Comparison of some properties of free and immobilized alpha-amylase by Aspergillus sclerotiorum in calcium alginate gel beads

Some properties of immobilized alpha-amylase by Aspergillus sclerotiorum within calcium alginate gel beads were investigated and compared with soluble enzyme. Optimum pH and temperature were found to be 5.0 and 40 degrees C, respectively, for both soluble and immobilized enzymes. The immobilized enzyme had a better Km value, but kcat/Km values were the same for both enzymes. Entrapment within calcium alginate gel beads improved, remarkably, the thermal and storage stability of alpha-amylase. The half life values of immobilized enzyme and soluble enzyme at 60 degrees C were 164.2, and 26.2 min, respectively. The midpoint of thermal inactivation (Tm) shifted from 56 degrees C (for soluble enzyme) to 65.4 degrees C for immobilized enzyme. The percentages of soluble starch hydrolysis for soluble and immobilized alpha-amylase were determined to be 97.5 and 92.2% for 60 min, respectively.     

Immobilization of Chloroperoxidase from Serratia marcescens

A bacterial non-heme chloroperoxidase from Serratia marcescensW 250 was immobilized in calcium alginate gel. Methods for stabilization of the immobilized enzyme were developed, and some kinetic parameters of the immobilized preparations were determined. The enzyme encapsulated into the gel granules in the presence of potassium ferricyanide followed by treatment with glutaraldehyde demonstrated the highest stability under the reaction conditions.     

Comparative study of controlled pore glass, silica gel and poraver for the immobilization of urease to determine urea in a flow injection conductimetric biosensor system

This study compared the responses of three enzyme reactors containing urease immobilized on three types of solid support, controlled pore glass (CPG), silica gel and Poraver. The evaluation of each enzyme reactor column was done in a flow injection conductimetric system. When urea in the sample solution passed though the enzyme reactor, urease catalysed the hydrolysis of urea into charged products. A lab-built conductivity meter was used to measure the increase in conductivity of the solution. The responses of the enzyme reactor column with urease immobilized on CPG and silica gel were similar and were much higher than that of Poraver. Both CPG and silica gel reactor columns gave the same limit of detection, 0.5 mM, and the response was still linear up to 150mM. The analysis time was 4-5 min per sample. The enzyme reactor column with urease immobilized on CPG gave a slightly better sensitivity, 4% higher than the reactor with silica gel. The life time of the immobilized urease on CPG and silica gel were more than 310h operation time (used intermittently over 7 months). Good agreement was obtained when urea concentrations of human serum samples determined by the flow injection conductimetric biosensor system was compared to the conventional methods (Fearon and Berthelot reactions). These were statistically shown using the regression line and Wilcoxon signed rank tests. The results showed that the reactor with urease immobilized on silica gel had the same efficiency as the reactor with urease immobilized on CPG.     

Kinetic studies of sepharose-and CH-sepharose-immobilized dihydrofolate reductase

Dihydrofolate reductase, purified to homogeneity from amethopterin-resistant Lactobacillus casei, was immobilized by coupling to cyanogen bromide-activated Sepharose or carbodiimide-activated CH-Sepharose. Coupling yields were determined by amino acid analysis following the hydrolysis of the gel. Enzyme activity was measured by the conventional spectrophotometric procedure, thus permitting the facile characterization of the immobilized enzyme. The pH optimum of the immobilized enzyme was shifted to 5.8 compared with pH 5.5 for the soluble enzyme. The immobilized enzyme retained greater than 90%of the initial activity over a six-month period and could be reused as many as ten times without loss of activity. As observed with the soluble enzyme, the activity of immobilized enzyme, which was lost on denaturation with 4M guanidine hydrochloride, was recovered rapidly and completely by washing the gel with buffer. The K(m) (app) values for dihydrofolate and NADPH for the immobilized enzyme were increased 15-164-fold over the K(m) values measured for soluble dihydrofolate reductase. Scatchard analysis of the interaction of amethopterin with the immobilized enzyme yielded linear plots and a K(d) (app) value of 0.56 x10(-8)M, and revealed that all of the immobilized enzyme molecules were capable of binding the ligand.     

Properties and application of immobilized beta-D-glucosidase coentrapped with Zymomonas mobilis in calcium alginate

The enzyme beta-D-glucosidase has been immobilized on concanavalin A-Sepharose to give a maximum loading of 2050 units/g dry weight of support material. The immobilized beta-D-glucosidase was also entrapped within calcium alginate gel spheres with apparently only 35% retention of activity when assayed with 10mM cellobiose. However, it was discovered that, unlike the immobilized enzyme, the entrapped immobilized enzyme was not subject to substrate inhibition up to 100mM cellobiose, suggesting that a concentration gradient of cellobiose existed between the bulk solution and the interior of the gel sphere. Thus, the activity of the entrapped immobilized enzyme was almost twice as high as that of the immobilized enzyme when assayed with 100mM cellobiose. Concanavalin A-Sepharose-immobilized beta-D-glucosidase and the bacterium Zymomonas mobilis coimmobilized in calcium alginate gel spheres converted cellobiose to ethanol in both batch and continuous-flow fermentation systems.     

