Initial Rate Of Hydrolysis Research Articles

Enzymatic hydrolysis of delignified bagasse polysaccharides

Sugarcane bagasse, consisting of cellulose, xylan, and lignin, was chemically treated to generate bagasse samples with continuously decreasing content of lignin. These bagasse samples were hydrolyzed by cellulase and xylanase enzymes, produced earlier by Penicillium janthinellum NCIM 1171 in the same bagasse polysaccharides production medium. The hydrolysis was carried out by using different concentrations of the enzymes at two different temperatures, 30 and 50 °C, taking hydrolysis of Avicel as control. It was found that while the maximum hydrolysis for Avicel was 70% that of some of the bagasse polysaccharides was as high as 95%. The products of hydrolysis were glucose, xylose, and arabinose, as confirmed by high pressure ion chromatography (HPIC). It is interesting to note that arabinose, which constitutes about 10% of the weight of bagasse xylan, could also be released easily by the enzymes. Also, the initial rates of hydrolysis was found to be much higher for the bagasse polysaccharides, and in some cases about 90% of the hydrolysis occurred within 20 h. Amongst all bagasse samples, the sample with (Kappa no. 1.2, lignin content 0.18%) gave the highest degree of hydrolysis at 50 °C. Even the bagasse polysaccharide with Kappa no. 16.8 (lignin content 2.5%) underwent greater extent of hydrolysis than Avicel. Apparently, the delignified bagasse medium appears to be a facile medium for the combined hydrolytic action of the cellulase and xylanase enzymes. Considering that sugarcane bagasse is a waste biomass material available in abundance annually, this methodology can be used to value-add to this biomass to produce sugars, which can be fermented to produce biofuels like ethanol.

Chemical Composition and Starch Hydrolysis of Acacia colei and Acacia tumida Seeds

ABSTRACTAcacia seed is a promising famine food due to its ability to thrive in dry zones of the world. In this study, some chemical compounds of nutritional importance as well as starch hydrolysis in three cultivars of Acacia tumida and A. colei were determined. The crude protein was 20.6–23.0%, while extractable protein was 11.5–17.5%. Total dietary fiber (TDF) (28.5–32.7%) and soluble dietary fiber (SDF) (2.2–5.8%) were higher than values reported for most legumes. Reducing sugar range was 31.0–54.5 mg/g, while total sugar range was 137–161 mg/g. The starch content of the seed was 25.6–32.3%. The samples did not contain any alkaloid but did contain saponins. Phytate and trypsin inhibitor contents were low; oxalate was fairly high (2.2–2.6 g/100 g), but tannin was on the high side 66.0–86.7 mg/g) compared with legumes. In vitro starch digestibility was highest in A. tumida (III). Using a first‐order kinetic equation, A. colei showed the highest initial rate of starch hydrolysis (t1/2 = 411.56 and 1,893.4 min), followed by A. tumida (III) (539.25 and 1,738.1 min); the lowest vales were for A. tumida (II) (1,764.99 and 4,249.8 min) for both methods of starch digestion.

Ammonia-catalyzed hydrolysis kinetics of mixture of tetraethoxysilane with methyltriethoxysilane by 29Si NMR

In situ liquid 29Si NMR was used to investigate the ammonia-catalyzed hydrolysis and condensation of tetraethoxysilane (TEOS) and methyltriethoxysilane (MTES) mixed systems with methanol as solvent. Varying the molar ratio of TEOS, MTES, water and ammonia in original solution, the initial hydrolysis rate constants of TEOS and MTES were calculated as well as the corresponding reactive orders. Under ammonia catalysis, the hydrolysis of TEOS and MTES in mixed system all retained the first-order reaction, similar to the cases of their individual systems. The hydrolysis rate constant of TEOS in mixed system is larger than that in its single system. Meanwhile, the hydrolysis rate constant of MTES in mixed system was smaller than that in its single system. Furthermore the condensation rate constants of TEOS and MTES in mixed system all decreased when compared to the individual condensation rate constants of MTES and TEOS. Another important result was: the reactive orders of both ammonia and water increased to different extent for TEOS and MTES in mixed system. Hydrolysis kinetics shows more compatible hydrolysis–condensation relative rates between TEOS and MTES. Assisted by the solid 29Si MAS NMR and XPS, the obtained hybrid gel showed weak cross-condensation between TEOS and MTES. With the small angle X-ray scattering (SAXS), the double fractal structure in particulate networks was disclosed to be resulted from the more compatible hydrolysis–condensation relative rates between TEOS and MTES.

