Isolation and properties of prostaglandin H synthetase immobilized in insoluble polyelectrolyte complexes

Immobilization of prostaglandin-H-synthetase (EC 1.14.99.1) of the microsomal fraction of ram vesicular gland in the microparticles of insoluble polyelectrolyte complexes by non-covalent incorporation was studied. It was shown that immobilization occurs with a high efficiency and high activity yield (40-70%). Non-specific reversible inhibition of the enzyme by the polycationic component of the complex was demonstrated. The dependencies of activity of the native and immobilized enzymes on pH, temperature and substrate (adrenaline and arachidonic acid) were studied. The immobilized enzyme had an increased thermal stability as compared to the native one; the thermoinactivation rate constant was decreased 3-7 times. The biexponential type of the curve of the rependence of activity vs. time for the native enzyme and the transformation of the curve into a simple exponential form for the immobilized enzyme were observed.     

Substrate protection of immobilized glucose isomerase

Using commercial immobilized glucose isomerase (SWETASE(R), Nagase Co.), the effect of substrate protection on enzyme deactivation has been studied in a batch manner. The data analysis was carried out based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrates was also considered. The protection factor was proposed to elucidate the dependence of the degree of substrate protection. The existence of the protection of glucose isomerase by the substrates has been verified experimentally. Also, the enzyme-substrate complex deactivates with a decay constant which is one-half that of the free enzyme. Theoretical analysis of enzyme deactivation with substrate protection offers an effective understanding which is essential for enzyme replacement and process optimization.     

Essential tryptophan residues of ribulose 1,5-bisphosphate carboxylase

Ribulose 1,5-bisphosphate carboxylase [3-phospho-D-glyceratecarboxy-lyase (dimerizing), EC 4.1.1.39] is rapidly and irreversibly inactivated by micromolar concentrations of dimethyl (2-hydroxy-5-nitrobenzyl) sulphonium bromide (DMHNB), a tryptophan selective reagent, after reversible protection of the reactive sulphydryl groups. The inactivation followed pseudo-first-order reaction kinetics. Replots of the kinetic data indicated that no reversible enzyme-inhibitor complex was formed prior to irreversible modification. Kinetic analysis and the correlation of the spectral data at 410 nm with enzyme activity indicated that inactivation by DMHNB resulted from modification of on an average one tryptophan per 67 kDa combination of large and small subunits. Several competitive inhibitors and substrate RuBP offered strong protection against inhibition. The k1/2 (protection) for RuBP was 1.3 mM, indicating that the tryptophan residues may be located at or near the substrate binding site. Free and total sulphydryl groups were not affected by the reagent. The modified enzyme exhibited significantly reduced intrinsic fluorescence, indicating that the microenvironment of the tryptophans at the active site is significantly perturbed. Tryptic peptide profiles and CD spectral analyses suggested that inactivation may not be due to the extensive conformational changes in the enzyme molecule during modification.     

Optimization of glucose isomerase reactor: optimum operating temperature mode

The optimum temperature operation mode required to achieve high fructose productivity is studied for immobilized glucose isomerase (GI) packed bed reactor. In this study, the reactor design equation based on reversible Michaelis-Menten kinetics assumes both thermal enzyme deactivation and substrate protection. The optimization problem is formulated as a discretized constrained nonlinear programming problem (NLP). The formulation is expressed in terms of maximization of fructose productivity as the objective function subject to reactor design equation, kinetic parameter equations, substrate protection factor equation and feasibility constraints. The constraints are discretized along the reactor operating period by employing piecewise polynomial approximations. Approximately 7% improvement in terms of fructose productivity is achieved when running the reactor at the optimum decreasing temperature operation mode as compared to the constant optimum isothermal operation.     

Kinetcs and decay of fumarase activity of immobilized Brevibacterium ammoniagenes cells for continuous production of L-malic acid

The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells.     

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.     

Analysis of substrate protection of an immobilized glucose isomerase reactor

The effect of substrate protection on enzyme deactivation was studied in a differential bed and a packed bed reactor using a commercial immobilized glucose isomerase (Swetase, Nagase Co.). Experimental data obtained from differential bed reactor were analyzed based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrate was considered. The deactivation constant of the enzyme-substrate complex was found to be about half of that of the free enzyme. The mathematical analysis describing the performance of a packed bed reactor under the considerations of the effects of substrate protection, diffusion resistance, and enzyme deactivation was studied. The system equations for the packed bed reactor were solved using an orthogonal collocation method. The presence of substrate protection and the diffusion effect within the enzyme particles resulted in an axial variation of effectiveness factor, eta(D), along the length of the packed bed. The axial distribution profile of eta(D) was found to be dependent on the operation temperature, Based on the effect of substrate protection, a better substrate feed policy could be theoretically found for promoting productivity in long-term operation. (c) 1993 John Wiley & Sons, Inc.     

