Bienzyme Electrode Compatible Behavior for Water-Soluble and-Insoluble Substrates

Abstract

A bienzymatic sensing layer containing two enzymes able to work sequentially, choline oxidase (ChOD) and phospholipase D (PLaseD), was used to design an electrochemical biosensor for the detection of either a water-soluble (choline) or insoluble (phosphatidylcholine) substrate. A photocrosslinkable polymer, poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), was used as host-matrix for enzyme immobilization. Controlled amounts of PVA-SbQ and of the two enzymes were directly coated on a platinum disk, then photopolymerized. The compatibility of working conditions for choline and phosphatidylcholine detection in the presence of Triton X-100 and CaCl₂ was investigated. The effect of the activity ratio PLaseD / ChOD on the sensor performance was determined. The sensitivities to choline and to phosphatidylcholine were 18 mA.1mol^?1 and 0.66 mA.1.mol^?1 respectively, the detection limit being 1.5.10^?8 M for choline and 1.5.10^?6 M for phosphatidylcholine. The linear range extended up to ca. 10^?4 M for choline and ca. 2.10^?5 M for phosphatidylcholine and the response time was close to 30 seconds for choline and ca. 2 min for phosphatidylcholine.     

Molecular and Kinetic Characterization of Two Extracellular Xylanases Isolated from Leucoagaricus gongylophorus

In this work, the xylanolytic profile of Leucoagaricus gongylophorus was studied, and two extracellular enzymes with xylanolytic activity (XyLg1 and XyLg2) were isolated, purified, and characterized. XyLg1 has a molecular mass of about 38 kDa and pI greater than 4.8. For beechwood xylan substrate, XyLg1 showed an optimum temperature of 40 °C, optimum pH between 8.5 and 10.5, and Km?=?14.7?±?7.6 mg mL^?1. Kinetic studies of the XyLg1 using polygalacturonic acid as substrate were developed, and the enzyme showed optimum pH 5.5, optimum temperature between 50 and 60 °C, and Km?=?2.2?±?0.5 mg mL^?1. XyLg2 has molecular weight of about 24 kDa and pI less than 4.8, and thus is an acid protein. Parameters such as optimum temperature (70 °C) and pH (4.0), as well as the kinetic parameters (Km?=?7.4?±?2.0 mg mL^?1) using beechwood xylan as substrate, were determined for XyLg2. This enzyme has no activity for polygalacturonic acid as substrate. XyLg1 and XyLg2 are the first native xylanases isolated and characterized from L. gongylophorus fungi and, due to their biochemistry and kinetic features, they have potential to be used in biotechnological processes.     

Biotransformation of iminodiacetonitrile to iminodiacetic acid by Alcaligenes faecalis cells immobilized in ACA-membrane liquid-core capsules

Biotransformation of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA) was investigated with a newly isolated Alcaligenes faecalis ZJUTBX11 strain showing nitrilase activity in the immobilized form. To reduce the mass transfer resistance and to increase the toleration ability of the microorganisms to the toxic substrate as well as to enhance their ability to be reused, encapsulation of the whole cells in alginate-chitosan-alginate (ACA) membrane liquid-core capsules was attempted in the present study. The optimal pH and temperature for nitrilase activity of encapsulated A. faecalis ZJUTBX11 cells were 7.5°C and 35°C, respectively, which is consistent with free cells. Based on the Michaelis-Menten model, kinetic parameters of the conversion reaction with IDAN as the substrate were: K _m = (17.6 ± 0.3) mmol L^?1 and V _max = (97.6 ± 1.2) μmol min^?1 g^?1 of dry cell mass for encapsulated cells and (16.8 ± 0.4) mmol L^?1 and (108.0 ± 2.7) μmol min^?1 g^?1 of dry cell mass for free cells, respectively. After being recycled ten times, the whole cells encapsulated in ACA capsules still retained 90 % of the initial nitrilase activity while only 35 % were retained by free cells. Lab scale production of IDA using encapsulated cells in a bubble column reactor and a packed bed reactor were performed respectively.     

