Catalytic properties of glucoamylase immobilized on the synthetic carbon material Sibunit

Glucoamylase (commercial preparation Glucavamorin) was immobilized by sorption on a carbon support Sibunit. Starch saccharification by the resulting biocatalyst (dextrin hydrolysis) was studied. Investigation of the effect of adsorptional immobilization on kinetic parameters of glucoamylase, including the rate constant of thermal inactivation, showed that immobilization of Glucavamorin on Sibunit resulted in a thousandfold increase in glucoamylase stability in comparison with the dissolved enzyme. Presence of the substrate (dextrins) in the reaction mixture had a considerable stabilizing effect. Increase in dextrin concentration increases the thermostability of the immobilized enzyme. The overall factor of glucoamylase stabilization adsorbed on Sibunit with the presence of 53% dextrin solutions in comparison with the dissolved enzyme approximated 10(5). The biocatalyst for starch saccharification made on the base of Subunit-adsorbed Glucavamorin had a high operational stability. Its half-inactivation time at 60 degrees C exceeded 30 days.     

Catalytic properties of thioredoxin immobilized on superparamagnetic nanoparticles

Thioredoxin (Trx1), a very important protein for regulating intracellular redox reactions, was immobilized on iron oxide superparamagnetic nanoparticles previously coated with 3-aminopropyltriethoxysilane (APTS) via covalent coupling using the EDC (1-ethyl-3-{3-dimethylaminopropyl}carbodiimide) method. The system was extensively characterized by atomic force microscopy, vibrational and magnetic techniques. In addition, gold nanoparticles were also employed to probe the exposed groups in the immobilized enzyme based on the SERS (surface enhanced Raman scattering) effect, confirming the accessibility of the cysteines residues at the catalytic site. For the single coated superparamagnetic nanoparticle, by monitoring the enzyme activity with the Ellman reagent, DTNB = 5,5′-dithio-bis(2-15 nitrobenzoic acid), an inhibitory effect was observed after the first catalytic cycle. The inhibiting effect disappeared after the application of an additional silicate coating before the APTS treatment, reflecting a possible influence of unprotected iron-oxide sites in the redox kinetics. In contrast, the doubly coated system exhibited a normal in-vitro kinetic activity, allowing a good enzyme recovery and recyclability.     

Catalytic properties of lipases immobilized on various mesoporous silicates

Lipases SP525, AK, LIP, and PS were immobilized on three kinds of mesoporous silicates (FMS, PESO, and SBA) with diameters of 27 to 92 A. The amount of lipase activity adsorbed on these supports was related to the pore size of the silicate. Enantioselectivities of immobilized lipases were similar to those of free lipases, and recycling could be done in both aqueous and organic solvents.     

Catalytic properties of a nitrile hydratase immobilized on activated chitosan

The catalytic properties of a nitrile hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters of acrylonitrile hydration catalyzed by immobilized nitrile hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate (V _max), whereas the Michaelis constant (K _M) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acrylonitrile transformation was shown, and the nitrile hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of nitrile hydratase inactivation. It was found that immobilized nitrile hydratases remain active at pH 3.0–4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acrylonitrile.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

糖化酶在丝素膜上的固定化及性质研究

利用丝素作糖化酶的固定化载体,应用包埋法和共价交联法两种方法,制备了固定化糖化酶丝素膜,研究结果表明,共价交联法制备的酶膜活力较高,且回收率可达50%以上;与溶液酶相比,固定化酶的最适温度提高了10℃,热稳定性与贮存稳定性也有了很大提高。     

Immobilization of enzymes on activated carbon: Properties of immobilized glucoamylase,glucose oxidase,and gluconolactonase

Apparent kinetics and pH–activity relationships have been determined for glucoamylase and glucose oxidase immobilized on activated carbon using a diimide method. Reaction rate expressions of Michaelis–Menten form adequately approximate the observed kinetics for both enzyme preparations over the ranges of substrate concentrations considered. Influences of external mass transfer as well as substrate and product adsorption on interpretation of the experimental data have been examined. Immobilization of a glucose oxidase–gluconolactonase enzyme mixture has been found to increase substantially the ratio of gluconolactonase to glucose oxidase activities compared to the corresponding activity ratio for these enzymes in solution.     

