Preparation of an immobilized two-enzyme system, beta-amylase-pullulanase, to an acrylic copolymer for the conversion of starch to maltose. II. Cocoupling of the enzymes and use in a packed bed column

β-Amylase (EC 3.2.1.2) and pullulanase (EC 3.2.1.9) have been covalently bound in a two-enzyme system to a crosslinked copolymer of acrylarmide-acrylic acid by using a water-soluble carbodiimide. The coupling yields based on the amounts of added β-amylase and pullulanase were 40% and 38%, respectively, with residual enzymic activities of 22% and 32% of those of free enzymes. A markedly increased operational stability was observed for the immobilized two-enzyme system compared to the free enzymes in solution. In order to find optimal operational conditions the influence of different pH values and temperatures on the conversion process was investigated. The action of the immobilized β-amylase-pullulanase derivative on partially hydrolyzed starch (DE 3.4–10.7) in a packed bed column was studied. Analysis of the product was performed using gas-liquid chromatography.     

Preparation of an immobilized two-enzyme system, Beta-amylase--pullulanase, to an acrylic copolymer for the conversion of starch to maltose. III. Process kinetic studies on continuous reactors

Kinetic studies on the parameters influencing the potential industrial application of an immobilized two-enzyme system of β-amylase and pullulanase for conversion of starch to a product with high maltose content, have been performed. The apparent Michaelis constant, the apparent product inhibitor constant, and the activation energy have been determined for the immobilized preparation and compared to the values for the corresponding soluble enzyme system. The catalytic activity of the immobilized enzymes was studied in a plug-flow reactor and a continuous feed stirred tank reactor. Mathematical models for these reactors have been formulated and adapted to fit the experimental data. Comparisons of the reactor efficiencies were made and the conditions were found to be such as to favor the plug-flow reactor. Results on operational stability tests at different temperatures and substrate concentrations are given.     

Preparation and properties of an immobilized Barley β-amylase

Barely β-amylase (α-1,4-glucan maltohydrolase, EC 3.2.1.2) has been immobilized by covalent fixation to amino derivatives of epichlorohydrin crosslinked Sepharose mediated by cyclohexyl isocyanide and acetaldehyde. The enzyme conjugates contain up to 35% of the total activity of the β-amylase added to the coupling mixture. The profiles of activity versus pH and ionic strength are essentially the same for free and immobilized β-amylase, whereas the resistance to inactivation during storage and use is considerably enhanced by immobilization. Columns with immobilized β-amylase have been used for continuous degradation of starch. At 45°C, half of the initial activity remains after seven weeks, and the corresponding figure at 23°C is 85 percent.     

Immobilization of enzymes based on hydrophobic interaction. I. Preparation and properties of a β-amylase adsorbate

Sweet potato β-amylase (α-1,4 glucan maltohydrolase, EC 3.2.1.2) was immobilized through adsorption onto an agarose gel to which nonpolar side chains had been introduced via ether bridges. The adsorbent showed evidence of saturation at an enzyme content of 35 mg per milliliter of packed gel. The adsorption was rapid and yielded a product whose operational stability depended on the initial content of β-amylase. Activity leakage was low. The relative activity of immobilized enzyme was inversely related to the amount of enzyme adsorbed to a given gel volume, having a maximal value of around 50% at low enzyme contents.     

