Production of gluconic acid by immobilized in pumice stones mycelium of Aspergillus niger using unconventional oxygenation of culture

Gluconic acid was produced in repeated batch processes with Aspergillus niger AM-11, immobilized in pumice stone particles using an unconventional oxygenation of culture media based on the addition of H₂O₂, decomposed by catalase to O₂ and water. The highest gluconic acid productivity of 8.2 g l^–1 h^–1 was reached with 30 g immobilized mycelium per 150 ml, 10% (w/v) glucose, at 24 °C and pH 6.5, with O₂ at 100% saturation. The immobilized mycelium was successfully reused up to 8 times in 1-h batches with only a slight loss (11%) of gluconic acid productivity.     

Coimmobilization of D-amino acid oxidase and catalase by entrapment ofTrigonopsis variabilis in radiation polymerised Polyacrylamide beads

Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells. However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide. The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO₂ was not effective in enhancing the operational life of the enzyme preparation.     

Immobilized xanthine oxidase: Kinetics, (in)stability,and stabilization by coimmobilization with superoxide dismutase and catalase

Milk xanthine oxidase was immobilized by covalent attachment to CNBr-activated Sepharose 4B and by adsorption to n-octylamine-substituted Sepharose 4B. The amounts of activity immobilized for the two preparations were 30 and 90%, respectively. The pH optima for free and adsorbed xanthine oxidase were at 8.6 and 8.2, respectively. Both free and immobilized xanthine oxidase show substrate inhibition. The apparent inhibition constant (K_i′) found for adsorbed xanthine oxidase with xanthine as substrate was higher than the K_i for the free enzyme, which was shown to be due to substrate diffusion limitation in the pores of the carrier beads (internal diffusion limitation). Higher substrate concentrations, as desirable for practical application in organic synthesis, can therefore be used with the immobilized enzyme without decreasing the rate. As a result of the internal diffusion limitation the apparent Michaelis constant (K_m′) for adsorbed xanthine oxidase was also higher than the K_m for the free enzyme. Immobilized xanthine oxidase was more stable than the free enzyme during storage at 4 and 30°C. Both forms rapidly lost activity during catalysis. The loss was proportional to the amount of substrate converted. Coimmobilization of xanthine oxidase with superoxide dismutase and catalase improved the operational stability, suggesting that O₂^? and H₂O₂ side-products of the enzymatic reaction were involved in the inactivation. Coimmobilization with albumin also had some stabilizing effect. Complete surrounding of xanthine oxidase by protein, however, by means of etrapment in a glutaraldehyde-crosslinked gelatin matrix, considerably enhanced the operational half-life. This system was less efficient than the Sepharose preparations either because much activity was lost during the immobilization procedure and/or because it had poor flow properties. Xanthine (15 mg)was converted by an adsorbed xanthine oxidase preparation and product (uric acid) was isolated in high yield (84%).     

Chainia Penicillin V Acylase: Strain Characteristics, Enzyme Immobilization, and Kinetic Studies

Aerobic cultures of an actinomycete were found to produce penicillin V acylase (PVA) (PA, EC-3.5.1.11) extracellularly. The presence of L-2-3 diamino-propionic acid in cell wall and formation of sclerotia on culture media led to its identification as Chainia, a sclerotial Streptomyces. Partially purified acylase was adsorbed on kieselguhr and entrapped in polyacrylamide gel. The immobilized preparation proved effective with respect to retention of enzyme and enzyme activity even after 15 successful cycles. The pH optimum for crude enzyme was in the range of pH 7.5–8.0, and for the (NH4)₂ SO₄ fraction it was pH 8.5. The immobilized enzyme showed maximal activity at pH 9.5. The optimum temperature for acylase activity was at 55°C. The crude enzyme, ammonium sulfate fraction, and immobilized enzyme showed K _m value for penicillin V of 6.13 mM, 14.3 mM, and 17.1 mM, respectively. Received: 11 December 1997 / Accepted: 9 April 1998     

Immobilization of Xylan-Degrading Enzymes from Scytalidium thermophilum on Eudragit L-100

Summary Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of activity between 55 and 65 °C as compared to 65 °C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared to 6.5 in case of free enzyme. Immobilization increased the t_1/2 of enzyme at 60 °C from 15 to 30 min with a stabilization factor of 2. The K_m and V_max values for the immobilized and free xylanase were 0.5% xylan and 0.89 μmol/ml/min and 0.35% xylan and 1.01 μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227 kcal/mol) as compared to free xylanase (210 kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification of xylan was also improved by xylanase immobilization.     

