Batch Enzyme Reaction Research Articles

Adsorption of cellulase Aspergillus niger on a commercial activated carbon: Kinetics and equilibrium studies

The adsorption kinetics of cellulase Aspergillus niger on a commercial activated carbon has been performed using a batch-adsorption technique. The effect of various experimental parameters such as initial enzyme concentration, contact time and temperature were investigated. The pseudo-first-order and pseudo-second-order kinetic models were used to describe the kinetic data which shows that the adsorption of the enzyme followed the pseudo-second-order rate expression and the rate constants were evaluated. The Langmuir and Freundlich adsorption isotherm models were applied to describe the equilibrium isotherms, and the isotherm constants were determined. It was found that Langmuir model was more suitable for our data. The activation energy of adsorption was also evaluated for the adsorption of enzyme onto activated carbon. It was found 11.37 kJ mol −1. Thermodynamic parameters Δ G 0, Δ H 0 and Δ S 0 were calculated, indicating that this process can be spontaneous and endothermic. The adsorption enthalpy and entropy were found 11.12 kJ mol −1 and 0.084 kJ mol −1 K −1, respectively. At 30 °C and at pH 4.8, 1 g activated carbon adsorbed about 1565 mg of cellulase, with a retention of 70% of the native enzyme activity up to five cycles of repeated batch enzyme reactions.

Characterization and application of immobilized lipase enzyme on different radiation grafted polymeric films: Assessment of the immobilization process using spectroscopic analysis

AbstractLipase has been immobilized onto different films, polypropylene and poly(tetrafluoroethylene‐perfluroro‐propyl vinyl ether) using glutalaradehyde as a crosslinker. Differential scanning calorimetery, Fourier transform infrared spectroscopic, x‐ray diffraction, and scanning electron microscopy measurements were carried out to confirm the structure of the polymer films as well as the immobilization process of the enzyme onto the polymeric carrier. The activity and stability of the resulting biopolymers produced by lipase have been compared to those for the native lipase. The experimental results showed that the optimum temperature and pH were 40°C and 8.0, respectively. The activity of the immobilized lipases varied with lipase concentration and with the yield of grafting. Subjecting the immobilized enzymes to a dose of γ‐radiation of (0.5–10 Mrad) showed complete loss in the activity of the free enzyme at a dose of 5 Mrad. A leakage of the enzyme from the irradiated membranes was not observed in the repeated batch enzyme reactions. The operational stability of the free and immobilized lipase in n‐hexane showed that the immobilized enzyme was much more stable than the free one. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 155–167, 2003

Immobilization of bromelain onto porous copoly(γ-methyl- l-glutamate/ l-leucine) beads

Water-insoluble bromelain was prepared by immobilizing bromelain onto the surface of porous copoly(γ-methyl- l-glutamate/ l-leucine) (ML) beads with and without spacer. The mode of the immobilization between bromelain and porous copolypeptide ML beads was covalent fixation. The relative activity and the stability of the immobilized bromelain was investigated. The retained activity of the bromelain covalently immobilized by the azide method was found to be excellent toward a small ester substrate, N-benzyl- l-arginine ethyl ester, but rather low toward casein, a high molecular weight substrate. The values of the Michaelis constant K m and the maximum reaction velocity V m for free and immobilized bromelain on the porous copolypeptide ML beads were estimated. Apparent K m was larger for immobilized bromelain than for the free one, while V m was smaller for the immobilized bromelain. The thermal stability of the covalently immobilized bromelain was higher than that of the free bromelain. The initial enzymatic activity of the immobilized bromelain remained approximately unchanged with storage time, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Preparation method of porous polymer materials by radiation technique and its application

AbstractThe radiation polymerization of hydrophilic hydroxyethyl methacrylate monomer solution at temperatures below 0 °C leads to the formation of a porous structure in the polymers. The melting peak of the eutectic water–monomer composition at the eutectic point (above −24 °C) could be distingushed and a glass transition temperature was observed at −96 °C.The porous structure was developed after melting small ice pieces in the polymers after polymerization. The porous structure formed above 0 °C contained discontinuous pores and that formed below 0 °C had continuous pores leading to reticular structure. In a mixture of water –dioxane –monomer, the pore diameter decreased with increasing monomer concentration. Replacing dioxane with decane led to a maximum pore diameter at 70% monomer concentration. The pore diameter in 70% monomer concentration using water and dioxane was 1∼4 µm, maximum activity in immobilized enzyme tablets was observed at this diameter. The porous structure was also varied by controlling the polymerization temperature. The durability of the immobilized enzyme tablets was demonstrated by the retention of high enzyme activities after repeated batch enzyme reactions. Copyright © 2001 John Wiley & Sons, Ltd.

