Stabilization of native and immobilized urease

The stability of native and immobilized urease isolated from Staphylococcus saprophyticus was studied at 4 degrees and 25 degrees C. The activity yield was 20% and 1.4% on the enzyme immobilization in albumin gel and latex membrane, respectively. Inactivation of native microbial urease proceeded 10 times slower in the solution containing 1 mM EDTA and 30 mM sodium sulfite. This solution contributed to a great extent to stabilization of immobilized urease both during storage in the phosphate buffer solution and in case of lyophilization.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.     

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.     

Tyrosine hydroxylase activity of immobilized tyrosinase on enzacryl-AA and CPG-AA supports: Stabilization and properties

Frog epidermis tyrosinase has been immobilized on Enzacryl-AA (a polyacrylamide-based support) and CPG(zirclad)-Arylamine (a controlled pore glass support) in order to stabilize the tyrosine hydroxylase activity of the enzyme; in this way, the immobilized enzyme could be used to synthesize L-dopa from L-tyrosine. The activity immobilization yield Y(IME) (act) (higher than 86%), coupling efficiency (up to 90%), storage stability (no loss in 120 days), and reaction stability (t(1/2) was higher than 20 h in column reactors) were measured for tyrosinase after its immobilization. The results showed a noticeable improvement (in immobilization yield, coupling efficiency, and storage and operational stabilities) over previous reports in which tyrosinase was immobilized for L-dopa production. The activity and stability of immobilized enzyme preparations working in three different reactor types have been compared when used in equivalent conditions with respect to a new proposed parameter of the reactor (R(p)), which allows different reactor configurations to be related to the productivity of the reactor during its useful life time. The characteristic reaction inactivation which soluble tyrosinase shows after a short reaction time has been avoided by immobilization, and the stabilization was enhanced by the presence of ascorbate. However, another inactivation process appeared after a prolonged use of the immobilized enzyme. The effects of reactor type and operating conditions on immobilized enzyme activity and stability are discussed.     

Macroporous gel particles as robust macroporous matrices for cell immobilization

A new design of robust matrices for cell immobilization is described. Macroporous gels (MGs) with immobilized microbial cells were prepared at subzero temperatures and were formed inside a plastic core (so-called, protective housing). Due to the protective housing the macroporous gel particles with immobilized cells can be used in well-stirred bioreactors. High retained activity of yeast (77-92%) and Escherichia coli (50-91%) cells immobilized in MGs after drying and storage in the dried state was due to the high structural stability and heterogeneous porous structure of the MGs.     

Immobilization of lipase within carbon nanotube–silica composites for non-aqueous reaction systems

The immobilization of lipases within sol–gel derived silica, using multi-walled carbon nanotubes (MWNTs) as additives in order to protect the inactivation of lipase during sol–gel process and to enhance the stability of lipase, was investigated. Three sol–gel immobilized lipases (Candida rugosa, Candida antarctica type B, Thermomyces lanuginosus) with 0.33% (w/w) MWNT showed much higher activities than lipase immobilized without MWNT. The influence of MWNT content and MWNT shortened by acid treatment in the sol–gel process on the activity and stability of immobilized C. rugosa lipase was also studied. In hydrolysis reaction, immobilized lipase containing 1.1% pristine MWNT showed 7 times higher activity than lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT showed 10 times higher activity in esterification reaction, compared with lipase immobilized without MWNT. The lipase coimmobilized with 2.7% shortened MWNT retained 96% of initial activity after 5 times reuse, while the lipase immobilized without MWNT was fully inactivated under the same condition.     

以Fe(III) 改性胶原纤维为载体固定过氧化氢酶

将胶原纤维用三价铁改性后作为载体,通过戊二醛的交联作用将过氧化氢酶固定在该载体上。制备的固定化过氧化氢酶蛋白固载量为16.7 mg/g,酶活收率为35％。研究了固定化酶与自由酶的最适pH、最适温度、热稳定性、贮存稳定性及操作稳定性。结果表明：过氧化氢酶经此法固定化后,最适pH及最适温度与自由酶相同,分别为pH 7.0和25 ℃;但固定化酶的热稳定性显著提高,在75 ℃保存5 h后,仍能保留30％的活力,而自由酶则完全失活;固定化酶在室温下保存12 d后,酶活力仍保持在88％以上,而自由酶在此条件下则完全失     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