Immobilization ofKluyvera citrophila penicillins acylase on controlled-pore ceramics

Penicillin acylase was purified fromKluyvera citrophila and immobilized on glutaraldehyde derivatives of silanized controlled-pore ceramics. The behaviour of the enzyme attached to TiO₂, Al₂O₃ and SiO₂ in the hydrolytic reaction are compared with that of the native enzyme as well as of the enzyme bound to CNBr-activated Sepharose 4B. The enzyme immobilized on TiO2 shows an efficiency of about 95% on the basis of protein bound. The penicillin acylase attached to SiO₂, unlike the enzyme immobilized on TiO₂, Al₂O₃ and Sepharose looses activity markedly in every cycle of use.     

Quantitating intraparticle O2 gradients in solid supported enzyme immobilizates: Experimental determination of their role in limiting the catalytic effectiveness of immobilized glucose oxidase

Enzymatic O₂‐dependent oxidations are receiving increased attention for use in fine chemicals synthesis. Solid supported oxidation catalysts often show poor efficiency due to pronounced O₂ diffusion restriction. Internal O₂ supply therefore constitutes a key parameter for optimizing the enzyme immobilization. We herein describe an optical sensing method for quantitation of space‐averaged intraparticle O₂ concentrations in porous Sepabeads carriers. The method applies phosphorescence lifetime measurements on Sepabeads labeled with an O₂ sensitive indicator dye. Using glucose oxidase immobilized at different loadings (0.005–12 mg/g) on labeled Sepabeads, we analyzed in real time during the enzymatic reaction the formation of O₂ concentration differences between bulk liquid and the intraparticle environment. We show that the O₂ gradient at apparent steady state increased with increasing enzyme loading, so that O₂ eventually became totally depleted from inside the highly loaded carriers. We also show that the residual intraparticle O₂ concentration was correlated with the catalytic effectiveness factor (η) of the enzyme immobilizate used, thus providing a direct measure of the magnitude of O₂ diffusion limitation. Once corrected for diffusional effect, η was no longer dependent on enzyme loading and its constant value now described the intrinsic activity of immobilized glucose oxidase. Three common procedures of enzyme immobilization, involving adsorption, cross‐linking, and covalent attachment, are shown to differ widely concerning the obtained intrinsic activity. Therefore, intraparticle O₂ concentration data enable distinction between diffusional restriction and activity loss as the two principal factors limiting the effectiveness of immobilized O₂ dependent enzymes, and thus they inform rational design of an optimally active oxidation biocatalyst on solid support. Biotechnol. Bioeng. 2013; 110: 2086–2095. © 2013 Wiley Periodicals, Inc.     

Enhanced biodesulfurization by magnetic immobilized <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> LSSE8-1-vgb assembled with nano-γ-Al<Subscript>2</Subscript>O<Subscript>3</Subscript>

Biodesulfurization activity can be enhanced by assembling nano-γ-Al₂O₃ particles on the magnetic immobilized Rhodococcus erythropolis LSSE8-1-vgb. The cells can be collected and reused conveniently by an external magnetic field. Firstly, cells were magnetic immobilized by coating with Fe₃O₄ nano-particles. The optimal ratio of cells to magnetic Fe₃O₄ nano-particles was determined to be 50:1 (g/g). Then nano-γ-Al₂O₃ adsorbents were assembled onto the cells to enhance the desulfurization activity. The nano-γ-Al₂O₃ adsorbent had the largest pore volume as well as specific surface area, and the strongest electrostatics interaction with microbial cell, and cells assembled with this nano-adsorbent performed the highest desulfurization activity. The activity of magnetic immobilized cells assembled with adsorbents was tested in desulfurization of model oil. The desulfurization rate was raised by nearly 20% when the amount ratio of magnetic particles to adsorbents was 1:5 (g/g). These cells can be reused. The activity decreased less than 10% through out three desulfurization-activation-reuse recycles.     

A Study on the Process of Lipase-catalyzed Synthesis of α-pinene Oxide in Organic Solvents

The synthesis of α-pinene oxide was studied in a three-phase system where immobilized Candida antarctica lipase B (Novozyme 435) was used to catalyze the formation of peroxyoctanoic acid from the parent carboxylic acid and hydrogen peroxide in toluene. The peroxycarboxylic acid formed was then used in situ for the oxidation of α-pinene to the corresponding epoxide. When hydrogen peroxide was added in the reaction mixture gradually over 6 h, conversions increased up to 31.6%. Initial rates of α-pinene oxidation increased from 85 to 708 mmol L^?1 h^?1 when the amount of H₂O₂ increased from 5 to 60 mmol. When the lipase was exposed to 75 mmol H₂O₂ for 0.5 h before its addition in the reaction mixture, its activity decreased to about 50%. The reusability of lipase was studied in five reaction cycles and was found to depend on the concentration of the hydrogen peroxide used.     