Aquaculture by-product: a source of proteolytic enzymes for detergent additives

Intestine proteases of Nile tilapia (Oreochromis niloticus) were partially purified by heat treatment (purification factor of 3.5, enzyme activity remained almost constant) to reach the maximum activity and stability within an alkaline pH range of 7.2–11.0. The optimum temperature and stability over a 120 min period were found to be at 55°C and at 35–45°C, respectively. The proteases’ activity was not affected by a 1 vol. % saponin surfactant, inactivated by 0.01 g mL^?1 sodium dodecylsulphate after 120 min, and it remained stable for 30 min in a 5 vol. % and 10 vol. % hydrogen peroxide solutions. The proteases were slightly activated by Ca²⁺, Mg²⁺, and K⁺ and the substrate most effectively hydrolysed was casein (40.0 U mg^?1). A 2⁴ full factorial design used to evaluated the influence of independent variables showed that the enzyme extract, detergent concentration and the incubation time had a significant influence on the enzymatic activity. The best conditions to be used concerning detergent additive were found with 0.3 mg mL^?1 of protein and 3.0 mg mL^?1 of detergent for 30 min in the presence of Astrus® detergent.     

Study On The Catalytic Reaction Of β-Cd-Hemin Using 2, 3, 4-Trichlorophenol As Substrate And Analytic Application

ABSTRACT|The catalytic activity of various mimetic enzymes instead of the peroxidase have been investigated by 4-aminoantipyrine (4-AAP) and 2, 3, 4-trichlorophenol (TCP) to form a dye utilizing hydrogen peroxide as hydrogen acceptor. The different Chlorophenolic derivatives, which act as a substrate in β-CD-hemin-H₂O₂-4-AAP catalytic reaction, have been systematically studied.|Meanwhile, the relationship of structure-effect for the β-CD-hemin as catalyst, and chlorphenols as substrate has been respectively discussed. The mechanism of catalytic reaction has been investigated. The results showed that β-CD-hemin was the best mimetic enzyme for peroxidase among those tested and TCP was a good substrate for the determination of hydrogen peroxide with β-CD-hemin. The method for the determination of hydrogen peroxide was proposed using 4-AAP-TCP system with β-CD-hemin as catalyst. A linear calibration graph was obtained over the H₂O₂ concentration of 4.8×10-^?8-7.7×10-^?5M, and the relative standard deviation at a H₂O₂ concentration of 2.8×10-^?5M was 2.5%. The apparent molar absorptivity of the chromogenic reaction for H₂O₂ was 1.54× 10⁴ L.mol-^?1.cm^?1. Satisfactory results were obtained in the determination of H₂O₂ in synthetic samples by this method.

Also, the method was coupled with the glucose oxidation reaction to determination glucose in human serum.     

Umbelliferone Phosphate as a Substrate for Acid and Alkaline Phosphatase

Abstract

The results of a complete study of 8 substrates for acid and alkaline phosphatase indicated 7-hydroxycoumarin (umbelliferone) phosphate to be the best substrate for the analysis of these enzymes. Using this ester, from 10^?6 to 2 × 10^?2 units per ml. of alkaline phosphatase and 10^?5 to 0.06 units per ml. of acid phosphatase can be determined with an accuracy and precision of about 1.5%. Samples of serum as small as 1 μ;1. can be assayed. Analysis is performed by a direct initial reaction rate method in 2–3 minutes.     

AP-GOD Biosensor Based on a Modified Poly(PHENOL) Film Electrode and Its Application in the Determination of Low Levels of Phosphate

Abstract

The features of a biosensor based on a platinum electrode modified with a poly(phenol) film coupled with two enzymes, alkaline phosphatase(AP) and glucose oxidase(GOD), were studied. The modification of the surface of the working electrode decreases interference from ascorbic acid, uric acid and some oxidizable organic materials (e.g. glycine), and decreases the noise of the current. The concentration of substrate, the activity ratio of the two enzymes, the applied potential, and the assembling of biosensor are important for the determination of low levels of phosphate.

The two enzymes have been immobilized on an Immobilon^(R) membrane. The linearity range appears in two sections with different sensitivity: one from 8-110 μmol I^?1 and another one from 0.1-1.0 mmol I^?1, with 1.2 μA/mmol/cm² and 0.4 μA/mmol/cm², respectively. Except for some heavy metal ions, ascorbic acid and some oxidizable organic materials, common anions and cations don't interfere with the determination of phosphate.

