Initial Stage Of Hydrolysis Research Articles

Montmorillonite Functionalized with Pralidoxime As a Material for Chemical Protection against Organophosphorous Compounds

Montmorillonite K-10 functionalized with α-nucleophilic 2-pralidoxime (PAM) and its zwitterionic oximate form (PAMNa) is introduced as a versatile material for chemical protection against organophosphorous (OP) compounds such as pesticides and chemical warfare agents (CWA). Upon inclusion into the montmorillonite interlayer structure, the pyridinium group of PAMNa is strongly physisorbed onto acidic sites of the clay, leading to shrinking of the interplanar distance. Degradation of diethyl parathion by PAMNa-functionalized montmorillonite in aqueous-acetonitrile solutions occurred primarily via hydrolytic conversion of parathion into diethylthio phosphoric acid, with the initial stages of hydrolysis observed to be pseudo-first-order reactions. Hydrolysis catalyzed by the clay intercalated by PAMNa was 10- and 17-fold more rapid than corresponding spontaneous processes measured at 25 and 70 °C, respectively. Hydrolytic degradation of diisopropyl fluorophosphate (DFP), a CWA simulant, was studied on montmorillonite clay functionalized by PAMNa and equilibrated with water vapor at 100% relative humidity by ³¹P high-resolution magic angle spinning NMR and was observed to be rather facile compared with the untreated montmorillonite, which did not show any DFP hydrolysis within 24 h. The incorporation of the functionalized clay particles into elastomeric film of polyisobutylene was shown to be a means to impart DFP-degrading capability to the film, with clay particle content exceeding 18 wt %.

Particulate structure of phytoglycogen nanoparticles probed using amyloglucosidase

Phytoglycogen is a plant-based, high-density carbohydrate nanoparticle that can be used as a starting material for preparing novel functional nano-constructs. In this study, the particulate structure of phytoglycogen was probed using amyloglucosidase hydrolysis, with amylopectin of waxy corn starch as a reference. Glucose release was monitored; molar mass, root mean square (RMS) radius, and dispersed molecular density were evaluated using multiangle laser-light scattering. The goal was to reveal the structural features of phytoglycogen nanoparticles. After extended amyloglucosidase hydrolysis, the total glucose release of phytoglycogen was lower than that of amylopectin. At the initial stage of hydrolysis, however, glucose was released at a higher rate from phytoglycogen, suggesting a dense distribution of nonreducing ends at the surfaces of nanoparticles. The reduction of molar mass and RMS radius caused by amyloglucosidase hydrolysis were much lower for phytoglycogen than for amylopectin. The dispersed molecular density of amylopectin remained at about 60 g/mol nm 3 during hydrolysis. In contrast, the density of phytoglycogen decreased from about 1200 to 950 g/mol nm 3, suggesting a density increment towards the external region of nanoparticle.

Purification of exo-1,3-beta-glucanase, a new extracellular glucanolytic enzyme from Talaromyces emersonii

The moderately thermophilic aerobic ascomycete Talaromyces emersonii secretes, under selected growth conditions, several β-glucan hydrolases including an exo-1,3-β-glucanase. This enzyme was purified to apparent homogeneity in order to characterise its biochemical properties and investigate hydrolysis of different β-glucans, including laminaran, a 1,3-β-glucan from brown algae. The native enzyme is monomeric with a molecular mass of ~40 kDa and a pI value of 4.3, and is active over broad ranges of pH and temperature, with optimum activity observed at pH 5.4 and 65 °C. At pH 5.0, the enzyme displays strict specificity for laminaran (apparent K(m) 1.66 mg mL⁻¹; V(max) 7.69 IU mL⁻¹) and laminari-oligosaccharides and did not yield activity against 1,4-β-glucans, 1,3;1,4-β-glucans or 4-nitrophenyl- and methylumbelliferyl-β-D: -glucopyranosides. Analysis of hydrolysis products formed during time-course hydrolysis of laminaran by high-performance anion exchange chromatography with pulsed amperometric detection revealed a strict exo mode of action, with glucose being the sole reaction product even at the initial stages of hydrolysis. The T. emersonii exo-1,3-β-glucanase was inhibited by glucono-δ-lactone (K(i) 1.25 mM) but at significantly higher concentrations than typically inhibitory for exo-glycosidases such as β-glucosidase. 'De novo' sequence analysis of the purified enzyme suggests that it belongs to family GH5 of the glycosyl hydrolase superfamily. The results clearly show that the exo-1,3-β-glucanase is yet another novel enzyme present in the β-glucanolytic enzyme system of T. emersonii.

