The effect of microwave irradiation on enzymatic hydrolysis of rice straw

A series of experiments involving microwave irradiation were carried out to evaluate the effect of microwave irradiation on enzymatic hydrolysis of rice straw. Compared with microwave irradiation free hydrolysis, rice straw pretreated by combining microwave irradiation with alkali could increase the initial hydrolysis rate but the hydrolysis yield remained unchanged. When the enzyme solution was treated by microwave irradiation, the initial hydrolysis rate increased slightly, but the yield was decreased remarkably. Its optimal hydrolysis conditions were temperature (45 degrees C), pH (4.8) and enzyme loading (20 mg g(-1) substrate), which was determined by an orthogonal experiment. When intermittent microwave irradiation was used, initial hydrolysis rate was greatly accelerated but the yield was decreased slightly. Its optimal hydrolysis conditions were temperature (50 degrees C), pH (4.8) and enzyme loading (20 mg g(-1) substrate), which was determined by another orthogonal experiment.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times

The kinetics of enzymatic hydrolysis of pure insoluble cellulose by means of unpurified culture filtrate of Trichoderma reesei was studied, emphasizing the kinetic characteristics associated with the extended hydrolysis times. The changes in the hydrolysis rate and extent of soluble protein adsorption during the progress of reaction, either apparent or intrinsic, were investigated. The hydrolysis rate declined drastically during the initial hours of hydrolysis. The factors causing the reduction in the hydrolysis rate were examined; these include the transformation of cellulose into a less digestible form and product inhibition. The structural transformation can be partially explained by changes in the crystallinity index and surface area. The product inhibition was caused by the deactivation of the adsorbed soluble protein by the products, which essentially represents the so-called "un-competitive" inhibition. The kinetics of beta-glucosidase were also studied. The result has shown that the action of beta-glucosidase is competitively inhibited by glucose. It has been found that the integrated form of the initial rate expression cannot be used in predicting the progress of reaction because the digestibility of cellulose changes drastically as the hydrolysis proceeds, and that the rate expression for enzymatic hydrolysis of cellulose cannot be simplified or approximated by resorting to the pseudo-steady-state assumption. A mechanistic kinetic model of cellulose hydrolysis should include the following major influencing factors: (1)mode of action of enzyme, (2) structure of cellulose, and (3) mode of interaction between the enzyme and cellulose molecules.     

Effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase in a capillary bed reactor

The effects of temperature on the hydrolysis of lactose by immobilized beta-galactosidase were studied in a continuous flow capillary bed reactor. Temperature affects the rates of enzymatic reactions in two ways. Higher temperatures increase the rate of the hydrolysis reaction, but also increase the rate of thermal deactivation of the enzyme. The effect of temperature on the kinetic parameters was studied by performing lactose hydrolysis experiments at 15, 20, 25, 30, and 40 degrees C. The kinetic parameters were observed to follow an Arrhenius-type temperature dependence. Galactose mutarotation has a significant impact on the overall rate of lactose hydrolysis. The temperature dependence of the mutarotation of galactose was effectively modelled by first-order reversible kinetics. The thermal deactivation characteristics of the immobilized enzyme reactor were investigated by performing lactose hydrolysis experiments at 52, 56, 60, and 64 degrees C. The thermal deactivation was modelled effectively as a first order decay process. Based on the estimated thermal deactivation rate constants, at an operating temperature of 40 degrees C, 10% of the enzyme activity would be lost in one year.     

Lipase-catalyzed hydrolysis of 2-naphtyl esters in biphasic system

The authors measured the rate of hydrolysis of the homologs of 2-naphtyl ester by using a Lewis cell with constant interfacial area to elucidate the kinetic mechanism of the lipase-catalyzed hydrolysis in biphasic system. On the basis of the two-film model, it was found from the analysis of experimental results that the hydrolysis of these substrates proceeds at the interface between the aqueous and organic phases. The interfacial reaction rate could be correlated by Michaelis-Menten mechanism. The values of the rate constant and the Michaelis constant were almost independent of the kinds of 2-naphtyl ester. The values of the interfacial kinetic parameters for 2-naphtyl ester were much greater than those for the hydrolysis in the aqueous phase.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Kinetics of chitinase production. I. Chitin hydrolysis

As part of the development of a comprehensive mathematical model for chitinase production by Serratia marcescens QMB 1466 growing on chitin, the different mass transport and kinetic steps involved during chitin hydrolysis were studied. The experimental results for the hydrolysis of chitin by a crude preparation of chitinase show a system kinetically limited by the overall rate of chitin hydrolysis. This rate is linearly related to the concentration of enzyme adsorbed on the chitin particle. Adsorbed and bulk enzyme concentration were found to be related through a Langmuir type of isotherm.     