The biosensor has been used to determine phosphate in some synthetic and real samples with a detection limit of 4 μmol I^?1 of phosphate. The results were compared with a standard spectrophotometric method. The accuracy and recovery of phosphate with the biosensor procedure are ±2% and 96 to 103%, respectively.

     

Litcrosomal Nadph Oxidase and Hybrid Electrodes

Abstract

The NADPH oxidizing activity of rat liver microsomes was investigated and found to be mainly due to the cytochrome P-450 system. The XADPH oxidase was utilized for the development of several organelle electrodes. Gelatin membrane immobilized microsomes were combined with an O₂ membrane sensor for bioelectrochemical measurement of NADPH. The dependence of the current on substrate concentration was linear up to 1 mmol·1^?1 To assemble hybrid electrodes for determination of glucose-6-phoaphate, ATP and isocitrate pure enzymes were coimmobilized with the microsomal fraction.     

Solubility studies in aqueous media: I. Solubility product of silver permanganate and standard electrode potentials of silver—silver permanganate electrode in aqueous media

The solubility and solubility product of silver permanganate in water have been determined at the temperatures ranging from 15 to 35°C over 5°C intervals in the presence of an added electrolyte, sodium perchlorate. The solubility of silver permanganate ranges from 0.966 x 10^?5 mol 1^?1 at 15°C to 1.420x10^?5 moll^?1 at 35°C and the corresponding solubility product 0.933 x 10^?10 mol² 1^?2 at 15°C to 2.017 x 10^?10 mol² 1^?2 at 35°C. The standard potentials of the Ag(s)/AgMnO₄(s)/ MnO^?₄ electrode have been calculated at these temperatures. The mean activity coefficients of silver permanganate at various rounded molarities of sodium perchlorate solutions, and the standard thermodynamic quantities for the process AgMnO₄(s)→Ag⁺ (aq)+MnO^?₄(aq) have been calculated at these temperatures.     

Integrated Process Production and Extraction of the Fibrinolytic Protease from Bacillus sp. UFPEDA 485

Fibrinolytic proteases are enzymes that degrade fibrin; these enzymes are a promising alternative for thrombolytic therapy, and microorganisms produce them. The aim of this study was to evaluate the optimum conditions for the integrated production and purification of fibrinolytic protease from Bacillus sp. UFPEDA 485. Extractive fermentation was carried out in a culture medium containing soybean flour and by adding polyethylene glycol (PEG) and Na₂SO₄ according to a 2³ experimental design. In all assays, the enzyme preferentially partitioned to the bottom phase (K?<?1), with an optimum activity of 835 U ml^?1 in the bottom phase (salt-rich phase). The best conditions for extractive fermentation were obtained with 18 % PEG 8000 and 13 % Na₂SO₄. Characterization showed that it is a metalloprotease, as a strong inhibition—residual activity of 3.13 %—occurred in the presence of ethylenediaminetetraacetic acid. It was also observed that enzymatic activity was stimulated in the presence of ions: CaCl₂ (440 %), MgCl₂ (440 %), FeSO₄ (268 %), and KCl (268 %). The obtained results indicate that the use of a low-cost substrate and the integration of fermentation with an aqueous two-phase system extraction may be an interesting alternative for the production of fibrinolytic protease.     

Gene Cloning,Expression, and Characterization of an Exo-inulinase from Paenibacillus polymyxa ZJ-9

An inulinase-producing strain, Paenibacillus polymyxa ZJ-9, was isolated from natural sources to produce R,R-2,3-butanediol via one-step fermentation of raw inulin extracted from Jerusalem artichoke tubers. The inulinase gene from P. polymyxa ZJ-9 was cloned and overexpressed in Escherichia coli BL21 (DE3), and the purified recombinant inulinase was estimated to be approximately 56 kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and gel filtration chromatography. This result suggests that the active form of the inulinase is probably a monomer. Terminal hydrolysis fructose units from the inulin indicate that enzymes are exo-inulinase. The purified recombinant enzyme showed maximum activity at 25 °C and pH 6.0, which indicate its extreme suitability for industrial applications. Zn²⁺, Fe²⁺, and Mg²⁺ stimulated the activity of the purified enzyme, whereas Co²⁺, Cu²⁺, and Ni²⁺ inhibited enzyme activity. The K _m and V _max values for inulin hydrolysis were 1.72 mM and 21.69 μmol min^?1 mg^?1 protein, respectively. The same parameters toward sucrose were 41.09 mM and 78.7 μmol min^?1 mg^?1 protein, respectively. Considering its substrate specificity and other enzymatic characteristics, we believe that this inulinase gene from P. polymyxa ZJ-9 could be transformed into other special bacterial strains to allow inulin conversion to other biochemicals and bioenergy through one-step fermentation.     

