Enzymatic conversion of invertase substrates in a water-organic medium

The behavior of intact and immobilized invertase in aqueous and water-organic media has been studied. In a water-organic medium, the transferase properties of the enzyme changed: pH optima of intact and immobilized invertase undergo shifts of 0.5 units; the temperature optimum decreases (50°C and 25°C in aqueous and water-organic media, respectively). The extent of conversion of isoamyl alcohol in water-organic medium shows a dependence on several factors (the enzyme and substrate concentrations; amount of the organic phase; and duration of the enzyme incubation with the substrate). Optimum parameters of isoamyl alcohol conversion were used for transforming fusel alcohols into alkyl fructosides. The results of this applied research have important practical implications (conversion of fusel oils of alcoholic beverages).     

Enzymatic conversion of invertase substrates in a water-organic medium

The behavior of intact and immobilized invertase in aqueous and water-organic media has been studied. In a water-organic medium, the transferase properties of the enzyme changed: pH optima of intact and immobilized invertase undergo shifts of 0.5 units: the temperature optimum decreases (50 degrees C and 25 degrees C in aqueous and water-organic media, respectively). The extent of conversion of isoamyl alcohol in water-organic medium shows a dependence on several factors (the enzyme and substrate concentrations; amount of the organic phase; and duration of the enzyme incubation with the substrate). Optimum parameters of isoamyl alcohol conversion were used for transforming fusel alcohols into alkyl fructosides. The results of this applied research have important practical implications (conversion of fusel oils of alcoholic beverages).     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Chemically surface modified gel (CSMG): an excellent enzyme-immobilization matrix for industrial processes

Invertase from S. cerevisiae has been immobilized on porous silica matrix, formed using sol-gel chemistry, with surface area of approximately 650 m(2)/g. The co-condensation of silica sol with 3-aminopropyl(triethoxy)silane produced an amino-chemically surface modified silica gel (N-CSMG) with a very high ligand loading of 3.6 mmol/g SiO(2); significantly higher than commercially available matrices. Surface amine groups were activated with glutaraldehyde to produce GA-N-CSMG, and invertase covalently attached by the aldehyde. Invertase was used as a model enzyme to measure the immobilizing character of the GA-N-CSMG material. Using an optimized immobilization protocol, a very high loading of 723 mg invertase per gram GA-N-CSMG is obtained; 3-200-fold higher than values published in literature. The reproducible, immobilized activity of 246,000 U/g GA-N-CSMG is also greater than any other in literature. Immobilized invertase showed almost 99% retention of free enzyme activity and no loss in catalytic efficiency. The apparent kinetic parameters K(M) and V(M) were determined using the Michealis-Menten kinetic model. K(M) of the free invertase was 1.5 times greater than that of the immobilized invertase--indicating a higher substrate affinity of the immobilized invertase. These findings show considerable promise for this material as an immobilization matrix in industrial processes.     

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

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 enzyme onto poly(ethylene-vinyl alcohol) membrane

Invertase was ionically bound to the poly(ethylene-vinyl alcohol) membrane surface modified with two aminoacetals with different molecular length, 2-dimethyl-aminoacetoaldehyde dimethylacetal (AAA) and 3-(N, N-dimethylamino-n-propanediamine) propionaldehyde dimethylacetal (APA). Immobilization conditions were determined with respect to enzyme concentration in solution, pH value, ionic strength in immobilization solution, and immobilization time. Various properties of immobilized invertase were evaluated, and thermal stability was found especially to be improved by immobilization. The apparent Michaelis constant, K(m), was smaller for invertase bound by APA with longer molecular lengths than for invertase bound by AAA. We attempted to bind glucoamylase of Rhizopus delemar origin in the same way. The amount and activity of immobilized glucoamylase were much less than of invertase.     

Optimization of invertase immobilization by adsorption in ionic exchange resin for sucrose hydrolysis

This work presents as a main objective to study the immobilization process of yeast invertase by adsorption in the ion exchanging resin Duolite A-568 for invert sugar production. Initially, a kinetic study of the soluble form of the enzyme was carried out. At the sequence was studied the immobilization process of yeast invertase in the weakly exchanging anionic resin Duolite A-568. The influences of the pH, enzyme concentration and temperature in the enzyme immobilization were analyzed through a central composite design (CCD). The results indicated that the retention of the catalytic activity in immobilization was strongly dependent of these variables, being maximum in a pH value of 5.0, with an enzyme concentration of 12.5 g/L (1.875 g of protein per liter) and temperature of 30 °C. The simultaneous influence of pH and temperature on the free and immobilized invertase activity was also studied through a CCD.     