Stabilization of d-amino acid oxidase from Rhodosporidium toruloides by immobilization onto magnetic nanoparticles

d-Amino acid oxidase from Rhodosporidium toruloides was immobilized onto glutaraldehyde-activated magnetic nanoparticles. Approximately four enzyme molecules were attached to one magnetic nanoparticle when the weight ratio of the enzyme to the support was 0.12. After immobilization, the T _m was increased from 45°C of the free form to 55°C. In the presence of 20 mM H₂O₂, the immobilized form retained 93% of its activity after 5 h while the free form was completely inactivated after 3.5 h.     

Immobilization of Rhizopus oryzae lipase on magnetic Fe3O4-chitosan beads and its potential in phenolic acids ester synthesis

A novel and simple method was developed for the preparation of magnetic Fe₃O₄ nanoparticles by chemical co-precipitation method and subsequent coating with 3-aminopropyltrimethoxysilane (APTMS) through silanization process. Magnetic Fe₃O₄-chitosan particles were prepared by the suspension cross-linking and covalent technique to be used in the application of magnetic carrier technology. The synthesized immobilization supports were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). Using glutaraldehyde as the coupling agent, the lipase from R. oryzae was successfully immobilized onto the functionalized magnetic Fe₃O₄-chitosan beads. The results showed that 86.60% of R. oryzae lipase was bound on the synthesized immobilization support. This immobilized lipase was successfully used for the esterification of phenolic acid which resulted in esterification of phenolic acid in isooctane solvent reaction system for 8 consecutive cycles (totally 384 h), 72.6% of its initial activity was retained, indicating a high stability in pharmaceutical and industrial applications.     

Production of α-keto acids: 2. Immobilized whole cells of Providencia sp. PCM 1298 containing -amino acid oxidase

A number of bacteria belonging to the genera Proteus, Providencia, Pseudomonas and Erwinia have been tested for their capacity to oxidize -amino acids to their corresponding α-keto acids. Members of the Proteus and the Providencia genera were active towards various -amino acids. Immobilized cell preparations of Providencia sp. PCM 1298 were shown to form up to 80 mg α-keto-γ-methiol butyric acid from -methionine per g of gel preparation (containing 4% w/w cells) per day. The productivity was highly dependent on the size of the beads. Oxygen appeared to be the rate-limiting substrate and oxygen transfer rates of 3–4 μmol cm⁻² h⁻¹ were calculated. The entrapment of activated charcoal to remove H₂O₂ formed during the oxidation extended the half-life of the immobilized biocatalyst considerably. A decrease in -amino acid oxidase [ -amino acid: oxygen oxidoreductase (deaminating); EC 1.4.3.2] activity during operation could be compensated for by reinoculation of the alginate-entrapped cells in fresh growth medium, allowing use of these preparations of immobilized bacterial cells for more than one month.     

Continuous production of L-aspartic acid by immobilized aspartase

Various methods were tried for the immobilization of aspartase, and the preparation having the highest activity was obtained when partially purified aspartase from Escherichia coli was entrapped into polyacrylamide gel Iattice. Enzymatic properties of the immobilized aspartase were investigated and compared with those of the native aspartase. With regard to optimum pH, temperature, concentration of Mn⁺⁺, kinetic constants and heat stability, no marked difference was observed between the native and immobilized aspartases. By employing an enzyme column packed with the immobilized aspartase, conditions for continuous production of L -aspartic acid from ammonium fumarate were investigated. When a solution of 1M ammonium fumarate (pH 8.5, containing 1mM MnCl₂) was passed through the aspartase column at the flow rate of SV = 0.08 at 37°C, the highest rate of reaction was attained. From the column effluents, L-aspartic acid was obtained in a good yield.     

The simultaneous production of sorbitol from fructose and gluconic acid from glucose using an oxidoreductase of Zymomonas mobilis

Summary The production of sorbitol and gluconic acid by toluene-treated, permeabilized cells of Zymomonas mobilis has been evaluated. From a 60% total sugar solution (300 g/l glucose and 300 g/l fructose), a sorbitol concentration of 290 g/l and a gluconic acid concentration of 283 g/l were achieved after 15 h in a batch process using free toluene-treated cells. A continuous process with immobilized cells was developed and only a small loss of enzyme activity (less than 5%) was evident after 120 h. With a strongly basic anion exchange resin and an eluent of 0.11 M Na₂B₄O₇/0.11 M H₃BO₃, good separation of sorbitol and gluconic acid was achieved.     

