Substrate For Enzymatic Hydrolysis Research Articles

Housefly larvae hydrolysate: orthogonal optimization of hydrolysis, antioxidant activity, amino acid composition and functional properties

BackgroundAntioxidant, one of the most important food additives, is widely used in food industry. At present, antioxidant is mostly produced by chemical synthesis, which would accumulate to be pathogenic. Therefore, a great interest has been developed to identify and use natural antioxidants. It was showed that there are a lot of antioxidative peptides in protein hydrolysates, possessing strong capacity of inhibiting peroxidation of macro-biomolecular and scavenging free redicals in vivo. Enzymatic hydrolysis used for preparation of antioxidative peptides is a new hot-spot in the field of natural antioxidants. It reacts under mild conditions, with accurate site-specific degradation, good repeatability and few damages to biological activity of protein. Substrates for enzymatic hydrolysis are usually plants and aqua-animals. Insects are also gaining attention because of their rich protein and resource. Antioxidative peptides are potential to be exploited as new natural antioxidant and functional food. There is a huge potential market in medical and cosmetic field as well.ResultProtein hydrolysate with antioxidant activity was prepared from housefly larvae, by a two-step hydrolysis. Through orthogonal optimization of the hydrolysis conditions, the degree of hydrolysis was determined to be approximately 60%. Fractionated hydrolysate at 25 mg/mL, 2.5 mg/mL and 1 mg/mL exhibited approximately 50%, 60% and 50% of scavenging capacity on superoxide radicals, 1, 1-Diphenyl-2-picrylhydrazyl radicals and hydroxyl radicals, respectively. Hydrolysate did not exhibit substantial ion chelation. Using a linoneic peroxidation system, the inhibition activity of hydrolysate at 20 mg/mL was close to that of 20 μg/mL tertiary butylhydroquinone, suggesting a potential application of hydrolysate in the oil industry as an efficient antioxidant. The lyophilized hydrolysate presented almost 100% solubility at pH 3-pH 9, and maintained nearly 100% activity at pH 5-pH 8 at 0°C- 4°C and room temperature during the first 6 months of storage. Essential amino acids in the hydrolysate accounted for 43% of the total amino acids.ConclusionsThe results suggesting that hydrolysate could be added to food oils as an efficient antioxidant. It might be useful for food additives, diet nutrients and pharmaceutical agents.

Enzyme Substrates Protective Encapsulation within Polymeric Microspheres

Determination of enzymes activity is an important task of analytical and biomedical oriented fluorimetry. Despite of a long track record of application, there is still some room for improvements in this field. In the case of hydrolases, spontaneous decomposition of substrate leads to substantial errors in determination of enzyme activity. An innovative and effective approach is proposed allowing protection of enzyme substrate within the lipophilic moiety of polyacrylate microspheres. It is shown that the introduction of substrate into microspheres is not only an effective method of prevention of unwanted spontaneous process, but also does not disturb the availability of substrate for enzymatic hydrolysis. The effect of presence of proteins in the sample on fluorimetric responses was studied. In contrary to previous studies related to application of lipophilic polymers in biomedical analysis, it is shown that the presence of bovine serum albumins enhances the sensitivity of fluorimetric determination. It is shown that this surprising effect is related to adsorption of proteins on the microspheres surface and change of surface charge of polymer.

Lupinine and epilupinine derivatives as substrates of Mammalian cholinesterases

Two series of lupinine and epilupinine acetates, the free bases and the N-methyl, N-ethyl, and N-propyl derivatives, were studied. Enzymatic hydrolysis of substrates by human erythrocyte acetylcholinesterase and horse blood serum butyrylcholinesterase was investigated in comparison with the corresponding dimethylalkyl derivatives of acetylcholine. The important role of non-productive substrate binding was taken into account in the data analysis. Additional information for a better understanding of the metabolism of lupinine derivatives in vivo in animals was obtained.

