Biphasic Hydrolysis Research Articles

Bi-phasic hydrolysis of corncobs for the extraction of total sugars and ethanol production using inhibitor resistant and thermotolerant yeast, Pichia kudriavzevii

In order to have sustainable energy and to reduce the reliance on fossil fuels, transition to renewable bioenergy is highly desired. Adequate biomass conversion to ethanol requires total carbohydrate extraction from the feedstock with minimum inhibitors. The present study aimed at revealing the fermentation potential of toxin resistant and thermotolerant Pichia kudriavzevii for ethanol production. Corncobs being one of the most abundant renewable biomass, was chosen for sequential bioconversion process to ethanol. Firstly, the biomass was pretreated using 2% NaOH which resulted in 83.12 ± 1.07% delignification. Subsequently biomass was used for biphasic hydrolysis (ultrasound assisted dilute acid followed by enzyme) which resulted in 27.03 ± 0.91 gL-1 of xylose, 0.33 ± 0.03 gL-1 furans in the first phase and 27.33 ± 0.9 gL-1 glucose in the later phase. Fermentation efficiency of P. kudriavzevii was evaluated using enzyme and acid hydrolysates (detoxified and undetoxified) which resulted in 11.98 gL-1 ethanol with glucose, 3.83 gL-1 and 3.64 gL-1 ethanol with detoxified and undetoxified acid hydrolysates with theoretical yield of 85.95, 31.89% and 28% respectively at 42 °C. Though the efficiency with xylose was low, the strain was found to be one among very few Pichia strains that have ability to ferment xylose. Furthermore, developing a potential biological process that utilizes the renewable biomass for energy development will not only save a lot of foreign exchange, but also helps in waste management in a sensible way.

Structural, thermal, and hydrolysis properties of large and small granules from C-type starches of four Chinese chestnut varieties

Large and small granules were separated from C-type starches of four Chinese chestnut varieties growing in the same environment. They had similar amylose contents from 17.7% to 20.2% and showed C-type crystallinity. The large granules had relative crystallinity from 19.2% to 20.3%, ordered degree from 0.672 to 0.706, and lamellar peak intensity from 233.2 to 267.1, but small granules had relative crystallinity from 16.2% to 18.2%, ordered degree from 0.635 to 0.663, and lamellar peak intensity from 201.6 to 213.1. The gelatinization peak temperatures ranged from 62.6 to 65.7 °C in large granules but from 60.3 to 61.7 °C in small granules, and enthalpy variation did from 12.5 to 13.7 J/g in large granules but from 10.1 to 11.7 J/g in small granules. Both large and small granules showed biphasic hydrolysis. Though small granules had significantly higher hydrolysis rate than large granules, but they had similar total hydrolysis extent during whole hydrolysis. The granule size had significantly positive relationships with relative crystallinity, ordered degree, lamellar peak intensity, and gelatinization temperature and enthalpy variation, but was negatively correlated to hydrolysis rate. The principal component analysis was conducted to reveal the interrelationships among different starch properties and the variations among different starches.

The relationship between enzyme hydrolysis and the components of rice starches with the same genetic background and amylopectin structure but different amylose contents

The relationship between starch components and hydrolysis previously focused on starches with different amylose contents (ACs) and amylopectin structures. In this study, starches were isolated from six rice seeds with the same genetic background but different activities of granule-bound starch synthase I. The starch components and enzyme hydrolysis were measured and analysed to reveal their relationships. The results showed that the six rice starches had the same amylopectin structure but different ACs ranging from approximately 1%–15%. The starch component parameters measured by different methods were significantly correlated with each other. The hydrolysis parameters of starch by both α-amylase and amyloglucosidase were analysed using three methods including a non-linear fit to the degree of hydrolysis, a first-order kinetics fit to the degree of hydrolysis, and a first-order kinetics fit to the hydrolysis rate, and they had significant correlation each other. Waxy starch had a fast hydrolysis rate and showed a single hydrolysis phase during early hydrolysis, while starch with a high AC had a bi-phasic hydrolysis process. Starch component parameters including the maximum absorption wavelength, iodine blue, AC, and low molecular weight fraction were significantly negatively correlated to the hydrolysis rate of starch. The above results indicated that starches with different ratios of amylose to amylopectin but the same amylopectin structure had significantly different hydrolysis properties. This study could provide some information for rice quality breeding and starch applications through changing the activity of granule-bound starch synthase I.