Biotransformation of 3-methoxy-8,14-seco-1,3,5(10),9(11) estratetraen-14,17-dione (secodione) to its 17beta-hydroxy derivative (secol) using immobilized yeast cells (Pichia farinosa Y-118)

The yeast cells of Pichia farinosa Y-118 were immobilized in polyacrylamide gel and used for 17 beta-oxidoreduction of secondione to secol. The loss of hydroxysteroid oxidoreductase activity of cells was found to be insignificant during immobilization. The preparation exhibited greater temperature stability as compared to free cells. The ratio of reaction volume to the volume of immobilized biocatalyst in the range 1.4-1.9 was found to be satisfactory for the reaction conditions studied. This ratio played a significant role in the stability of the catalyst particle, since beyond a critical value the disintegration of gel granules was rapid resulting in sharp decline of activity. The immobilized cell preparation could be used 50 times over a period of 100 days without loss of activity. However, the activity declined in further reuses, leaving the preparation 50 and 35% active after its 60th and 70th uses, respectively.     

Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems

Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83-93%) and efficiency (51-53%) were obtained when chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of chlorophyllase as compared to that of the free one. The immobilization of chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.     

磁性聚乙二醇载体固定化α淀粉酶的研究

α淀粉酶广泛应用于粮食加工、食品、酿造、发酵、纺织品和医药工业［1］．由于固定化酶的优点,国内外研究人员对固定化糖化酶［2,3］和固定化α淀粉酶[4]的制备及在淀粉酶法生产葡萄糖方面的应用作了大量的研究,显示了工业应用前景．然而,迄今为止,用磁性载体固定化α淀粉酶尚未见报道．我们用磁性聚乙二醇胶体粒子作载体,制备出具有磁响应性强、稳定性强、活力高的固定化α淀粉酶．由于具有磁响应性,可借助外部磁场方便简单地回收酶,为该酶工业化生产葡萄糖提供了一种新的途径．而且,由于磁性的优点,也为该酶在食品、医药、纺织…     

Covalent immobilization of a hydroperoxide lyase from mung beans (Phaseolus radiatus L.)

A fatty acid hydroperoxide lyase of mung beans has been covalently immobilized on different commercially available gels which represents the first immobilization of this type of enzyme from a higher plant. UltraLink Iodoacetyl possessed optimum coupling properties and yielded a maximum activity of 1.3 U ml–1 gel and a yield of 84%. The effect of various protective reagents (e.g. thiols, antioxidants) and of the substrate concentration on the re-usability of the immobilized enzyme was investigated. Compared to a control, the relative activity during re-use was enhanced 1.8- to 2.3-fold in the presence of dithiothreitol. As the hydroperoxide lyase was irreversibly inhibited by the substrate, its re-usability depended strongly on the hydroperoxide concentration. The lowest inactivation was with 55 M hydroperoxide which resulted in a relative activity of 73% after the third cycle. The storage stability of the hydroperoxide lyase was significantly improved by immobilization and resulted in a relative activity of 86% after 18 days, whereas the soluble enzyme lost 68% of its initial activity. © Rapid Science Ltd. 1998     

Immobilization of lipase on epoxy activated (1-->3)-alpha-D-glucan isolated from Penicillium chrysongenum

A water-insoluble linear (1-->3)-alpha-D-glucan was isolated from Penicillium mycelia. Three kinds of epoxy-activated microspheres of this glucan were prepared as supports for Candida sp. lipase (EC3.1.1.3) immobilization. The highest immobilization yield was 36.4%. The specific activity was 26.85 U/mg, and only 4.1% of activity was lost in comparison with the free enzyme used for immobilization. The higher thermal stability, storage stability, and reusability of the immobilized lipase make it a potential candidate for wide application.     

Influence of ionic liquids as additives on sol–gel immobilized lipase

The immobilization of lipase from Candida rugosa, using ionic liquids as additives to protect the inactivation of lipase by released alcohol and shrinking of gel during sol–gel process, was investigated. The influence of various factors, such as structure of ionic liquids, content of ionic liquids and types of precursor in the sol–gel process on the activity and stability of immobilized lipase was also studied. The highest hydrolytic activity of immobilized lipase was obtained when the hydrophilic ionic liquid, [C₂mim][BF₄], was used as an additive, while the highest stability of immobilized lipase was obtained by using hydrophobic ionic liquid, [C₁₆mim][Tf₂N]. Therefore, the binary mixtures of these ionic liquids as additives were used to obtain the optimal immobilized lipase, which shows both high activity and stability. The hydrolysis and esterification activities of lipase co-immobilized with the mixture of 1:1 at molar ratio of [C₂mim][BF₄] and [C₁₆mim][Tf₂N] were 10-fold and 14-fold greater than in silica gel without ionic liquids (ILs), respectively. After 5 days incubation of this immobilized lipase in n-hexane at 50 °C, 84% of initial activity was remained, while the residual activity of the lipase immobilized without ILs was 28%.     

