Characterization of polymeric buffers for operating membrane-trapped enzyme reactors in an electric field

A novel class of amphoteric, polymeric buffers, is described, consisting of grafting onto growing polyacrylamide chains weakly acidic and basic acrylamido-monomers (called Immobilines; protolytic groups as N-substituents on the nitrogen of the amido bond), for operating a membrane-immobilized enzyme reactor (MIER) in an electric field. With these soluble, polymeric buffers, it is possible to operate the membrane reactor at any optimum of pH activity, for any given enzyme, in the pH 3-10 scale. Such buffers, being amphoteric, are confined in the enzyme reaction chamber by the same isoelectric trapping mechanism. The best buffers were found to be those polymerized in presence of 9% neutral monomer (acrylamide) and containing 20 mM Immobiline as buffering ion. To decrease their viscosity in solution, the polymeric buffers are synthesized at high temperatures (70 degrees C) and in presence of a chain-transfer agent. The weight average molecular size in these conditions has been found to be ca. 200,000 Da. These buffers exhibited excellent performance in a variety of enzyme reactions in the MIER, such as in the case of penicillin G acylase and histidine decarboxylase and were found to greatly stabilize enzyme activity, permitting operation of the MIER over extended periods of time. As an example, in a penicillin G acylase reactor, >75% enzyme activity was maintained over a 10-d cycle of operation, while with conventional buffers more than 90% inactivation was experienced over the same period of time. This novel class of macromolecular, amphoteric buffers could also be exploited in other types of conventional bioreactors not based on an isoelectric trapping mechanism.     

Enzyme reactions in a multicompartment electrolyzer with isoelectrically trapped enzymes

The possibility of performing bioconversions under an electric field is here reported. A system is described by which the enzyme is trapped by an isoelectric mechanism between two zwitterionic membranes having pI values encompassing the isoelectric point of the enzyme. The enzyme is loaded into a multicompartment electrolyzer and kept operating under an electric field, which will continuously harvest the reaction product. Since, under focusing conditions, all buffering ions will vacate the reaction chamber at steady state, the buffering ion is trapped into the enzyme chamber by using amphoteric buffers co-isoelectric with the enzyme. As an example of such ‘isoelectrically immobilized’ reactor, the enzyme β-hydroxysteroid dehydrogenase is blocked into an isoelectric trap delimited by a pI 8.0 and a pI 6.5 membranes. 100 mM histidine (pI 7.47) is co-immobilized by the same isoelectric mechanism into the enzyme chamber. The dehydrocholic acid substrate (3,7,12-trioxo-5β-cholanoic acid) and reduced co-factor (NADH) are continuously infused into the enzyme chamber and the product (3β-hydroxy-7,12-dioxo-5β-cholanoic acid, a compound of pharmaceutical interest) and the oxidized co-factor (NAD⁺) collected, separately, into the two neighbouring chambers at the anodic side. Advantages: in a soluble form, the enzyme maintains the reaction kinetics of the free soluble form. Additionally, the reaction product and exhausted co-factor can be recovered by electrophoretic transport.     

Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina

Alcohol oxidase from Pichia pastoris was immobilized on nanoporous aluminium oxide membranes by silanization and activation by carbonyldiimidazole to create a flow-through enzyme reactor. Kinetic analysis of the hydrogen peroxide generation was carried out for a number of alcohols using a subsequent reaction with horseradish peroxidase and ABTS. The activity data for the immobilized enzyme showed a general similarity with literature data in solution, and the reactor could generate 80 mmol H₂O₂/h per litre reactor volume. Horseradish peroxidase was immobilized by the same technique to construct bienzymatic modular reactors. These were used in both single pass mode and circulating mode. Pulsed injections of methanol resulted in a linear relation between response and concentration, allowing quantitative concentration measurement. The immobilized alcohol oxidase retained 58 % of initial activity after 3 weeks of storage and repeated use.     