A novel approach to biphasic strategy for intensification of the hydrothermal process to give levulinic acid: Use of an organic non-solvent

Levulinic acid is a platform chemical obtained from acid-catalyzed hydrothermal conversion of cellulose-rich biomass. The low amounts of solid biomass which can be handled in the reactor limit the levulinic acid concentration in the aqueous stream, making the economic viability of the aqueous phase process unsuitable for large scale applications. Now a novel approach to biphasic process has been proposed, where a mineral oil has been used as non-solvent for levulinic acid, thus concentrating it in the water phase, reducing the water volume to be processed downstream but at the same time maintaining enough liquid phase to sustain the slurry processability. The work has studied: i) the optimization of the biphasic hydrolysis of corn grain to levulinic acid; ii) the characterization of the recovered oil; iii) the evaluation of the energetic properties of the recovered hydrochar for its exploitation, thus smartly closing the biorefinery cycle.

Comparison of enzymatic hydrolysis in a centrifugal partition chromatograph and stirred tank reactor

Recently the Centrifugal Partition Chromatography (CPC) device is investigated as a reactor for biocatalytic reactions, as it enables biocatalyst immobilization without solid support and continuous operation of biphasic reaction systems. However, a detailed determination of the enzymes behavior in the CPC reactor and comparison to a classical stirred tank reactor (STR) for biphasic enzyme catalysis is not shown yet. In this study, the performance of an enzymatic biphasic hydrolysis reaction using lipase from Candida rugosa is systematically evaluated using a STR. The results are compared to different experiments conducted in the CPC reactor and used to evaluate the reaction performance in each. The same characteristics and limitations were observed in STR and CPC. At all states the CPC provided a similar reaction performance. However, the reaction in the CPC runs faster into limitations and was not easily scalable due to complex effects of the flow pattern. Although the enzyme was immobilized successfully and the activity of the lipase was preserved during CPC operation optimizations are needed to make the CPC reactor more competitive. For instance, scaling up the chamber geometry seems to be mandatory to increase the reaction performance, which may promote this reactor concept as an alternative to common devices for continuous biphasic biocatalytic reactions.

A novel diffusion–biphasic hydrolysis coupled kinetic model for dilute sulfuric acid pretreatment of corn stover

Kinetic experiments on the dilute sulfuric acid pretreatment of corn stover were performed. A high xylan removal and a low inhibitor concentration were achieved by acid pretreatment. A novel diffusion–hydrolysis coupled kinetic model was proposed. The contribution to the xylose yield was analyzed by the kinetic model. Compared with the inhibitor furfural negatively affecting xylose yield, the fast and slow-hydrolyzing xylan significantly contributed to the xylose yield, however, their dominant roles were dependent on reaction temperature and time. The impact of particle size and acid concentration on the xylose yield were also investigated. The diffusion process may significantly influence the hydrolysis of large particles. Increasing the acid concentration from 0.15M to 0.30M significantly improved the xylose yield, whereas the extent of improvement decreased to near-quantitative when further increasing acid loading. These findings shed some light on the mechanism for dilute sulfuric acid hydrolysis of corn stover.