Enzymatic degradation of monolayer for poly(lactide) revealed by real-time atomic force microscopy: effects of stereochemical structure, molecular weight, and molecular branches on hydrolysis rates

The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.     

Role of water activity on the rates of acetyl phosphate and ATP hydrolysis

The rates of hydrolysis of acetyl phosphate in the presence of 0.1 M NaOH and of ATP in the presence of either 1 M HCl or 1 M NaOH were measured at different temperatures and in the presence of different concentrations of the organic solvents dimethyl sulfoxide or ethylene glycol. Under all conditions tested, there was a progressive increase in the rate constant of hydrolysis of both phosphate compounds as the water activity of the medium was decreased by the addition of organic solvents. At 25 degrees C, substitution of 70% of the water of the medium by dimethyl sulfoxide promoted an increase of two orders of magnitude in the rate constant of acetyl phosphate hydrolysis. In the presence of 80% and 90% dimethyl sulfoxide the rate of acetyl phosphate hydrolysis increased by more than two orders of magnitude and was so fast that it could not be measured with the method used. The effect of organic solvents on the rate of ATP hydrolysis was less pronounced than that observed for acetyl phosphate hydrolysis. At 30 degrees C, substitution of 90% of water by an organic solvent promoted a 4-6-fold increase of the rate of ATP hydrolysis. Acceleration of either acetyl phosphate or ATP hydrolysis rates was promoted by a decrease in both activation energies (Ea) and in entropies of activation delta S. The data obtained are discussed with reference to the mechanism of catalysis of enzymes involved in energy transduction such as the Ca2+-ATPase of sarcoplasmic reticulum and the F1-ATPase of mitochondria.     

Asparaginase production by human clinical isolates of Vibrio succinogenes.

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.     

Enzymatic hydrolysis of ATP and e-ATP by F-actin

Enzymatic hydrolysis of e-ATP by F-actin with and without application of sonic vibration at various pHs was investigated and compared with that of ATP. These was no significant difference on enzymatic activity between F-actin-bound e-ADP and F-actin-bound ADP. The hydrolysis rate of e-ATP under sonic vibration decreases monotonically with decreasing pH, similar to that of ATP. The magnitude of e-ATP hydrolysis rate was, however, about one third of that of ATP hydrolysis rate in the pH range between 6.3 and 8.5. Enzymatic hydrolysis of e-ATP without sonic vibration at room or higher temperatures decreases monotonically with increasing pH and becomes almost negligible at pH 8.5. The pH profile and the magnitude of enzymatic hydrolysis without sonic vibration were similar with ATP. Since the fluorescence intensity of e-ATP at 410 nm is enhanced by the binding with G-actin, the exchange binding affinity of e-ATP to G-actin which can be measured fluorophotometrically was about one third of that of ATP.     

Kinetic study on enzymatic hydrolysis of cellulose by cellulose from Trichoderma viride

Pure cellulose (Avicel) was hydrolyzed batchwise at 50 degrees C and pH 4.8 by cellulase from Trichoderma viride (Meicelase CEP). Then the effects of the crystallinity of cellulose as well as the thermal deactivation and product (cellubiose and glucose) inhibition to cellulose on the hydrolysis rate were quantitatively investigated. While these factor had evidently retarded the enzymatic hydrolysis of cellulose to a significant extent, the hydrolysis rates observed could not be explained. For practical purposes, an empirical, simple rate expression was developed which included only one parameter: a overall rate retardation constant. This empirical rate expression held for the hydrolysis of at least two kind of cellulosic materials: Avicel and tissue paper.     

Asparaginase production by human clinical isolates of Vibrio succinogenes

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.     

Factors affecting the hydrolysis of ceramide-3 by alpha-galactosidase A from human liver.