Hydrolysis of amylopectin by amylolytic enzymes: level of inner chain attack as an important analytical differentiation criterion

Differences in amylase action pattern on amylopectin were demonstrated by the relation between the decrease in potassium iodide–iodine binding of waxy maize starch and the increase in reducing value during hydrolysis, as expressed by the RV 80 value (i.e., the reducing value for a potassium iodide–iodine binding value of 80% of that of the starting material). In the initial stages of the hydrolysis, the ratio of the increase in the level of reducing polysaccharides to the increase in the total level of reducing sugars formed during amylolysis of amylopectin can be considered as a measure of the level of inner chain attack (LICA) in the overall hydrolysis of the amylopectin structure and correlated with the respective RV 80 value. Bacillus amyloliquefaciens α-amylase and Aspergillus oryzae α-amylase, with the lowest RV 80 and the highest LICA values, hydrolysed the inner chains of amylopectin to a greater extent than did porcine pancreatic α-amylase. In the initial stages of hydrolysis, Bacillus stearothermophilus maltogenic amylase, like the Bacillus cereus β-amylase, did not display any significant degree of internal hydrolysis of amylopectin, in line with the high RV 80 and very low LICA values for these enzymes. However, at the later stages of hydrolysis, the maltogenic amylase probably exhibited a significant degree of internal hydrolysis of amylopectin, which itself seems to depend on temperature. The temperature dependence of the hydrolysis pattern of this enzyme is relevant for interpretation of its action as antifirming enzyme in bread-making applications.

Kinetic Control of Intralayer Cobalt Coordination in Layered Hydroxides: Co1−0.5xoctCoxtet(OH)2(Cl)x(H2O)n

We report the synthesis and characterization of new structural variants of the isotypic compound with the generic chemical formula, Co(1-0.5x)(oct) Co(x)(tet) (OH)2 (Cl)x (H2O)n, all modifications of an alpha-Co(OH)2 lattice. We show that the occupancy of tetrahedrally coordinated cobalt sites and associated chloride ligands, x, is modulated by the rate of formation of the respective layered hydroxide salts from kinetically controlled aqueous hydrolysis at an air-water interface. This new level of structural control is uniquely enabled by the slow diffusion of a hydrolytic catalyst, a simple technique. Independent structural characterizations of the compounds separately describe various attributes of the materials on different length scales, revealing details hidden by the disordered average structures. The precise control over the population of distinct octahedrally and tetrahedrally coordinated cobalt ions in the lattice provides a gentle, generic method for modulating the coordination geometry of cobalt in the material without disturbing the lattice or using additional reagents. A mechanism is proposed to reconcile the observation of the kinetic control of the structure with competing interactions during the initial stages of hydrolysis and condensation.

An ab initio study of the first stage of catalysis in the monomeric aspartic proteinases

We have studied the initial stages of hydrolysis in the monomeric aspartic proteinases by performing ab initio calculations (Hartree–Fock, MP2, DFT) on the active site of endothiapepsin. The crystallographic coordinates of a difluorostatone inhibitor complexed with endothiapepsin were used to generate a template for the initial enzyme/substrate complex. The active site was modelled as a formic acid–formate anion moiety (representing the catalytic aspartates, Asp-32 and Asp-215) and a bound water molecule. Acetamide was used to represent the substrate, and residues Gly-34, Ser-35, Gly-217, and Thr-218, which all interact with the active site, were modelled using formamide and methanol. The calculations predict that T218 plays a direct role in catalysis by forming a strong hydrogen bond with the water oxygen during the initial stages of nucleophilic attack. S35 plays an indirect role by lowering the p K a of Asp-32 and facilitating the formation of the gem–diol intermediate. The predicted reaction pathway is unusual because it involves two local minima, with very short O water...C substrate distances, which are stabilised by T218.