Preparation of immobilized enzymes by N-vinylpyrrolidone and the general properties of the glucoamylase gel

Immobilized glucoamylase, invertase, and β-galactosidase were prepared by using N-vinylpyrrolidone monomer (VP) under γ-ray irradiation. The enzyme-VP solutions were gelled by irradiation with 2.9 Mrad and the added enzymes were almost completely entrapped. Activity losses on entrapping were 55% for the VP-glucoamylase gel, and more than 90% in the case of VP-invertase and VP-β-galactosidase gels. No leakage of enzyme from these gels could be detected within 1 hr. The VP-glucoamylase gel was capable of hydrolyzing dextrin (mol wt 10,400) to glucose and the glucose equivalent was equal to that obtain able with native enzyme. The optimum temperature, heat stability, pH activity curve, and pH stability of VP-glucoamylase gel were slightly inferior to those of native enzyme, while Km was a little larger than that of native enzyme.     

Catalytic properties of lipases immobilized onto ultrasound-treated chitosan supports

Ultrasound sonication has been utilized to produce fragmentation of chitosan polymer and hence increase the chitosan surface area, making it more accessible to interactions with proteins. In this context, we have investigated the catalytic properties of lipases from different sources immobilized onto ultrasound-treated chitosan (ChiS) pre-activated with glutaraldehyde (ChiS-G). Atomic force microscopy indicated that ChiS-G displays a more cohesive frame without the presence of sheared/fragmented structures when compared with ChiS, which might be attributed to the cross-linking of the polysaccharide chains. The immobilization efficiency onto ChiS-G and ChiS were remarkably higher than using conventional beads. In comparison with the free enzymes, lipases immobilized onto ChiS show a slight increase of apparent K_m and decrease of apparent V_max. On the other hand, immobilization onto ChiS-G resulted in an increase of V_max, even though a slight increase of K_m was also observed. These data suggest that the activation of chitosan with glutaraldehyde has beneficial effects on the activity of the immobilized lipases. In addition, the immobilization of the lipases onto ChiS-G displayed the best reusability results: enzymes retained more than 50% of its initial activity after four reuses, which might be attributed to the covalent attachment of enzyme to activated chitosan. Overall, our findings demonstrate that the immobilization of lipases onto ultrasound-treated chitosan supports is an effective and low-cost procedure for the generation of active immobilized lipase systems, being an interesting alternative to conventional chitosan beads.     

Substrate thermostabilization of soluble and immobilized glucoamylase]

A new kinetic approach to the study of enzyme thermal inactivation in the presence of a substrate, which influences the rate of inactivation has been developed. The method was applied to investigation of inactivation kinetics of soluble and porous silica-immobilized glucoamylase. It was found that the binding of a substrate (maltose or maltodextrines Star-Dri 24-R) increases the thermal stability of glucoamylase, the stabilizing effect being more pronounced in the case of the soluble enzyme (40-fold stabilization) as compared to the immobilized one (15-fold stabilization). The stabilizing effect does not depend on the length of the substrate (maltose, d. p. 2 or dextrines, d. p. 7). Glucose, a product of the enzymatic hydrolysis, has a much lower stabilizing effect. It was concluded that the main role in the glucoamylase thermostabilization is played by the substrate stabilization rather than by the immobilization itself (3-fold stabilization). However, a combined effect of thermostabilization of glucoamylase due to both immobilization and/or substrate stabilization is restricted by the same limit of value for immobilized and soluble enzymes, which is equal to 40--50-fold in comparison with the soluble enzyme in the absence of the substrate.     

Effect of pore diffusion limitation on dextrin hydrolysis by immobilized glucoamylase

Data reported here and previously indicate that when dextrin is hydrolyzed in the presence of immobilized glucoamylase, use of a larger average molecular weight substrate leads to lower overall rates of hydrolysis, while the maltose concentration during the bulk of the reaction and the maximum glucose concentration are lower than when the soluble form of the enzyme is employed under the same conditions. Computer simulation of the system demonstrated that all three observations were caused by pore diffusion limitation: the first by slow diffusion of substrate, the second by slow diffusion of intermediates, and the third by slow diffusion of glucose. Follow-up experiments with glucoamylase immobilized to particles of different sizes confirmed this finding, as results with the smallest beads were identical to those with soluble glucoamylase.     

Kinetic and thermodynamic properties of an immobilized glucoamylase from a mesophilic fungus, Arachniotus citrinus

Purified glucoamylase from Arachniotus citrinus was immobilized on polyacrylamide gel with 70% yield of immobilization. The immobilization improved the pH optima, temperature optima, values of K(m), V(max), and activation energy. Irreversible thermal denaturation studies of soluble and immobilized glucoamylase indicated that immobilization decreased the entropy and enthalpy of deactivation by magnitudes and made the immobilized glucoamylase thermodynamically more stable.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

A heat transfer study on packed bed reactors of immobilized glucoamylase

Enzymes are generally sensitive to temperature changes. Porous glass particles used for glucoamylase immobilization are poor thermal conductors and a non-uniform temperature distribution can conceivably develop in a packed bed reactor of immobilized glucoamylase on porous beads. This study was made to determine experimentally the temperature and concentration profiles in an immobilized glucoamylase column. This work provides a procedure for examining possible heat effects on reactor column performance in enzyme applications.     