A carbon nanotube/silica sol–gel architecture for immobilization of horseradish peroxidase for electrochemical biosensor

A novel third-generation biosensor for hydrogen peroxide (H₂O₂) has been constructed based on horseradish peroxidase (HRP) immobilized by the sol–gel (SG) technology on carbon nanotube (CNT)-modified electrode. CNT has good promotion effects on the direct electron transfer between HRP and the electrode surface and the SG network provides a biocompatible microenvironment for enzyme. The immobilized HRP retained its bioelectrocatalytic activity for the reduction of hydrogen peroxide and can respond to the change of concentration of H₂O₂ rapidly. The heterogeneous electron transfer rate constant was evaluated to be 2.8 ± 0.4 s⁻¹. The amperometric response to H₂O₂ shows a linear relation in the range from 0.5 to 300 μmol l⁻¹ and a detection limit of 0.1 μmol l⁻¹ (S/N = 3). The K _M^app value of HRP immobilized on the electrode surface was found to be 1.35 mmol l⁻¹. The biosensor exhibited high sensitivity, rapid response and excellent long-term stability.     

Improvement of activity and stability of Rhizomucor miehei lipase by immobilization on nanoporous aluminium oxide and potassium sulfate microcrystals and their applications in the synthesis of aroma esters

In this study, Rhizomucor miehei lipase (RML) was immobilized on the hexagonally-ordered nanoporous aluminium oxide membranes (RML-Al₂O₃-NP) by adsorption and as protein-coated microcrystals (RML-PCMCs) by simultaneously precipitating RML on micron-sized potassium sulfate crystals (K₂SO₄) in pre-chilled acetone. The hydrolytic activities of immobilized lipase preparations were investigated in terms of p-nitrophenyl palmitate hydrolysis and their esterification activities were examined for the synthesis of some aroma esters such as butyl acetate, isoamyl acetate, hexyl acetate, heptyl acetate, and geranyl acetate. The immobilization yields were 33.8 and 25.1%, respectively for RML immobilized on Al₂O₃-NP membranes and potassium sulfate crystals. The catalytic efficiency ratios of RML-Al₂O₃-NP and RML-PCMCs were 2.3- and 3.9-fold higher than that of the free lipase, respectively in terms of hydrolytic activity. The free lipase was stabilized as 4.1- and 10.5-fold, respectively at 40 and 50?°C when immobilized on Al₂O₃-NP. The corresponding stabilization factors were 4.6- and 12.8-fold higher for RML-PCMCs. RML-Al₂O₃-NP and RML-PCMCs maintained 84 and 86% of their initial hydrolytic activities, respectively after 10 reuses. Of the synthesized aroma esters, the highest yield was obtained for the geranyl acetate. After 4?h reaction time, no geraniol was detected in the preparative-scale (196?g/L) synthesis of geranyl acetate for both the immobilized lipases when the initial geraniol amount, vinyl acetate amount, RML-PCMCs amount, and reaction temperature values were 1?mmol, 3?mmol, 100?mg (or 300?mg RML-Al₂O₃-NP), and 50?°C, respectively. These results show that the immobilization of R. miehei lipase by adsorption on nanoporous aluminium oxide and as protein-coated microcrystals leads to the obtention of highly stable, catalytically more active, and reusable lipase preparations.     

Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

An investigation of the properties of glucoamylase immobilized on glass beads involving 5-diazosalicylic acid bonded to a titanium (IV) oxide film

Optimum conditions have been determined for the immobilization of glucoamylase on glass involving diazotized 5-aminosalicylic acid bonded to a deposited imperfectly crystallized film of TiO₂. The changes in the kinetic and thermodynamic characteristics of the enzyme on immobilization have been determined. There are significant differences in the behaviour of the immobilized enzyme towards its substrates, maltose and starch. The apparent K_m for starch increased on immobilization whereas that for maltose decreased. The pH optimum for the immobilized preparation showed a shift to acid pH relative to that of the free enzyme.     

The properties of subtilisin,Novo type,immobilized on porous glass

Studies of the properties of subtilisin, Novo type, immobilized on porous glass with the aid of hexamethylene diisocyanate were carried out. The immobilized proteinase preparation shows optimum activity at a pH value of 10.7 and at a temperature between 60–65°C. It was stable in a wider range of pH and temperature values than the native subtilisin. The K_M values for hemoglobin and BAEE were 9.2 × 10^?5 [M] and 139 × 10^?5 [M], respectively. Under relatively non-aqueous conditions, immobilised subtilisin was able to synthesize phenylacetic acid ethyl ester.     

Evidence for conformational changes in grape catechol oxidase

A rapid, 4–10-fold, activation of grape catechol oxidase by a short exposure to acid pH or urea is demonstrated. Activation was either reversible or irreversible, depending on length and type of treatment. The change in activity of the enzyme is due primarily to an increase in V_max, while the affinity for 4-methylcatechol decreases and that for O₂ increases. Activation occurs in intact chloroplasts as well as in a partially purified enzyme preparation. Activation was apparently due to conformational changes in the enzyme. O₂ concentration appeared to control enzyme activity, presumably by an O₂ induced conformational change. Irreversible activation was accompanied by changes in the electrophoretic mobility of the enzyme.