Screening ofExiguobacterium acetylicum from soil samples showing enantioselective and alkalotolerant esterase activity

About 3,000 bacterial colonies with esterase activities were isolated from soil samples by enrichment culture and halo-size on Luria broth-tributyrin (LT) plates. The colonies were assayed for esterase activity in microtiter plates using enantiomerically pure (R)- and (S)-2-phenylbutyric acid resorufin ester (2PB-O-res) as substrates. Two enantioselective strains (JH2 and JH13) were selected by the ratio of initial rate of hydrolysis of enantiomerically pure (R)- and (S)-2-PB-O-res. When cell pellets were used, both strains showed hgh apparent enantioselectivity (Eapp>100) for (R)-2PB-O-res and were identified asExiguobacterium acetylicum. The JH13 strain showed high esterase activity onp-nitrophenyl acetate (pNPA), but showed low lipase activity onp-nitrophenyl palmitate (pNPP). The esterase was located in the soluble fraction of the cell extract. The crude intracellular enzyme preparation was stable at a pH range from 6.0 to 11.0.

In vitro digestion rate and resistant starch content of tortillas stored at two different temperatures

In vitro indicators of starch bioavailability were evaluated in freshly prepared maize tortillas and compared to those exhibited by 24, 48 or 72 h-stored samples. Storage took place either at room temperature (approx. 25 °C) or under refrigeration (4 °C). Potentially available starch (AS) content decreased from 670 g kg −1 in the control tortilla to 583 g kg −1 in 72 h-stored preparations. Concomitant increases in total resistant starch (RS) and retrograded resistant starch (RRS) were recorded upon storage. RRS content in 72 h-stored samples (35–39 g kg −1) doubled that of freshly prepared tortillas. Changes in AS, RS and RRS were not affected by storage temperature. Both initial rate and final point of starch hydrolysis by pancreatic amylase were reduced in samples kept for 48 and 72 h, without influence of storage temperature. Storage length is suggested as a major determinant of the bioavailability of starch in tortillas.

Inhibition of Autotaxin by Lysophosphatidic Acid and Sphingosine 1-Phosphate

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Mechanistic Plasticity of DNA Topoisomerase IB: Phosphate Electrostatics Dictate the Need for a Catalytic Arginine

Four conserved amino acids of type IB topoisomerases (Arg130, Lys167, Arg223, and His265 in vaccinia topoisomerase) catalyze the attack by tyrosine on the scissile phosphodiester to form a DNA-(3'-phosphotyrosyl)-enzyme intermediate. The mechanism entails general acid catalysis (by Lys167 and Arg130) and transition-state stabilization (via contact of His265 with the pro-Sp oxygen). Here we query the function of Arg223, which accelerates transesterification by a factor of 10(5). The requirement for Arg223 is alleviated by a neutral Sp methylphosphonate (MeP) linkage at the cleavage site. Arg223 is not required for the 30,000-fold activation of the latent endonuclease activity of topoisomerase by the Sp MeP. The rate of autohydrolysis by the DNA-(3'-MeP)-topoisomerase intermediate approaches 10% of the rate of religation to a 5'-OH DNA strand. These findings underscore the importance of transition-state electrostatics in determining the composition of the active site and dictating the balance between strand transferase and hydrolase functions.

The effect of inorganic pyrophosphate on the activity and Pi-binding properties of mitochondrial F1-ATPase

Interaction of F1-ATPase from beef heart mitochondria with PPi has been investigated. The presence of PPi in the ATPase assay medium does not affect the initial rate of ATP hydrolysis by F1-ATPase, but slows down the decrease of enzyme activity in the course of ATP hydrolysis and increases the steady-state rate of ATP hydrolysis. Being present in the ATPase assay medium, PPi accelerates the ATP-dependent reactivation of an inactive complex formed by F1-ATPase and ADP. This inactive complex is also reactivated after preincubation with PPi. F1-ATPase, preincubated with PPi, is inactivated by azide much more slowly than is the non-preincubated enzyme. PPi stimulates the binding of Pi to F1-ATPase by decreasing mainly the Kd for Pi and only slightly raising the stoichiometry of high-affinity Pi binding. It follows from the results obtained that PPi interacts with the non-catalytic site(s) of F1-ATPase.

Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate

The initial rate and enantioselectivity of enzymatic asymmetric hydrolysis of amino acid esters were examined in methylimidazolium-based ionic liquids with anions including tetrafluoroborate, chloride, bromide and bisulfate and in typical organic solvents. Papain displayed much higher enantioselectivity but lower activity in phosphate buffer solution of 1-butyl-3-methylimidazolium tetrafluoroborate BMIM·BF4 than in other media tested (i.e. E=100, V0=0.21 mM min−1 in BMIM·BF4, E=2, V0=0.43 mM min−1 in phosphate buffer, E=14–92, V0=0.22–0.25 mM min−1 in organic solvents for D,L-phenylglycine methyl ester). The influence of BMIM·BF4 on enzyme activity and enantioselectivity also varied with the substrate and the enzyme used. All of the enzymes assayed showed no activity or low enantioselectivity in the ILs with anions including chloride, bromide and bisulfate.

Cutinase activity in supercritical and organic media: water activity, solvation and acid–base effects

We performed a comparative study on the activity of Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY, in n-hexane, acetonitrile, supercritical ethane (sc-ethane) and sc-CO 2, at two different water activity ( a W) values set by salt hydrate pairs in situ and at acid–base conditions fixed with solid-state buffers of aqueous p K a between 4.3 and 10.6. The reaction studied was the transesterification of vinyl butyrate by ( R,S)-2-phenyl-1-propanol. The transesterification activity of cutinase was highest and similar in sc-ethane and in n-hexane, about one order of magnitude lower in acetonitrile and even lower in sc-CO 2. Activity coefficients ( γ) generated for the two substrates indicated that they were better solvated in acetonitrile and thus less available for binding at the active site than in the other three solvents. γ data also suggested higher reaction rates in sc-ethane than in n-hexane, as observed, and provided evidence for a direct negative effect of sc-CO 2 on enzyme activity. Manipulation of the acid–base conditions of the media did not afford any improvement of the initial rates of transesterification relative to the blanks (no added acid–base buffer, only salt hydrate pair), except in the case of cutinase immobilized on zeolite NaA in sc-ethane at a W = 0.7. The poor performance of the blank in this case and the great improvement observed in the presence of a basic buffer suggest a deleterious acidic effect in the medium which, an experiment without additives confirmed, was not due to the known acidic character of the salt hydrate pair used to set a W = 0.7. In acetonitrile, increasing a W was accompanied by a decrease in initial rates of transesterification, unlike in the other solvents. There was considerable hydrolysis in acetonitrile, where initial rates of hydrolysis increased about 20-fold from a W = 0.2 to 0.7. Hydrolysis was less pronounced in sc-ethane and in n-hexane, and only at a W = 0.7, and in sc-CO 2 butyric acid was detected only at very long reaction times, in agreement with a generally low catalytic activity. Cutinase enantio-selectivity towards the alcohol substrate was low and unaffected by any manipulation of medium conditions.

Effects of high pressure and microwave on pronase and α-chymotrypsin hydrolysis of β-lactoglobulin

The effects of hydrostatic high pressure, microwave irradiation, and conventional heating on hydrolysis of bovine β-lactoglobulin AB by pronase and α-chymotrypsin were studied. The high pressure treatments were performed between 100 and 300 MPa, the microwave irradiation was applied at different power conditions, pronase (30 W) and α-chymotrypsin (15 W). All enzymatic reactions were carried out during 10 and 20 min, at 40 °C. The initial rate of pronase and α-chymotrypsin hydrolysis of protein was increased by using either physical treatment during the enzymatic digestion. The microwave-assisted digestion by pronase was more effective than those performed under conventional heating. The only apparent microwave induced difference on protein digestion by α-chymotrypsin was in a lower relative concentration of peak eluting at 24.5 min, and the conversion of this fraction into final peaks eluting from 24.5 to 28.5 min (F1 fraction) after 20 min of incubation. Under high pressure, the β-lactoglobulin AB is completely hydrolyzed by pronase and α-chymotrypsin.

Enzymatically Amplified Surface Plasmon Resonance Imaging Method Using RNase H and RNA Microarrays for the Ultrasensitive Detection of Nucleic Acids

A novel surface enzymatic amplification method that utilizes RNA microarrays in conjunction with the enzyme RNase H is developed for the ultrasensitve detection and analysis of target DNA molecules. The enzyme RNase H is shown to selectively and repeatedly destroy RNA from RNA-DNA heteroduplexes on gold surfaces; when used in conjunction with the label-free technique of surface plasmon resonance imaging, multiple DNA targets can be detected at a concentration of 10 fM on a single chip. In addition, this method is utilized for the sequence-specific detection of the TSPY gene in both purified and unpurified PCR products. Finally, in a series of kinetics measurements, the initial rate of hydrolysis is shown to depend directly on the surface concentration of DNA-RNA heteroduplexes.