Encapsulation of β-amylase in water-oil-water enzyme emulsion liquid membrane (EELM) bioreactor for enzymatic conversion of starch to maltose

Abstract

The β-amylase was encapsulated in emulsion liquid membrane (ELM), which acted as a reactor for conversion of starch to maltose. The membrane phase was consisted of surfactant (span 80), stabilizer (polystyrene), carrier for maltose transport (methyl cholate) and solvent (xylene). The substrate starch in feed phase entered into the internal phase by the process of diffusion and hydrolyzed to maltose by encapsulated β-amylase. Methyl cholate present in the membrane acts as a carrier for the product maltose, which helps in transport of maltose to feed phase from internal aqueous phase. The residual activity of β-amylase after the five-reaction cycle was found to decrease to ～70%, which indicated possibility to recycle the components of the emulsion and enzyme. The pH and temperature of the encapsulated enzyme were found to be optimum at 5.5 and 60?°C, respectively. The novelty of the present work lies in the development of Enzyme Emulsion Liquid Membranes (EELM) bioreactor for the hydrolysis of starch into maltose mediated by encapsulated β-amylase. The attempt has been made for the first time for the successful encapsulation of β-amylase into EELM. The best results gave the highest residual enzyme activity (94.1%) and maltose production (29.13?mg/mL).     

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.     

Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747.

The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B.     

Enhancement of starch conversion efficiency with free and immobilized pullulanase and alpha-1,4-glucosidase

Glucoamylase and pullulanase were immobilized on reconstituted bovine-hide collagen membranes using the covalent azide linkage method. A pretanning step was incorporated into the immobilization procedure to enable the support matrix to resist proteolytic activity while accommodating an operating temperature of 50 degrees C. The immobilized glucoamylase and pullulanase activities were 0.91 and 0.022 mg dextrose equivalent (DE) min(-1) cm(-2) of membrane, respectively. Immobilized glucoamylase had a half-life of 50 days while the immobilized pullulanase had a half-life of 7 days. This is a considerably improved stability over that reported by other researchers. The enzymes were studied in their free and immobilized forms on a variety of starch substrates including waxy maize, a material which contains 80% alpha-1-6-glucosidic linkages. Substrate concentrations ranged from 1% to a typical commercial concentration of 30%. Conversion efficiencies of 90-92% DE were obtained with free and immobilized glucoamylase preparations. Conversion enhancements of 4-5 mg of DE above this level were obtained by the use of pullulanase in its free or immobilized forms. Close examination of free pullulanase stability as a function of pH indicated improved thermal stability at higher pH values. At 50 degrees C and pH 5.0, the free enzyme was inactivated after 24 h. At pH 7.0, the enzyme still possessed one-half its activity after 72 h. Studies were conducted in both batch and continuous total recycle reactors. All experiments were conducted at 50 degrees C. Experiments conducted with coimmobilized enzymes proved quite promising. Levels of conversion equivalent to those obtained with the individually immobilized enzymes were realized.     

Production of maltose and maltotriose from starch and pullulan by a immobilized multienzyme of pullulanase and β-amylase

Pullulanase was immobilized on tannic acid and TEAE-cellulose, and β-amylase was covalently immobilized on p-aminobenzylcellulose. Both the immobilized enzymes showed similar properties in pH and temperature optima and heat stability. On passing the pullulan solution at high temperature (50°C) through a column packed with immobilized pullulanase, only maltotriose was obtained for ten days and the half-life was about 15 days. In a continuous reaction using immobilized multienzyme, starch was completely converted into maltose at 50°C and at a space velocity of 1.2, a comparative longer half-life (20 days) was obtained. It was concluded that starch was smoothly converted into maltose with the aid of α-amylase contaminated in the immobilized pullulanase and the operational stability of the column increased with 2-5mM Ca²⁺.     

Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

Pepsin immobilized by covalent fixation to hydroxyalkyl methacrylate gels: preparation and characterization.

Insoluble active derivatives of pepsin (EC 3.4.23.1) were prepared by covalent binding of this enzyme to hydroxyalkyl methacrylate gels modified with 1,6-diaminohexane or epsilon-aminocaproic acid in an acid medium by means of water-soluble carbodiimide. The amount of attached enzyme, its proteolytic activity, pH activity curves of the preparations obtained and the time and pH dependence of their stability were determined.     

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.     