Development of simple methods for preparation of yeast and plant protoplasts immobilized in alginate gel beads

A simple method for preparation of yeast and plant protoplasts immobilized in alginate gel beads was developed. Yeast cells were first immobilized in strontium alginate gel beads and then treated with protoplast isolation enzyme so that the protoplasts are formed inside the beads. In the case of plant cells, degassing treatment was necessary in order to facilitate enzyme penetration into the cell aggregates. A mixture of the degassing treated plant cells and sodium alginate solution was dropped into SrCl2 containing the protoplast isolation enzymes. Thus protoplasts isolation and gel solidification proceeded simultaneously. With these methods, the required time was shorter while the viability of the immobilized protoplasts were higher than when the conventional method is used.     

Catalytic properties and stability of horseradish peroxidase immobilized in polyacrylamide gel]

Effect of polyacrylamide (PAA) gel on properties of horseradish peroxidase, immobilized by means of the incorporation into PAA gel is studied. Catalytic properties of immobilized enzyme are studied. Km value and pH-dependency of the enzyme activity are found to be close to those of soluble enzyme, kcat value is 3 times lower at pH 7.0. PH-stability of immobilized peroxidase at 20 degrees C and thermostability of soluble and immobilized peroxidases at pH 7.0 within the temperature range from 20 to 81 degrees C are studied. The stability of peroxidase in PAA gel is found to decrease (in 3 times at 20 degrees C, and in 17 times at 56 degrees C). A mechanism of the effect of PAA gel on catalytic properties and stability of peroxidase is discussed.     

Polyacrylamide gel immobilization of porcine liver esterase for the enantioselective production of levofloxacin

Porcine liver esterase was immobilized in polyacrylamide gel for the enantioselective production of levofloxacin from ofloxacin butyl ester. The initial activity of immobilized esterase was found to be significantly affected by the polyacrylamide gel composition. The optimum concentrations of monomer and crosslinker were determined to be 20% and 8.3%, respectively. The activity of immobilized esterase was 55.4% compared to a free enzyme. Enantiomeric excess was maintained at 60%, almost the same level as that of free enzyme. In addition, the immobilized esterase could be used repeatedly up to 10 times without experiencing any severe loss of activity and enantioselectivity.     

Amylase from Lactobacillus cellobiosus D-39 isolated from vegetable wastes: characteristics of immobilized enzyme and whole cell

Cells and partially purified α-amylase (EC 3.2.1.1) of the producer strain, Lactobacillus cellobiosus D-39 were immobilized on acrylamide gel. The enzyme showed marked improvement in operational stability. Both immobilized cells and enzyme were stable for a long period and no appreciable loss activity was detected on keeping at 4°C for 4 months. The amylase activity of immobilized cells and enzyme attained maximum at pH 6.0 and 7.6 respectively and at temperature 60°C for both cases. The effects of various solvents, detergent and metal ions were tested; Triton X-100 gave maximum stimulation of the enzyme activity of immobilized cells whereas metal ions exhibited no such enhancement for either of immobilized cells or enzyme.     

Immobilization of pectawamorine G10x by gel entrapment

Polyacrylamide gel immobilization of pectawamorine G10x was investigated. Its pectinesterase and polygalacturonase activity and stability in storage were measured. The degree of pectawamorine binding during gel immobilization was 80--90%, 55% of initial activity being retained. Thermal stability of the immobilized and native preparations was equal. Pectinesterase activity of the gel immobilized enzyme increased during storage.     

Comparative study of immobilized and soluble NADH:FMN-oxidoreductase-luciferase coupled enzyme system

The properties of a coupled enzyme system (NAD(P)H:FMN-oxidoreductase and luciferase) from luminous bacteria were studied. The enzymes and their substrates were immobilized in polymer gels of different types: starch (polysaccharide) and gelatin (polypeptide). Maximum activity yield (100%) was achieved with the enzymes immobilized in starch gel. An increase in K _{m app} was observed in both immobilized systems as compared with the soluble coupled enzyme system. Immobilization in starch and gelatin gels increased the resistance of the NAD(P)H:FMN-oxidoreductase and luciferase coupled enzyme system to the effects of external physical and chemical factors. The optimum pH range expanded both to the acidic and alkaline regions. The resistance to concentrated salt solutions and high temperature also increased. The coupled enzyme system immobilized in starch gel (with activation energy 30 kJ/mol) was characterized by the best thermostability. The immobilized coupled enzyme system can be used to produce a stable and highly active reagent for bioluminescent analysis.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

Production and immobilization of D-aminoacylase of Alcaligenes faecalis DA1 for optical resolution of N-acyl-DL-amino acids.