Rapid estimation of activation enthalpies for cytochrome-P450-mediated hydroxylations

Cytochrome P450 (CYP) enzymes play a critical role in detoxication and bioactivation of xenobiotics; thus, the ability to predict the biotransformation rates and regioselectivity of CYP enzymes toward substrates is an important goal in toxicology and pharmacology. Here, we present the use of the semiempirical quantum chemistry method SAM1 to rapidly estimate relative activation enthalpies (ΔH^?) for the hydroxylation of aliphatic carbon centers of various substrates. The ΔH^? were determined via a reaction path calculation, in the reverse direction (RRP), using the iron‐hydroxo‐porphine intermediate and the substrate radical. The SAM1 ΔH^? were calculated via unrestricted Hartree‐Fock (UHF) and configuration interaction (CI) formalisms for both the doublet and quartet spin states. The SAM1 RRP ΔH^?, after subtracting a correction factor, were compared with density functional theory (DFT) B3LYP activation energies for two sets of substrates and showed R² ranging from 0.69 to 0.89, and mean absolute differences ranging from 1.2 ± 1.0 to 1.7 ± 1.5 kcal/mol. SAM1 UHF and CI RRP calculation times were, on average, more than 200 times faster than those for the corresponding forward reaction path DFT calculations. Certain key transition‐state (TS) geometry measurements, such as the forming O···H bond length, showed good correlation with the DFT values. These results suggest that the SAM1 RRP approach can be used to rapidly estimate the DFT activation energy and some key TS geometry measurements and can potentially be applied to estimate substrate hydroxylation rates and regioselectivity by CYP enzymes. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Organotellurium-bridged cyclodextrin dimers as artificial glutathione peroxidase models

To elucidate the importance of the goodness of fit in complexes between substrates and glutathione peroxidise (GPX) mimics, we examined the decomposition of a variety of structurally distinct hydroperoxides at the expense of glutathione (GSH) catalyzed by 2,2′-ditellurobis(2-deoxy-γ-cyclodextrin) (2-Te-γ-CD), and by the corresponding derivatives of β-cyclodextrin (β-CD) and α-cyclodextrin. The good fit of the cumene group into the γ-CD binding cavity reflected the result of well-defined reaction geometry, leading to the most excellent peroxidase activity with high substrate specificity. Furthermore, the catalytic constant and the combination with the best binding also exhibited the highest regioselectivity in the substrate decomposition. Saturation kinetics were observed and the catalytic reaction agreed with a ping-pong mechanism, in analogy with natural GPX, and might exert its thiol peroxidase activity via tellurol, tellurenic acid, and tellurosulfide. The stoichiometry of the inclusion complex was determined to be of 2:1 host-to-guest. The value of stability constant K _c for (2-Te-γ-CD)₂/GSH at room temperature was calculated to be 3.815?×?10⁴?M^?2, which suggested that 2-Te-γ-CD had a moderate ability to bind GSH. Importantly, the proposed mode of the (2-TeCD)₂/GSH complex was the possible important noncovalent interactions between enzymes and substrates in influencing catalysis and binding.     

Purification, Characterization, and Specificity Determination of a New Serine Protease Secreted by Penicillium waksmanii

