Estimation of intrinsic kinetic constants for pore diffusion-limited immobilized enzyme reactions

A simple method is presented that establishes intrinsic rate parameters when slow pore diffusion of substrate limits immobilized enzyme reactions that obey Michaelis-Menten kinetics. The Aris-Bischoff modulus is employed. Data at high substrate concentrations, where the enzyme would be saturated in the absence of diffusion limitation, and at low substrate concentrations, where effectiveness factors are inversely proportional to reaction modulus, are used to determine maximum rate and Michaelis constant, respectively. Because Michaelis-Menten and Langmuir-Hinshelwood kinetics are formally identical, this method may be used to estimate intrinsic rate parameters of many heterogeneous catalysts. The technique is demonstrated using experimental data from the hydrolysis of maize dextrin with diffusion-limited immobilized glucoamylase. This system yields a Michaelis constant of 0.14%, compared to 0.11% for soluble glucoamylase and 0.24% for immobilized glucoamylase free of diffusional effects.     

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.     

Influence of glucose on the kinetics of maltodextrin hydrolysis using free and immobilized glucoamylase

The study of the effect of glucose addition to the kinetics of maltodextrin hydrolysis catalyzed with free and immobilized Aspergillus niger glucoamylase does not show any significant glucose inhibitory effect. This result is in contradiction with data previously reported in the literature. On the contrary, a slight glucose-activating effect was observed. This effect was greater in the case of the immobilized enzyme. The glucose inhibitory effect may thus not be involved in the case of practical saccharification conditions catalyzed with glucoamylase when maltodextrins are used as substrate.     

A semimechanistic mathematical model for growth of Rhizopus oligosporus in a model solid-state fermentation system

A Semimechanistic mathematical model is developed which describes the growth of Rhizopus oligosporus in a model solid-state fermentation system. Equations are presented for the release of glucoamylase, the diffusion of glucoamylase, the hydrolysis of starch, the generation and diffusion of glucose, and the uptake of glucose and conversion into new biomass. Good agreement of the model with the experimental data was obtained only after the glucoamylase diffusivity and the maximum specific glucose uptake rate were altered from their originally determined values. The model recognizes the distributed nature of the solid-state fermentation and therefore is able to predict the concentration profiles of the system components within the substrate. The model provides an insight into the possible rate-limiting steps in solid-state fermentation-the generation of glucose within the substrate and the resulting availability of glucose at the surface.     

Isoglucose production from raw starchy materials based on a two-stage enzymatic system

A new low-cost glucoamylase preparation for liquefaction and saccharification of starchy raw materials in a one-stage system was developed and characterized. A non-purified biocatalyst with a glucoamylase activity of 3.11 U/mg, an alpha-amylase activity of 0.12 WU/mg and a protein content of 0.04 mg protein/mg was obtained from a shaken-flask culture of the strain Aspergillus niger C-IV-4. Factors influencing the enzymatic hydrolysis of starchy materials such as reaction time, temperature and enzyme and substrate concentration were standardized to maximize the yield of glucose syrup. Thus, a 90% conversion of 5% starch, a 67.5% conversion of 5% potato flour and a 55% conversion of 5% wheat flour to sweet syrups containing up to 87% glucose was reached in 3 h using 1.24 glucoamylase U/mg hydrolyzed substrate. The application of such glucoamylase preparation and a commercially immobilized glucose isomerase for the production of glucose-fructose syrup in a two-stage system resulted in high production of stable glucose/fructose blends with a fructose content of 50%. A high concentration of fructose in obtained sweet syrups was achieved when isomerization was performed both in a batch and repeated batch process.     

Immobilized glucoamylase on porous glass

A study was made to determine the controlling mass transfer resistance in the overall reaction rate for conversion of maltose to glucose, catalyzed by glucoamylase immobilized onto porous glass. For normal operation of a packed column and air-stirred batch reactor, the rate controlling step was found to be the internal resistance of simultaneous pore diffusion and chemical reaction. Experimental effectiveness factors were determined and are compared with those derived from a theoretical diffusion model based on Michaelis-Menten kinetics. Also given are temperature and pH relationships for the free and immobilized glucoamylase.     

Immobilization of Enzyme into Poly(vinyl alcohol) Membrane

Glucoamylase, invertase, and cellulase were entrapped within poly(vinyl alcohol) (PVA) membrane cross-linked by means of irradiation of ultraviolet light. The conditions for immobilization of glucoamylase were examined with respect to enzyme concentration in PVA, sensitizer (sodium benzoate) concentration in PVA, irradiation time, and membrane thickness. Various characteristics of immobilized glucoamylase were evaluated. Among them, the pH activity curve for the immobilized enzyme was superior to that for the native one, and thermal stability was improved by immobilization with bovine albumin. The apparent K(m) was larger for immobilized glucoamylase than for the native one, while V(max) was smaller for the immobilized enzyme. Also, the apparent K(m) appeared to be affected by the molecular size of the substrate. Further, immobilized invertase and cellulase showed good stabilities in repeating usage.     