Immobilization of cane invertase on bentonite

Sugar-cane invertase (β-d-fructofuranoside fructohydrolase, EC 3.2.1.26) immobilized on bentonite clay in 0.05 m acetate buffer, pH 4.5, has been shown to be capable of hydrolysing sucrose. The bentonite-invertase (BI) complex gave 55.5% retention of enzyme activity on the surface. A further 17 and 22% increase in retention of enzyme activity was obtained using the covalent linking agents, cyanuric chloride and thionyl chloride, giving bentonite-cyanuric chloride-invertase (BCCI) and bentonite-thionyl chloride-invertase (BTCI) complexes. Concentrations of acetate buffer >0.2 M disrupt the bentonite-invertase complexes. The immobilized invertase complexes showed high temperature optima (60–65°C) and high thermal stability compared to the free enzyme. The pH profiles of the free and immobilized enzyme were the same. The rate of hydrolysis of sucrose was increased using immobilized enzymes, which required a higher substrate concentration than the free enzyme. The insoluble enzyme conjugate-carrier complexes when used for sucrose hydrolysis in a batch process showed 53.1 (BI), 57.4 (BCCI) and 59.6% (BTCI) conversions, respectively, in 12 h, compared to 42.3% conversion in 24 h with the free enzyme. The immobilized invertase complexes can be used for sucrose inversion for about five cycles. The application of this immobilization procedure may help in the removal of invertase from cane juice to reduce sugar losses in industry.     

Clues to the origin of high external invertase activity in immobilized growing yeast: prolonged SUC2 transcription and less susceptibility of the enzyme to endogenous proteolysis.

Expression of the SUC2 gene encoding invertase was studied using free and gelatin-immobilized yeast cells to try to explain the high activity of this enzyme exhibited by immobilized cells when allowed to grow in a nutrient medium. The results indicated that at least two factors are probably responsible for the accumulation of invertase in immobilized cells. First, the expression of the SUC2 gene was maintained throughout growth in immobilized cells, whereas its expression was only transient in free cells. Second, invertase of immobilized cells was shown to be less susceptible to endogenous proteolytic attack than that of the corresponding free cells. These results have been interpreted, respectively, in terms of diffusional limitations and changes in the pattern of invertase glycosylation due to growth of yeast in an immobilized state.     

Enhancement of operational stability of an enzyme biosensor for glucose and sucrose using protein based stabilizing agents

With the incorporation of lysozyme during the immobilization step, considerable enhancement of the operational stability of a biosensor has been demonstrated in the case of an immobilized single enzyme (glucose oxidase) system for glucose and multienzyme (invertase, mutarotase and glucose oxidase) system for sucrose. Thus an increased number of repeated analyses of 750 samples during 230 days for glucose and 400 samples during 40 days of operation for sucrose have been achieved. The increased operational stability of immobilized single and multienzyme system, will improve the operating cost effectiveness of the biosensor.     

Immobilization and stabilization of invertase on Cajanus cajan lectin support

Use of lectins as ligands for the immobilization and stabilization of glycoenzymes has immense application in enzyme research and industry. But their widespread use could be limited by the high cost of their production. In the present study preparation of a novel and inexpensive lectin support for use in the immobilization of glycoenzymes containing mannose or glucose residues in their carbohydrate moiety has been described. Cajanus cajan lectin (CCL) coupled covalently to cyanogen bromide activated Seralose 4B could readily bind enzymes such as invertase, glucoamylase and glucose oxidase. The immobilized and glutaraldehyde crosslinked preparations of invertase exhibited high resistance to inactivation upon exposure to enhanced temperature, pH, denaturants and proteolysis. Binding of invertase to CCL-Seralose was however found to be readily reversible in the presence of 1.0 M methyl alpha-D mannopyranoside. In a laboratory scale column reactor the CCL-Seralose bound invertase was stable for a month and retained more than 80% of its initial activity even after 60 days of storage at 4 degrees C. CCL-Seralose bound invertase exhibited marked stability towards temperature, pH changes and denaturants suggesting its potential to be used as an excellent support for the immobilization of other glycoenzymes as well.     