Protease Immobilization onto Poly(.GAMMA.-methyl-D-glutamate) Fibers.

Water-insoluble protease, such as papain or ficin, was fixed by covalent bond onto the surface of poly (γ-methyl-D-glutamate) [PMDG] fibers, and the relative activity and the stability of the immobilized protease were investigated. The retained activity of protease covalently immobilized onto PMDG fibers was found to be excellent toward a small ester substrate, N-benzyl-L-arginine ethyl ester [BAEE]. The values of the Michaelis constant (Km) and the maximum reaction velocity (Vm) for free and the immobilized protease onto the PMDG fibers were estimated. The apparent Km was larger for immobilized protease than for the free enzyme, while Vm was smaller for the immobilied protease. The thermal stability of the covalently immobilized protease was higher than that of the free one. The initial enzymatic activity of the covalently immobilized protease remained approximately unchanged with storage time, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Preparation of immobilized cellulase beads and their application to hydrolysis of cellulosic materials

Cellulase was immobilized on aluminium oxide beads with a polymer matrix by radiation polymerization at low temperature using various monomers. The optimum conditions for the preparation of the immobilized enzyme beads were obtained, and the characteristics of the immobilized enzyme beads investigated by repeated batch enzyme reactions. The cellulose component of chaff, pretreated by electron beam irradiation and subsequent pulverization, was effectively hydrolysed by the immobilized enzyme beads, in which the conversion (hydrolysis) yield in the repeated batch enzyme reactions with 10% chaff slurry was 53%.

Preparation of polymeric urease discs by an electron beam irradiation technique

A preparation method of immobilized urease discs by an electron beam irradiation technique was developed, and the relationship between enzyme activity and preparation conditions was investigated. The immobilized urease disc was a thin circular film (200μm, 5mm φ) that is useful for biomedical applications. The activity of urease irradiated with 1 Mrad at room temperature was protected by the presence of cysteine. The activity of the immobilized urease discs was studied as a function of monomer concentration (80–90%) and the thicker disc gave a high activity. The durability of the immobilized urease discs gave a high activity. The durability of the immobilized urease discs was evaluated by repeated batch enzyme reactions, and a high activity yield (80–85%) was obtained.

Immobilization of urease on grafted starch by radiation method

The acrylamide was grafted by radiation onto starch which is a kind polymeric biomaterial. The urease was immobilized on the grafted starch. Some experiments to observe the quantitative relationships between the percent grafl and the activity of immobilized enzyme were determined. The enzyme activity was maintained by more than seven batch enzyme reactions.

Papain immobilization onto porous poly(λ‐methyl L‐glutamate) beads

AbstractWater‐insoluble papain was prepared by immobilizing papain onto the surface of porous poly(λ‐methyl L‐glutamate) (PMLG) beads with and without spacer. The mode of the immobilization between papain and porous PMLG beads was covalent fixation. The relative activity and the stability of the immobilized papain was investigated. The retained activity of the papain covalently immobilized by the azide method was found to be excellent toward a small ester substrate, N‐benzyl L‐arginine ethyl ester (BAEE), compared with that of the peptide binding method. The values of the Michaelis constant Km and the maximum reaction velocity Vm for free and immobilized papain on the PMLG beads were estimated. The apparent Km was larger for immobilized papain than for the free enzyme, while Vm was smaller for the immobilized papain. The thermal stability of the covalently immobilized papain was higher than that of the free papain. The initial enzymatic activity of the covalently immobilized papain remained approximately unchanged with storage time, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Protease immobilization onto porous chitosan beads