1. The effect of detergents on the catalytic properties of alpha-galactosidase from human liver was studied using p-nitrophenyl-alpha-galactoside and galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosylceramide (ceramide-3) as substrates. 2. The hydrolysis of p-nitrophenyl-alpha-galactoside by alpha-galactosidase was inhibited by commercial preparations of sodium taurocholate and by taurocholate purified from these preparations by thin-layer chromatography. The extent of inhibition was dependent on the concentration of the detergent and on the amount of protein present. The impurities present in the preparation also inhibited the hydrolysis. 3. The inhibition of taurocholate preparations of p-nitrophenyl-alpha-galactoside hydrolysis was pH-dependent. 4. The inhibition by taurocholate of p-nitrophenyl-alpha-galactoside hydrolysis can be partly overcome by adding glycosphingolipids. 5. No significant hydrolysis of ceramide-3 occurs in the absence of detergent. Upon adding increasing concentrations of taurocholate, the rate of hydrolysis increases to a maximum value. At still higher taurocholate concentrations the activity decreases. 6. The concentrations of taurocholate giving a maximal rate of hydrolysis of ceramide-3 is dependent on the amount of protein present and independent of the ceramide-3 concentration. 7. When the pH dependence of the rate of hydrolysis of ceramide-3 was measured in the presence of a commercially available preparation of pure taurocholate or of crude taurocholate, curves with different shapes were obtained.     

Kinetic fluorescence measurement of fluorescein di-beta-D-galactoside hydrolysis by beta-galactosidase: intermediate channeling in stepwise catalysis by a free single enzyme

Kinetic fluorescence measurements were employed to quantitative to stepwise hydrolysis of fluorescein di-beta-D-galactoside (FDG) by beta-galactosidase and the intermediate fluorescein mono-beta-D-galactoside (FMG) channeling. The kinetic parameters, Michaelis-Menten constant Km and enzymatic catalysis rate k2, for FDG hydrolysis to FMG by beta-galactosidase were obtained as 18.0 microM and 1.9 mumol.(min-mg)-1, respectively. The FMG intermediate is hydrolyzed via two modes: (1) FMG that is in free solution binding to the enzyme substrate binding site in competition with FDG and then being hydrolyzed (binding mode); (2) FMG being directly hydrolyzed into the final products of fluorescein and galactose before the FMG can diffuse away from the enzyme active site (channeling mode). The extent of the FMG channeling mode was found to depend on the FDG hydrolysis rate but to be independent of the free enzyme concentration. A channeling factor, defined as the ratio of the real FMG hydrolysis rate with both binding and channeling modes over that which would be observed with an exclusive binding mode, was used to quantitate the effect of the intermediate channeling. The FMG channeling factor was determined to be close to 1 at low FDG concentration (about 5.1 microM), where the slow FDG hydrolysis rate gives an ineffective channeling and where the FMG is then hydrolyzed mainly via the binding mode. However, the channeling factor dramatically increases at higher FDG concentrations (greater than Km), strongly indicating that the effective FMG channeling mode, resulting from the considerable FDG hydrolysis rate at high FDG concentrations, becomes a primary pathway to channel a steady system hydrolysis with a high rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

从葫芦巴种子中提取薯蓣皂甙元方法的研究

为探明葫芦巴种子中药用活性成分薯蓣皂甙元提取的最佳方法,设置了不同的酸解液浓度及酸解时间以了解葫芦巴粉酸解的适宜条件;并在酸化工艺前将脱脂的葫芦巴粉进行自然发酵或接种曲霉发酵以提高薯蓣皂甙元的提取率。结果表明葫芦巴粉酸解液最佳浓度为15%的硫酸溶液,最适宜的酸解时间为6 h。在酸化工艺前增加发酵工艺,可以显著提高有效成份薯蓣皂甙元的提取率,接霉菌发酵比直接酸化提高62%,自然发酵比直接酸化提高48%。     

Changes in the enzymatic hydrolysis rate of Avicel cellulose with conversion

The slow down in enzymatic hydrolysis of cellulose with conversion has often been attributed to declining reactivity of the substrate as the more easily reacted material is thought to be consumed preferentially. To better understand the cause of this phenomenon, the enzymatic reaction of the nearly pure cellulose in Avicel was interrupted over the course of nearly complete hydrolysis. Then, the solids were treated with proteinase to degrade the cellulase enzymes remaining on the solid surface, followed by proteinase inhibitors to inactive the proteinase and successive washing with water, 1.0 M NaCl solution, and water. Next, fresh cellulase and buffer were added to the solids to restart hydrolysis. The rate of cellulose hydrolysis, expressed as a percent of substrate remaining at that time, was approximately constant over a wide range of conversions for restart experiments but declined continually with conversion for uninterrupted hydrolysis. Furthermore, the cellulose hydrolysis rate per adsorbed enzyme was approximately constant for the restart procedure but declined with conversion when enzymes were left to react. Thus, the drop off in reaction rate for uninterrupted cellulose digestion by enzymes could not be attributed to changes in substrate reactivity, suggesting that other effects such as enzymes getting "stuck" or otherwise slowing down may be responsible.     