Immobilization of chitosan-invertase neoglycoconjugate on carboxymethylcellulose-modified chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on a carboxymethylcellulose-coated chitin support via polyelectrolyte complex formation. The yield of immobilized protein was determined to be 72% and the enzyme retained 68% of the initial invertase activity. The optimum temperature for invertase was increased by 5 degrees C and its thermostability was enhanced by about 9 degrees C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 12.6-fold more resistant to thermal treatment at 65 degrees C than the native counterpart. The prepared biocatalyst retained 98% and 100% of the original catalytic activity after 10 cycles of reuse and 70 h of continuous operational regime in a packed bed reactor, respectively. The immobilized enzyme retained 95% of its activity after 50 days of storage at 37 degrees C.     

Polyaniline grafted polyacylonitrile conductive composite fibers for reversible immobilization of enzymes: Stability and catalytic properties of invertase

Polyacylonitrile fibers (PAN) surfaces were modified with chemical polymerization of conductive polyaniline (PANI) in the presence of potassium dichromate as an oxidizing agent. The effect of aniline concentration on the grafting efficiency and on the electrical surface resistance of PAN/PANI composite fibers was investigated. The surface resistance of the conductive composite fibers in this work was found to be between 8.0 and 0.5 kΩ/cm. As the amount of grafted PANI increased on the PAN fibers the electrical resistance of composite fibers decreased. The PAN/PANI composite fibers were characterized by SEM and FTIR studies. Composite PAN/PANI fibers were used for reversible immobilization of invertase. The immobilization efficiency and the activity of the immobilized invertase (from 1.0 mg/mL invertase solution at pH 5.5) were increased with increasing PANI contents of the composite fibers. The maximum amount of immobilized enzyme onto composite fibers containing 2.0% PANI was about 76.6 mg/g. The optimum pH for the free enzyme was observed at 5.0. On the other hand, immobilized invertase yielded a broad optimum pH profile between pH 5.0 and 7.0. Immobilized invertase exhibited 83% of its original activity even after two months storage at 4 °C while the free enzyme showed only 7% of its initial activity.     

Invertase covalent grafting onto corn stover

The covalent coupling of an invertase from baker's yeast onto an agricultural by-product, corn grits, has been developed. The optimal conditions for each step of the chemical modification of the support have been determined: oxidation with sodium metaperiodate, amination with ethylenediamine, reduction with sodium cyanoborohydride, and activation with glutaraldehyde. Activities up to 7.2 x 10(4) mumol reducing sugars produced/min g support could thus be achieved. Invertase coupling onto corn grits yields a derivative with a 25 times higher activity than when coupling this enzyme onto porous silica. The operational stability of invertase immobilized onto corn stover was found to be very high, with a half-life of up to 365 days at 40 degrees C when using a 2M sucrose solution as substrate. This immobilization method could be easily scaled up to the preparation of 10 kg of invertase derivative.     

Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 10 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively.     

Properties of invertase immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber membrane

Invertase was ionically immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2-dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 mug/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K(m) (') for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined.     

Preparation of Membranous Immobilized Invertase and It’s Characteristics

Invertase was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was conducted aerobically while the mixture of monomers and enzyme was frozen. Retained activity was 51~76%. Immobilized invertase shifted its optimum pH by about 0.7 to the acidic site.

The optimum reaction temperature of enzyme became a little higher (Ca 5°C) by immobilization. Heat stability was improved by immobilization. Release of fixed enzyme was found to be considerably low (1.2~4.1%) and release of several immobilized proteins decreased as the molecular weight increased.     

Immobilization of chitosan-modified invertase on alginate-coated chitin support via polyelectrolyte complex formation

Saccharomyces cerevisiae invertase was chemically modified with chitosan and further immobilized on sodium alginate-coated chitin support. The yield of immobilized protein was determined as 85% and the enzyme retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 °C and its thermostability was enhanced by about 9 °C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was four-fold more resistant to thermal treatment at 65 °C than the native counterpart. The biocatalyst prepared retained 80% of the original catalytic activity after 50 h under continuous operational regime in a packed bed reactor.     

Immobilization of invertase via carbohydrate moiety on chitosan to enhance its thermal stability

A new technique using chitosan as support for covalent coupling of invertase via carbohydrate moiety improved the activity and thermal stability of immobilized invertase. The best preparation of immobilized invertase retained 91% of original specific activity (412 U mg^–1). The half-life at 60°C was increased from 2.3 h (free invertase) to 7.2 h (immobilized invertase). In contrast, the immobilization of invertase via protein moiety on chitosan or using Sepharose as support resulted in less thermostable preparations. Additionally, immobilization of invertase on both supports caused the optimal reaction pH to shift from 4.5 to 2.5 and the substrate (sucrose) concentration for maximum activity to increase from 0.5 M to 1.0 M.