Immobilization of Β-D-galactosidase fromLactobacillus bulgaricus on polyacrylamide in the presence of protective agents and properties of the immobilized enzyme

Β-D-Galactosidase (EC 3.2.1.23) fromLactobacillus bulgaricus (1373) was immobilized in a Polyacrylamide gel lattice in the presence of dithiothreitol, glutathione, cysteine, bovine serum albumin, casein, lactose and glucono-δ-lactone. Cysteine, bovine serum albumin, and lactose were found very effective in preserving the activity. With cysteine, bovine serum albumin and lactose, the activity yields were 61, 60 and 66% respectively, as compared to 31% without protective agents. The yield improved upto 85% when all the three protective agents, cysteine, bovine serum albumin and lactose were added during immobilization. The addition of protective agents did not have any effect on optimum pH, optimum temperature, kinetic constants and pH stability when compared with Β-galactosidase immobilized without the use of protective agents; however the heat and storage stabilities were found to increase.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Immobilization of hydrogenase in nylon gel containing electron mediator and application to an artificial photosystem

Hydrogenase of Desulfovibrio vulgaris was immobilized in nylon gel containing an electron mediator. With the immobilization, the stability for storage and repeated use increased and the optimal pH was shifted to the acidic side. Photoinduced hydrogen production was observed in an artificial photosystem consisting of the hydrogenase gel, metal porphyrin, and mercaptethanol.     

Direct immobilization of tyrosinase enzyme from natural mushrooms (Agaricus bisporus) on D-sorbitol cinnamic ester

Mushroom tyrosinase was immobilized from an extract onto the totally cinnamoylated derivative of D-sorbitol by direct adsorption as a result of the intense hydrophobic interactions that took place. The immobilization pH value and mass of lyophilized mushrooms were important parameters that affected the immobilization efficiency, while the immobilization time and immobilization support concentration were not important in this respect. The extracted/immobilized enzyme could best be measured above pH 3.5 and the optimum measuring temperature was 55 degrees C. The apparent Michaelis constant using 4-tert-butylcatechol as substrate was 0.38+/-0.02 mM, which was lower than for the soluble enzyme from Sigma (1.41+/-0.20 mM). Immobilization stabilized the extracted enzyme against thermal inactivation and made it less susceptible to activity loss during storage. The operational stability was higher than in the case of the tyrosinase supplied by Sigma and immobilized on the same support. The results show that the use of p-nitrophenol as enzyme-inhibiting substrate during enzyme extraction and immobilization made the use of ascorbic acid unnecessary and is a suitable method for extracting and immobilizing the tyrosinase enzyme, providing good enzymatic activity and stability.     

Stabilization ofd-amino-acid oxidase fromTrigonopsis variabilis by manganese dioxide

Stabilization of immobilized D-amino-acid oxidase was achieved as follows. Yeast Trigonopsis variabilis producing D-amino-acid oxidase was used to deaminate cephalosporin C to glutaryl-7-aminocephalosporanic acid. Permeabilized cells were co-immobilized with manganese dioxide by entrapment in (poly)acrylamide gel so that hydrogen peroxide, liberated in the reaction, could be partially deactivated and both the enzyme and the substrate could be stabilized. Activity of entrapped cells was determined by HPLC and enzyme flow microcalorimetry. The process was evaluated in terms of activity, immobilization yield, storage stability and oxo-product formation by immobilized preparations. The storage stability of immobilized biocatalysts with MnO2 was nearly doubled and production of 2-oxoadipyl-7-aminocephalosporanic acid was 2-3-fold higher than by entrapped cells without MnO2. Glutaryl-7-aminocephalosporanic acid can be easily obtained from the resulting oxo-product by a non-enzymic reaction via externally added hydrogen peroxide.