AbstractWater‐insoluble proteases were prepared by immobilizing papain, ficin, and bromelain onto the surface of porous chitosan beads with any length of spacer by covalently fixation. The activity of the immobilized proteases was found to be still high toward small ester substrate, N‐benzyl‐L‐arginine ethyl ester (BAEE), but rather low toward casein, a high‐molecular‐weight substrate. The relative activity of the immobilized proteases with spacer gave an almost constant value for the substrate hydrolysis within the surface concentration region studied. The values of the Michaelis constant Km and the maximum reaction velocity Vm for free and immobilized proteases on the porous chitosan beads are estimated. The apparent Km values were larger for immobilized proteases than for the free ones, while Vm values were smaller for the immobilized proteases. The pH, thermal, and storage stability of the immobilized proteases were higher than those of the free ones. The initial enzymatic activity of the immobilized protease maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Lipoprotein lipase immobilization onto polyacrolein microspheres

A lipoprotein lipase (LPL) was made water insoluble by immobilizing onto the surface of polyacrolein (PAA) microspheres with and without oligoglycines as spacer. The activity of the immobilized LPL was found to remain high toward a small ester substrate, p-nitrophenyl laurate (pNPL). The relative activity of the immobilized LPL without spacer decreased gradually with the decreasing surface concentration of the immobilized LPL on the PAA microsphere. On the contrary, the immobilized LPL with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than that without any spacer. The Michaelis constant K(m) and the maximum reaction velocity V(m) were estimated for the free and the immobilized LPL. The apparent K(m) was larger for the immobilized LPL than for the free one, while V(m) was smaller for the immobilized LPL. The pH, thermal, and storage stabilities of the immobilized LPL were higher than those of the free one. The initial enzymatic activity of the immobilized LPL maintained almost unchanged without any leakage and inactivation of LPL when the batch enzyme reaction was performed repeatedly, indicating the excellent durability of the immobilized LPL.

Protease immobilization onto polyacrolein microspheres

Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Protease immobilization onto copoly(ethylene/acrylic acid) fiber

AbstractWater‐insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of the copoly(ethylene/acrylic acid) fiber. The effect of the surface area of the copolymer fiber on the amount of the immobilized protease was investigated. The mode of the immobilization between protease and copolymer such as a covalent fixation or an ionic interaction on the enzymatic activity and the stability of the immobilized protease was also investigated. The stability of the immobilized protease was also investigated. The stability of the immobilized protease in this study meant thermal stability, durability for repeated use, stability in urea, and durability for repeated washing. The activity of covalently immobilized proteases was found to be still higher toward small ester substrates, but rather low toward casein, a high molecular weight substrate. The covalently immobilized proteases onto the copolymer fiber gave an almost constant specific activity, suggesting less structural deformation of the protease molecule than the conventional immobilized enzyme system in the lower surface concentration. The thermal stability of the immobilized proteases was higher than that of the respective ionic interaction or native proteases. The initial enzymatic activity of the covalently immobilized proteases was maintained almost unchanged without any elimination and inactivation of proteases when the batch enzyme reaction was performed repeatedly, indicating excellent durability.

Lipoprotein lipase immobilization onto copolypeptide fibers.

Water insoluble lipoprotein lipase was prepared by immobilizing lipoprotein lipase (LPL) onto the surface of copolypeptide fibers with and without oligoglycine spacer. As sample fiber, copoly (L-glutamic acid/L-leucine) (PLGA-co-PLL) and copoly (γ-methyl-L-glutamate/L-leucine) (PMLG-co-PLL) were prepared. The mode of the immobilization between LPL and copolypeptide fibers such as a covalent fixation or an ionic interaction on the enzymatic activity and stability of the immobilized LPL was investi-gated. The retained activity of the LPL covalently immobilized by the azide method was found to be ex-cellent toward the small ester substrate, p-Nitrophenyl laurate (pNPL), compared to that by the peptide binding method. The values of the Michaelis constant Km and the maximum reaction velocity Vm for free and the immobilized LPL on the polypeptide fibers are estimated. The apparent Km was larger for immo-bilized LPL than for the free one, while Vm was smaller for the immobilized LPL. The thermal stability of the covalently immobilized LPL was higher than that of free LPL. The initial enzymatic activity of the covalently immobilized LPL almost unchanged without any definite elimination and inactivation of LPL, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Preparation of immobilized enzyme surfaces by radiation technique

Immobilized cellulase surfaces were prepared by a simple radiation technique, coating 2-hydroxyethyl methacrylate and tetraethyleneglycol diacrylate on the inner surface of a polyethylene tube. The durability of the surfaces was examined by repeated batch enzyme reactions as a function of monomer and enzyme concentration. The amount of the enzymes located on the surface was about 0.5 mg/cm 2, in which the enzymes did not leak from the polymer matrix. The optimum monomer concentration in the immobilization was 60–70%.