Immobilization of mold and bacterial amylases on silica carriers

The immobilization of alpha-amylase and glucoamylase was investigated by several coupling methods on silica carriers, different types of Silokhroms, and silica gels. The most active immobilized mold and bacterial alpha-amylases and mold glucoamylase were obtained with titanium salts. These activities were twice the value of that obtained by glutaraldehyde or azo coupling. The half-lives of A. oryzae alpha-amylase, B. subtilis alpha-amylase, and A. niger glucoamylase, immobilized to silica carriers at 45 degrees C and under continuous operation at a high concentration of substrate, were 14, 35, and 65 days, respectively.     

Enzymatic hydrolysis of soluble starch in a polyethylene glycol-dextran aqueous two-phase system

Hydrolysis of soluble starch by glucoamylase and β-amylase was investigated as a model reaction in an aqueous two-phase system consisting of polyethylene glycol (PEG) and dextran (DEX). Changes in glucose concentration observed in the batch reaction experiments with glucoamylase were almost identical for the aqueous two-phase and pure water systems, showing that the enzymic reactions investigated were not influenced by the presence of PEG and DEX. The partition of β-amylase into the DEX phase was insufficient compared to that of glucoamylase. Hence, the former enzyme was crosslinked with glutaraldehyde to increase its apparent molecular weight and, as a consequence, the partition coefficient, defined as the concentration ratio of the component partitioned into the PEG phase to that into the DEX phase, was decreased to 17% of that of the original enzyme. In the operation in which the enzyme and substrate are partitioned selectively into the DEX phase and allowed to react there while the product, thus transferring to the PEG phase, is recovered, the aqueous two-phase system with a smaller partition coefficient provided longer operational stability.     

Properties of urease immobilized on the functional organic silica surface

The paper deals with kinetics of the urea hydrolysis by microbial-origin urease dissolved and immobilized on the organic silica surface. It is shown that hydrolysis kinetics for soluble urease is described by the Michaelis-Menten equation until the concentration of urea reaches 1 M. Two fractions differing in the Michaelis constant are revealed for silochrome immobilized urease. The rate of urea hydrolysis by native and immobilized urease was studied depending on the pH value in presence of the substrate in the 1 M and 5 mM concentration. The hydrolysis rate of 1 M urea in the buffer-free solution by silochrome-immobilized urease is practically independent of pH within 4.5-6.5. Application of a 2.5 mM phosphate-citrate buffer as a solvent causes an increase in the hydrolysis rate within this pH range. For a soluble urease the 1 M urea hydrolysis rate dependence on pH is ordinary at pH 5.8-6.0. If the substrate concentration is 5 mM, the pH-dependences for the rate of the urea hydrolysis by silochrome- and aerosil-immobilized urease are close and at pH above 6.0 coincide with those for a soluble enzyme. The found differences in the properties of soluble and immobilized ureases are explained by the substrate and reaction products diffusion.     

红曲AS 3．3491葡萄糖淀粉酶生物合成的调节

红曲 Monascus AS 3.978的变异株 AS 3.3491曾是葡萄糖淀粉酶的工业生产菌株。本文报告的实验结果表明该菌株的葡萄糖淀粉酶是构成酶,它的形成受降解物阻遏的调控。AS 3.3491葡萄糖淀粉酶形成的时间过程属典型的产物形成与生长呈负相关的类型,即菌丝体生长停止后酶才开始大量形成。从培养基中除去过量易利用碳源可使葡萄糖淀粉酶形成消阻遏。各种可利用碳源以低浓度分别加到洗涤的菌丝体悬浮液中都能促进葡萄糖淀粉酶的形成,其中蜜二糖和半乳糖的促进作用最甚。在限制生长的条件下,即向洗涤菌丝悬浮液连续缓慢供给低浓度葡萄糖,则在整个培养过程中,葡萄糖淀粉酶的活力稳步上升,不需要任何诱导物,并且比酶形成值达到蜜二糖培养基的水平。因此,高浓度葡萄糖阻遏葡萄糖淀粉酶形成,但低浓度葡萄糖却促进酶的形成。     

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.     

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.     

Crosslinked enzyme crystals of glucoamylase as a potent catalyst for biotransformations

Glucoamylase (E.C: 3.2.1.3, alpha-(1-->4)-glucan glucohydrolase) mainly hydrolyzes starch and has been extensively used in the starch, glucose (dextrose), and fermentation industries. Immobilized glucoamylase has an inherent disadvantage of lower conversion rates and low thermostability of less than 55 degrees C when used in continuous operations. We have developed crosslinked enzyme crystals (CLEC) of glucoamylase that overcome the above disadvantages, possess good thermal stability and retain 98.6% of their original activity at 70 degrees C for 1h, 77% activity at 80 degrees C for 1h, and 51.4% activity at 90 degrees C for 0.5h. CLEC glucoamylase has a specific activity of 0.0687 IU/mg and a yield of 50.7% of the original activity of the enzyme under optimum conditions with starch as the substrate. The crystals obtained are rhombohedral in shape having a size approximately 10-100 microm, a density of 1.8926 g/cm(3) and a surface area of 0.7867 m(2)/g. The pH optimum of the glucoamylase crystals was sharp at pH 4.5, unlike the soluble enzyme. The kinetic constants V(max) and K(m) exhibited a 10-fold increase as a consequence of crystallization and crosslinking. The continuous production of glucose from 10% soluble starch and 10% maltodextrin (12.5 DE) by a packed-bed reactor at 60 degrees C had a productivity of 110.58 g/L/h at a residence time of 7.6 min and 714.1g/L/h at a residence time of 3.4 min, respectively. The CLEC glucoamylase had a half-life of 10h with 4% starch substrate at 60 degrees C.     

