dissociation, and redispersion of the conjugates at the interface. The effect of other factors, including the polarity of organic solvent and pH and ionic strength of the aqueous phase, was evaluated. It was found that the polar solvent slightly facilitated the assembling, whereas pH and ionic strength showed minimal effects.     

Experimental confirmation of calculated phases and electron density profile for wet native collagen.

An experimental procedure is developed to phase the reflections obtained in x-ray diffraction investigations of collagen in native wet tendons. Phosphotungstic acid was used for isomorphous addition in phase determination and was located by electron microscopy. Structure factors (with phases) were obtained from the electron microscopy data for the heavy metal. Structure-factor magnitudes for collagen with and without the heavy metal were obtained from the x-ray diffraction data. The first 10 orders were investigated. Standard Argand diagrams provided two solutions for each of these, except the weak sixth order. In each case, one of the two possible solutions agrees well with the phases proposed on theoretical grounds by Hulmes et al. The present results suggest that their other proposed phases are probably correct. An electron density profile along the unit cell of the fibril is presented that shows a distinct step, as expected on the basis of the hole-overlap model. The overlap region is 48% of the length of the unit cell.     

Enzyme-carrying polymeric nanofibers prepared via electrospinning for use as unique biocatalysts

Improvement of catalytic efficiency of immobilized enzymes via materials engineering was demonstrated through the preparation of bioactive nanofibers. Bioactive polystyrene (PS) nanofibers with a typical diameter of 120 nm were prepared and examined for catalytic efficiency for biotransformations. The nanofibers were produced by electrospinning functionalized PS, followed by the chemical attachment of a model enzyme, alpha-chymotrypsin. The observed enzyme loading as determined by active site titration was up to 1.4% (wt/wt), corresponding to over 27.4% monolayer coverage of the external surface of nanofibers. The apparent hydrolytic activity of the nanofibrous enzyme in aqueous solutions was over 65% of that of the native enzyme, indicating a high catalytic efficiency as compared to other forms of immobilized enzymes. Furthermore, nanofibrous alpha-chymotrypsin exhibited a much-improved nonaqueous activity that was over 3 orders of magnitude higher than that of its native counterpart suspended in organic solvents including hexane and isooctane. It appeared that the covalent binding also improved the enzyme's stability against structural denaturation, such that the half-life of the nanofibrous enzyme in methanol was 18-fold longer than that of the native enzyme.     

Correlation between the stability of alpha-chymotrypsin at high temperatures and "salting in" of a strong solution]

A correlation was found between the thermal stability of alpha-chymotrypsin and the coefficient Ks of the Sechenov equation as a quantitative measure of the "salting-in" or "salting-out" capacity of solutes. At high temperatures, an increase in the concentration of "salting-in" agents (KSNC, GuHCl, urea, formamide) resulted in thermal stabilization of alpha-chymotrypsin. The maximal (about 100-fold) stabilizing effect in concentrated solutions of salting-in agents was comparable with those induced by covalent modification with hydrophilic reagents or immobilization. Conversely, an increase in the concentration of "salting-out" agents stabilized the enzyme only marginally at high temperatures. An additivity of solutes' action on the thermal stability of the protein has been demonstrated. The observed correlation was explained in terms of the solutes' action on the reversible conformational transition of the enzyme native form into a much more stable form existing at high temperatures.     

Cell surface charge. Correlation of partition with electrophoresis

Critical mixtures of aqueous solutions os polymers separate into two or more immiscible phases. Particulate materials distribute in such phase systems generally between one bulk phase and the interface between bulk phases. The distribution is described by a simple partition law, and is qunatitatively determined by, inter alia, the nature of the particle surface, particularly net electrical charge. The partition behaviour of various cells, native or modified by treatment with trypsin, neurominidase or maleic anhydride, correlate strongly with electrophoretic mobility. Partition behaviour and electrophoretic mobility are both dependent upon cell surface charge. Thus, in appropriate conditions, changes in surface charge may be registered as changes in partition.     