The production of D-aminoacylase by Alcaligenes faecalis DA1 was induced 5- to 50-fold by N-acetyl-D-amino acids. This strain produced about 443 units of D-aminoacylase and 52 units of L-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-DL-leucine as the carbon source. The D-aminoacylase was partially purified by Fractogel DEAE 650 column chromatography and then immobilized on another Fractogel DEAE 650 column. The catalytic activity of the immobilized D-aminoacylase was 2,650 units per milliliter of gel. The Km values for the free and the immobilized enzymes were found to be 1.00 and 0.22 mM, respectively, using N-acetyl-D-methionine as a substrate. The optimal reaction pH and temperature for both soluble and immobilized enzyme were around 8.0 and 45 degrees C, respectively. The free enzyme was stable in the pH range from 5.0 to 11.0, whereas the immobilized enzyme tended to detach from the gel at pH values higher than 9.0. Both forms of enzyme were stable up to 40 degrees C. When used for the optical resolution of N-acetyl-DL-methionine, the immobilized enzyme maintained 90% initial activity after 17 days of continuous operation at 45 degrees C. The process of purification and immobilization of D-aminoacylase described in this report is very effective and easy to scale up.     

A new immobilization of microbial cells

Summary A new immobilization of microbial cells based on the growth of cells in gel is presented. The cells grew very well in carrageenan gel when fed nutrients required for growth. The growing cells immobilized in gel formed a dense layer of cells near the gel surface. Because the cells were near the gel surface, they efficiently catalyzed single enzyme reactions. In addition, the immobilized growing cell system was applied to the complicated multienzyme reactions since large numbers of cells in the gel could constantly be maintained for long periods.     

Trypsin immobilization in silica gel

Trypsin immobilization in an inorganic polymer, i.e. silica gel, was investigated. Properties of the immobilized enzyme were examined. It was found that the enzyme retained its activity, depending on the gel genesis and dehydration of the carrier. The effect of stabilizing supplements on the enzyme activity was studied. The enzyme immobilization was accompanied by an increase in its thermostability.     

Isolation and characteristics of immobilized preparations of prostaglandin synthetase

Enzymatic preparations of prostaglandin synthetase were immobilized by various methods, i.e., covalent binding to the carrier, incorporation into the matrix of polyacrylamide gel and adsorption on ion-exchangers. The most representative results were obtained with the use of the absorption technique. In this case, the enzyme activity either decreased very insignificantly or remained unaffected. The stability of immobilized enzyme preparations was studied. Some kinetic characteristics of immobilized prostaglandin synthetase were obtained.     

Depolymerization of chondroitin C Sulfate by immobilized Proteus vulgaris cells

Chondroitinase ABC catalyzing the depolymerization of chondroitin sulfate was induced by incubating the Proteus vulgaris cells in a medium containing chondroitin C sulfate as an inducer. Incubation of P. vulgaris cells for 12 h in the presence of 0.3% inducer was optimal to obtain the cells with highly active chondroitinase ABC. Such cells were immobilized in k-carrageenan gel lattice, and some properties of chondroitinase ABC in immobilized cells were studied in comparison with those of the enzyme without immobilization (free enzyme). The stabilities of the enzyme toward heat and storage were remarkably improved by immobilizing the cells in k-carrageenan gel lattice. Optimal pH and temperature for activity of the enzyme were slightly shifted to the alkaline region and higher temperature by immobilization and were 9.0 and 35 degrees C, respectively.     

Penicillin amidase from E. coli. A comparative study of the stability of penicillin amidase immobilized by various means]

The properties of immobilized penicillinamidases prepared by different methods were studied. Immobilization of penicillinamidase was achieved by using the covalend binding ion exchange sorption, incorporation into gel and other procedures. The effect of the carrier type, purification level of the native enzyme and other factors on stability of the immobilized preparations was studied. In 4 cases significant stabilization of the enzyme in the immobilized state was observed, while in 3 cases destabilization was registered.     

Improved immobilized enzyme systems using spherical micro silica sol-gel enzyme beads

Spherical micro silica sol-gel immobilized enzyme beads were prepared in an emulsion system using cyclohexanone and Triton-X 114. The beads were used for thein situ immobilization of transaminase, trypsin, and lipase. Immobilization during the sol to gel phase transition was investigated to determine the effect of the emulsifying solvents, surfactants, and mixing process on the formation of spherical micro sol-gel enzyme beads and their catalytic activity. The different combinations of sol-gel precursors affected both activity and the stability of the enzymes, which suggests that each enzyme has a unique preference for the silica gel matrix dependent upon the characteristics of the precursors. The resulting enzyme-entrapped micronsized beads were characterized and utilized for several enzyme reaction cycles. These results indicated improved stability compared to the conventional crushed form silica sol-gel immobilized enzyme systems.     

Novel sol-gel immobilization of horseradish peroxidase employing a detergentless micro-emulsion system

A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.