Properties of β-Galactosidase from Verticillium albo-atrum

An intracellular, inducible β-galactosidase [EC 3.2.1.23] was partially purified from Verticillium albo-atrum. The activity was associated with a particle of about one million molecular weight and required polyhydroxyl compounds for stabilization and activation. It was inhibited by various sulfhydryl inhibitors and EDTA. The latter inhibition could be overcome by adding Mn²⁺ to reaction mixtures. The β- galactoside (ONPG) activity toward lactose (apparent K_m= 0.08 M) and o-nitrophenyl-β-D-galactoside (ONPG) (apparent K_m= 2×10^-23M) purified in parallel. Lactose competitively inhibited the degradation of ONPG with a K_i of 0.1 M. When activated by glycerol, the enzyme produced not only glucose and galactose from lactose, but also other unidentified products, perhaps by transglycosylation.     

Proteomics of the milk fat globule membrane from Camelus dromedarius

Camel milk has been widely characterized with regards to casein and whey proteins. However, in camelids, almost nothing is known about the milk fat globule membrane (MFGM), the membrane surrounding fat globules in milk. The purpose of this study was thus to identify MFGM proteins from Camelus dromedarius milk. Major MFGM proteins (namely, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already evidenced in cow milk were identified in camel milk using MS. In addition, a 1D‐LC‐MS/MS approach led us to identify 322 functional groups of proteins associated with the camel MFGM. Dromedary MFGM proteins were then classified into functional categories using DAVID (the Database for Annotation, Visualization, and Integrated Discovery) bioinformatics resources. More than 50% of MFGM proteins from camel milk were found to be integral membrane proteins (mostly belonging to the plasma membrane), or proteins associated to the membrane. Enriched GO terms associated with MFGM proteins from camel milk were protein transport (p‐value = 1.73 × 10^?14), translation (p‐value = 1.08 × 10^?11), lipid biosynthetic process (p‐value = 6.72 × 10^?10), hexose metabolic process (p‐value = 1.89 × 10^?04), and actin cytoskeleton organization (p‐value = 2.72 × 10^?04). These findings will help to contribute to a better characterization of camel milk. Identified MFGM proteins from camel milk may also provide new insight into lipid droplet formation in the mammary epithelial cell.     

Response Surface Optimization of Lyoprotectant for Lactobacillus bulgaricus During Vacuum Freeze-Drying

The individual and interactive effects of skimmed milk powder, lactose, and sodium ascorbate on the number of viable cells and freeze-drying survival for vacuum freeze-dried powder formulation of Lactobacillus bulgaricus were studied by response surface methodology, and the optimal compound lyoprotectant formulations were gained. It is shown that skim milk powder, lactose, and sodium ascorbate had a significant impact on variables and survival of cultures after freeze-drying. Also, their protective abilities could be enhanced significantly when using them as a mixture of 28% w/v skim milk, 24% w/v lactose, and 4.8% w/v sodium ascorbate. The optimal freeze-drying survival rate and the number of viable cells of Lactobacillus bulgaricus were observed to be (64.41 ± 0.02)% and (3.22 ± 0.02) × 10¹¹ colony-forming units (CFU)/g using the optimal compound protectants, which were very close to the expected values 64.47% and 3.28 × 10¹¹ CFU/g.     

β-Galactosidase from Lactobacillus pentosus: Purification,characterization and formation of galacto-oligosaccharides

A novel heterodimeric β-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U_oNPG/mg protein. The K_m, k_cat and k_cat/K_m values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) were 38 mM, 20 s^-1, 530 M^-1·s^-1 and 1.67 mM, 540 s^-1, 325 000 M^-1·s^-1, respectively. The temperature optimum of β-galactosidase activity was 60–65°C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal β-galactosidases. Mg²⁺ ions enhanced both activity and stability significantly. L. pentosus β-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of β-(1→3) and β-(1→6) linkages, and the main transgalactosylation products identified were the disaccharides β-D-Galp-(1→6)-D -Glc, β-D-Galp-(1→3)-D -Glc, β-D -Galp-(1→6)-D -Gal, β-D -Galp-(1→3)-D -Gal, and the trisaccharides β-D -Galp-(1→3)-D -Lac, β-D -Galp-(1→6)-D -Lac.     

