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.     

Iron-shortage-induced increase in citric acid content and reduction of cytosolic aconitase activity in Citrus fruit vesicles and calli

Aconitase, which catalyses the conversion of citrate into isocitrate, requires Fe for its activity. The yeast and animal enzyme loses its enzymatic activity under Fe shortage and binds to RNA of genes involved in Fe homeostasis, altering their expression. Thus, the enzyme provides a regulatory link between organic acid metabolism and Fe cellular status. Roots and leaves of Fe-deficient plants show induction in organic acids, especially citrate. Although no RNA-binding activity has been so far demonstrated for the plant aconitase, whether alternations in enzyme activity by Fe could play a role in this induction remain unanswered. This question was investigated in lemon fruit [ Citrus limon (L.) Burm var Eureka ], characterized by the accumulation of citrate to about 0.3 M in the juice vesicles cells (pulp). Calli and isolated juice vesicles showed two- to three-fold induction in citrate level when subjected to Fe shortage. The mRNA level of aconitase exhibited no changes under reduced Fe concentrations. Analysis of aconitase isozymes demonstrated that out of two aconitase isozymes, typically detected in citrus fruit, only the cytosolic form displayed a reduced activity under low Fe concentrations. Our data support the notion of a limited Fe-availability-induced reduction in cytosolic aconitase, resulting in a slower rate of citrate breakdown and a concomitant increase in citrate levels.     

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.     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Iron Binding and Autoreduction by Citrate: Are These Involved in Signalling by Iron Regulatory Protein-l?

Ferric ions bind to citrate and undergo an autoreduction to form a ferrous-citrate complex, greatly increasing the redox activity of the iron complex. Ferrous ions and citrate are also essential for the enzymic activity of aconitase. Aconitase, with its iron-sulphur cluster has a versatile structure which allows it to act as an iron regulatory protein (IW-1). The purpose of this study was to see whether iron binding, and its autoreduction by citrate, could play a physiological signalling role in iron regulation. Significant amounts of ferrous ions were associated with citrate, when measured using ferrozine, however, these did not appear to activate iron-requiring aconitase.     

Inhibition of aconitase in citrus fruit callus results in a metabolic shift towards amino acid biosynthesis

Citrate, a major determinant of citrus fruit quality, accumulates early in fruit development and declines towards maturation. The isomerization of citrate to isocitrate, catalyzed by aconitase is a key step in acid metabolism. Inhibition of mitochondrial aconitase activity early in fruit development contributes to acid accumulation, whereas increased cytosolic activity of aconitase causes citrate decline. It was previously hypothesized that the block in mitochondrial aconitase activity, inducing acid accumulation, is caused by citramalate. Here, we investigated the effect of citramalate and of another aconitase inhibitor, oxalomalate, on aconitase activity and regulation in callus originated from juice sacs. These compounds significantly increased citrate content and reduced the enzyme’s activity, while slightly inducing its protein level. Citramalate inhibited the mitochondrial, but not cytosolic form of the enzyme. Its external application to mandarin fruits resulted in inhibition of aconitase activity, with a transient increase in fruit acidity detected a few weeks later. The endogenous level of citramalate was analyzed in five citrus varieties: its pattern of accumulation challenged the notion of its action as an endogenous inhibitor of mitochondrial aconitase. Metabolite profiling of oxalomalate-treated cells showed significant increases in a few amino acids and organic acids. The activities of alanine transaminase, aspartate transaminase and aspartate kinase, as well as these of two γ-aminobutyrate (GABA)-shunt enzymes, succinic semialdehyde reductase (SSAR) and succinic semialdehyde dehydrogenase (SSAD) were significantly induced in oxalomalate-treated cells. It is suggested that the increase in citrate, caused by aconitase inhibition, induces amino acid synthesis and the GABA shunt, in accordance with the suggested fate of citrate during the acid decline stage in citrus fruit.     

Urease Immobilization on Arylamine Glass Beads and its Characterization

Jack bean urease has been immobilized on arylamine glass beads (200–400 mesh size, 75–100 Å pore size) and its properties compared with soluble enzyme. The binding of urease was 13.71 mg per gram beads. The K_m for soluble and immobilized urease for urea was 4.20 mM and 8.81 mM, respectively. V_max values of urease decreased from 200 to 43.48 μmol of ammonia formed per min per mg protein at 37°C on immobilization. Both pH and buffer ions influenced the activities of soluble as well as immobilized urease. Soluble urease exhibited pH optima at 5.5 and 8.0. However, immobilized urease showed one additional pH optimum at 6.5. In comparison to phosphate buffer, citrate buffer was inhibitory to urease activity. Immobilization of urease on arylamine glass beads resulted in improved thermal, storage and operational stability. Because of inertness of support and stability of immobilized urease, the preparation can find applications in ‘artificial kidney’ and urea estimation in biological fluids viz., blood, milk etc.     