Characterization of bis-[triethoxysilylpropyl] tetrasulfide layers on aluminum based on water-based silanization solution

In this work, a water-based silanization solution was prepared using a biphasic hydrolysis system composed of 85% (V/V) water and 15% (V/V) bis-[triethoxysilylpropyl] tetrasulfide (BTESPT)/n-heptane/ethanol mixture for efficiently coating aluminum with silane layer against corrosion. The BTESPT-based coatings on several pretreated aluminum samples were characterized by scanning electron microscopy and X-ray energy dispersive spectroscopy, and their electrochemical behaviors were assessed in 0.1M NaCl neutral solution by means of electrochemical impedance spectroscopy and Tafel polarization. The BTESPT-based coating of about 180nm thick was found to be uniform and compact, and the maximum corrosion resistance of 106Ω of the BTESPT-treated aluminum samples was observed, which is larger than that of bare aluminum by two orders of magnitude. Durability tests in NaCl solution demonstrated that the BTESPT coating can provide superior protection of alumina substrate from corrosion for 10-day immersion in the corrosive media.

Ceftazidime-hydrolysing -lactamase OXA-145 with impaired hydrolysis of penicillins in Pseudomonas aeruginosa

To describe a novel extended-spectrum oxacillinase, named OXA-145, differing from narrow-spectrum OXA-35 (from the OXA-10 group) by deletion of residue Leu-165. The genetic environment of bla(OXA-145) and the biochemical properties of OXA-145 are reported. We also assessed the impact of the Leu-165 deletion on the hydrolysis spectrum of the ancestor OXA-10. Extended-spectrum β-lactamase OXA-145 was identified in the multidrug-resistant clinical Pseudomonas aeruginosa 08-056, and characterized by isoelectric focusing, PCR and DNA sequencing. Antibiotic susceptibility tests were performed by agar dilution. The resistance profiles conferred by cloned bla(OXA-10), bla(OXA-35), bla(OXA-145) and a bla(OXA-10) derivative obtained by site-directed mutagenesis were determined in Escherichia coli. Kinetic parameters of OXA-35 and OXA-145 were established after purification of His-tagged proteins. The sequence of OXA-145, encoded by a class 1 integron-borne gene in strain 08-056, differed from that of narrow-spectrum penicillinase OXA-35 by a single amino acid deletion (Leu-165) located in the highly conserved omega loop. Deletion of Leu-165 from OXA-35 (yielding OXA-145) or OXA-10 (the progenitor of OXA-35) extended the hydrolysis spectrum to third-generation cephalosporins and to monobactams, while reducing that for penicillins. OXA-145 showed biphasic hydrolysis curves for all the substrates tested. Its activity against nitrocefin was 10-fold higher in the presence of sodium hydrogen carbonate. OXA-145 is a new extended-spectrum β-lactamase from the OXA-10 group. The deletion of Leu-165 is responsible for a shift in the hydrolysis spectrum from penicillins to third-generation cephalosporins, as well as monobactams. The loss of penicillin hydrolysis was due to a non-carboxylated Lys-73.

Kinetic modeling analysis of maleic acid‐catalyzed hemicellulose hydrolysis in corn stover

Maleic acid-catalyzed hemicellulose hydrolysis reaction in corn stover was analyzed by kinetic modeling. Kinetic constants for Saeman and biphasic hydrolysis models were analyzed by an Arrhenius-type expansion which include activation energy and catalyst concentration factors. The activation energy for hemicellulose hydrolysis by maleic acid was determined to be 83.3 +/- 10.3 kJ/mol, which is significantly lower than the reported E(a) values for sulfuric acid catalyzed hemicellulose hydrolysis reaction. Model analysis suggest that increasing maleic acid concentrations from 0.05 to 0.2 M facilitate improvement in xylose yields from 40% to 85%, while the extent of improvement flattens to near-quantitative by increasing catalyst loading from 0.2 to 1 M. The model was confirmed for the hydrolysis of corn stover at 1 M maleic acid concentrations at 150 degrees C, resulting in a xylose yield of 96% of theoretical. The refined Saeman model was used to evaluate the optimal condition for monomeric xylose yield in the maleic acid-catalyzed reaction: low temperature reaction conditions were suggested, however, experimental results indicated that bi-phasic behavior dominated at low temperatures, which may be due to the insufficient removal of acetyl groups. A combination of experimental data and model analysis suggests that around 80-90% xylose yields can be achieved at reaction temperatures between 100 and 150 degrees C with 0.2 M maleic acid.