Continuous conversion of starch to glucose with immobilized glucoamylase

Partially purified glucoamylase from Aspergillus awamori NRRL 3112 was immobilized on diethylaminoethyl cellulose in the presence of low ionic-strength acetate buffers at pH 4.2. The active enzyme–cellulose complex was used to convert starch substrates continuously to glucose in stirred reactors. Substrate concentrations as high as 30% could be quantitatively converted to glucose at a rate of more than 25 mg/min/liter at 55°C for periods of 3 to 4 weeks in a 4-liter reactor. Shutdowns were due to mechanical problems and not to loss of enzymes, which could be recovered with no appreciable loss of specific activity. Transfer products, such as isomaltose and panose, were present in immobilized enzyme-produced syrups but to no greater degree than in soluble glucoamylase digests of starch.     

Pineapple stem bromelain immobilized on different supports: Catalytic properties in model wine

Bromelain from pineapple stem has been covalently immobilized on different supports to select the more efficient biocatalyst that should be applied toward unstable proteins in real white wine. In this preliminary study, catalytic properties of different immobilized bromelain forms were compared under wine‐like conditions, against a synthetic substrate (Bz‐Phe‐Val‐Arg‐pNA).Covalent immobilization affected protease kinetic properties, even if all immobilized forms presented both a better substrate affinity and higher half‐life (with the exception of a few procedures) with respect to the free enzyme. Stem bromelain was successfully immobilized on chitosan beads without glutaraldehyde thus yielding a food‐safe and promising biocatalyst for unstable real wine future application. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Continuous production of glucoamylase by immobilized growing cells ofAureobasidium pullulans

Summary Yeast-like cells ofAureobasidium pullulans were immobilized in Ca-alginate gel beads and employed for continuous production of glucoamylase in a fluidized-bed reactor (250 ml working volume). After an activation time of 48 h, to allow the in situ germination of the fungal blastospores, the reactor was operated continuously for over 150 h. A steady state enzyme concentration of 1.2–1.3 U ml^–1 of glucoamylase activity and an enzyme volumetric productivity of ca. 130 U ml^–1 h^–1 were obtained at a medium flow rate of 26 ml h^–1. Enzyme activity and volumetric productivity were influenced by fermentation conditions such as inoculum size and airflow rate.     

Hydrolysis of soluble starch by rotary disc of immobilized glucoamylase

Immobilized glucoamylase sheet was prepared using soluble collagen prepared from cow hide powder as the support material. The immobilized glucoamylase sheet was attached to the rotary disc and the rates of hydrolysis of maltose and soluble starch in the tank were measured. Qualitative discussions are made of the effect of stirring speed of immobilized enzyme disc on the overall reaction rate.     

Recycle use of immobilized glucoamylase by tangential flow filtration unit

Various properties of glucoamylase immobilized onto corn stover supporting material and separation of immobilized enzyme by tangential flow filtration unit were studied. Optimum pH and temperature of immobilized enzyme were 3.5 and 60 degrees C, respectively. Enzyme stability was studied in a packed-bed column. The starch conversion rate was attained at 81% for 15 days; after that, the hydrolysis rate gradually decreased. Size of supporting material proved to be an important factor, with higher activity and good loading yield resulting from smaller supporting material. Glucoamylase immobilized onto supporting material less than 44 mum was used for hydrolysis of 10% soluble starch at pH 3.5 and 40 degrees C for 3 h. Then immobilized glucoamylase was separated from the product by means of a tangential flow filtration unit using a 0.2-mum pore size Nylon 66 membrane filter. This operation was continued until 180 ml filtrate was obtained from a 260-mL starting volume. Then, the next batch was started by adding 180 mL starch substrate into the reactor. The batchwise experiments were repeated 20 times. The average filtration rate of each batch was determined and found to sharply decline during the first four batches. Thereafter, it gradually decreased from batch to batch. The cause of decreasing filtration rate appeared to be due to retrogradation of starch. The percentage of starch hydrolysis within 20 batches was in the range 89-96%. The filtration rate becomes higher if the hydrolyzation time is extended to 14 h. Resistance to filtration was also investigated. Almost all of the total resistance is related to insoluble materials, with the significant part of this from the resistance due to insoluble materials deposited on a surface of membrane and boundary layer resistance. Using a microscopic method, no microorganisms were found in the filtrate.