The purpose of this work was to purify a protease from Penicillium waksmanii and to determine its biochemical characteristics and specificity. The extracellular protease isolated that was produced by P. waksmanii is a serine protease that is essential for the reproduction and growth of the fungus. The protease isolated showed 32 kDa, and has optimal activity at pH 8.0 and 35 °C towards the substrate Abz-KLRSSKQ-EDDnp. The protease is active in the presence of CaCl₂, KCl, and BaCl, and partially inhibited by CuCl₂, CoCl₂ and totally inhibited by AlCl₃ and LiCl. In the presence of 1 M urea, the protease remains 50 % active. The activity of the protease increases 60 % when it is exposed to 0.4 % nonionic surfactant-Triton X-100 and loses 10 % activity in the presence of 0.4 % Tween-80. Using fluorescence resonance energy transfer analysis, the protease showed the most specificity for the peptide Abz-KIRSSKQ-EDDnp with k _cat/K _m of 10,666 mM^?1?s^?1, followed by the peptide Abz-GLRSSKQ-EDDnp with a k _cat/K _m of 7,500 mM^?1?s^?1. Basic and acidic side chain-containing amino acids performed best at subsite S₁. Subsites S₂, S₃, S^′ ₂, and S^′ ₁, S^′ ₃ showed a preference for binding for amino acids with hydrophobic and basic amino acid side chain, respectively. High values of k _cat/K _m were observed for the subsites S₂, S₃, and S^′ _2. The sequence of the N-terminus (ANVVQSNVPSWGLARLSSKKTGTTDYTYD) showed high similarity to the fungi Penicillium citrinum and Penicillium chrysogenum, with 89 % of identity at the amino acid level.     

Purification of chloroperoxidase from Musa paradisiaca stem juice

Chloroperoxidase from Musa paradisiaca stem juice has been purified to homogeneity using a concentration obtained by ultrafiltration and anion exchange chromatography on diethylaminoethyl (DEAE) cellulose. The purified enzyme gave a single protein band in SDS‐PAGE analysis corresponding to molecular mass of 43 kDa. The native PAGE analysis result has also given a single protein band, confirming the purity of the enzyme. The purified enzyme was chlorinated and brominated with monochlorodimedone, the substrate used for measuring the halogenating activity of chloroperoxidases. The K_m and k_cat values using monochlorodimedone as the substrate were 20 μM and 1.64 s^?1, respectively, giving a k_cat/K_m value of 8.2 × 10⁴ M^?1 s^?1. The pH and temperature optima of the chlorinating activity were 3.0 and 25°C, respectively. The K_m values for the peroxidase activity using pyragallol and H₂O₂ as the variable substrates were 89 and 120 μM, respectively. The pH and temperature optima of the peroxidase activity using pyrogalllol as the substrate were the same as the pH and temperature optima of the halogenating activity. The peroxidase activity of the enzyme is competitively inhibited by sodium azide, indicating that it is a hemeperoxidase different from nonheme peroxidases. © 2012 Wiley Periodicals, Inc. Int J Chem Kinet 45: 92–100, 2013     

Solid-supported single- and multi-enzyme amplified assays for clostridiopeptidase A

A rapid and sensitive spectrophotometric assay for Clostridium histolyticum clostridiopeptidase A (collagenase) was accomplished by measuring the activity of an alkaline phosphatase indicator enzyme released into solution from insoluble, covalently linked alkaline phosphatase indicator enzyme released into solution from activity of the alkaline phosphatase was monitored spectrophotometrically using either p-nitrophenyl phosphate as substrate or more sensitively by a signal amplification system consisting of NAD⁺, alcohol dehydrogenase, diaphorase and INT-Violet. Under the reaction conditions the amount of indicator enzyme produced is directly proportional to the concentration of collagenase. With p-nitrophenyl phosphate as substrate the magnitude of the signal was 0.003 abs. min^?1 per 100 ng ml^?1 collagenase whereas with the multienzyme amplification system it was 0.035 abs. min^?1, i.e. approximately as 12-fold increase. The method consists in first incubating the substrate with the bacterial collagenase for 20 min, then up to 96 samples of the released alkaline phosphatase can be analysed in 2 min using a microtitre plate reader run in the kinetic mode.     

Studies on Schiff Base Complexes with Aza-Crown Ether Pendants as Synthetic Hydrolases for p-Nitrophenyl Picolinate in Brij35 Surfactant Micellar Solution

Schiff base complexes with aza-crown ether pendants have been synthesized and employed as models for hydrolase enzymes by studying the kinetics of their hydrolysis reactions with p-nitrophenyl picolinate (PNPP) in Brij35 surfactant micellar solution. A kinetic model of PNPP cleavage catalyzed by these complexes is proposed. The effects of complex structures and reaction temperature on the rate of catalytic PNPP hydrolysis have also been examined. The rate increases with pH of the buffered Brij35 micellar solution under 25°C; all four complexes exhibited high activity in the catalytic PNPP hydrolysis. The catalytic activity of the phenyl-bridged Schiff base complex is larger than that of ethyl-bridged Schiff base complex for the same substituent and metal. The catalytic activity of manganese(III) complex is superior over cobalt(II) complex in catalyzing hydrolysis of PNPP under the same ligand. The pseudo-first-order rate for PNPP hydrolysis catalyzed by CoL¹ containing aza-crown ether is 2.96 × 10⁴ times that of spontaneous hydrolysis of PNPP in Brij35 surfactant micellar solution at pH = 7.60, [S] = 2.0 × 10^?4 mol dm^?3.     

