Kinetics and mechanism of the hydrolysis of sodium carboxymethylcellulose (Na-CMC) by a cellulase complex

TheSomogyi-Nelson colorimetric method is applied in a new manner more suitable for evaluating the kinetics of the enzyme hydrolysis of sodium carboxymethylcellulose (Na-CMC) catalyzed by the cellulase complex. By means of selective inhibition of a chosen enzyme from the cellulase complex it became possible to trace the effect of the other enzymes included in its composition.

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Kinetik und Mechanismus der Hydrolyse von Natriumcarboxymethylcellulose (Na-CMC) durch einen Cellulase-Komplex

Zusammenfassung Die kolorimetrische Methode nachSomogyi undNelson wird nach einem neuen Verfahren zur Verfolgung der Kinetik der hydrolytischen Spaltung von Natriumcarboxymethylcellulose (Na-CMC), katalysiert durch den Cellulase-Komplex, angewandt. Durch selektive Inhibierung eines bestimmten Enzyms des Cellulase-Komplexes kann man die Wirkung der anderen zu seiner gesamten Zusammensetzung gehörenden Enzyme verfolgen.

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Symbols Used E enzyme (E—cellulase;E—exo-cellobiohydrolase;E—-glucosidase) - [E] _w weight concentration of enzymeE - S substrate (Na-CMC—sodium carboxymethylcellulose) - [S]₀ weight concentration of substrateS - I inhibitor (I—lactose;I—calcium chloride;I—condurrite-B-epoxide) - P product (P—oligosaccharides;P—cellobiose;P—D-glucose) - P end product (K , K , K ) - DP degree of polymerization - DS degree of substitution - ES enzyme-substrate complex (E S, E S, E S) - EP enzyme-product complex (E P, E P) - EI enzyme-inhibitor complex (E I, E I, E I) - M _s molecular mass of substrateS - K _s substrate constant (K _s , K _s , K _s ) - K _I inhibitor constant (K _I , K _I , K _I ) - K _m Michaelis-Menten constant - k ₊₁,k ₊₂ (k ₊₂ ,k ₊₂ ,k ₊₂ ) forward rate constants - k _–1 reverse rate constant - ₀ initial rate of reaction - V maximal reaction rate - A change in absorbance - molar absorption coefficient - wavelength Herrn Prof. Dr.Hans Tuppy zum 60. Geburtstag herzlichst gewidmet.     

A kinetic method for the direct determination of cellobiose hydrolysis by β-glucosidases

TheSomogyi—Nelson colorimetric method is applied in a new manner which is more suitable for following the kinetics of cellobiose hydrolysis catalyzed by -glucosidase (EC 3.2.1.21). TheSomogyi—Nelson colour reagent, which is a mixture of the solutions of the reagent ofSomogyi and that ofNelson in a volume ratio of 1:1, is added to the enzyme-substrate solution at the very start of the reaction. The colour reagent reacts with the product (D-glucose). Under the reaction conditions (0.1M acetate buffer,pH = 5.0 and temperature 37°C) the colour reagent does not affect the enzyme activity. The method excludes any inhibition of the product, owing to the continuous removal of the latter by the colour reagent. The method suggested has been applied to monitor cellobiose hydrolysis with -glucosidase, contained in four cellulase enzyme preparations from various fungal sources. The values of theMichaelis parameters (Km, V) were determined.

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Eine kinetische Methode zur Verfolgung der Hydrolyse von Cellobiose durch ß-Glucosidasen

Zusammenfassung Die kolorimetrische Methode nachSomogyi undNelson wird nach einem neuen Verfahren zur Verfolgung der Kinetik der hydrolytischen Spaltung von Cellobiose, katalysiert durch -Glucosidase (EC 3.2.1.21), angewandt. Das Farbreagenz nachSomogyi undNelson (Mischung der Reagenzien vonSomogyi undNelson im Volumenverhältnis 1:1) wird der Enzym-Substrat-Lösung zu Beginn der Reaktion hinzugefügt. Das Farbreagenz tritt mit derD-Glukose in Reaktion, wobei unter den gegebenen Reaktionsbedingungen (0,1M Azetatpuffer,pH = 5,0 und 37°C) die Enzymaktivität nicht beeinflußt wird. Die entwickelte Methode wurde zur Verfolgung der Hydrolyse von Cellobiose durch ß-Glucosidasen, die in vier Enzympräparaten aus verschiedenen Pilzstämmen enthalten waren, angewandt. Es wurden dieMichaelis-Parameter (Km, V) bestimmt.

     

Flow-Microcalorimetric Determination of Cellobiase Activity and Its Inhibition by Glucose

Abstract

A method for measuring cellobiase activity of the Trichoderma reesei CCF 1853 cellulase complex using a Thermal Activity Monitor and a flow - mix mode is described. The kinetic constant K_M and the linear dependence of dQ_max/dt (the maximum heat flow at the total saturation of enzyme with substrate) on the enzyme concentration were determined. The process of the end product inhibition of cellobiase activity by glucose has been observed too. The obtained results allow to determine the mechanism of the inhibition and an inhibition constant for glucose.

The procedure is completely general in nature and is applicable to other enzymatic systems.     

Influence of Silica-Derived Nano-Supporters on Cellobiase After Immobilization

Core shell magnetite nanoparticle (CSMN) was successfully synthesized with diameter around 125 nm according to the determination with scanning electronic microscopy. SBA-15 with diameter around 31 nm was synthesized in our previous work as another supporter for immobilized degradation enzymes. The aim of this study was to investigate the influence of silica-derived nano-supporters on cellobiase after immobilization. With covalent method, glutaraldehyde was introduced to immobilize cellobiase. The immobilized enzyme efficiency, specific activity, and its characterization, including optimum pH, pH stability, optimum temperature for enzyme reaction, and enzyme thermal stability were investigated. Results show that the method of enzyme immobilization on both nano-supporters could improve cellobiase stability under low pH and high temperature conditions compared with the free enzyme. In the aspect of immobilization efficiency, SBA had higher amount of bounded protein than that of CSMN, but had lower specific enzyme activity than CSMN, assumably due to the change in silica surface properties caused by process of supporter synthesis.     

Modeling cellobiose hydrolysis with integrated kinetic models

The enzyme cellobiase Novozym 188, which is used for improving hydrolysis of bagasse with cellulase, was characterized in its commercial available form and integrated kinetic models were applied to the hydrolysis of cellobiose. The specific activity of this enzyme was determined for pH values from 3.0–7.0, and temperatures from 40–75°C, with cellobiose at 2 g/L. Thermal stability was measured at pH 4.8 and temperatures from 40–70°C. Substrate inhibition was studied at the same pH, 50°C, and cellobiose concentrations from 0.4–20 g/L. Product inhibition was determined at 50°C, pH 4.8, cellobiose concentrations of 2 and 20 g/L, and initial glucose concentration nearly zero or 1.8 g/L. The enzyme has shown the greatest specific activity, 17.8 U/mg, at pH 4.5 and 65°C. Thermal activation of the enzyme followed Arrhenius equation with the Energy of Activation being equal to 11 kcal/mol for pH values 4 and 5. Thermal deactivation was adequately modeled by the exponential decay model with Energy of Deactivation giving 81.6 kcal/mol. Kinetics parameters for substrate uncompetitive inhibition were: Km=2.42 mM, V _max=16.31 U/mg, Ks=54.2 mM. Substrate inhibition was clearly observed above 10 mM cellobiose. Product inhibition at the concentration studied has usually doubled the time necessary to reach the same conversion at the lower temperature tested.