Effect of segmented fluid flow, sonication and phase transfer catalysis on biphasic reactions in capillary microreactors

The contact between immiscible liquids in a microfluidic system creating segmented flow offers great potential in the study of biphasic reactions in organic chemistry with significant advantages with respect to conventional flask techniques. For a simple biphasic hydrolysis, we show that the application of various reaction conditions in microreactors using segmented flow can dramatically increase the reaction rate, especially when sonication and phase transfer catalysis are combined with segmentation.

Enhancement of Reaction Rates by Segmented Fluid Flow in Capillary Scale Reactors

AbstractFor a simple biphasic hydrolysis, we show that the application of various reaction conditions in microreactors using segmented flow can dramatically increase the reaction rate. A tandem diazotation/Heck reaction served as an example that even homogeneous reactions can be accelerated by segmentation in microreactors.

Relationship between α-amylase degradation and the structure and physicochemical properties of legume starches

Starch from cultivars of black bean, pinto bean, smooth pea, lentil and wrinkled pea starches were isolated and their composition, physicochemical properties and susceptibility towards porcine pancreatic α-amylase determined. The yield of starch ranged from 16.4 to 34.1% on a whole seed basis. The shape of the granules varied from round to oval to irregular. Bound and total lipids ranged from 0.26 to 0.80% and 0.35 to 0.84%, respectively. The total amylose content ranged from 30.5 to 78.4%, of which 10.3 to 12.2% was complexed by native starch lipids. The X-ray diffraction pattern was of the ‘B’ type in wrinkled pea starch and of the ‘C’ type in other starches. The relative crystallinity and the ‘B’ polymorphic content ranged from 17.7 to 33.4% and 27.1 to 92.2%, respectively. The starches differed in the degree of swelling, extent of amylose leaching, gelatinization temperatures and gelatinization enthalpy. The above differences were more pronounced between cultivars of black bean and lentil. The results showed that starch chain interactions within the amorphous domains were more pronounced in wrinkled pea starch. All starches exhibited a biphasic hydrolysis pattern, a relatively rapid rate at the initial stage followed by a progressively decreased rate thereafter. Wrinkled pea starch exhibited a much higher initial velocity than the other starches. Cultivars of black bean and lentil showed significant differences in their initial velocities. However, differences in initial velocity between cultivars of smooth pea and pinto bean were not significant. Black bean, lentil and wrinkled pea starches showed a plateau at 93, 85 and 65% hydrolysis, respectively. The time taken for the appearance of the plateau was identical for black bean cultivars, but was different for cultivars of lentil. Pinto bean and smooth pea cultivars showed no plateau. At the end of the assay period (120 h), cultivars of each legume species were hydrolyzed to the same extent, and the extent of hydrolysis among the legume species followed the order: black bean>lentil>smooth pea>pinto bean>wrinkled pea. The X-ray pattern and the ‘B’ polymorphic content of all starches remained unchanged on hydrolysis. However, the relative crystallinity increased in wrinkled pea, but remained unchanged in the other starches. On hydrolysis, the apparent amylose content decreased in all starches. The extent of this decrease was most pronounced in wrinkled pea. In all starches, the enthalpy of gelatinization decreased, and the gelatinization transition temperatures increased slightly on hydrolysis. This study showed that the rate and extent of hydrolysis was influenced by structural organization of the starch chains within the native granule, and by the extent of interaction between hydrolyzed amylose chains.

Unfunctionalized, alpha-epimerizable nonracemic ketones and aldehydes can be accessed by crystallization-induced dynamic resolution of imines.