Preparation of maltotriitol-rich malto-oligosaccharide alcohol from starch

Malto-oligosaccharide alcohols (MOSA) are one of the most important sugar alcohols widely used as sweetener in food, cosmetic, and pharmaceutical industries in recent years, of which maltotriitol-rich MOSA is much more recognized. With the aim of preparing maltotriitol-rich MOSA from starch, a novel process was developed and optimized. Starch was first liquefied with thermostable Bacillus licheniformis α-amylase. The liquefied starch was then saccharified to yield maltotriose-rich malto-oligosaccharides under the cooperative actions of Bacillus naganoensis pullulanase, Bacillus amyloliquefaciens α-amylase, and barley bran β-amylase. The maltotriitol-rich MOSA was finally prepared by chemical hydrogenation. Under the optimized conditions, maltotriitol-rich MOSA containing 42.18% maltotriitol was obtained with a conversion rate of 104.57% from starch. The process can be employed for large-scale preparation of maltotriitol-rich MOSA, and a further modification of the process can lead to the formulation of various types of MOSA with different percentages of components of sugar alcohols.     

Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices

Pectinlyase, present in different commercial pectinases used in juice technology, was immobilized on alginate beads. The optimal conditions were: 0.17 g alginate ml(-1), 1.2% (w/v or v/v) enzyme concentration and acetic-HCl/glycine-HCl buffer at pH 3.6 or tris-HCl/imidazole buffer at pH 6.4. Maximum percentage of immobilization (10.6%) was obtained with Rapidase C80. Kinetic parameters of free and immobilized pectinlyase were also determined. The pH and temperature at which activity of soluble and immobilized enzyme was maximum were 7.2 and 55 degrees C. Thermal stability was not significantly altered by immobilization, especially at 40 degrees C, showing two periods of different stability. Free and immobilized preparation reduced the viscosity of highly esterified pectin from 1.09 to 0.70 and 0.72 mm(2) s(-1), respectively, after 30 min at 40 degrees C. Furthermore, the immobilized enzyme could be re-used through 4 cycles and the efficiency loss in viscosity reduction was found to be only 9.2%.     

Starch conversion by immobilized glucoamylase

Glucoamylase bound to DEAE-cellulose in 0.05 M sodium acetate, pH 4.0, is active in the conversion of starch to glucose. The activity of the DEAE-cellulose-bound enzyme ranges from 16 to 55% of the activity of the free enzyme. Binding of the enzyme narrows the pH optimum to approximately 4.0 and lowers the temperature optimum to 40–50°C as compared to a 60°C temperature optimum for the free enzyme. Concentrations of acetate buffer above 0.1 M disrupt the DEAE-cellulose-enzyme complex. Columns were used with some success for the continuous conversion of starch. Pretreatment of the starch with α-amylase and clarification were necessary to prevent blocking of the column. Columns maintained activity for more than 3 weeks of continuous operation.     

Coupling of the Penicillium duponti acid protease to ethylene-maleic acid (1 : 1) linear copolymer. Preparation and properties of the water-soluble derivative.

The coupling of the thermostable acid protease (EC 3.4.23.-) of Penicillium duponti K 1014 to ethylene-maleic acid (1 : 1) linear copolymer in the presence of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide at pH 3.0, afforded a soluble enzyme derivative with a protein incorporation yield of 67% under optimal conditions. The protein content of the enzyme-polymer complex, the molecular weights of the reactants, and the mean value of 2.2 lysine residues per mol of enzyme found in amide linkage to the matrix, support a structure consisting of two polymer chains per mol of protease, each chain acylating a single lysine residue of the enzyme. The isoelectric point of the coupled enzyme was found to be 3,47, a value lower than that measured on the free protease (3.81). The specific activity of the bound protease against casein, at pH 3.7 and 30 degrees C, was 34% of that of the free enzyme, and at 75 degrees C increased to 70%. The increased size of the coupled enzyme resulted in an improved retention of activity by ultrafiltration membranes over that observed with free protease, alone or in admixture with ethylene-maleic acid copolymer. A water-soluble, coupled pepsin was prepared in 43% yield on protein basis by using the aminoethylmonoamide of ethylene-maleic acid copolymer and the same water-soluble carbodiimide.     