Determination of enzyme kinetic parameters of cyclic CMP-specific phosphodiesterase by quantitative fast atom bombardment tandem mass spectrometry

The determination of cytidine 3′,5′-cyclic monophosphate-specific phosphodiesterase activity by means of fast-atom bombardment (FAB) mass Spectrometry with mass-analysed ion kinetic energy (MIKE) spectrum scanning is described. Initial efforts to determine the activity of the enzyme by this method were unsuccessful owing to the obfuscation of sample-related peaks by peaks emanating from the incubation buffer and cation adducts; dilution of buffer and a desalting procedure overcame these difficulties. In the resulting positive-ion FAB mass spectra, characteristic peaks of the enzyme substrate and product could be readily identified and the protonated molecular ions selected for MIKE scanning. By spiking enzyme incubates with known amounts of substrate and product, and measuring peak heights in the MIKE spectra of both spiked and unspiked samples, the substrate/product ratio at the end of a series of phosphodiesterase incubations was determined. From the data obtained, the K_m and V_max of the phosphodiesterase were calculated as 6.08 mM and 11 μmol min^?1 mg^?1, respectively, showing good agreement with the analogous values of 8.06 mM and 5.8 μmol^?1 min^?1 mg^?1 obtained by radioactive assay.     

Extract from Eugenia punicifolia is an Antioxidant and Inhibits Enzymes Related to Metabolic Syndrome

The present study aimed to investigate in vitro biological activities of extract of Eugenia punicifolia leaves (EEP), emphasizing the inhibitory activity of enzymes related to metabolic syndrome and its antioxidant effects. The antioxidant activity was analyzed by free radicals scavengers in vitro assays: DPPH·, ABTS^·+, O₂ ^·?, and NO· and a cell-based assay. EEP were tested in inhibitory colorimetric assays using α-amylase, α-glucosidase, xanthine oxidase, and pancreatic lipase enzymes. The EEP exhibited activity in ABTS^·+, DPPH·, and O₂ ^·? scavenger (IC₅₀?=?10.5?±?1.2, 28.84?±?0.54, and 38.12?±?2.6 μg/mL), respectively. EEP did not show cytotoxic effects, and it showed antioxidant activity in cells in a concentration-dependent manner. EEP exhibited inhibition of α-amylase, α-glucosidase, and xanthine oxidase activities in vitro assays (IC₅₀?=?122.8?±?6.3; 2.9?±?0.1; 23.5?±?2.6), respectively; however, EEP did not inhibit the lipase activity. The findings supported that extract of E. punicifolia leaves is a natural antioxidant and inhibitor of enzymes, such as α-amylase, α-glucosidase, and xanthine oxidase, which can result in a reduction in the carbohydrate absorption rate and decrease of risks factors of cardiovascular disease, thereby providing a novel dietary opportunity for the prevention of metabolic syndrome.     

L-leucyl-sarcosyl-L-phenylalanine, synthesis and effect on aminopeptidases I and II from Bacillus stearothermophilus

The synthesis of L-leucyl-sarcosyl-L-phenylalanine is described. This tripeptide resists hydrolysis by the aminopeptidases AP I and AP II from Bacillus stearothermophilus. The peptide is a reversible inhibitor of both enzymes. The inhibition constant for AP I is 5 × 10^?3 mol/l and 1.5 × 10^?3 mol/l for AP II. In the hydrolysis of leucine p-nitroanilide we observed noncompetitive inhibition by leucylsarcosyl-phenylalanine with both enzymes. With dipeptide substrates however, the inhibitory effect of leucyl-sarcosyl-phenylalanine was drastically reduced by saturating substrate concentrations.