Flow Injection Analysis of Lactose in Milk Using a Chemically Modified Lactose Electrode

β-Galactosidase and glucose oxidase were immobilized with bovine serum albumin using glutaraldehyde on to a glassy carbon electrode silanized with 3-aminopropyltriethoxysilane. The laboratory-constructed lactose electrode was used for flow injection analysis to determine the lactose content in milk. Electrochemical interference could be detected by a non-enzymatic electrode (W₂) and the current was subtracted from that of the enzymatic electrode (W₁), providing an accurate measurement of the hydrogen peroxide that was enzymatically generated. The peak current was linearly related to the lactose concentration in the range 10^?4~ 1.5 × 10^?3 M (original concentration) and 40 samples/hr could be analyzed. The relative standard deviation for 10 assays was less than 2%. The proposed method was compared with the chloramine T method and the values determined by both methods were in good agreement.     

Optimization of galacto-oligosaccharide production by Bifidobacterium infantis RW-8120 using response surface methodology

Oligosaccharide (OS) production, cell concentration (2×10⁹ colony-forming unit/ml), lactose concentration (25% wt/vol), reaction time (6 h), and temperature (50°C) were chosen as the central condition of the central composite design (CCD) for optimizing the production process using Bifidobacterium infantis RW-8120 in skim milk. Statistical analysis (P<0.01) revealed that the most relevant variable concerning OS production and yield was the lactose concentration. The coefficient of determination (R ²) is good for the second-order OS production model (0.92) and fairly good for the second-order nonlinear OS yield model (0.816). An increase of lactose concentration and temperature resulted in a higher OS production. The optimal values for OS production appear to be near the area associated with the central points of the modeling design except for the lactose concentration, which was 40% (wt/vol) of the final volume. Received 29 March 2002/ Accepted in revised form 09 August 2002     

Production of Metarhizium anisopliae spores using nutrient‐impregnated membranes and its economic analysis

Metarhizium anisopliae spores were produced on nutrient‐impregnated membranes (NIMs). The NIM system involved wetting the membrane with a spore and nutrient suspension, followed by harvesting the spores produced after incubation. The cost efficiency of spore production was assessed for a range of nutrient sources and membrane types. Skim milk powder (20 g l^‐1) was found to be the most cost‐effective nutrient source of the nine nutrients examined. Yield was 5.7 × 10⁶ spores/cm² after 28 days incubation on a paper membrane. Supplementation of the skim milk with either sucrose (2 g l^‐1) or dextrose + KNO₃ maximized yield. Superwipe, an absorbent fibrous material, was the most efficient of 16 membranes tested which ranged from fibreglass mesh to paper and cloth. A series of small pilot plants were built, but the cost efficiency of spore production decreased as the size of the membrane increased from 24 × 24 cm to 270 × 15 cm and up to 100 × 80 cm. Yield on the two smaller pilot plants was over 10⁷spores/cm², but the cost (nutrient and membrane only) of producing 10¹³ spores (standard dose required per hectare) was around $A37 and was found not to be competitive with spore production on grain.     

Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos ofPicea abies (Norway spruce)

Summary Embryogenic callus was initiated from radicles of mature embryos removed from imbibed seeds (24 h). Embryogenic and other nonembryogenic types of callus proliferated on a modified half-strength Murashige-Skoog medium (MS) basal medium (BM) supplemented withmyo-inositol, casein hydrolysate (CH), L-glutamine (gln) and growth regulators kinetin (KN), N⁶-benzyladenine (BAP) each (20×10⁻⁶ M), 2,4-dichlorophenoxyacetic acid (2,4-D) (50×10⁻⁶ M) Embryogenic callus bearing suspensor-like cells in a mucilaginous gel matrix was isolated and maintained by subculture every 10 to 12 days on BM with KN, BAP each (2×10⁻⁶ M) and 2,4-D (5×10⁻⁶ M). Somatic embryos developed spontaneously from the callus on this medium at 23±1° C. Closer examination revealed that numerous polyembryonic clusters, comprised of elongated cells (suspensors) and small dense cells with large nuclei (somatic embryos), occurred in the viscous gel. When this enriched embryonal-suspensor mass was subcultured to low 2,4-D (1×10⁻⁶ M), globular embryos developed by 40 to 60 days. Upon transfer to a liquid medium without growth regulators, the embryos elongated and developed cotyledons and shoots with needles. Plantlet development was completed by 30 days in a basal medium without CH, gln and growth regulators. The total culture time was 150 days. Approximately 40±10 embryos were formed from 500 mg of initial callus. Somatic embryogenesis became aberrant if embryos remained attached to the callus mass and were not subcultured within 10 to 12 days according to the described protocol. Somatic embryos were encapsulated in an alginate gel and stored at 4° C for nearly two months without visible adverse effects on viability. Editor's Statement This paper presents advances in the in vitro regeneration of a commercially useful plant species from stored seeds. In addition, data is presented on short-term storage of the plantlets, and long-term proliferation of the embryonal mass in vitro.     