Regulation of Aconitase Synthesis in Bacillus subtilis: Induction, Feedback Repression, and Catabolite Repression

The synthesis of aconitase in Bacillus subtilis wild-type and different citric acid cycle mutants has been studied and the influence of various growth conditions examined. Aconitase is induced by citrate and precursors of citrate and repressed by glutamate. Induction and repression counteract each other, and at equimolar concentrations of citrate and glutamate, aconitase synthesis is unaffected. Induction by citrate can partly overcome catabolite repression of aconitase. Isocitrate dehydrogenase show endogenous induction of aconitase due to citrate accumulation. Leaky mutants defective in citrate synthase and aconitase cannot be induced by citrate, which indicates that they carry a regulatory mutation. The complex regulation of aconitase is discussed with reference to the participation of this enzyme in glutamate biosynthesis and energy metabolism.     

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.     

Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: activity, stability and co-immobilization

In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.     

Activities and subcellular localization of enzymes responsible for lipolysis and gluconeogenesis during the germination of Brassica campestris cv. esculenta seeds

During the growth of turnip seedlings, two new lipases have been demonstrated, one with a maximum activity at pH 4.5 (acid lipase) and the other with a maxima at pH 8.6 (alkaline lipase). Many different enzymes are involved in gluconeogenesis: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase, aconitase, citrate synthetase, fumarase, glycolate oxidase, phosphoenol-pyruvate carboxykinase. All of these show maximum activity coinciding with the stage in which lipid hydrolysis is maximal and when the accumulation of soluble carbohydrates has also reached its peak. The alkaline lipase as found to be located mainly in the spherosomes, whereas the glyoxysomes contained the following main activities: catalase, isocitrate lyase, malate synthetase, malate dehydrogenase and citrate synthetase. Aconitase, together with cytochrome oxidase and fumarase showed their highest activity in the mitochondria, and the presence of malate dehydrogenase, citrate synthetase and glycolate oxidase was also observed in these organelles. In the membrane-bound fraction, the activities of cytochrome reductase, glycolate oxidase and phosphoenol-pyruvate kinase were marked, although the latter enzyme was even more active in the soluble fraction.     

Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.     

Repeated use of immobilized Gluconobacter oxydans cells for conversion of glycerol to dihydroxyacetone

Dihydroxyacetone (DHA) is of great interest in the fine chemical and pharmaceutical industry; therefore, the discovery of suitable biocatalysts for the efficient production of it is very necessary. In the experiment, Gluconobacter oxydans was immobilized in polyvinyl alcohol (PVA). Various parameters of the immobilized cells were investigated. The results have shown that the optimal conversion conditions by the immobilized cells were at 30 degrees C and pH 6.0. The immobilized cells remained very active over the period of 14 days for storage and only lost 10% of its original activity. Repeated use of immobilized cells for conversion of glycerol to DHA was carried out in a 1.5 L stirred tank reactor, the average conversion rate was about 86%. Despite the high shear stress, bead shape was not affected, even after five consecutive conversion cycles. The regenerated biocatalyst could recover 90% of its initial activity.     

Resistance to deactivation of enzyme-collagen membranes

Amongst the attractive properties of immobilized enzymes, an enhanced stability is very often underlined. In our case, the covalent attachment of numerous enzymes from different classes to water-insoluble collagen films allowed us to study their resistance to inactivation or denaturation after coupling. The influence of heat, denaturing reagents like concentrated urea or guanidinium chloride, the incubation in the presence of glutaraldehyde, have been tested on aspartate amino-transferase either in soluble form or bound on collagen films. The fact that diffusional effects can lead to an apparent enhancement of stability after immobilization has been taken into account and their influence studied for both thermal and storage stability : diffusional limitations are partly responsible for the enhanced stability of the bound enzyme but the binding to the collagen membrane itself increases its storage stability. The resistance of proteolytic enzymes to autolysis has also been checked.     

Immobilization of lipase to chitosan beads using a natural cross-linker

Genipin, a reagent of plant origin was used for the immobilization of lipase by cross-linking to chitosan beads. The catalytic properties and operational and storage stabilities of the immobilized lipase were compared with the soluble lipase. Under optimum conditions, 198 microg protein was bound per g chitosan with a protein-coupling yield of 35%. The hydrolytic activity was 10.8 U/g chitosan and the relative specific activity was 108%. The immobilized lipase showed better thermal and pH stabilities compared to the soluble form. The immobilized enzyme exhibited mass transfer limitations as reflected by a higher apparent K(m) value and a lower energy of activation. The immobilized enzyme retained about 74% of its initial activity after five hydrolytic cycles.     