This paper describes an operationally simple deracemization process of aldehydes and ketones. This new crystallization-induced dynamic resolution (CIDR) protocol allows for nearly complete conversion of the racemic mixture into one enantiomer. Crystallization of imines derived from racemic ketones or aldehydes 1 and trans-(1R,2R)-1-amino-6-nitroindan-2-ol (2) afforded diastereomerically pure, crystalline imines 3. Biphasic hydrolysis of 3 then affords recovered 2 and enantiomerically enriched 1 in high yield and er (substrate, yield/ee: 2-methylcyclohexanone, 97%/92; 2-ethylhexanal, 94%/98; 2-methylcyclopentanone, 94%/98; 2-cyclohexylcyclohexanone, ND/98; 3-methyl-2-pentanone, ND/76). The scope, limitations, and industrial perspective of this process are discussed. This highly effective CIDR process is likely due to pi-stacking of 2 and a hydrogen bonding of the imine with the free hydroxyl of 2 in the solid state.

Solubilization Processes in Autocatalytic Biphasic Reactions

The kinetics of solubilization of ethyl hexanoate into an aqueous phase was investigated in the presence of various additives (NaCl, ethanol, and sodium hexanoate). The salting-out effect of NaCl and the solvent effect observed in the presence of ethanol were quantified. Solubilization by sodium hexanoate shows a dramatic enhancement when the critical micellar concentration is exceeded. This effect was interpreted by the formation of mixed oil/surfactant aggregates. The kinetic rate constants of solubilization of ethyl hexanoate into the water phase were determined in the presence of sodium hexanoate using a model including salting-in, salting-out, and aggregation effects. When NaCl was replaced by NaOH, ethyl hexanoate was hydrolyzed to sodium hexanoate and ethanol. The kinetics of this reaction was recorded in the same geometrical setup as for previous solubilization experiments. The solubilization kinetic model was able to describe the hydrolysis reaction by just adding a rapid bulk hydrolysis step. The model quite accurately predicts the autocatalytic effect of sodium hexanoate formation and the overall reaction time. This result clearly shows the crucial role of solubilization processes on this biphasic hydrolysis reaction.

Ester containing aggregates in the autocatalytic biphasic hydrolysis of ethyl alkanoate

Autocatalytic kinetics are observed during the biphasic alkaline hydrolysis of C-4, C-6 and C-8 (butanoate, hexanoate and octanaoate) ethyl esters. Ethanol and alkanoate anions are the products of the reaction. A kinetic model including spontaneous dissolution of the organic phase into the aqueous phase, aqueous hydrolysis of the ester, formation of ester containing aggregates and micellisation has been proposed. For C-4, taking into account salting-in and solvent effects is sufficient to reproduce the autocatalytic behaviour. For C-8, the major autocatalytic process is an ester containing aggregates (ECA) mediated phase transfer. For C-6, it appears that the role of ECA is double. Small sized ECA which are formed at the beginning of the reaction do not perform rapid phase transfer and trap surfactant molecules. Larger ECA which appear at the end of the reaction are the main phase transfer agent. Using an empirical formula to describe the size of the interface, the model is able to reproduce the effect of a variation of the volumic ratio of the two phases as well as the effect of stirring rate and non-emulsified experiments.

Physical properties and hydrolysis kinetics of (μ-oxo)bis [tris(2pyridylmethyl)aminechromium(III)] dimers with complementary sulfate or molybdate bridging ligands