Properties of enzymes immobilized by the diazotized m-diaminobenzene method.

Some properties of a number of enzymes immobilized by the diazotized m-diaminobenzene (dDAB) method are described. The pH-activity profiles of beta-D-glucosidase, glucoamylase, peroxidase, uricase, and D-glucose oxidase were virtually unchanged on immobilization while those of catalase and dextranase were significantly altered. beta-D-Glucosidase, glucoamylase, and glucose oxidase were found to be more susceptible to denaturation on lyophilization when immobilized than in the native state; however, sorbitol had a marked protective effect in every case examined. Sorbitol was also found to exert a stabilizing effect when lyophilized immobilized preparations were stored. Immobilization marginally improved the stabilities of a number of enzymes to heating at 60 degrees at pH 8.0. The usefulness for continuous reaction of a column of glucoamylase attached to celite was established. The reuse of the solid supports was demonstrated.     

Fermentation of corn starch to ethanol with genetically engineered yeast

Expression of the glucoamylase gene from Aspergillus awamori by laboratory and distiller's strains of Saccharomyces cerevisiae allowed them to ferment soluble starch. Approximately 95% of the carbohydrates in the starch were utilized. Glycerol production was significantly decreased when soluble starch was used instead of glucose. Ethanol yield on soluble starch was higher than that on glucose. The rate of starch fermentation was directly related to the level of glucoamylase activity. Strains with higher levels of glucoamylase expression fermented starch faster. The decline in starch fermentation rates toward the end of the fermentation was associated with accumulation of disaccharides and limit dextrins, poor substrates for glucoamylase. The buildup of these products in continuous fermentations inhibited glucoamylase activity and complete utilization of the starch. Under these conditions maltose-fermenting strains had a significant advantage over nonfermenting strains. The synthesis and secretion of glucoamylase showed no deleterious effects on cell growth rates, fermetation rates, and fermentation products.     

Glucose oxidation in a dual hollow fiber bioreactor with a silicone tube oxygenator

A dual hollow fiber bioreactor, consisting of an outer silicone membrane for oxygen supply and an inner polyamide membrane for substrate permeation, was used as an immobilized enzyme reactor to carry out enzymatic glucose oxidation. Attaching a silicone tube oxygenator to provide an additional oxygen supply improved the conversion in glucose oxidation when the oxygen supply was rate-limiting. The reactor was operated in both diffusion and ultrafiltration modes. In the latter case, the conversion was much higher, but the stability of the immobilized enzyme was better maintained in the diffusion mode. As the inlet glucose concentration increased from 10mM to 500mM, the conversion decreased from 70 to 20%.     

Enzymatic processes for the purification of trehalose

A dual‐enzyme process aiming at facilitating the purification of trehalose from maltose is reported in this study. Enzymatic conversion of maltose to trehalose usually leads to the presence of significant amount of glucose, by‐product of the reaction, and unreacted maltose. To facilitate the separation of trehalose from glucose and unreacted maltose, sequential conversion of maltose to glucose and glucose to gluconic acid under the catalysis of glucoamylase and glucose oxidase, respectively, is studied. This study focuses on the hydrolysis of maltose with immobilized glucoamylase on Eupergit® C and CM Sepharose. CM Sepharose exhibited a higher protein adsorption capacity, 49.35 ± 1.43 mg/g, and was thus selected as carrier for the immobilization of glucoamylase. The optimal reaction temperature and reaction pH of the immobilized glucoamylase for maltose hydrolysis were identified as 40°C and 4.0, respectively. Under such conditions, the unreacted maltose in the product stream of trehalose synthase‐catalyzed reaction was completely converted to glucose within 35 min, without detectable trehalose degradation. The conversion of maltose to glucose could be maintained at 0.92 even after 80 cycles in repeated‐batch operations. It was also demonstrated that glucose thus generated could be readily oxidized into gluconic acid, which can be easily separated from trehalose. We thus believe the proposed process of maltose hydrolysis with immobilized glucoamylase, in conjunction with trehalose synthase‐catalyzed isomerization and glucose oxidase‐catalyzed oxidation, is promising for the production and purification of trehalose on industrial scales. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Immobilization of enzymes in porous supports: Effects of support-enzyme solution contacting

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.