Effect of Formic Acid and Furfural on the Enzymatic Hydrolysis of Cellulose Powder and Dilute Acid-Pretreated Poplar Hydrolysates

Biomass pretreatment often leads to the formation of compounds that are inhibitory to enzymatic hydrolysis. To remove inhibitory compounds prior to enzymatic hydrolysis, pretreated biomass is washed with at least 3 volumes of water. However, this washing step would be difficult to manage in commercial operations because of the unsustainable water consumption. This study reports on the effects of formic acid and furfural on Accellerase 1500 with cellulose powder and dilute acid-pretreated poplar as substrates. Using cellulose powder as the substrate for enzymatic hydrolysis with the addition of 5 or 10 mg/mL formic acid, glucose recovery was reduced by 34% and 81%, respectively, in comparison to the control. The addition of furfural, at 2 or 5 mg/mL, to the enzymatic system reduced glucose recovery by 5% and 9%, respectively. When 5 mg/mL of formic acid was combined with 5 mg/mL of furfural, glucose recovery in the cellulose powder enzymatic system was reduced by 59%. Inhibition of sugar recovery was more pronounced when dilute acid-pretreated poplar was used as a substrate for enzymatic hydrolysis. At 24 h incubation, recovery reductions were 94%, 97%, and 93% in the presence of 5 or 10 mg/mL formic acid or the 5 mg/mL combination.

Efeito do teor de umidade sobre o pré-tratamento a vapor e a hidrólise enzimática do bagaço de cana-de-açúcar

EFFECT OF MOISTURE CONTENT IN THE STEAM TREATMENT AND ENZYMATIC HYDROLYSIS OF SUGARCANE BAGASSE. The effect of moisture content in the steam treatment and enzymatic hydrolysis of sugarcane bagasse was evaluated. Steam treatment was perfomed at 195-210 oC for 4-8 min using cane bagasse with moisture contents in the range 16-100 wt% (dry basis). Increased moisture contents not only had a positive influence in recovery of main cane biomass components but also resulted in better substrates for enzymatic hydrolysis. As a result, drying is not required for optimal pretreatment and enzymatic hydrolysis of sugarcane bagasse, which can be processed into second generation ethanol immediately after crushing and hot water washing.

Valorization of residual woody biomass (Olea europaea trimmings) based on aqueous fractionation

AbstractBACKGROUND: Olive tree trimmings, a widely available agricultural residue lacking added value applications, were subjected to treatments with hot, compressed water under a variety of operational conditions. As a result of treatments, hemicelluloses were solubilized, and the treated solids were enriched in cellulose and lignin. Spent solids from autohydrolysis were assayed as substrates for enzymatic hydrolysis and for bioethanol production by simultaneous saccharification and fermentation.RESULTS: Liquors from the aqueous fractionation stage resulted in the formation of soluble hemicellulose‐derived saccharides (mainly of oligomeric nature) at yields up to 26.2 g per 100 g oven‐dry raw material. Enzymatic hydrolysis of spent solids from the aqueous fractionation step led to solutions containing up to 58.8 g glucose L−1 (corresponding to cellulose to glucose conversions up to 83.2%). Simultaneous saccharification and fermentation assays using spent solids as substrates enabled the production of media containing up to 38.2 g ethanol L−1, corresponding to 72% of the stoichiometric amount.CONCLUSION: Aqueous (or hydrothermal) processing is a technology enabling the recovery of hemicelluloses (as soluble saccharides) and the production of spent solids with high susceptibility to enzymatic hydrolysis (suitable for bioethanol production by simultaneous saccharification and fermentation). Copyright © 2011 Society of Chemical Industry

High pressure: a tool to improve the enzymatic production of glycosides

Naringinase, an enzyme complex that expresses α -l-rhamnosidase and β -d-glucosidase activities in native state, can be used to deglycosylate natural glycosides. The selective inactivation of one of these activities will allow the biosynthesis of different bioactive compounds in a simple, effective and cheap way. In this work, pressure and temperature were the tools used to selectively inactivate the activities expressed by naringinase. The main goal was the identification of pressure–temperature conditions to acquire conditions for the maximization of enzymatic hydrolysis of substrates with different numbers of glycosidic residues. α -l-Rhamnosidase was 32-fold more resistant against inactivation at 250 MPa than at atmospheric pressure. The best pressure condition to reduce β -d-glucosidase inactivation at 75°C was 173 MPa, while in the case of α -l-rhamnosidase inactivation at 85°C, it was above 250 MPa. Moreover, a selective inactivation of β -d-glucosidase activity of naringinase was attained, allowing an easy and cheap method with which to produce prunin and other expensive glycosides. The present work highlights the effect of high pressure on enzyme protection against thermal inactivation, demonstrating its potential as a powerful tool in biosynthesis.