The complexes [(tmpa)Cr(μ-O)(μ-EO 4)Cr(tmpa)](ClO 4) 2 · nH 2O (E = S, n = 1; E = Mo, n = 0; tmpa = tris(2-pyridylmethyl)amine) have been prepared through the displacement of water from [Cr(tmpa)(OH)]2(ClO 4) 4 · 4H 2O by the tetrahedral oxyanions in CH 3CN solution. Electronic spectra, asymmetric CrOCr stretching frequencies, half-wave Cr(III,IV/III,III) reduction potentials and 1H NMR features of the O 2−, SO 4 2− and O 2−, MoO 4 2−-bridged dimers are essentially identical to those of [Cr(tmpa)CI] 2O 2+, consistent with structures having near-linear CrOCr bridging units and symmetric tmpa chelation placing both apical nitrogen atoms trans to oxyanion oxygen atoms. The title complexes undergo biphasic hydrolysis reactions in the pH 4.80–7.80 interval. The fast phase rate exhibits no pH dependence, while k slow increases with pH in a way that suggests the presence of both protonated and deprotonated reactant species. Since these findings mirror those reported previously for [Cr(tmpa)CI] 2O 2+ hydrolysis, we propose a closely related mechanism featuring H 2O-induced ring-opening of the bridging EO 4 2− group in the first step ( k fast) followed by loss of EO 4 2− from two intermediates, [(EO 4)(tmpa)CrOCr(tmpa)(H 2O)] 2+ ( k a) and [(EO 4)(tmpa)CrOCr(tmpa)(OH)] + ( k b), related by a rapid ionization equilibrium ( K a). Reported k fast, k a, k b and p K a parameters (25.0°C, I = 0.1 M) are 1.7 × 10 −1, 6.8 × 10 −3, 7.98 × 10 −2 s −1, 6.66 (SO 4 2−) and 1.6 × 10 −1, 6.4 × 10 −3, 8.01 × 10 −2 s −1, 6.76 (MoO 4 2−), respectively. Our findings for both fast and slow steps in the hydrolysis of Cl −, SO 4 2− and MoO 4 2− are consistent with a pathway in which bond-making to the incoming water ligand and bond-breaking is promoted specifically by a cis-labilizing influence of the oxo bridge.

Asymmetry of lipid organization in cholinergic synaptic vesicle membranes.

The lipid composition of purified Torpedo cholinergic synaptic vesicles was determined and their distribution between the inner and outer leaflets of the vesicular membrane was investigated. The vesicles contain cholesterol and phospholipids at a molar ratio of 0.63. The vesicular phospholipids are (mol% of total phospholipids): phosphatidylcholine (40.9); phosphatidylethanolamine (24.6); plasmenylethanolamine (11.5); sphingomyelin (12); phosphatidylserine (7.3); phosphatidylinositol (3.7). The asymmetry of the synaptic vesicle membranes was investigated by two independent approaches: (a) determining accessibility of the amino lipids to the chemical label trinitrobenzenesulphonic acid (TNBS); (b) determining accessibility of the vesicular glycerophospholipids to phospholipase C (Bacillus cereus). TNBS was found to render the vesicles leaky and thus cannot be used reliably to determine the asymmetry of Torpedo synaptic vesicle membranes. Incubation of the vesicles with phospholipase C (Bacillus cereus) results in biphasic hydrolysis of the vesicular glycerophospholipids. About 45% of the phospholipids are hydrolysed in less than 1 min, during which no vesicular acetylcholine is released. In the second phase, the hydrolysis of the phospholipids slows down markedly and is accompanied by loss of all the vesicular acetylcholine. These findings suggest that the lipids hydrolysed during the first phase are those comprising the outer leaflet. Analysis of the results thus obtained indicate that the vesicular membrane is asymmetric: all the phosphatidylinositol, 77% of the phosphatidylethanolamine, 47% of the plasmenylethanolamine and 58% of the phosphatidylcholine were found to reside in the outer leaflet. Since phosphatidylserine is a poor substrate for phospholipase C (B. cereus), its distribution between the two leaflets of the synaptic vesicle membrane is only suggestive.

Kinetic mechanism of mitochondrial adenosine triphosphatase. ADP-specific inhibition as revealed by the steady-state kinetics.