Changes in the Ovalbumin Proteolysis Profile by High Pressure and Its Effect on IgG and IgE Binding

Egg proteins are responsible for one of the most common forms of food allergy, especially in children, and one of the major allergens is ovalbumin (OVA). With the aim to examine the potential of high pressure to enhance the enzymatic hydrolysis of OVA and modify its immunoreactivity, the protein was proteolyzed with pepsin under high-pressure conditions (400 MPa). Characterization of the hydrolysates and peptide identification was performed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS). The antigenicity (binding to IgG) and binding to IgE, using the sera of patients with specific IgE to OVA, were also assessed. The results showed that, upon treatment with pepsin at 400 MPa, all of the intact protein was removed in minutes, leading to the production of hydrolysates with lower antigenicity than those produced in hours at atmospheric pressure. However, the exposure of new target residues only partially facilitated the removal of allergenic epitopes, because the hydrolysates retained residual IgG- and IgE-binding properties as a result of the accumulation of large and hydrophobic peptides during the initial stages of hydrolysis. These peptides disappeared at later stages of proteolysis, although reactivity toward IgG and IgE was not completely abolished. Some fragments identified in the hydrolysates (such as Leu124-Phe134, Ile178-Ala187, Leu242-Leu252, Gly251-Ile259, Lys322-Gly343, Phe358-Phe366, and Phe378-Pro385) carried previously identified IgE-binding epitopes. Because some of the peptides found, such as Phe358-Phe366, probably contain only one binding site for IgE, the possibility to use high pressure to tailor hydrolysates that contain mostly peptides with only one IgE-binding site, which may help the immune system to tolerate egg proteins, is suggested.

Amylolytic Hydrolysis of Native Starch Granules Affected by Granule Surface Area

Initial stage of hydrolysis of native starch granules with various amylolytic enzymes, alpha-amylase from Bacillus subtilis, glucoamylase I (GA-I) and II (GA-II) from Aspergillus niger, and beta-amylase from sweet potato showed that the reaction was apparently affected by a specific surface area of the starch granules. The ratios of the reciprocal of initial velocity of each amylolytic hydrolysis for native potato and maize starch to that for rice with the amylolytic enzymes were nearly equivalent to the ratio of surface area per mass of the 2 starch granules to that of rice, that is, 6.94 and 2.25, respectively. Thus, the reciprocal of initial velocity of each enzymatic hydrolysis as expressed in a Lineweaver-Burk plot was a linear function of the reciprocal of surface area for each starch granule. As a result, it is concluded that amylolytic hydrolysis of native starch granules is governed by the specific surface area, not by the mass concentration, of each granule.

Phase Transformation of TiO<sub>2</sub> in the Preparation of Pearlescent Pigment

The TiO2/mica pearlescent pigments were prepared by the hydrolysis of TiOCl2 on the natural mica (muscovite) followed by a calcinations process. The phase transformation of anatase to rutile during calcination and their proportion in the TiO2 thin layer have been analyzed by XRD measurements. The pH controlling during the initial stage of hydrolysis of TiOCl2 showed a pronounced effect on the phase transformation of TiO2 on the substrate. This result may be due to the fact that the pH controlling during the hydrolysis affects the crystal structure and size of TiO2. The decrease in crystallinity and crystallite size of TiO2 resulted in lowering the temperature of phase transformation from anatase to rutile.