Enhanced enzymatic activity and stability of trypsin by reductive alkylation in solid phase

Amino groups of trypsin (EC 3.4.21.4) were reductively alkylated in solid phase to obtain a surface-active and biologically active enzyme in an o/w emulsion system. Trypsin adsorbed on a benzamidine-sepharose column was reductively alkylated with n-octanal in the presence of sodium borohydride, i.e., trypsin-C8. Activity of trypsin-C8 against Nalpha-benzoyl-L-arginine-p-nitroanilide was three times higher than that of native trypsin. Activities of trypsin and trypsin-C8 against casein were almost the same. After incubating the trypsin solution at 40 degrees C for 1 h, residual activities in the emulsion and solution systems were 64.2 and 57.4%, respectively. On the other hand, residual activities of native trypsin following incubation were 21.8% in the emulsion system and 33.2% in the solution system. Enhancement of trypsin-C8 stability in the emulsion system may derive from interaction between the hydrophobic areas of trypsin-C8 molecules and the hydrophobic phase of the emulsion.     

Enzymes in polyelectrolyte complexes. The effect of phase transition on thermal stability

Penicillin amidase, alpha-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution in equilibrium precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: the enzyme is in the precipitate; (b) the enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60 degrees C, and 300-fold at pH 3.1, 25 degrees C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70 degrees C. The localization of enzyme on N-PEC has been established by titration of alpha-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an alpha-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of alpha-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.     

A new CSLM-based method for determination of the phase behaviour of aqueous mixtures of biopolymers

On mixing different types of high molecular weight (bio)polymers in an aqueous solution, phase separation often occurs. In some cases, the occurrence of phase separation may be readily observed, because due to density differences the heavier of the two phases is accumulated at the bottom of the vessel in which the mixture is contained. By using classical techniques, the composition of the two phases may then be determined. In the case where the density differences are not so large, and the viscosity of the system is high, the two phases remain intimately mixed. An alternative route to determine the phase behaviour of these systems might be a microscopic technique (Confocal Scanning Laser Microscopy, CSLM), using the fluorescence intensity of labelled biopolymers to quantify their concentration and phase volume in the system. Experiments were performed with several mixtures of sodium alginate, labelled with fluorescein, and sodium caseinate, fluorescently labelled with Texas Red. The viscosity of the mixtures studied was low enough to allow bulk phase separation of the phases by using an ultracentrifuge. Results of the phase volumes, and the composition of the phases, obtained independently by applying the two different methods (CSLM, or analysis of the separate phases after centrifugation) were compared and found to be in reasonable agreement.     

Conformations of N-(2-fluorophenyl)-N-phenylcarbamoyl-alpha-chymotrypsin

N-(2-Fluorophenyl)-N-phenylcarbamoyl chloride is shown to react with alpha-chymotrypsin to give a catalytically inactive material. A crystal structure determination shows that the chloride exists in the solid state in two conformations. In both of these the aromatic rings are tilted substantially relative to the plane through the atoms of the carbamoyl chloride group; the structures differ by a 180 degrees rotation of the 2-fluorophenyl ring. Fluorine NMR studies of alpha-chymotrypsin modified with this carbamoyl chloride show that, when bound to the enzyme, one aromatic ring of the diphenylcarbamoyl group likely rotates slowly while the other rotates much more rapidly or else is frozen in one dominant conformation. In the denatured enzyme (8 M urea) at room temperature and above, both aromatic rings of the diphenylcarbamoyl group appear to be rapidly rotating although differential linewidth changes observed at lower sample temperatures suggest that rotation of one ring becomes slow under these conditions. Rotation about the carbamoyl carbon-nitrogen bond is detected in fluorine NMR spectra of both the native and the denatured modified enzymes as the sample temperature is increased. Rates of carbamoyl rotation in the chloride, in the native modified enzyme, and in the denatured enzyme at 25 degrees C are approximately 66, 10, and 200 s-1, respectively.     

Interfacial inactivation of epoxide hydrolase in a two-liquid-phase system

Enantioselective epoxide hydrolases are useful biocatalysts for the preparation of enantiopure epoxides and diols. The kinetic resolution of racemic epoxides can be carried out in an organic/aqueous biphasic system to allow use of high epoxide concentrations. Enzyme inactivation in such a system, however, may occur by contact with the interface. In this study, we investigated the factors which influence the interfacial inactivation of Agrobacterium radiobacter epoxide hydrolase in an octane/water biphasic system. Rates of interfacial inactivation were measured both in a stirred-cell, which has a planar interface, and in an emulsion reactor. Interfacial inactivation rates measured in the stirred-cell at a fixed interfacial area increased with mixing intensity. Interfacial inactivation rates per unit area were lower in the emulsion reactor than in the stirred-cell and increased with bulk aqueous enzyme concentration. Circular dichroism measurements showed that during biphasic incubation all unadsorbed soluble enzyme existed in the native conformation. Activity assays showed that the dissolved enzyme was also fully active, indicating that inactivated enzyme precipitated from solution. Using an inactive epoxide hydrolase mutant structurally similar to the wild-type enzyme in order to avoid the conversion of the epoxide, it was found that high concentrations of epoxide in the organic phase increased the rate of interfacial inactivation.     