Sulfite Pretreatment (SPORL) for Robust Enzymatic Saccharification of Corn Stalks

This study demonstrates sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) for robust bioconversion of corn stalks. With only about 3% sodium bisulfite charge on corn stalks and 30 min pretreatment at temperature 180°C, SPORL can achieve near complete cellulose conversion to glucose in 48h enzymatic hydrolysis. Sulfuric acid charge has great effect on SPORL pretreatment. Enzymatic cellulose conversion of substrate increased with the increase in acid charge under the studied conditions. Bisulfite with different counter-ions can be used in the SPORL. Magnesium as counter ion was found to be more effective on enzymatic hydrolysis of substrate than sodium or ammonium.

Lipase-catalyzed dynamic kinetic resolution of racemic ibuprofen ester via hollow fiber membrane reactor: Modeling and simulation

The enantio-separation of racemic ibuprofen ester via enzymatic membrane reactor is studied. The lipase-catalyzed resolution technique integrates kinetic resolution (KR) with in situ racemization resulting in 100% optically pure product. A mathematical model of lipase-immobilized hollow fiber membrane reactor incorporating dynamic kinetic resolution (DKR) of racemic ibuprofen ester is proposed. In the process of developing theoretical models for DKR, two zones were considered: (i) enzymatic hydrolysis of substrate in membrane matrix support and (ii) simultaneous racemization of unreacted substrate outside the membrane. The first part of the modeling work emphasized on the derivation of DKR rate equations for both enantiomers, based on the enzymatic resolution mechanism. The second part of the DKR model was derived by considering the mass transfer in the DKR rate equation. The model was solved using two numerical methods by means of MATLAB ® build-in solver. The first numerical technique was based on the explicit Runge–Kutta to solve the system of non-linear first-order ordinary differential equations (ODEs) of DKR reaction rate. The second approach was a collocation technique for solving the non-linear second-order ODEs of the convective hydrolysis–racemization phenomena in the membrane layer. A number of process parameters were studied in order to investigate their effects on the concentration profiles and separation efficiency in terms of enantiomeric excess, i.e. ee s and ee p by simulating the models. The model parameters include Bodenstein number, B 0, Thiele modulus, Φ 2 and dimensionless racemization constant, γ. The simulation results showed that the hollow fiber membrane operates effectively at B 0 = 8.68, γ = 10, Φ 2 = 1 with ee s = 2% and ee p = 98.5%.

Pretreatment and enzymatic hydrolysis of recovered fibre for ethanol production

Recovered fibre from pulp mills represents a potentially significant feedstock for conversion to ethanol. Enzymatic hydrolysis of untreated recovered fibre (86.5 Kappa, 13% lignin) resulted in a hexose yield of approximately 23%, which highlighted the need for an effective pretreatment. Recovered fibre was pretreated as a substrate for enzymatic hydrolysis using oxygen delignification. An experimental design was used to optimize temperature (90–150 °C), caustic loading (2–10%), and reaction time (20–60 min). The post-delignification Kappa values ranged from 76.7 (11.5% lignin) under the mildest pretreatment conditions, to 20 (3% lignin) under the most severe pretreatment conditions. The effect of caustic load appears to have an increased effect at higher temperatures, with the Kappa numbers ranging from 76.7 (90 °C, 2% caustic, 20 min) to 56.0 (150 °C, 2% caustic, 20 min) and from 64.7 (90 °C, 10% caustic, 20 min) to 38.0 (150 °C, 10% caustic, 60 min). These changes in Kappa number reflect changes in the lignin fraction of 3.1% and 4%, respectively. Increasing the caustic load from 2% to 10% decreased the oxygen delignification yield from 93.5% to 87.9% at 90 °C and 20 min reaction time, and 80.3% to 74.7% at 150 °C. The effect of time on oxygen delignification yield was found to be most significant in the first twenty minutes, which correlates with the drop in Kappa number that was observed. The pretreated fibre was subjected to enzymatic hydrolysis using commercial enzymes Celluclast (80 FPU/mL, 20.1 CBU/mL) and Novozym (640.5 CBU/mL). A series of enzyme loadings ranging from 19 to 77 FPU/g were utilized on solids loading ranging from 20 to 100 g (dry fibre)/L. Based on the pretreatment and hydrolysis results an empirical model was developed that can predict hydrolysis sugar concentrations based on the Kappa number, enzyme loading, and initial recovered fibre concentration.