Kinetics and mechanism of adenosine 5′-triphosphate hydrolysis catalyzed by the Cu2+ ion: The role of conformation and the catalytic effect of the OH− ion

The kinetics of 5′-ATP hydrolysis catalyzed by the Cu2+ ion has been investigated by HPLC in the pH range 5.6–7.8 at 25°C. Two series of experiments differing in the initial [Cu · ATP]0 (1: 1) concentration have been carried out. The reaction was being conducted up to ≈40% ATP conversion. The (CuATP2−)2OH−⨑ub;DOH−⫂ub; complex, which consists of two monomeric Cy(CuATP2−) molecules (in which the N7 atom and the γ-phosphate group are coordinated to Cu2+), is responsible for the formation of CuADP− + Pi (Pi is an inorganic phosphate). The highest possible DOH− concentration at a given pH is reached at the initial stage of hydrolysis. The pH value at which the highest initial rate of ADP formation is reached (pHmax (w 0, ADP)) decreases as the D concentration increases. At pH > pHmax, the decrease in the ADP formation rate in the course of the processes is pH-independent and, once an ATP conversion of 20–26% is reached, hydrolysis proceeds in a steady-state regime such that ADP and AMP form from ATP by parallel reactions. The participation of the OH− ion in the catalysis of the formation of hydrolysis intermediates is considered.

Simulation of the kinetics of adenosine 5′-triphosphate hydrolysis catalyzed by the Cu2+ ion: The role of conformation and the catalytic effect of the OH− ion

The hydrolysis kinetics of the dimeric complex (CuATP2− · OH2)2 {D} up to ≈40% ATP conversion at 25°C, pH 5.7–7.8, and [Cu · ATP]0 = (2.07 ± 0.03) × 10−3 mol/l is analyzed by numerical simulation. CuADP− + Pi (Pi is an inorganic phosphate) form from DOH−, and the latter forms rapidly from D. The abstraction of H+ from the coordinated H2O molecule is an irreversible reaction involving an OH− ion from the medium. The maximum possible DOH− concentration at a given pH is reached at the initial stage of hydrolysis (0.3–6.0 min after the initiation of hydrolysis). CuADP− + Pi form from D via two consecutive irreversible steps. The ADP buildup rate in the process is determined by the reversible conformational transformation of DOH− resulting in a pentacovalent intermediate (IntK). OH− ions from the medium are involved both in IntK formation and in the reverse reaction and are a hydrolysis inhibitor. AMP forms from the intermediate IntK3, which forms reversibly from DOH−, OH− ions from the medium being involved in the forward and reverse reactions. This is followed by irreversible (AMPH)− formation involving H3O+ ions from the medium. The rate and equilibrium constants are determined for the formation and decomposition of hydrolysis intermediates. The concentrations of the intermediates are plotted versus time for various pH values. The structures of the intermediates are suggested. The causes of a peak appearing in the initial ADP formation rate versus pH curve are analyzed.

Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1-->6)-galactanase gene.

The nucleotide sequence depicted in Figure 1 has been submitted to the DDBJ nucleotide sequence database under the accession no. AB104898. A gene encoding endo-beta-(1-->6)-galactanase from Trichoderma viride was cloned by reverse transcriptase-PCR and expressed in Escherichia coli. The gene contained an open reading frame consisting of 1437 bp (479 amino acids). The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases. A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa. The gene product expressed in E. coli as a recombinant protein fused with thioredoxin and His(6) tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T. viride, i.e. recombinant enzyme endo-hydrolysed beta-(1-->6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse alpha-L-arabinofuranosidase-treated arabinogalactan protein from radish. It produced beta-(1-->6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and beta-(1-->6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-beta-(1-->6)-galactanase. As far as we know, this is the first time an endo-beta-(1-->6)-galactanase has been cloned.

Purification and characterization of an endo-β-(1→6)-galactanase from Trichoderma viride

An endo-β-(1→6)-galactanase from Onozuka R-10, a commercial cellulase preparation from Trichoderma viride, was purified 57-fold. Apparent M r values of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were 47,000 and 17,000, respectively. The enzyme was assayed with a galactan from Prototheca zopfii, which has a high proportion of β-(1→6)-linked galactosyl residues . It exhibited maximal activity toward the galactan at pH 4.3. The enzyme hydrolyzed specifically β-(1→6)-galactooligosaccharides with a degree of polymerization higher than 3 and their acidic derivatives with 4- O-methyl-glucosyluronic or glucosyluronic groups at the nonreducing terminals. The methyl β-glycoside of β-(1→6)-galactohexaose was degraded to reducing galactooligomers with a degree of polymerization 2–5 as the products at the initial stage of hydrolysis, and galactose and galactobiose at the final stage, indicating that the enzyme can be classified as an endo-galactanase. The extent of hydrolysis of the carbohydrate portion of a radish root arabinogalactan-protein (AGP) increased when α- l-arabinofuranosyl residues attached to β-(1→6)-linked galactosyl side chains of the AGP were removed in advance. The enzyme released galactose, β-(1→6)-galactobiose, and 4- O-methyl-β-glucuronosyl-(1→6)-galactose as major hydrolysis products when allowed to act exhaustively on the modified AGP.