Immobilization of cellulase using porous polymer matrix

AbstractA new method is discussed for the immobilization of cellulase using porous polymer matrices, which were obtained by radiation polymerization of hydrophilic monomers. In this method, the immobilized enzyme matrix was prepared by enzyme absorbtion in the porous polymer matrix and drying treatment. The enzyme activity of the immobilized enzyme matrix varied with monomer concentration, cooling rate of the monomer solution, and hydrophilicity of the polymer matrix, taking the change of the nature of the porous structure in the polymer matrix. The leakage of the enzymes from the polymer matrix was not observed in the repeated batch enzyme reactions.

Immobilization of microbial cells in membrane form by radiation‐induced cast‐polymerization

AbstractA method of cell immobilization by radiation‐induced cast‐polymerization is reported. Immobilization parameters which influence the enzymatic activity of immobilized cell membranes have been studied in batch enzyme reactions. These parameters are monomer concentration, membrane thickness and additive concentration. The Michaelis constant of immobilized cell membranes depends on the membrane thickness and additive concentration. The heat and pH stability of immobilized cell membranes was studied.

Immobilization of microbial cells by radiation-induced polymerization of glass-forming monomers and immobilization ofStreptomyces phaeochromogenes cells by polymerization of various hydrophobic monomers

The immobilization ofStreptomyces phaeochromogenes cells was studied by the radiation-induced polymerization of various hydrophobic glass-forming monomers at low temperatures. The glucose isomerase activity of cells immobilized in hydrophobic polymers showed no decrease in activity with repeated use (batch enzyme reaction). Activity increased with increasing monomer concentration in contrast to results with the immobilized enzyme. The hydrophobic polymer composite was microspheric in form. The particle diameter of the composite increased with the increasing monomer concentration.K m values of the immobilized cells were close to that of intact cells. It was deduced that the cells were trapped on the surface part of the hydrophobic polymer ready to react with the substrate, and not within the matrix where diffusion would play an important role in reaction rates.

Immobilization of enzymes by radiation-induced polymerization of glass-forming monomers. double entrapping of enzymes in the presence of various additives

Immobilization of enzymes by a double entrapping method was investigated, α-Amylase was trapped by radiation-induced low temperature polymerization of 2-hydroxyethyl methacrylate after being absorbed by various inorganic additives. Drierite showed the most remarkable effect by increased enzymatic activity and decreased activity loss in batch enzyme reactions. Double entrapping of cellulase was tried by preparing an entangled mixture of enzyme and various natural polymers as additives and then polymerizing with the monomeric system at low temperatures. The composite, including enzyme-additive mixture prepared by coprecipitating and coevaporating methods, showed a considerable enhancing effect on enzymatic activity and durability in batch enzyme reactions.

Studies on Continuous Enzyme Reactions

The kinetics of hydrolysis of acetyl-dl-methionine in DEAE-cellulose-aminoacylase (EC 3.5.1.14) column and DEAE-Sephadex-aminoacylase column was studied. The rate of hydrolysis of substrate was shown to be dependent on the flow rate and independent to the dimension of the enzyme column. The rate of hydrolysis of the substrate was equal in cases of down-ward flow and of up-ward flow. The deteriorated aminoacylase columns by long period operation were reactivated by the recharge of aminoacylase to them. The continuous enzyme reaction using an aminoacylase column was superior to the batch enzyme reaction using native aminoacylase. The enzymatic properties of the water-insoluble aminoacylase prepared by linking mold aminoacylase (EC 3.5.1.14) to DEAE-Sephadex were studied and compared with those of the native aminoacylase. Optimum pH values for hydrolysis of several substrates by the DEAE-Sephadex-amino-acylase complex (DSA-complex) shifted about 0.5~1.5 pH units more to the acid side than those by the native enzyme. On the effects of metal ions and inhibitors, substrate specificity, optical specificity and kinetic constants, no marked difference was observed between the native enzyme and the DSA-complex. Heat stability, optimum temperature and resistance towards proteases were increased by conversion from the native form to the insoluble enzyme. It was also observed that the DSA-complex was activated by urea.