Influence of solid loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated olive tree biomass

Olive tree pruning biomass, pretreated by either liquid hot water or steam explosion under selected conditions, was used as a substrate for enzymatic hydrolysis. The pretreated material was further submitted to alkaline delignification, the objective being to improve hydrolysis yields as well as increasing cellulose content in the pretreated feedstock. The enzymatic hydrolysis of pretreated residues was performed using a commercial cellulase mixture supplemented with β-glucosidase, using a solid loading range from 2 to 30% (w/v). The influence of substrate concentration on the enzymatic hydrolysis yield and on glucose concentration was studied. Comparative results with and without a delignification step are presented. Enzymatic hydrolysis at high substrate concentration (≥20%) is possible, yielding a concentrated glucose solution (>50 g/L). Nevertheless, a cellulose fraction of the pretreated residue remains unaltered.

Immobilization of the recombinant xylanase B (XynB) from the hyperthermophilic Thermotoga maritima on metal-chelate Eupergit C 250L

Immobilization of XynB from the hyperthermophilic Thermotoga maritima on nickel-chelate IDA (iminodiacetic acid disodium salt monohydrate)-Eupergit C 250L (Ni-E, nickel-chelate Eupergit C 250L) resulted in high immobilization yield (76.3%), catalytic efficiency (77.9%) and bond protein (96.6%). Thermostability of Ni-E XynB was significantly improved by 1 mol l −1 phosphate buffer (pH 7.0) treatment. Chemical surface modifications were followed by FT-NIR (Fourier transform near infrared) spectroscopy, strongly indicated that a chemical reaction between the amines or some other groups and epoxy groups took place in Eupergit C 250L during immobilization. Characterization of the immobilized XynB was further evaluated. The optimum pH was not affected by immobilization, but the optimum pH range of the immobilized XynB was pH 4.6–6.6 while that of the free XynB was pH 5.6–7.5. The free XynB had an optimum temperature of 90 °C, whereas that of the immobilized XynB was shifted to 100 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Autohydrolysis liquor of corncob (ALC) was used as the substrate for enzymatic hydrolysis by the immobilized XynB in a packed-bed enzyme reactor. The major hydrolysis product is xylobiose. The immobilization procedure developed provides a promising solution for application of xylanases in continuous hydrolysis of ALC.

Enzymatic saccharification of hydrogen peroxide-treated solids from hydrothermal processing of rice husks

Rice husks were subjected to hydrothermal processing in aqueous medium to cause the degradation of xylan to xylooligosaccharides. The resulting solid phase, with enhanced contents of cellulose and lignin, was assayed as a substrate for enzymatic hydrolysis either directly or after treatments with hydrogen peroxide in alkaline media under a variety of operational conditions. Solid yield, solid composition and susceptibility to enzymatic hydrolysis were assessed by empirical modelling. The susceptibility of the solids to enzymatic hydrolysis was greatly improved by peroxide treatments. Under selected conditions (oxidizing treatments with 50 g hydrogen peroxide/100 g solids at 80 °C for 4 h using a liquor to solid ratio of 15 g/g) lignin was removed in high proportion, and cellulose was almost quantitatively recovered in solid phase. In the subsequent enzymatic hydrolysis (using an enzyme/substrate ratio of 25 FPU/g at liquor to solid ratio of 15 g/g for 48 h), 67.9% of cellulose was converted into glucose, leading to a medium containing 43.5 g glucose/L. Higher cellulose conversions were achieved when the liquor to solid ratio employed in the enzymatic hydrolysis was increased, but then the final glucose concentrations decreased owing to the higher amount of liquid.