Comparative study on the hydrolysis kinetics of substituted ethoxysilanes by liquid-state 29Si NMR

Liquid-state 29Si NMR was used to investigate the ammonia-catalyzed initial hydrolysis of different substituted ethoxysilanes dissolved in methanol. The following ethoxysilanes were used: tetraethoxysilane (TEOS), methyltriethoxysilane (MTES) and dimethyldiethoxysilane (DBS). Their reaction expresses were obtained by assuming first-order in the reactant ethoxysilanes and varying other reactant ammonia and water concentrations. Individual kinetic rate constants for the initial hydrolysis of each ethoxysilanes, the rate-limiting step for total reactions, were calculated, which were independent of any reactant concentration. The results indicated that the steric factors have more important effect on the reactive reactivity than inductive factors, so that the initial hydrolysis rate constants for the studied ethoxysilanes decreased in an unusual order DDS > TEOS > MTES. For these three ethoxysilanes, a common conclusion was drawn that the substitution of the hydroxyl group for the ethoxyl group resulted in a downfield shift. In the case of DDS, however, the trend was opposite to the previous studies in which the hydrolyzed resultants had upfield shift compared with DDS monomer.

Molecular engineering of Rhizopus oryzae lipase using a combinatorial protein library constructed on the yeast cell surface

We constructed a combinatorial yeast library through cell-surface display of the pro- and mature region of lipase from Rhizopus oryzae (ProROL) and obtained clones retaining lipase activity in fluorescent plate assay. The initial reaction rates of hydrolysis and methanolysis could be measured directly as whole-cell biocatalyst without complex treatments such as concentration, purification, and immobilization. The selected clones showed wide-ranging variation of reaction specificity. The K138R mutant showed a 1.3-fold shift of reaction specificity toward methanolysis compared to the wild type, while the V-95D, I53V, P-96S/F196Y, and Q128H/Q197L mutants showed shifts toward hydrolysis of 1.6–5.9-fold. Predictions of the mutants’ three-dimensional structure suggested that the hydrogen-bond distance between threonine 83 and aspartic acid 92 may influence reaction specificity, which shifted toward hydrolysis in mutants where this distance was shorter than in the wild type, but toward methanolysis where it was longer. The positions of amino acid residues (aa) 53, 138 and 196 in ProROL are considered the sites that influence hydrogen-bond distance and change reaction specificity. Construction of a surface-displayed combinatorial library in yeast cells is thus a powerful tool in accelerating the combinatorial approach to enzyme engineering and novel whole-cell biocatalyst development.

Molecular engineering of Rhizopus oryzae lipase using a combinatorial protein library constructed on the yeast cell surface

We constructed a combinatorial yeast library through cell-surface display of the pro- and mature region of lipase from Rhizopus oryzae (ProROL) and obtained clones retaining lipase activity in fluorescent plate assay. The initial reaction rates of hydrolysis and methanolysis could be measured directly as whole-cell biocatalyst without complex treatments such as concentration, purification, and immobilization. The selected clones showed wide-ranging variation of reaction specificity. The K138R mutant showed a 1.3-fold shift of reaction specificity toward methanolysis compared to the wild type, while the V-95D, I53V, P-96S/F196Y, and Q128H/Q197L mutants showed shifts toward hydrolysis of 1.6–5.9-fold. Predictions of the mutants’ three-dimensional structure suggested that the hydrogen-bond distance between threonine 83 and aspartic acid 92 may influence reaction specificity, which shifted toward hydrolysis in mutants where this distance was shorter than in the wild type, but toward methanolysis where it was longer. The positions of amino acid residues (aa) 53, 138 and 196 in ProROL are considered the sites that influence hydrogen-bond distance and change reaction specificity. Construction of a surface-displayed combinatorial library in yeast cells is thus a powerful tool in accelerating the combinatorial approach to enzyme engineering and novel whole-cell biocatalyst development.