Characterization of alkaline thermoactive cellulase-free xylanases from alkalophilic Bacillus (NCL 87-6-10).

Two alkaline xylanases designated as "A" and "C", respectively, were isolated from the culture filtrates of the alkalophilic Bacillus grown on a wheat bran-yeast extract medium. The two xylanases occurred in the culture filtrate in a ratio of 10:90. These xylanases were purified to homogeneity on a CM-Sephadex matrix followed by further separation of Xylanase "A" on a phenyl sepharose column and preparative electrophoresis. The two xylanases differed considerably in their physico-chemical properties, kinetics and in their mode of action. Xylanase "C" had a molecular weight of 25,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and was a cationic protein with a pI of 8.9. In contrast xylanase "A" had a molecular weight of 45,000 with a pI of 5.3. The two xylanases showed distinct differences in their hydrolysis pattern. Xylanase "A" produced comparatively larger amounts of small molecular weight oligosaccharides and xylose namely xylotriose (X(3)), xylobiose (X(2)) and xylose even in the initial stages of hydrolysis (2 and 5 h) while xylanase "C" produced negligible amounts of X(2) and no xylose for the same period of incubation. At 24 h only traces of xylose was produced by xylanase "C" while substantial amounts of the monomer was produced by xylanase A in 24 h. Xylanase "A" had a broad pH optimum ranging from pH 6.0-10.0 at 40-60 degrees C while xylanase "C" had an optimum pH of 8.0 at 40-60 degrees C. Xylanases "A" and "C" differed in their K(m) and V(max) values. Xylanase "A" had a K(m) of 1.67 mg/ml and a V(max) of 3.85 x 10(2) micromol/ml/min, whereas xylanase "C" had a K(m) of 10 mg/ml and a V(max) of 1.43 x 10(4) micromol/ml/min.

Alkaline Hydrolysis of Solution-Grown Poly(L-lactic acid) Single Crystals.

The changes in morphology and crystalline state of solution-grown single crystals of poly(L-lactic acid)(PLLA) in aqueous NaOH solution of various strengths were investigated by means of transmission electron microscopy, atomic force microscopy and gel permeation chromatography. The degradations of hexagonal and lozenge shaped PLLA single crystals started from the lateral side of the crystal to generate the notched morphology at initial stage of hydrolysis. As hydrolysis proceeded, the molecular weight of degraded PLLA crystals corresponded to equal the value calculated from lamellar thickness measured by atomic force microscopy, suggesting that the tight chain-folding region of PLLA molecules on crystal surface was degraded by alkaline hydrolysis. Based on the results, alkaline hydrolysis with NaOH solution firstly occurs at the loosely chain-packing region on crystal edges, and then gradually progresses from both crystal edges and tight chain-packing region with chain-folding on the crystal surface.

Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process

Past technoeconomic modeling work has identified the relatively large contribution that enzymatic hydrolysis adds to the total cost of producing ethanol from lignocellulosic substrates. This cost was primarily due to the high concentration of enzyme and long incubation time that was required to obtain complete hydrolysis. Although enzyme and substrate concentration and end-product inhibition influenced the rate of hydrolysis, the effect was less pronounced during the initial stages of hydrolysis. During this time most of the cellulases were adsorbed onto the unhydrolyzed residue. By recycling the cellulases adsorbed to the residual substrate remaining after an initial 24 h, a high rate of hydrolysis, with low overall residence time and minimal cellulase input, could be achieved for several rounds of enzyme recycle. A comparison of the front end (pretreatment, fractionation, and hydrolysis) of a softwood/hardwood to ethanol process indicated that the lignin associated with the softwood-derived cellulose stream limited the number of times the cellulose containing residue could be recycled. (c) 1996 John Wiley & Sons, Inc.