Covalent coupling of pullulanase to an acrylic copolymer using a water soluble carbodi-imide

Pullulanase (EC 3.2.1.9) prepared from a culture of Acrobacter aerogeneshas been covalently bound to an inert crosslinked copolymer of aerylamide-acrylic acid by using a water-soluble carbodi-imide. The binding yield based on the amount of added pullulanase was 34%. The residual enzymic activity was 43%, of that of free enzyme. Coupling in the presence of the substrate pullulan gave a 5-fold increase in activity over that obtained when substrate was lacking. The effect of different carbodi-imide concentrations on the coupling has been investigated. The isoelectric point of the pullulanase preparation (3.5–4.0) was determined using isoelectric, focusing, in order to find optimal pH conditions for the coupling procedure. The immobilized pullulanase in a packed bed column was used to debranch amylopeetin to low molecular weight amylose.     

Preparation and characterization of enzymes immobilized by graft copolymerization to different polysaccharides

A method of enzyme immobilization by graft copolymerization on polysaccharides is reported. Glycidylmethacrylate was used as a vinylating reagent and the reaction product with enzymes (HRP, GOD, Am, ChT) was copolymerized with different matrices (cellulose, Sepharose, Sephadex, Starch). Various factors affect the final activity of copolymers; these include the redox system, the type of support, and the quantity and type of vinyl monomer added. Using a fixed quantity of enzyme and support (3 mg enzyme, 100 mg support), the coupling efficiency varied from 2 to 50%. The most important characteristics in these immobilized systems were tested (stability in continuous washing, kinetic characteristics, storage, thermal, and lyophilization stability). Immobilized-enzyme graft copolymers have very similar kinetic behavior to that of the free enzyme. Diffusion is not seriously limited, as shown by kinetic parameters and energy activation values, and this indicates that the immobilization reaction does not alter the enzymatic activity.     

Preparation of immobilized soybean β-amylase on porous cellulose beads and continuous maltose production

Immobilized soybean β-amylase was prepared by using porous cellulose beads. The expressed activity of the β-amylase–cellulose beads conjugated below 35 mesh was 59–69% of the initial activity and the protein content was 10–13%. General properties of the conjugate were almost identical with those of the native enzyme except for the K_m value. The K_m value of the conjugate was 40mM and the K_m value of the native enzyme was 0.6mM. This large difference was probably caused by pore structure, i.e., a pore diffusion problem. The film diffusion problem occurred at the flow rate below a linear velocity of 3 cm/min. Maximum maltose contents of the hydrolyzates prepared by the conjugate and the native enzyme were 69 and 71%, respectively. After a continuous column operation at 50°C for 17 days, the activity of the column was 60% of the activity. The half-life of the column at 40°C was 40 days.     

Immobilization of enzymes on Spheron: 2. β-Amylase — The development of a column reactor—separator

The titanium-chelation method has been used to immobilize β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) on to Spheron. On various grades of Spheron, protein coupling yields of 56–76% were obtained with barley and sweet-potato β-amylases. The specific enzymic activities of the immobilized enzymes fell in the range 3.7–7.6% of those of the soluble enzymes. The immobilized enzymes were more stable than the soluble, especially in the presence of l-cysteine and serum albumin. The presence of cysteine and serum albumin brought about increases in activity in the preparations, presumably by regenerating essential thiol groups in the enzyme which had been oxidized during the operations. Maltose could be separated from amylopectin and other large polysaccharides by chromatography on Spheron P100, and a system was developed in which maltose, produced by hydrolysis of amylopectin applied in pulses to a column of immobilized β-amylase, was separated from starting material and by-products on a second column of Spheron P100.