Effect of Additional Hydroxyl Functionalities on the Hydrolytic Stability of Oligoesters

AbstractSummary: The addition of a trifunctional polyol (trimethylolpropane) to oligoesters comprised of hexahydrophthalic anhydride, cyclohexane‐1,4‐dicarboxylic acid, cyclohexane‐1,3‐dicarboxylic acid, neopentyl glycol, and cyclohexane‐1,4‐dimethanol was investigated via an accelerated hydrolysis environment. The oligoesters were dissolved in an acetone/water solution, and the hydrolysis was evaluated by monitoring carboxylic acid formation and by measurement of the molecular weight shift by size exclusion chromatography. Aliquots of the oligoester solution were evaluated intermittently over a 36‐week period. Both the initial rate and the overall rates of hydrolysis were reported as hydrolysis velocities. The influence of hydroxyl concentration is apparent in the initial velocity of hydrolysis. After the induction period, steric effects appear to be the controlling factor for the retardation of main chain scission.A comparison of the initial and final rates of hydrolysis as a function of oligoester composition.imageA comparison of the initial and final rates of hydrolysis as a function of oligoester composition.

Effect of operating variables on the hydrolysis rate of palm oil by lipase

Hydrolysis of palm oil by lipase enzyme in aqueous phase was studied in a well stirred bioreactor under defined mixing conditions. Agitation speed, concentration of the surfactant, palm oil, and the enzyme were varied to study their effect on the initial rate of hydrolysis. Addition of surfactant, gum arabic, increased the rate of hydrolysis and its optimum amount was found to be 25 mg l −1. The optimum enzyme loading was found to be 92.5 kLU l −1. Studies on the effect of agitation speed and substrate concentration showed that the initial rate of hydrolysis is dependent on the interfacial area between the oil phase and the aqueous phase containing the enzyme. No substrate inhibition was observed in the range of palm oil concentration 0–262 g l −1, used in this study.

Humicola lanuginosa lipase hydrolysis of mono-oleoyl-rac-glycerol at the lipid–water interface observed by atomic force microscopy

A new type of planar lipid substrate for Humicola lanuginosa lipase (HLL) has been prepared by depositing a monolayer of 1-mono-oleoyl-rac-glycerol (MOG) on top of a monolayer of dipalmitoyl-phosphatidylcholine (DPPC) on mica by the Langmuir–Blodgett (LB) technique. The bilayer was subsequently exposed to HLL in a liquid cell of an atomic force microscope (AFM) allowing the time course of the lipolytic degradation to be observed. By analysing a series of AFM images, we find that enzymes are preferentially activated at the edge of nano-scale defects present in the bilayer prior to enzyme injection, while defect-free areas of the substrate are surprisingly stable towards enzyme degradation. The initial rate of hydrolysis is found to be proportional to the perimeter length, P, of the initial nano-scale defects as well as the bulk enzyme concentration, c HLL; d(lipid)/d t= k P c HLL. We estimate the specific rate of MOG hydrolysis by HLL to be 2.5×10 4 MOG molecules/(minute·molecule of HLL).

Nested cooperativity and salt dependence of the ATPase activity of the archaeal chaperonin Mm-cpn

The properties of the ATPase activity of the type II chaperonin from Methanococcus maripaludis (Mm-cpn) were examined. Mm-cpn can hydrolyze not only ATP, but also CTP, UTP, and GTP, albeit with different effectiveness. The ATPase activity is dependent on magnesium and potassium ions, and is effectively inhibited by sodium ions. Maximal rates of ATP hydrolysis are achieved at 600 mM potassium. Initial rates of ATP hydrolysis by Mm-cpn were determined at various ATP concentrations, revealing for the first time the presence of both positive intra-ring and negative inter-ring cooperativity in the archaeal chaperonin.

Effects of water activity and alcohol concentration on the kinetic resolution of lipase-catalyzed acyl transfer in organic solvents

A lipase-catalyzed enantioselective thiotransesterification between ( R, S)-naproxen 2,2,2-trifluoroethyl thioester (STF S ) and 4-morpholine ethanol in isooctane was employed to produce ( S)-naproxen ester prodrug by controlling the initial water activity and considering the hydrolysis and esterification side-reactions. Good enzyme enantioselectivity with more than 90% of enantiomeric excess for the desired product was obtained when initial water activity and alcohol concentration were controlled at 0.113 and 10 mM, respectively. A detailed analysis of the apparent equilibrium constant and ratio of initial rates of thiotransesterification and hydrolysis varied with the water activity and alcohol concentration indicated that water and alcohol might act as modifiers in manipulating the enzyme active center aside from their conventional roles as acyl acceptor and enzyme inhibitor.