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta

A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 mumol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10-unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1-unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent K(m) for the immobilized enzyme was 3-4 times greater than the K(m) (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.     

Inactivation kinetics of mushroom tyrosinase in the dimethyl sulfoxide solution

Mushroom tyrosinase (EC 1.14.18.1) is a kind of copper-containing oxidase that catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones and then forms brown or black pigments. In the present paper, the effects of dimethyl sulfoxide on the enzyme activity for the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) have been studied. The results show that low concentrations of dimethyl sulfoxide (DMSO) can lead to reversible inactivation of the enzyme, and the IC ₅₀ is estimated to be 2.45 M. Inactivation of the enzyme by DMSO is classified as mixed type. The kinetics of inactivation of mushroom tyrosinase at low concentrations of DMSO solution has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The results show the free enzyme molecule is more fragile than the enzyme–substrate complex in the DMSO solution. It is suggested that the presence of the substrate offers marked protection of this enzyme against inactivation by DMSO.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Optical solid-state detection of organophosphates using organophosphorus hydrolase

We have developed a sensor surface for optical detection of organophosphates based on reversible inhibition of organophosphorus hydrolase (OPH) by copper complexed meso-tri(4-sulfonato phenyl) mono(4-carboxy phenyl) porphyrin (CuC1TPP). OPH immobilized onto glass microscope slides retains catalytic activity for more than 232 days. CuC1TPP is a reversible, competitive inhibitor of OPH, binding at the active site of the immobilized enzyme. The absorbance spectrum of the porphyrin-enzyme complex is measured via planar waveguide evanescent wave absorbance spectroscopy using a blue LED as a light source and an Ocean Optics USB2000 as the spectrophotometer. The characteristics of the absorbance spectrum of CuC1TPP are specific and different when the porphyrin is bound to the enzyme or is bound non-specifically to the surface of the slide. Addition of a substrate of OPH such as one of the organophosphates paraoxon, coumaphos, diazinon, or malathion displaces the porphyrin from the enzyme resulting in reduced absorbance intensity at 412 nm. Absorbance changes at 412 nm show log-linear dependence on substrate concentration. Paraoxon concentrations between 7 parts per trillion (ppt) and 14 parts per million (ppm) were investigated and a 3:1 S/N detection limit of 7 ppt was determined. Concentrations of 700 ppt to 40 ppm were investigated for diazinon, malathion, and coumaphos with detection limits of 800 ppt, 1 part per billion, and 250 ppt, respectively. This optical technique does not require the addition of reagents or solutions other than the sample and absorbance spectra can be collected in less than 6 s.     

多聚半乳糖醛酸内切酶的固定化及其性质研究

棉花枯萎病菌多聚半乳糖醛酸内切酶在pH大于7时不稳定,故对它进行多种化学修饰而又不影响其活性,必须在pHd小于7的体系中进行。本文报道将PGAUase在还原剂存在下,与稀酸处理的Sepharose 4B交联,获得较高活力的固定化酶。固定化酶催化动力学表明,最适pH为4,4,最适温度为55℃,在pH1至8.0范围内稳定。和溶液酶比较,对热稳定性提高,但对碱稳定性下降。以多聚半乳糖醛酸为底物,Km为0.27mmol/L,Vmax为66.67nmol/L·min,均大于溶液酶(Km=0.07mmol/L,Vmax=28.00nmol/L·min)。在pH4.8,30℃,聚半乳糖醛酸在固相酶的柱中循环水解不同的时间降解产物经圆盘电泳和等电聚焦测定,得到不同大小的寡糖片段混合物,证明固相酶和溶液酶的作用方式相同,同时使以酶解法制备一定大小的有生物活性的寡糖分子成为可能。     

Optimum temperature operation mode for glucose isomerase reactor operating at constant glucose conversion

The optimum temperature operation mode required to achieve constant outlet glucose conversion is determined for immobilized glucose isomerase continuous packed bed reactor. The reactor design equation assumes reversible Michaelis-Menten kinetics with both enzyme deactivation and substrate protection. An increasing temperature profiles are determined for different operating periods, residence times and glucose conversions. The temperature increase with time is very small at low degree of glucose conversion and at relatively long residence time. The temperature rise with time increases at high degree of conversion and at relatively short residence time.     

Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ～pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent K_m value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ～pH 6.0.     