Limited modifications of soya proteins by immobilized subtilisin: comparison of products from different reactor types

A comparison of different immobilized enzyme reactors has been made for the limited modification of soya storage proteins and the products compared with those from action of the soluble enzyme. Clarified total water extracts of soya protein were subjected to the action of subtilisin in a soluble and immobilized form. The sodium dodecyl sulfate (SDS) electrophoresis patterns of soya proteins modified by enzyme in the two forms differed for unbuffered soya protein at the same pH of 8.0. However, identical patterns could be obtained by a downward adjustment of the pH of soya protein treated with immobilized enzyme. The same SDS electrophoresis pattern could be obtained for a packed column of immobilized enzyme and a well-mixed vessel by buffering. Operation of the column reactor at higher superficial linear velocities (above 1.47 cm/min), higher protein concentrations (8.8% w/v), and prolonged periods (24 h) led to a bed compression attributed to the protein coating of the support.     

Distribution and change of microbial activity in combined UASB and AFB reactors for wastewater treatment

A thermophilic upflow anaerobic sludge blanket (UASB) reactor was combined with a mesophilic aerobic fluidized bed (AFB) reactor for treatment of a medium strength wastewater with 2,700?mg COD?l^?1. The COD removal efficiency reached 75% with a removal rate of 0.2 g COD?l^?1 h^?1 at an overall hydraulic retention time 14 hours. The distribution of microbial activity and its change with hydraulic retention time in the two reactors were investigated by measuring ATP concentration in the reactors and specific ATP content of the biomass. In the UASB reactor, the difference in specific ATP was significant between the sludge bed and blanket solution (0.02?mg ATP g VS^?1 versus 0.85?mg ATP g VS^?1) even though the ATP concentrations in these two zones were similar. A great pH gradient up to 4 was developed along the UASB reactor. Since a high ATP or biological activity in the blanket solution could only be maintained in a narrow pH range from 6.5 to 7.5, the sludge granules showed a high pH tolerance and buffering capacity up to pH 11. The suspended biomass in AFB reactor had a higher specific ATP than the biomass fixed in polyurethane carriers (1.6?mg ATP g VS^?1 versus 1.1?mg ATP g VS^?1), which implies a starvation status of the immobilized cells due to mass transfer limitation. The aerobes had to work under starvation conditions in this polishing reactor. The anaerobic biomass brought into AFB reactor contributed to an increase in suspended solids, but not the COD removal because of its fast deactivation under aerobic conditions. A second order kinetic model was proposed for ATP decline of the anaerobes. The results on distribution of microbial activity in the two reactors as well as its change with hydraulic retention time lead to further performance improvement of the combined anaerobic/aerobic reactor system.     

The Mode of Action of Organdie-Damaging Factor from Mung Bean Leaves

Organelle-damaging factor, which damages cell organelles andcauses them to release their matrix enzymes at pH 7.5, had noability to actively hydrolyze carbohydrates, proteins and lipidsat pH 7.5. When the factor was incubated with spinach chloroplastsin the presence of calcium ions, the matrix enzyme, triosephosphateisomerase, was released from chloroplasts after a lag period.A significant amount of activity of the factor was lost duringthe release. We suggest that the factor is not an enzyme andbounds tightly to organelle membranes to break them. (Received October 6, 1980; Accepted December 3, 1980)     

吸附-交联法固定D-泛解酸内酯水解酶的研究

选择6种吸附树脂和离子交换树脂对D-泛解酸内酯水解酶进行固定化,筛选出了固定化效果较好的大孔弱碱性丙烯酸系阴离子交换树脂D-380为载体,用先吸附后交联的方法固定化。通过实验对固定化条件进行了优化,得出最佳的固定化条件为：加酶量6U／g树脂、吸附pH7．5、吸附时间4h、吸附温度30℃、交联剂戊二醛终浓度0．1％、交联时间2h。实验表明在此条件下制得的固定化酶有很好的稳定性：固定化酶在连续20次的底物水解反应后,剩余酶活达到71％。当温度达到80℃时游离酶几乎失去酶活,而固定化酶剩余酶活为60％以上。游离酶的pH稳定性范围为pH7～8,而固定化酶为pH6．5～8．5。     

A comparative study of immobilized amyloglucosidase in a packed bed reactor and a continuous feed stirred tank reactor

The catalytic activity of amyloglucosidase covalently attached to DEAE-cellulose was studied in a packed bed reactor and a continuous feed stirred tank reactor (CSTR) for the reaction maltose → glucose. At low flow rates mass-transfer limitations in the bed reactor lead to lower conversions for this reactor compared to the CSTR. Simple theoretical expressions for these reactors were compared with the experimental results. There are significant differences between the kinetic parameters and pH profile of the immobilized and free enzyme. The immobilized enzyme also showed greater stability at 50°C than did free amyloglucosidase. The temperature dependence of the reaction rate was the same for immobilized and free enzyme.     