A new spectrofluorimetric method for the determination of some tetracyclines based on their interfering effect on resonance fluorescence energy transfer

A rapid, simple, inexpensive and highly sensitive spectrofluorimetric method was developed for the determination of trace amounts of some tetracyclines (TCs), namely tetracycline hydrochloride (TCH), oxytetracycline hydrochloride (OTCH) and minocycline hydrochloride (MCH). Binding rhodamine B (RhB) to gold nanoparticles (Au NPs) resulted in quenching of the fluorescence of RhB by a resonance energy transfer (FRET) mechanism, with Au NPs as the energy acceptors. The presence of TCs caused the release of RhB molecules and recovered their fluorescence, and this was used as a basis for the quantitative determination of TCs. The reaction was monitored spectrofluorimetrically by measuring the increase in fluorescence of RhB at 572 nm starting 5 min after mixing the reagents in Tris buffer solution (pH 6.5). The effect of various experimental factors such as buffer type, pH, concentrations of the involved reagents and reaction time were studied to optimize the reaction conditions. Under optimum conditions, the calibration graphs were linear within the ranges 2.08 × 10^?9–1.04 × 10^?6 mol/L, 2.01 × 10^?9–1.00 × 10^?6 mol/L and 2.02 × 10^?9–1.01 × 10^?6 mol/L and detection limits (LODs) of 0.61 × 10^?9, 0.32 × 10^?9 and 0.66 × 10^?9 mol/L were calculated for TCH, OTCH and MCH, respectively, with corresponding percent relative standard deviations (%RSDs) of 1.18, 1.21 and 1.54 (n = 5). The method was successfully applied to the determination of TCs in drinking water, human urine, bovine milk and breast milk samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Biochemical Studies on Cycasin

Purification of β-glucosidase from the seeds of Japanese cycad, and properties of the purified preparation are described. The enzyme activity was determined by colorimetry using ONPG as substrate. Crude preparation was obtained easily by adsorption on fibrous CMC pulp. It was further purified by chromatography on CMC powder, and a preparation which showed an activity of 135-folds of the original extract was obtained. Influences of pH, temperature, and substrate concentration upon the enzyme activity were examined. Michaelis constant of the enzyme for ONPG was 3.3×10^–3M.     

Influence of pH and impeller tip speed on the cultivation of<Emphasis Type="Italic">Bifidobacterium pseudocatenulatum</Emphasis> G4 in a milk-based medium

Biomass production ofBifidobacterium pseudocatenulatum G4 in a milk-based medium was carried out in a 2- and 10-L stirred tank fermenters. The effects of impeller tip speed (0.28, 0.56, and 0.83 m/s) and pH control (6.0, 6.5, and 7.0) on the biomass production were investigated. The growth performance in the 2-L fermenter was significantly improved when the impeller tip speed was held constant at 0.56 m/s and the pH was controlled at 6.5. These conditions yielded a maximum biomass of 1.687×10⁹ cfu/mL, a maximum specific growth rate of 0.504 h⁻¹, a biomass productivity of 9.240×10⁷ cfu/mL·h, and a biomass yield of 9.791×10¹⁰ cfu/g lactose. The consumption of milk lactose resulted in the accumulation of 7.353 g/L acetic acid and 6.515 g/L lactic acid, with an acetic:lactic ratio of 1.129. Scale-up of the fermentation process to a 10-L fermenter based on a constant impeller tip speed of 0.56 m/s yielded reproducible results with respect to biomass production and cell viability.     

Effects of various inhibitors on β-galactosidase purified from the thermoacidophilic <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis> subsp. <Emphasis Type="Italic">Rittmannii</Emphasis> isolated from Antarctica

β-Galactosidase purified from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica is a member of the GH42 family. The enzyme was not effected by various concentrations of its reaction product glucose, but was greatly inhibited by the other reaction product galactose using both substrates, ONPG and lactose. Linewever-Burk plot analysis derived from both ONPG and lactose hydrolysis results showed that galactose is a mixed-type inhibitor of the purified β-galactosidase. The enzyme was slightly activated by Mg²⁺ (13% at 20 mM), while inhibited at higher concentrations of Ca⁺² (33% at 10 mM), Zn⁺² (86% at 8 mM) and Cu⁺² (87% at 4 mM). The enzyme activity was not significantly altered by the metal ion chelators EDTA and 1,10-phenanthroline up to 20 mM, indicating that this enzyme is not a metalloenzyme. 2-Mercaptoethanol and DTT were found to enhance β-galactosidase activity, while p-chloromercuribenzoic acid (PCMB) completely inhibited enzymatic activity (97% at 1 mM; 99.7% at 2 mM), indicating at least one essential Cys residue modified by the reagents in the active site of β-galactosidase. Iodoacetamide and Nethylmaleimide had little effect on the β-galactosidase. Phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme strongly (19.8% at 1 mM; 71.9% at 10 mM), also showing the participation of serine for enzyme activity.     