Enzymatic production of pentoses from the hemicellulose fraction of corn residues

Three commercial xylanase preparations were evaluated as a sole enzyme source for the enzymatic production of pentoses from the hemicellulose fraction of corn husks and corn cobs. The hemicellulose fraction was obtained by extracting corn husks or corn cobs with 1.25 mol/l NaOH, and the alkaline extract was used as a substrate for enzymatic hydrolysis. Rapidase Pomaliq (Gist-brocades) was found to yield significantly more pentoses from corn husks and corn conb than Clarex ML (Genenor) or Validase × (Valley Research). Rapidase Pomaliq was capable of increasing the concentration of pentoses from an initial value of 106.5 to 210.6 g/kg dry matter of corn husks or 8.6 to 141.6 g/kg dry matter of corn cobs, respectively, under favorable conditions (480 min of reaction at pH 5.0 and 50 °C). Products of the enzymatic reaction were identified as arabinose, xylose, xylobiose, and xylotriose. The results of this study indicate that Rapidase Pomaliq, an enzyme preparation derived from Aspergillus niger and Trichoderma reesei, could serve as a sole enzyme source for the production of pentoses and xylooligosaccharides from corn residues.

Fractionation and utilisation of corn fibre carbohydrates

Corn fibre contains starch, cellulose, hemicellulose, lignin, oil, protein, etc. The separation and the utilisation of its carbohydrate components were studied. A 99.8% of the starch was removed by a two-step enzymtic hydrolysis. The remaining solid fraction was pretreated with alkali (2.5% NaOH) and alkaline peroxide (2.5% NaOH, 0.6% H 2O 2), in order to isolate corn fibre gum (CFG). The highest yield could be obtained under the most severe conditions (alkaline peroxide, 120 °C, 120 min). Following the pretreatment 51.3% of the corn fibre gum could be isolated by alcoholic precipitation from the soluble fraction. The solid fraction was used as carbon source for cellulase enzyme fermentation and as substrate for enzymatic hydrolysis. A 30% higher filter paper activity was obtained using pre-treated corn fibre as carbon source compared to the activities reached on original and destarched corn fibre. However, 15% higher filter paper activities were obtained on Solka Floc than on the pre-treated corn fibre samples. The enzymes produced were used for the hydrolysis of the pre-treated corn fibre substrate. The results of hydrolysis were compared to the performance of industrial enzymes. After 24 h of hydrolysis, the carbohydrate yields showed that the enzyme produced on pre-treated corn fibre could degrade the corn fibre substrate approximately 15% more efficiently.

Synthesis and evaluation of B-, C-, and D-ring-substituted estradiol carboxylic acid esters as locally active estrogens.

We have synthesized derivatives of estradiol that are structurally modified to serve as "soft" estrogens and act within a geographically limited area of the body; estrogens without systemic action. We have previously shown with 16alpha-substituted analogues of estradiol that carboxylates proximal to the steroid ring neither bind to the estrogen receptor nor activate estrogen-responsive genes. However, when the carboxylic acid is masked as an ester, they bind to the receptor and stimulate estrogenic responses. Enzymatic hydrolysis through nonspecific esterases can inactivate these estrogens and thereby limit their area of action. Here, we describe our continued studies to design "soft" estrogens by synthesizing carboxylic acid esters of estradiol at the 7alpha-, 11beta-, and 15alpha-positions in the steroid nucleus at which bulky substituents are accommodated by the estrogen receptor. These compounds were tested for estrogen receptor binding (estrogen receptors alpha and beta), stimulation of an estrogen sensitive gene in Ishikawa cells in culture, and as substrates for enzymatic hydrolysis. Likely candidates were tested in in vivo assays for systemic and local estrogenic action. The biological studies showed that regardless of the point of attachment, all of the short-chain carboxylic acids, C-1 to C-3, were devoid of hormonal action, while many of the esters were estrogenic. The site on the steroid nucleus had great influence on hormonal activity and esterase hydrolysis. Formate esters at 7alpha and 15alpha were good estrogens, but lengthening the chain to acetate dramatically decreased hormonal activity. However, the 7alpha-formate esters were not enzymatically hydrolyzed. At 11beta, the acetate (methyl ester) was an effective estrogen, but increasing the chain length to propionate dramatically reduced hormonal activity. In general, the length of the alcohol from methyl to butyl had only a small effect on receptor binding, and as the size of the alcohol increased, so did esterase hydrolysis. One exception was the 11beta-acetate esters where increasing the alcohol moiety from methyl to ethyl eliminated estrogenic activity (Ishikawa cells) without affecting estrogen receptor binding. Several of the esters were tested in vivo, and two, the methyl and ethyl esters of estradiol-15alpha-formate, appeared to have the requisite properties (high local and low systemic activity) of superior "soft" estrogens.

Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases

Three types of N-acetylated chitosans (NACs) with different degrees of acetylation (DA) were prepared and used as a substrate for enzymatic hydrolysis with a commercially available pectinase and a modified one. Pectinase modification was conducted using polyalkyleneoxide–maleic anhydride copolymer (PEO–MA copolymer). The effects of DA on enzymatic reaction with native and modified pectinases were investigated experimentally. Initial hydrolysis rate and Michaelis–Menten kinetic parameters were measured by analysis of reducing sugars. DA of NAC strongly affected the hydrolytic characteristics of native and modified pectinases. N-acetylation of chitosan increased the initial hydrolysis rate and the enzyme-substrate affinity with respect to both pectinases: NACs with DA over 0.3 showed high initial hydrolysis rate and strong affinity between enzyme and substrate. Especially, when NAC with DA over 0.3 was treated with modified pectinase, the affinity became much stronger than the native pectinase.

Development of a generalized phenomenological model describing the kinetics of the enzymatic hydrolysis of NaOH-treated pine wood

Pinus pinaster wood samples were treated during 3 h with alkaline solutions (containing 1, 5.5, or 10 weight percent NaOH) at 100, 115, or 130 degrees C using liquor/wood ratios of 6, 8, or 10 g/g. The solid residues obtained in treatments were used as substrates for enzymatic hydrolysis. In the hydrolysis assays, the reaction time (in the range 0-48 h) was considered as an operational variable. The cellulose conversion achieved at the end of assays were highest for samples pretreated at high temperature and high alkali concentration using low liquor/wood ratios. The experimental results obtained in each hydrolysis trial were fitted to an empirical model based on the assumption that the cellulose contained in substrates was composed by two fractions having different susceptibility to hydrolysis. The kinetic parameters obtained for the various experiments performed were correlated with the operational variables by means of empirical, statistically significant equations, which provided a generalized interpretation of the process.

Enzymatic hydrolysis of wood: an engineering assessment

Eucalyptus wood samples were delignified with HCl-catalysed acetic acid solutions under selected experimental conditions and treated with NaClO solutions. The solid residues obtained were employed as substrates for enzymatic hydrolysis. The NaClO concentration used in the pretreatment step, the liquor/solid ratio and the enzyme/substrate ratio were considered as operational variables. The experimental data allowed the development of generalized kinetic models which provided the necessary information for design calculations. The operational conditions were compared from an engineering viewpoint on the basis of economic estimates. Optimum conditions were established from these estimates.

Human milk bile-salt stimulated lipase: further investigations on the amino-acids residues involved in the catalytic site

The bile-salt-stimulated lipase purified from human skim milk was modified with diisopropyl phosphofluoridate (DFP), N-ethyl-5-phenylisoxazolium-3′-sulfonate and ethoxyformic anhydride. These chemical modifications lead to the following results: (1) the inhibition of the enzyme by DFP is due to the phosphorylation of a single residue, probably a serine residue, which may represent the acylable group of the enzyme; (2) carbethoxylation of histidine residues leads to inhibition of the enzyme activity. Among the nine modified histidine residues, only one is essential for enzyme activity; (3) a free carboxyl group with a p K a of 5.4 is also involved in catalysis. These three essential results residues are involved in the enzymatic hydrolysis of substrates whatever their physical state (soluble or emulsified). Upon treatment with DFP as well as with ethoxyformic anhydride, the enzyme remains able to bind to the model interface formed by siliconized glass-beads with almost the same efficiency ( K d between 4.1 and 7.4·10 −8 M) than the native bile-salt-stimulated lipase ( K d = 6.3·10 −8 M). Moreover, the modified and native enzymes occupy the same interfacial area (4000–4600 Å 2/molecule). By contrast, the enzyme modified by N-ethyl-5-phenylisoxazolium-3′-sulfonate reagent presents an interfacial area close to that of a denatured protein of size ( ≈ 18 300 A ̊ 2/ molecule ) and a K d more than 20-fold higher than that of the native enzyme. Solvent isotope effects measured on k cat/ K m and K cat indicate that the catalytic mechanism of bile-salt-stimulated lipase involves transitions states that are stabilized by hydrogen bonds as described in the two-step acylation-deacylation mechanism of serine enzymes.