Kinetic specifics of the synthesis of luminescent materials using the sol-gel method

Based on the sol-gel method for producing luminescent silicate materials, the process kinetics is studied for the initial stages of hydrolysis and polycondensation of tetraethoxysiloxane for different temperatures and component ratios. A model of the process is proposed, and reaction rate constants are determined. A conclusion is made on the limiting effect of the polycondensation stage up to the moment of gel formation.

Changes in Breaking Strength and Water Retention of Cellulosic Fibers hydrolyzed by Commercial Cellulases.

The changes in weight loss, breaking strength and water retention of the cellulosic fibers; cotton, viscose rayon filament and cuprammonium rayon filament yarns, by the enzymatic hydrolysis were investigated with the commercial cellulase A and cellulase B from Aspergillus niger and from Trichoderma viride, respectively. Under the hydrolysis conditions used in this study, the surface fibrils of cotton and rayon yarns did not easily eliminated. The cellulase B lost weight at faster rate than the cellulase A for all samples: the weight loss increased in the order viscose rayon < cotton < cuprammonium rayon. In all cases, although the fall in breaking strength was totally progressive with increasing weight loss, the relationship between the breaking strength and weight loss appears to be markedly affected by the fine structure of cellulosic fiber. Retention of strength after 240 min incubation time increased in the order cotton < cuprammonium rayon < viscose rayon. It was especially noted, in the case of cellulase B, that there was the range of weight loss which the strength remains constant for all samples. Water retention value of hydrolyzed yarns was found to reach a minimum at initial stages of hydrolysis and then increase at more advanced stages of hydrolysis in all cases.

Alkaline Hydrolysis of Solution-Grown Poly[(R)-3-hydroxybutyrate] Single Crystals

The changes in morphology and crystalline state of solution-grown single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) in NaOH solution of various strengths were investigated by means of transmission electron microscopy, atomic force microscopy, and gel permeation chromatography. Lath-shaped P(3HB) single crystals, which had widths of 2−3 μm, were split to narrow ribbonlike crystals of around 0.1 μm at the initial stage of hydrolysis without a decrease in lamellar thickness. As hydrolysis time increased, ribbonlike crystals were degraded from the lateral side of the crystal to generate the notched morphology. Furthermore, the molecular weight of degraded P(3HB) crystals corresponded to equal the value calculated from lamellar thickness measured by atomic force microscopy, suggesting that the tight chain-folding region of P(3HB) molecules on the crystal surface was degraded by alkaline hydrolysis. This surface degradation was further supported by the disappearance in the electron micrograph of straight lines on the surface of the degraded crystal decorated with polyethylene. Despite the unselective degradation of alkaline hydrolysis from both crystal edges and crystal surfaces, narrow ribbonlike crystals were generated at the initial stage of hydrolysis. This observation suggests that single crystals with chain-folding have two regions of tight chain-packing region with adjacent reentry folds and a loosely chain-packing region with random reentry folds with interval of 100 nm or less. The model developed in this paper suggests that alkaline hydrolysis with NaOH solution first occurs at the loosely chain-packing region on crystal surface and then gradually progresses from both crystal edges and tight chain-packing region with chain-folding on the crystal surface.

The effect of fiber characteristics on hydrolysis and cellulase accessibility to softwood substrates

The effect of gross fiber characteristics on enzyme accessibility and hydrolysis of Douglas fir kraft pulp substrates was investigated. The average fiber size and coarseness of the substrate had a significant effect on the enzyme adsorption capacity. This was primarily due to the increased specific surface area of small fibers and fines. The observed adsorption capacities were in agreement with the hydrolysis rates and yields because the substrates with the lower average fiber size were hydrolyzed both at a faster rate and more completely. The observed changes in fiber-length distribution and fiber coarseness suggested that the effect of fiber size was most influential during the initial stages of hydrolysis. The small fibers and fines present in heterogeneous, lignocellulosic substrates were hydrolyzed rapidly, yielding a high initial rate of hydrolysis.