Anionic exchange nylon laminated membranes for enzyme immobilization

Summary This work presents the optimization of the chemical steps involved in nylon modification with dimethyl sulphate, polyethyleneimine, glutaraldehyde and 2-diethyl aminoethylamine to obtain a weak basic anion exchange support. Activated nylon laminated membranes were utilized for aminoacylase immobilization, allowing an ionically adsorbed enzyme derivative with high activity (0.16 U/mg E·cm²) and low removed activity (<1%). Optimum immobilization conditions and kinetic parameters were also determined. This immobilized enzyme can be used in laminated enzyme membrane reactors.     

Integration of purification with immobilization of Candida rugosa lipase for kinetic resolution of racemic ketoprofen

The two processes for the partial purification and for the immobilization of a crude lipase preparation (Candida rugosa Lipase OF) have been successfully integrated into one by simple adsorption of the enzyme onto a cation ion exchanger resin (SP-Sephadex C-50) at pH 3.5. Due to selective removal of the unfavorable lipase isoenzyme (L1), the enzyme components (mainly L2 and L3) that are tightly fixed on the resin displayed a significantly improved enantioselectivity (E value: 50 versus 13 with addition of Tween-80) in the biocatalytic hydrolysis of 2-chloroethyl ester of rac-ketoprofen. The activity yields of the immobilized lipase were 48 and 70%, respectively when emulsified and non-emulsified substrates were employed for enzyme assay. Moreover, the concentration of Tween-80 was found to be a factor affecting the lipase enantioselectivity. By using such an immobilized enzyme as biocatalyst, the process for preparing enantiopure (S)-ketoprofen becomes simpler and more practical as compared with the previously reported procedures and the product was obtained with >94% ee at 22.3% conversion in the presence of an optimal concentration (0.5 mg/ml) of Tween-80 at pH 3.5. Furthermore, the operational stability of the immobilized biocatalyst was examined in different types of reactors. In an air-bubbled column reactor, the productivity was much higher than that in a packed-bed column reactor, in spite of a slightly lower stability. Under optimal conditions, the air-bubbled column reactor could be operated smoothly for at least 350 h, remaining nearly 50% activity.     

Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient

A new method is described for preparative protein purification, based on isoelectric focusing on immobilized pH gradients. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow-chamber, and focusing the impurities in the Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. The sample flow-chamber is built in the centre of the apparatus, and is coupled to an upper and lower segment of an immobilized pH gradient. The protein to be purified is kept isoelectric in the flow-chamber and prevented from leaving it by arranging for the extremities of the immobilized pH gradient, forming the ceiling and the floor of this chamber, to have isoelectric points just higher (e.g. +0.05 pH units, on the cathodic side) and just lower (e.g. -0.05 pH units, on the anodic side) than the known pI of the species of interest. Macromolecules and small ions leave the flow chamber at a rate corresponding to a first order reaction kinetics (the plot of log C vs. time being linear). In general, for macromolecules, 12 h of recycling under current allow removal of 95% impurities. After 24 h of recycling, the protein of interest is more than 99.5% pure. The recoveries are very high (approaching 100%) as the sample under purification never enters the Immobiline gel and thus does not have to be extracted from a hydrophilic matrix, as typical of preparative gel electrophoresis.     

Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles

Abstract

The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two enzymatic reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of enzymatic reactors operated in sequential mode: a membrane reactor using fsMP-laccase and a reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.     