Interaction of methylchymotrypsin with Streptomyces subtilisin inhibitor

The effect of methylation of histidine-57 of alpha-chymotrypsin with Streptomyces subtilisin inhibitor was examined. Methylchymotrypsin was isolated by affinity chromatography on inhibitor-Sepharose, and the interaction of this inactive enzyme with inhibitor was quantitatively analyzed by two different methods: the spectrophotometric titration of difference spectrum resulted in the complex formation and the application of competitive enzyme assay by using substrates of large Km values. The former method gave values of 8.6 . 10(-6) M as dissociation constant (Kd) of methylchymotrypsin . inhibitor complex and 0.91 as the number of binding sites (n) per inhibitor monomer, both of which were almost equivalent to those for native enzyme . inhibitor complex. By the latter novel method, values of 7.9 . 10(-6) M and 1.08 were obtained for Kd and n, respectively, for interaction of inhibitor with alpha-chymotrypsin, and 8 . 10(-6) M as Kd for methylchymotrypsin . inhibitor complex. These results indicate that methylation of histidine-57 of active site in alpha-chymotrypsin molecule does not affect essentially the binding ability to inhibitor and the modified enzyme binds stoichiometrically to inhibitor, as the native enzyme does, with a molar ratio of 1:1 per inhibitor monomer.     

1H NMR of compounds with low water solubility in the presence of erythrocytes: effects of emulsion phase separation

When lipophilic compounds like diethyl phthalate (DEP) were added to water, two sets of resonances appeared in the 1H NMR spectrum, whereas when added in concentrations above approximately 3.5 mM to erythrocytes in a high haematocrit suspension, only one set of resonances was observed at the low-frequency position. The appearance of one set of resonances at lower frequency was found to be common to a series of lipophilic compounds in erythrocytes. The appearance of the NMR spectra is ascribed to the existence of an emulsion, meaning two different phases of a compound: a "droplet" (resonances to lower frequency) and aqueous dissolved phase (resonances to higher frequency). The absence of the resonances from the dissolved phase in erythrocyte solution is ascribed to exchange broadening. The absolute chemical shift of the compound in its "droplet" phase was also measured using a cylindrical/spherical microcell. This arrangement mimicked the geometry of the dissolved versus the phase-separated species and thus obviated the effect of a difference in magnetic susceptibility between the "droplet" solute and its aqueous solution. Factors influencing the formation of emulsion phases such as erythrocytes, haemoglobin and smaller proteins were investigated; they are found to be effective in the order given.     

Two- and three-phase mixing in a concentric draft tube gas-lift fermentor

Two- and three-phase mixing studies were carried out in a 44-L concentric draft tube gas-lift fermentor. It was proposed to use the fermentor for the production of solvents using immobilized bacteria. Bubble size, gas holdup, liquid velocities, circulation, and mixing times were determined for various superficial gas velocities in distilled water, starch, carboxymethyl cellulose, and ethanol solutions. The observed trends for two phase mixing were similar to other studies but the results were found to be more sensitive to liquid properties. This was possibly due to the large value of downcomer to riser area used in this study. Mixing in three phases highlighted the difficulty in predicting the effect of adding solids to the gas-liquid system. Results showed that the gas-lift fermentor was ideally suited to dealing with three phases but more work is necessary before accurate models can be developed to account for the effect of solids.     

Bilayer permeability-based substrate selectivity of an enzyme in liposomes

Liposomes were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which contained the water soluble proteinase alpha-chymotrypsin. This liposome entrapped enzyme showed selectivity for externally added substrates in that only small substrates (benzoyl-l-Tyr-p-nitroanilide or acetyl-l-Phe-p-nitro-anilide)-for which the liposome bilayer was permeable-were transformed into products. Large substrates (succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide or casein) could not penetrate from the external aqueous phase into the liposomes, and were not hydrolyzed. This substrate selectivity is entirely based on the compartimentation and permeability properties of the liposome microreactor.     