酶解鹿茸血ACE抑制活性及其与抗氧化活性关系的研究

实验采用木瓜蛋白酶和Alcalase碱性蛋白酶水解鹿茸血,在不同的时间取样,分别测定血管紧张素转化酶(ACE)抑制率、二苯代苦味肼基自由基(DPPH.)清除率和羟自由基(.OH)清除率。结果表明Alcalase蛋白酶水解产物ACE抑制活性和DPPH.清除活性较强,其水解3h的产物ACE抑制率为90.63%,DPPH.清除率为14.40%;木瓜蛋白酶水解5h的产物.OH清除率最高,为96.7%。用SPSS10.0软件分析结果,发现ACE抑制率与DPPH.清除率具有显著相关性。     

Effects of N-acetylation degree on N-acetylated chitosan hydrolysis with commercially available and modified pectinases

Three types of N-acetylated chitosans (NACs) with different degrees of acetylation (DA) were prepared and used as a substrate for enzymatic hydrolysis with a commercially available pectinase and a modified one. Pectinase modification was conducted using polyalkyleneoxide-maleic anhydride copolymer (PEO-MA copolymer). The effects of DA on enzymatic reaction with native and modified pectinases were investigated experimentally. Initial hydrolysis rate and Michaelis-Menten kinetic parameters were measured by analysis of reducing sugars. DA of NAC strongly affected the hydrolytic characteristics of native and modified pectinases. N-acetylation of chitosan increased the initial hydrolysis rate and the enzyme-substrate affinity with respect to both pectinases: NACs with DA over 0.3 showed high initial hydrolysis rate and strong affinity between enzyme and substrate. Especially, when NAC with DA over 0.3 was treated with modified pectinase, the affinity became much stronger than the native pectinase.     

Enzymatic hydrolysis of nitriles by an engineered nitrile hydratase (papain Gln19Glu) in aqueous-organic media

A mutant of the cysteine protease papain, displaying nitrile hydratase and amidase activities, was expressed in Pichia pastoris and used for the hydrolysis of peptide nitriles in aqueous-organic media. The rate of hydrolysis of these nitriles is lowered by increasing acetone concentration. This is caused by an increase of the Michaelis constant, and a variation of Vmax proportional to the amount of water in the mixture. The hydrolysis of the amide is less affected by the increase in co-solvent, which results in lower accumulation of this intermediate product. With the peptide nitrile tested, high nitrile concentrations could be used to promote the production of the amide and prevent its hydrolysis to the acid by diminishing the relative rate of amide hydrolysis. A number of non-peptidyl nitriles were also tested as potential substrates but activity was detected for only one compound with structural resemblance to peptide nitriles.     

The effect of process conditions on the alpha-amylolytic hydrolysis of amylopectin potato starch: An experimental design approach

The hydrolysis of amylopectin potato starch with Bacillus licheniformis alpha-amylase (Maxamyl) was studied under industrially relevant conditions (i.e. high dry-weight concentrations). The following ranges of process conditions were chosen and investigated by means of an experimental design: pH [5.6-7.6]; calcium addition [0-120 microg/g]; temperature [63-97 degrees C]; dry-weight concentration [3-37% [w/w]]; enzyme dosage [27.6-372.4 microL/kg] and stirring [0-200 rpm]. The rate of hydrolysis was followed as a function of the theoretical dextrose equivalent. The highest rate (at a dextrose equivalent of 10) was observed at high temperature (90 degrees C) and low pH (6). At a higher pH (7.2), the maximum temperature of hydrolysis shifted to a lower value. Also, high levels of calcium resulted in a decrease of the maximum temperature of hydrolysis. The pH, temperature, and the amount of enzyme added showed interactive effects on the observed rate of hydrolysis. No product or substrate inhibition was observed. Stirring did not effect the rate of hydrolysis. The oligosaccharide composition after hydrolysis (at a certain dextrose equivalent) did depend on the reaction temperature. The level of maltopentaose [15-24% [w/w]], a major product of starch hydrolysis by B. licheniformis alpha-amylase, was influenced mostly by temperature.