Amphoteric, isoelectric immobiline membranes for preparative isoelectric focusing

Amphoteric, isoelectric agarose membranes, as devised by Martin and Hampson [Martin, A.J.P. and Hampson, F. (1978) J. Chromatogr. 159, 101-110], are found unsuitable for blocking electroendosmosis in multi-compartment electrolysers during preparative isoelectric focusing, due to the poor and highly unpredictable incorporation of carboxyls and amino groups on the polysaccharide moiety. New, polyacrylamide-based membranes are described, containing as buffers and titrants the Immobiline chemicals used to produce immobilized pH gradients. These new membranes are supported on both faces by a non-woven polypropylene cloth, a material exhibiting minimal adsorption properties for proteins. Due to the extensively developed Immobiline technology, membranes with highly predictable isoelectric points, well-defined buffering capacity and conductivity can be synthesized at any pH value along the pH 3-10 scale. They are effective in blocking electroendosmosis even when the delta pH on either side of the membrane is as high as 1.5 pH unit.     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

Activity studies of glucose oxidase immobilized onto poly(N-vinylimidazole) and metal ion-chelated poly(N-vinylimidazole) hydrogels

Poly(N-vinylimidazole), PVIm, gels were prepared by γ-irradiation polymerization of N-vinylimidazole in aqueous solutions. These affinity gels with a water swelling ratio of 1800% for plain polymeric gel and between 30 and 80% for Cu(II) and Co(II)-chelated gels at pH 6.0 in phosphate buffer were used in glucose oxidase (GOx) adsorption–desorption studies. Different amounts of Cu(II) and Co(II) ions (maximum 3.64 mmol/g dry gel for Cu(II) and 1.72 mmol/g dry gel for Co(II)) were loaded onto the gels by changing the initial concentration of Cu(II) and Co(II) ions, and pH. GOx adsorption on these gels from aqueous solutions containing different amount of GOx at different pH was investigated in batch reactors. Immobilized glucose oxidase activity onto the poly(N-vinylimidazole), and Cu(II) and Co(II)-chelated poly(N-vinylimidazole) were investigated with changing pH and the initial glucose oxidase concentration. Maximum activity of immobilized glucose oxidase onto the PVIm, Cu(II) and Co(II)-chelated PVIm gels was investigated and pH dependence was observed to be at pH 6.5 for free enzyme, pH 7.0 for PVIm, pH 7.5 for Cu(II) and Co(II)-chelated PVIm gels, respectively. The stability of the immobilized enzyme is very high for all gels and the residual activity was higher than 93% in the first 10 days.     

Immobilization of alpha-chymotrypsin on poly(urethane-graft-acrylic acid)

A study was made of the immobilization of alpha-chymotrypsin (alpha-CT) onto a previously well characterized synthetic polyurethane grafted with acrylic acid P(U-g-AA). The P(U-g-AA) had previously been prepared using 2,2'-azo-bis-isobutyronitrile (AIBN) as a radical initiator and acrylic acid as monomer in the presence of an unsaturated polyurethane in solution at 60 degrees C. Some kinetic parameters of both the native enzyme and the enzyme immobilized on the P(U-g-AA) were evaluated. Using a Lineweaver-Burk plot (double reciprocal), it was found that the Michaelis-Menten constant (Km(for the immobilized enzyme was (4.0 +/- 0.9) x 10(-3) M and that of the free enzyme was (3.0 +/- 0.2) x 10(-3) M. The enzyme alpha-chymotrypsin was immobilized on the grafted polyurethane micelles/aggregates with about 45% retention of activity. Also the immobilized alpha-CT retained this activity for at least 6 weeks. The immobilized enzyme was found to have a maximum stability at 43 degrees C compared with 36 degrees C in the case of free enzyme, and the pH optimum was shifted from pH 6.6 to pH 8.2. The long-term operational stability of the enzyme was investigated and this is of interest since the enzyme is probably trapped physically in a micellar environment. The assay of the enzyme was carried out in 0.01 M phosphate buffer, pH 7.5, using p-nitrophenyl acetate as a substrate. No inhibition of alpha-CT in the presence of the synthetic ungrafted and grafted polyurethane was observed.     