1. A substantial increase of the initial rate of ATP hydrolysis was observed after preincubation of bovine heart submitochondrial particles with phosphoenolpyruvate and pyruvate kinase. 2. The activation was accompanied by an increase of Vmax, without change of Km for ATP. 3. The activated particles catalysed the biphasic hydrolysis of ATP in the presence of an ATP-regenerating system; the initial rapid phase was followed by a second, slower, phase in a time-dependent fashion. 4. The higher the ATP concentration used as a substrate, the higher is the rate of transition between these two phases. 5. The particles catalysed the hydrolysis of ITP with a lag phase; after preincubation with phosphoenolpyruvate and pyruvate kinase, ITP was hydrolysed at a constant rate. 6. Qualitatively the same phenomena were observed when soluble mitochondrial ATPase (F1-ATPase) prepared by the conventional method in the presence of ATP was used as nucleotide triphosphatase. 7. A kinetic scheme is proposed, in which the intermediate active enzyme-product complex (E.ADP) formed during ATP hydrolysis is in slow equilibrium with the inactive E*.ADP complex forming as a result of dislocation of ADP from the active site of ATPase to the other site, which is not in rapid equilibrium with the surrounding medium.

The isolation and properties of pig submandibular kallikrein

The kallikrein from pig submandibular glands was highly purified, with an overall yield of 31%. Affinity chromatography on bovine basic pancreatic trypsin inhibitor linked to Sepharose 4B was an especially effective step in the purification procedure, giving a purification factor of 80. The enzyme is a single-chain molecule, occurring, as does pig urinary kallikrein, as a major B-form of apparent mol.wt. 39600 and minor amounts of an A-form of apparent mol.wt. 35900; the two forms can be separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid composition of pig submandibular kallikrein is very similar to, but not quite identical with, that of the two-chain beta-kallikrein isolated from pig pancreatic autolysates. Submandibular kallikrein contains notably more glucosamine and hexoses than does pancreatic beta-kallikrein. Submandibular kallikrein, and also urinary kallikrein, exhibit an unusual biphasic hydrolysis of substrate esters that is not shared by pancreatic beta-kallikrein. For the submandibular enzyme, the K(m) for the initial reaction phase of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester is 0.15+/-0.01mm (mean+/-s.e.m.), but rises to 0.69+/-0.04mm (mean+/-s.e.m.) in the stationary reaction phase; the V(max.) does not differ significantly between the two phases. The esterolytic activities of submandibular and urinary kallikreins on a number of esters of different amino acids resemble each other much more closely than those of pancreatic beta-kallikrein.

A Thermodynamic Study of the Hydrolysis of Cytidine 2‘,3‘-Cyclic Phosphate

From the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 (Received for publication, March 17, 1965) Bovine pancreatic ribonuclease catalyzes the hydrolysis of cytidine 2’,3’-cyclic phosphate according to the following equa- tion (1): Cytidine 2’ ,3’-cyclic phosphate + Hz0 --t cytidine 3’-phosphate (1) This reaction is generally considered to be a model for the second step of the biphasic hydrolysis of ribonucleic acid and conse- quently has been subjected to considerable scrutiny in recent years. A number of kinetic studies have been made of this reaction (2-5). The over-all reaction is generally stated to be “irreversible” (2, 3, 6, 7); however, no estimates of the equilib- rium constant have appeared in the literature. In fact, very little thermodynamic data related to cytidine 2’,3’-cyclic phos- phate and cytidine 3’-phosphate are available although the usefulness of such data is readily apparent. In the present study, the equilibrium constant of the hy- drolysis reaction has been determined by two independent methods. The first method utilizes the thermodynamic cycle librium constant utilizes radioactive tracers: is added to a solution of cytidine 3’-phosphate-%, and, after equilibrium has been est.ablished, the concentration of cytidine 2’,3’-cyclic phosphate-% present. is determined by the isotope dilution technique. From a knowledge of the equilibrium constant and the steady state kinetic parameters of the forward reaction some of the steady state kinetic parameters characterizing the reverse re- action can be calculated.