Characterization of an active site peptide modified by the substrate analogue 3-bromo-2-ketoglutarate on a single chain of dimeric NADP+-dependent isocitrate dehydrogenase

The substrate analogue 3-bromo-2-ketoglutarate reacts with pig heart NADP+-dependent isocitrate dehydrogenase to yield partially inactive enzyme. Following 65% inactivation, no further inactivation was observed. Concomitant with this inactivation, incorporation of 1 mol of reagent/mol of enzyme dimer was measured. The dependence of the inactivation rate on bromoketoglutarate concentration is consistent with reversible binding of reagent (KI = 360 microM) prior to irreversible reaction. Manganous isocitrate reduces the rate of inactivation by 80% but does not provide complete protection even at saturating concentrations. Complete protection is obtained with NADP+ or the NADP+-alpha-ketoglutarate adduct. By modification with [14C]bromoketoglutarate or by NaB3H4 reduction of modified enzyme, a single major radiolabeled tryptic peptide was obtained by high performance liquid chromatography with the sequence: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Evidence in the following paper (Bailey, J.M., Colman, R.F. (1987) J. Biol. Chem. 262, 12620-12626) indicates that X is glutamic acid. Enzyme modified at the coenzyme site by 2-(bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the presence of manganous isocitrate is not further inactivated by bromoketoglutarate. Bromoketoglutarate-modified enzyme exhibits a stoichiometry of binding isocitrate and NADPH equal to 1 mol/mol of enzyme dimer, half that of native enzyme. These results indicate that bromoketoglutarate modifies a residue in the nicotinamide region of the coenzyme site proximal to the substrate site and that reaction at one catalytic site of the enzyme dimer decreases the activity of the other site.     

The properties of invertase,covalently immobilized at activated carbon

The covalent immobilization of yeast invertase with glutaraldehyde at activated carbon, modified preliminarily by urea and dimethyl formamide treatment, has been established. Some physicochemical properties of the immobilized and native enzyme in water and water-organic solutions have been studied. Hydrolytic, as well as transferase enzyme characteristics have changed after immobilization. The optimal conditions for hydrolytic and transferase activity of immobilized invertase are pH 6.0 and 7.0, respectively. The optimum temperature for the immobilized enzyme is 30°C. The conversion degree of isoamyl alcohol depends on the substrate and enzyme concentrations in medium, holdup time and organic phase quantity in the reaction medium.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

Kinetics of inhibition of beta-glucosidase from Ampullarium crossean by bromoacetic acid

The kinetics of inhibition of beta-glucosidase from Ampullarium crossean by bromoacetic acid (BrAc) has been studied. The results show that the enzyme can be irreversibly and completely inactivated at high BrAc concentration, while at low BrAc concentration, inhibition of the enzyme is a slow, reversible reaction. The microscopic rate constants for the reactions of BrAc with the enzyme were determined. The presence of the substrate offers obvious protection of the enzyme against inhibition by BrAc. The above results suggest that the histidine residue is essential for activity and is situated at or near the active site of the enzyme.     

Optimization of Operating Temperature for Continuous Immobilized Glucose Isomerase Reactor with Pseudo Linear Kinetics

In this work, the optimal operating temperature for the enzymatic isomerization of glucose to fructose using a continuous immobilized glucose isomerase packed bed reactor is studied. This optimization problem describing the performance of such reactor is based on reversible pseudo linear kinetics and is expressed in terms of a recycle ratio. The thermal deactivation of the enzyme as well as the substrate protection during the reactor operation is considered. The formulation of the problem is expressed in terms of maximization of the productivity of fructose. This constrained nonlinear optimization problem is solved using the disjoint policy of the calculus of variations. Accordingly, this method of solution transforms the nonlinear optimization problem into a system of two coupled nonlinear ordinary differential equations (ODEs) of the initial value type, one equation for the operating temperature profile and the other one for the enzyme activity. The ODE for the operating temperature profile is dependent on the recycle ratio, operating time period, and the reactor residence time as well as the kinetics of the reaction and enzyme deactivation. The optimal initial operating temperature is selected by solving the ODEs system by maximizing the fructose productivity. This results into an unconstrained one‐dimensional optimization problem with simple bounds on the operating temperature. Depending on the limits of the recycle ratio, which represents either a plug flow or a mixed flow reactor, it is found that the optimal temperature of operation is characterized by an increasing temperature profile. For higher residence time and low operating periods the residual enzyme activity in the mixed flow reactor is higher than that for the plug flow reactor, which in turn allows the mixed flow reactor to operate at lower temperature than that of the plug flow reactor. At long operating times and short residence time, the operating temperature profiles are almost the same for both reactors. This could be attributed to the effect of substrate protection on the enzyme stability, which is almost the same for both reactors. Improvement in the fructose productivity for both types of reactors is achieved when compared to the constant optimum temperature of operation. The improvement in the fructose productivity for the plug flow reactor is significant in comparison with the mixed flow reactor.     

Immobilization of glucooligosaccharide oxidase of Acremonium strictum for oligosaccharic acid production

Summary The glucooligosaccharide oxidase was covalently immobilized to chitosan with polyethyleneimine and glutaraldehyde. Immobilization improved thermal stability. When used for conversion of starch hydrolysate to oligosaccharic acids, the immobilized enzyme maintained 75% initial activity after 60 days of continuous operation. Strong substrate inhibition was seen at high concentrations of cellobiose and lactose for free enzyme but not for immobilized enzyme.