Continuous preparation of (S)-3-hydroxy-3-phenylpropionate by asymmetric reduction of 3-oxo-3-phenylpropionic acid ethyl ester with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> CGMCC No.2266 in a membrane reactor

In this study, (S)-3-hydroxy-3-phenylpropionate was prepared continuously by coupling microbial transformation and membrane separation. The effect of several factors on membrane flux, reactor capacity, and reaction conversion were investigated. A kinetic model of the continuous reduction process was also developed. The appropriate molecular weight cut-off of the ultrafiltration membrane was 30 kDa. The reactor capacity reached a maximum of 0.136/h at a biomass concentration and membrane flux of 86 g/L (dry weight/reaction volume) and 20 mL/h, respectively. The (S)-3-hydroxy-3-phenylpropionate yield was 3.68 mmol/L/day after continuous reduction over seven days. The enantiometric excess of (S)-3-hydroxy-3-phenylpropionate reached above 99.5%. The kinetic constants of continuous reduction were as follows: r _m = 3.00 × 10⁻³ mol/L/h, k _cat = 3.49 × 10⁻⁴ mol/L/h, k ₁ = 3.09 × 10⁻² mol/L, and k ₂ = 5.00 × 10⁻⁷ mol/L. The kinetic model was in good agreement with the experimental data obtained during continuous reduction. Compared with batch reduction, continuous reduction can significantly improve the catalytic efficiency of microbial cells and increase the reactor capacity.     

Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis

β-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of β-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8–7.0 with more than 97% activity. Purified β-galactosidase was optimally active at 50 °C. Kinetic parameters K_m and V_max for purified enzyme were 400 µM and 1.22 × 10⁻¹ U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca²⁺, Mg²⁺ and Mn²⁺ slightly activated the enzyme whereas NH₄⁺, Co²⁺ and Fe³⁺ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that β-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min⁻¹ and t_1/2 of 14.74 min. This is better than other studied β-galactosidases. Both sonicated Pediococcus acidilactici cells and purified β-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of β-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca²⁺ shows that it is suitable for milk processing.     

Comprehensive optimization of composite cryoprotectant for Saccharomyces boulardii during freeze-drying and evaluation of its storage stability

Abstract

Saccharomyces boulardii (S. boulardii) is widely adopted in the diarrhea treatment for humans or livestock, so guaranteeing the survival rate of S. boulardii is the critical issue during freeze-drying process. In this study, the survival rate of S. boulardii with composite cryoprotectants during freeze-drying procedure and the subsequent storage were investigated. With the aid of response surface method, the composite cryoprotectants were comprehensively optimized to be lactose of 21.24%, trehalose of 22.00%, and sodium glutamate of 4.00%, contributing to the supreme survival rate of S. boulardii of 64.22?±?1.35% with the viable cell number of 9.5?±?0.07?×?10⁹ CFU/g, which was very close to the expected rate of 65.55% with a number of 9.6?×?10⁹ CFU/g. The accelerated storage test demonstrated that the inactivation rate constant of the freeze-dried S. boulardii powder was k_?18?=?8.04?×?10^?6. In addition, the freeze-dried goat milk powder results exhibited that the inactivation rate constants were k₄?=?4.48?×?10^?4 and k₂₅?=?9.72?×?10^?3 under 4 and 25?°C, respectively. This work provides a composite cryoprotectant formulation that has a good protective effect for the probiotic S. boulardii during freeze-drying process, possessing the potential application prospect in food, medicine, and even feed industry.     

Sorbitol and Gluconate Production in a Hollow Fibre Membrane Reactor by Immobilized Zymomonas Mobilis

This study describes the results of a hollow fibre membrane reactor with immobilized treated cells of Zymomonas mobilis which produced sorbitol and gluconic acid continuously from fructose and glucose respectively. A productivity of 10–20 g sorbitol · L^-1 · h^-1 and 10–20 gluconate · L^-1 · h^-1 (based on total bioreactor volume) from a feed of 100 g · L^-1 each of glucose and fructose was possible at high dilution rates. Kinetic parameters describing the reaction rate of treated cells in batch reactors were used to analyse the performance of the hollow fibre membrane reactor employing significant convective mass transfer. No significant mass transfer limitation was apparent.     