The hydrodynamics of a Graesser ("raining bucket") contactor with a reverse micellar phase

A variety of contactor types have been assessed for the liquid-liquid extraction of proteins using reversed micelles; however, many of these contactors suffer from drawbacks such as emulsion formation and poor mass transfer performance. In this study, a small (1.25 L) Graesser "raining bucket" contactor was assessed for use with this system since it has the potential to ameliorate many of these problems. The aim of the work was to evaluate the hydrodynamics of the contactor in order to use this information for future work on mass transfer performance. Hydrodynamic characteristics such as the axial mixing coefficient were determined by residence time distribution studies using a tracer injection method. The effect of rotor speed and flow rate of each phase on axial mixing was investigated, and as a result of its unusual structure, i.e., falling/rising sheet, the interfacial mass transfer area in the Graesser was determined by image analysis. It was found that rotor speed had more influence on the axial mixing coefficient in the aqueous phase than in the reverse micellar phase. The axial mixing coefficient in each phase increased by increasing the flow rate of the same phase. The images obtained in a dropping cell showed that under the conditions of this study (3 rpm, 22 degrees C), the bucket pours one phase through the other in the form of a curtain or sheet. A new image technique was developed to determine the interfacial area of both phases, and it was found that the specific area was 8.6 m(2)/m(3), which was higher than in a spray column but considerably lower than in a RDC or a Graesser run at high rotational speed (50 rpm) without the addition of a surfactant.     

Do membrane-bound enzymes access their substrates from the membrane or aqueous phase: interfacial versus non-interfacial enzymes

For membrane-bound enzymes that act on substrates that partition between the membrane and aqueous phases, it is possible to imagine two fundamentally different mechanisms. Interfacial enzymes must access their substrate from the membrane phase, in other words substrate in the membrane binds directly to the active site of the enzyme at the membrane without mixing with substrate molecules in the aqueous phase. On the other hand, non-interfacial enzymes, either bound to membranes or present in the aqueous phase, must access their substrates from the aqueous phase, i.e. substrate in the aqueous phase binds directly to the enzyme without mixing with substrates in the membrane phase. An interfacial mechanism for some enzymes including secreted and cytosolic phospholipase A(2) and phosphoinositide 3'-hydroxykinase was rigorously proven by demonstrating that these enzymes processively hydrolyze many phospholipids without desorbing from the surface of vesicles (scooting mode). The non-interfacial mechanism is more difficult to establish because it cannot be addressed by steady-state kinetics. Using a pre-steady-state method in which the enzymatic velocity is measured during the time it takes for substrate to exchange between vesicles, a non-interfacial mechanism was proven for vesicle-bound plasma platelet activating factor acetylhydrolase. This enzyme prefers more water-soluble phospholipids such as those with sn-2 acetyl or oxidatively truncated fatty acyl chains, and this is readily explained by the mandatory access of substrate from the aqueous phase.     

A new method for determining the absolute molarity of solutions of trypsin and chymotrypsin by using p-nitrophenyl N2-acetyl-N1-benzylcarbazate

1. p-Nitrophenyl N(2)-acetyl-N(1)-benzylcarbazate (NPABC) was synthesized and shown to acylate alpha-chymotrypsin stoicheiometrically; reaction at 25 degrees occurs almost instantaneously at pH7.04 and within 2min. at pH5.04 and there is no observable turnover during 10min. 2. The absolute molarity of solutions of alpha-chymotrypsin can be determined by spectrophotometric measurement of the p-nitrophenol liberated during the acylation step; the results obtained at pH5.04 and pH7.04 agree with one another and with those determined by the method of Erlanger & Edel (1964). 3. Trypsin reacts stoicheiometrically, but more slowly than alpha-chymotrypsin, with NPABC, and it, like chymotrypsin, can be spectrophotometrically titrated at pH7.04. At pH5.04, however, reaction between trypsin and NPABC is sufficiently slow for the reagent to be nearly specific for alpha-chymotrypsin. Specificity for one or other enzyme can be ensured by using soya-bean trypsin inhibitor or the chymotrypsin inhibitor l-1-chloro-3-toluene-p-sulphonamido-4-phenylbutan-2-one. Bovine thrombin does not react with NPABC. 4. Evidence is presented that indicates that acylation of alpha-chymotrypsin and trypsin by NPABC occurs at the active centres of the enzymes. 5. Evidence was obtained that indicates that one or more tryptophan residues move into a more hydrophobic environment when alpha-chymotrypsin and trypsin are acylated by NPABC.