Influence of immobilization procedure and salt environment on functional stability of chloroplast membranes: experimental data and numerical analysis

The interactions between chloroplast membranes and their microenvironment within artificial matrices (albumin-glutaraldehyde matrix, polyurethane foam) where investigated. Particularly, the influence of a high-ionic-strength medium (0.75 M potassium citrate) on the stability of the photosynthetic ferricyanide reduction by immobilized thylakoids has been studied. A method of data analysis based on a nonlinear identification method combined with the numerical integration of the equation of the transient state of the continuous stirred tank reactor (CSTR) is proposed to estimate the actual degradation of the photosynthetic electron transfer. A statistical analysis achieved on the parameter values has allowed a quantitative assessment of the global behavior of immobilized chloroplast membranes. From the mathernatical analysis of the experimental data, we demonstrate that citrate used in the reaction media prevents the photoinactivation of the electron transfer chain whatever the nature of the matrix or the type of the reactor. The use of an albumin-glutaraldehyde matrix or an open reactor during experiments also has allowed a better stabilization of the photosystems under operational conditions.     

Iron deficiency induces changes in metabolism of citrate in lateral roots and cluster roots of Lupinus albus

This paper describes the first measurement of enzyme activities in cluster roots under –Fe stress, at different stages of cluster root development and function. In Lupinus albus L., Cluster roots are produced both under iron- and phosphorus-deficient conditions. In both cases the structure is similar, but the level of exudation is much greater in iron-deficient plants. Much work has been done on the enzyme kinetics of P-deficient cluster roots, but none on enzyme activities of Fe-deficient cluster roots. The enzymes investigated were citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), isocitrate dehydrogenase [IDH(NAD) (EC 1.1.1.41) and IDH (NADP) (EC 1.1.1.42)] and lactate dehydrogenase (LDH) (EC 1.1.1.27). In cluster roots, citrate synthase activity was initially lower than in lateral roots but, after 5 days, recovered to the lateral root level. Cluster root aconitase levels initially increased, but fell sharply on day 3, and no activity was detected after day 5. IDH (NAD) levels were much lower in cluster roots than in laterals, dropping to a low on day 3, and then rising throughout development. IDH (NADP) levels were always higher in cluster roots than in lateral roots, increasing throughout development. LDH levels in cluster roots fell throughout development. Internal tissue concentrations of citrate were markedly higher in –Fe laterals than in +Fe lateral roots and in cluster roots. Cluster root levels of citrate increased dramatically after day 3. Results are discussed within the context of previous work on enzyme kinetics under –P, and the importance of a block in aconitase activity is highlighted.     

胰蛋白酶的固定化及其性质研究

 以自制的脱乙酰壳多糖作载体,戊二醛为交联剂,对胰蛋白酶的固定化条件及其固定化酶的性质进行了研究。考查了交联剂的用量、pH值、以及载体与酶的比例等因素对胰蛋白酶固定化的影响。在所选择的固定化条件下,固定化酶的活性回收可达50％以上。同时研究了固定化胰蛋白酶的一些性质;最适温度60℃,最适PH8.0,Km值比可溶性酶升高,热稳定性、pH贮存稳定性以及在乙醇水溶液中的稳定性明显高于可溶性胰蛋白酶。在柱式反应器内,以2％酪蛋白为底物对,操作半衰期为40天。     

The potential of immobilized bacterial α-amylase on coconut coir,a smart carrier for biocatalysts

Purified α-amylase from a soil bacterium Bacillus sp. SKB4 was immobilized on coconut coir, an inexpensive cellulosic fiber, with the cross-linking agent glutaraldehyde. The catalytic properties and stability of the immobilized enzyme were compared with those of its soluble form. The enzyme retained 97.2% of its activity and its catalytic properties were not drastically altered after immobilization. The pH optimum and stability of the immobilized enzyme were shifted towards the alkaline range compared to the free enzyme. The optimum temperature for enzymatic activity was 90°C in both forms of the enzyme. The soluble and immobilized enzyme retained 19% and 70% of original activity, respectively, after pre-incubation for 1 h at 90°C. Immobilized amylase was less susceptible to attack by heavy metal ions and showed higher K_m and V_max values than its free form. The bound enzyme showed significant activity and stability after 6 months of storage at 4°C. All of these characteristics make the new carrier system suitable for use in the bioprocess and food industries.