Immobilization by Polyurethane of Pseudomonas dacunhae Cells Containing l-Aspartate beta-Decarboxylase Activity and Application to l-Alanine Production

Whole cells of Pseudomonas dacunhae containing l-aspartate beta-decarboxylase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol; W. R. Grace & Co., Lexington, Mass.). The immobilized cell preparation was used to convert l-aspartic acid to l-alanine. Properties of the immobilized P. dacunhae cells containing aspartate beta-decarboxylase activity were investigated with batch reactors. Retention of enzyme activity was observed to be as much as 100% when cell lysis was allowed to occur before immobilization. The pH and temperature optima were determined to be 5.5 and 45 degrees C, respectively. Immobilized P. dacunhael-aspartate beta-decarboxylase activity was stabilized by the addition of 0.1 mM pyridoxal-5-phosphate and 0.1 mM alpha-ketoglutaric acid to a 1.7 M ammonium aspartate (pH 5.5) substrate solution. Under conditions of semicontinuous use in a batch reactor, a 2.5% loss in immobilized l-aspartate beta-decarboxylase activity was observed over a 31-day period.     

Bioconversion of D-galactose to D-tagatose: continuous packed bed reaction with an immobilized thermostable L-arabinose isomerase and efficient purification by selective microbial degradation

The continuous enzymatic conversion of D-galactose to D-tagatose with an immobilized thermostable L-arabinose isomerase in packed-bed reactor and a novel method for D-tagatose purification were studied. L-arabinose isomerase from Thermoanaerobacter mathranii (TMAI) was recombinantly overexpressed and immobilized in calcium alginate. The effects of pH and temperature on D-tagatose production reaction catalyzed by free and immobilized TMAI were investigated. The optimal condition for free enzyme was pH 8.0, 60°C, 5 mM MnCl(2). However, that for immobilized enzyme was pH 7.5, 75°C, 5 mM MnCl(2). In addition, the catalytic activity of immobilized enzyme at high temperature and low pH was significantly improved compared with free enzyme. The optimum reaction yield with immobilized TMAI increased by four percentage points to 43.9% compared with that of free TMAI. The highest productivity of 10 g/L h was achieved with the yield of 23.3%. Continuous production was performed at 70°C; after 168 h, the reaction yield was still above 30%. The resultant syrup was then incubated with Saccharomyces cerevisiae L1 cells. The selective degradation of D-galactose was achieved, obtaining D-tagatose with the purity above 95%. The established production and separation methods further potentiate the industrial production of D-tagatose via bioconversion and biopurification processes.     

Immobilization of urease onto chemically modified acrylonitrile copolymer membranes

Poly (acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were subjected to seven different chemical modifications. The amounts of new groups incorporated in the membranes with the modifications were determined. Urease was covalently immobilized on the modified membranes. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity was found for urease bound to membranes modified with hydroxylammonium sulfate (68%) and hydrazinium sulfate (67%). Optimum pH of free urease was determined to be 5.8. For positively charged membranes, pH optimum was shifted to higher values, while for negatively charged membranes-to lower pH. The charge of the matrix affected also the rate of the enzyme reaction. The highest rate was measured with urease immobilized on membranes modified with hydroxylammonium sulfate and hydrazinium sulfate. The major part of the immobilized enzyme on different modified membranes remained stable-only ca. 20% of enzyme activity was lost for 4 h at 70 degrees C while the free enzyme was totally inactivated.     

Characterization of glucose oxidase immobilized on collagen

Summary The enzyme glucose oxidase (E.C. 1.1.3.4) was immobilized on collagen — a proteinaceous material found in biological systems as a structural material for a wide variety of cells and membranes. The novel technique of electrocodeposition, which utilizes the principles of electrophoresis, was used to deposit the enzyme-collagen complex on stainless steel helical supports. This technique has been developed in our laboratory. The mechanism of complex formation between collagen and enzyme involves multiple salt linkages, hydrogen bonds and van der Waals interactions.As a first step toward examining its feasible technical use, the kinetic behavior of the collagen-supported glucose oxidase was studied in a batch recycle type reactor and was compared with that for the soluble form. A novel reactor configuration consisting of multiple concentric electrocodeposited helical coils was used. The reactor was found to attain a stable level of activity which was maintained for several months under cyclic testing. The optimum levels of pH and temperature for the immobilized form of the enzyme were the same as those of the soluble enzyme, but the immobilized enzyme was more active than the soluble form at higher temperatures and pH. The values of the Michaelis-Menten parameters indicate that the overall reaction rate of the immobilized enzyme may be partially restricted by bulk and matrix diffusion.