Characterization of exopolysaccharide (EPS) produced by Weissella hellenica SKkimchi3 isolated from kimchi

Weissella hellenica SKkimchi3 produces the higher exopolysaccharide (EPS) on sucrose than lactose, glucose, and fructose at pH 5 and 20°C. Sucrose was exclusively used to cultivate SKkimchi3 in all experiments base on the EPS production tests. The molecular mass of EPS, as determined by gel permeation chroma-tography, was 203,000. ¹H and ¹³C NMR analysis indicated that the identity of EPS may be a glucan. When EPS, starch, and cellulose was treated with a-amylase, glucoamylase, glucosidase, and cellulase, glucose was produced from starch and cellulose but was not produced from EPS. Based on HPLC analysis, elemental analysis, ¹H and ¹³C NMR analysis, and enzymatic hydrolysis tests, EPS was estimated to be a glucan. EPS suspension was not precipitated even by centrifugation at 10,000×g for 60 min, and EPS made the fermented milk and bacterial culture viscous.     

Lipase-catalyzed kinetic resolution of (R,S)-1-phenylethyl propionate in an enzyme membrane reactor

Enzymatic stereoselective hydrolysis of (R,S)-1-phenylethyl propionate was performed in a stirred tank and in a biphasic enzyme membrane reactor. Lipase from Pseudomonas sp. was proved to be a good enantioselective catalyst for this reaction. The enzyme was covalently immobilized in a porous polyamide membrane (flat sheet as well as hollow-fibres) via glutaraldehyde. An influence of membrane hydrophobicity on reactor performance was observed. Initial lipase activity and productivity in the processes were equal to 1.05 × 10^?4, 1.3 × 10^?5 and 1.0 × 10^?5 mole/(h × mg of enzyme) in the case of native lipase, in the aromatic polyamide hydrophobic membrane reactor and in the hydrophilic polyamide-6 membrane reactor, respectively. The influence of some factors such as temperature, pH, buffer concentration, initial substrate concentration and addition of β-cyclodextrin derivatives on reaction rate and enantioselectivity was investigated and discussed. In the enzyme membrane reactor both organic and aqueous phases circulated countercurrently on both sides of the membrane. At a conversion degree of under 55–60%, pure enantiomer of the remaining ester (i.e. > 98%) was obtained.     

E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro

Microencapsulation enhances the oral delivery of probiotic bacteria. In this study, the probiotic Escherichia coli Nissle 1917 (EcN) was microencapsulated using alginate and chitosan nanoparticles. The result showed 90% encapsulation yield of EcN, and the encapsulated EcN displayed significantly (P < 0.05) increased survival in low pH (1.5), high bile salt concentration (4%), and high temperature (70 °C). The most effective cryopreservatives of EcN during freezing and thawing was skim milk and sucrose. Exposure to microencapsulated EcN significantly (P < 0.05) reduced the Campylobacter jejuni growth by 2 log CFU. The rate of EcN release from microcapsule was 9.2 × 10⁵ cell min⁻¹, and the appropriate model to describe its release kinetics was zero order. Importantly, the entrapment of EcN inside the microcapsule did not eliminate the exterior diffusion of EcN produced antioxidant compounds. In addition, the EcN microcapsule efficiently adhered to intestinal HT-29 cells and the pre-treatment of HT-29 cells with EcN-microcapsule for 4 h significantly (P < 0.05) reduced the invasion (1.9 log) of C. jejuni; whereas, completely abolished the intracellular survival. Furthermore, HT-29 cells pre-treated with encapsulated EcN in PCR array showed decreased expression (> 1.5-fold) of genes encoding chemokines, toll-like receptors, interleukins, and tumor necrosis factors. In conclusion, the alginate-chitosan microcapsule can provide effectual platform to deliver probiotic EcN and thereby can reduce the Campylobacter infection in chickens and humans.

     

Conformation of Alcohols Having Apopinane Skeleton

Galactostatin obtained from the fermentation broth of Streptomyces lydicus PA-5726 strongly inhibited β-galactosidase. Its derivatives, galactostatin-lactam and 1-deoxygalactostatin, were also inhibitors. Galactostatin and 1-deoxygalactostatin were fully competitive inhibitors with high affinities for Penicillium multicolor β-galactosidase, and their Ki values were 4.0 × 10^?9 and 3.3 × 10^?8m at pH 6.0, respectively, using ONPG as substrate. In their presence, the steady-state velocities of the enzyme were reached in a matter of minutes. Galactostatin-lactam, in contrast, showed no detectable lag time on interaction with the enzyme, and the type of inhibition was also competitive with a Ki value of 1.3 × 10^?5 m. These three inhibitors bound to the enzyme in the same molar ratio (1:1).