Thiol Group Reagents Research Articles

Purification and Biochemical Characterization of α-Amylase from Radish (Raphanus sativus L.) Seeds Using Response surface methodology

The α-amylase from radish seeds has been purified to apparent homogeneity with specific activity of 821.66 U/mg of protein with 36 folds purification. The purified enzyme displayed single protein band on native PAGE (polyacrylamide gel electrophoresis) confirmed by activity staining. The SDS PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) revealed protein band of approximately 43 and 90 kDa. The calcium content of enzyme preparation was around 1.442 µg/mg of protein as analyzed by AAS (atomic absorption spectrophotometer). The energy of activation for enzyme was 3.82 kcal/moles with Km and Vmax values 4.8 mg/mL and 0.377, respectively, using starch as the substrate. The metal ions such as Ca2+ and Mg2+ showed augmented activity whereas Ni2+, Fe2+ and Cu2+ showed slight inhibition in the amylase activity. The thiol group reagents also activated the enzyme. Further, the effect of pH and temperature on α-amylase activity were analyzed and optimized utilizing response surface methodology. The value of regression coefficients was found significant (R2 = 92.62%) showing the suitability of the proposed model. The multiple regression and analysis of variance showed the individual and cumulative effect of pH and temperature on the activity of enzyme with optimum pH of 6.5 and temperature of 60 °C. Additionally, contour plot, 3D surface plot and optimization plot were used to predict the effect of each variable with minimum set of experiments.

Role of 4‐hydroxy‐2,3‐nonenal in the pathogenesis of fibrosis

Transient activation of fibroblasts or fibroblast-like cells to proliferate and to produce elevated quantities of extracellular matrix is essential to fibrosis. This activation is regulated by several cytokines produced by various inflammation-associated cells. Among these, transforming growth factor beta1 (TGFbeta1) is considered of major importance. Many studies have shown that lipid peroxidation play a key role in the initiation and progression of fibrosis in different organs. In fact, 4-hydroxy-2,3-nonenal (HNE), the major aldehydic product of lipid peroxidation, is able to induce TGFbeta1 expression and synthesis, and activation of activator protein-1 (AP-1) transcription factor. In this study, using the murine macrophage line J774-A1, we show that these effects are strictly related to the chemical structure of HNE, since neither 2-nonenal nor nonanal are biologically active to the same extent. Moreover, we demonstrate that HNE can indeed contribute to the onset of fibrosis by stimulating AP-1 binding to DNA and consequently inducing TGFbeta1 expression, since thiol-group reagents, such as N-ethylmaleimide and 4-(chloro-mercuri)-benzenesulfonic acid, that down-modulate HNE entrance and localisation inside the cell, prevent both phenomena. The possibility to control fibrogenic cytokine levels by means of antioxidant or dietetic treatments opens new potential pharmacological and nutritional horizons in the treatment of many chronic diseases characterised by excessive fibrosis.

Pharmacology of the human red cell voltage-dependent cation channel. Part II: inactivation and blocking

Pharmacological modulation of the nonselective voltage-dependent cation (NSVDC) channel from human erythrocytes was studied. Using the inorganic cations ruthenium red and La3+, as well as the organic thiol group reagents iodoacetamide (IAA) and N-ethylmaleimide (NEM), it was possible to demonstrate a concentration-dependent decrease in the voltage-activated conductance, reflecting an inhibition or inactivation of the channel. Initial voltage activation was achieved by injecting human red cells into sucrose-substituted Ringers with a low chloride concentration, which causes a strongly positive membrane potential to develop, initially determined by the equilibrium potential for Cl- ( approximately +100 mV). Due to the voltage- and time-dependent activation of the cation channel, net effluxes, minimized by addition of a chloride conductance blocker, occurred and Vm gradually decreased and stabilized at a value less positive than E(Cl), reflecting the increased cation conductance, g+, reaching 1.5-2.0 microS/cm2. In the presence of inhibitors of the NSVDC channel, both the membrane potential repolarization and the cation efflux were diminished.

Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents

Among the oxidative breakdown products of ω-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor β1 (TGFβ1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFβ1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFβ1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.

Kinetics of inactivation of glutamate decarboxylase by cysteine-specific reagents.

Mercuric chloride, p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid) irreversibly inhibited the activity of Escherichia coli glutamate decarboxylase. Their second order rate constants for inactivation are 0.463 microM(-1) min(-1), 0.034 microM(-1) min(-1), 0.018 microM(-1) min(-1), respectively. The characteristics of the inhibition by the three thiol-group reagents supports the idea that cysteinyl residues at the binding sites for the cofactor and/or the substrate are important for enzyme activity in E. coli.

Cysteine residues in the C-terminus of the neutral- and basic-amino-acid transporter heavy-chain subunit contribute to functional properties of the system b0,+-type amino acid transporter

The neutral- and basic-amino-acid-transport glycoprotein NBAT (rBAT, D2) expressed in renal and jejunal brush-border membranes interacts with the b0,+AT permease to produce a heteromeric transporter effecting amino acid and cystine absorption. NBAT mutations result in type I cystinuria. The b0,+AT permease is presumed to be the catalytic subunit, but we have been investigating the possibility that cysteine residues within the C-terminus of NBAT are also important for expression of transport function. NBAT mutants were produced with combinations of Cys664/671/683 → Ala substitutions. Mutants with Cys664 → Ala show decreased arginine and cystine transport and specifically lose sensitivity to inhibition of transport by the thiol-group reagent N-ethylmaleimide (NEM). We suggest that the C-terminus of NBAT may have a direct role in the mechanism of System b0,+ transport (the major transport activity defective in type I cystinuria) and that Cys664 of NBAT is the major target for NEM-induced inactivation of the transport mechanism.

Cysteine residues in the C-terminus of the neutral- and basic-amino-acid transporter heavy-chain subunit contribute to functional properties of the system b0,+-type amino acid transporter

The neutral- and basic-amino-acid-transport glycoprotein NBAT (rBAT, D2) expressed in renal and jejunal brush-border membranes interacts with the b(0,+)AT permease to produce a heteromeric transporter effecting amino acid and cystine absorption. NBAT mutations result in type I cystinuria. The b(0,+)AT permease is presumed to be the catalytic subunit, but we have been investigating the possibility that cysteine residues within the C-terminus of NBAT are also important for expression of transport function. NBAT mutants were produced with combinations of Cys(664/671/683)-->Ala substitutions. Mutants with Cys(664)-->Ala show decreased arginine and cystine transport and specifically lose sensitivity to inhibition of transport by the thiol-group reagent N-ethylmaleimide (NEM). We suggest that the C-terminus of NBAT may have a direct role in the mechanism of System b(0,+) transport (the major transport activity defective in type I cystinuria) and that Cys(664) of NBAT is the major target for NEM-induced inactivation of the transport mechanism.

Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide.

The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.

Interactions between the thiol-group reagent N-ethylmaleimide and neutral and basic amino acid transporter-related amino acid transport

The neutral and basic amino acid transport protein (NBAT) expressed in renal and jejunal brush-border membranes is involved in amino acid and cystine absorption. NBAT mutations result in Type 1 cystinuria. A C-terminal myc-tagged NBAT (NBATmyc) retains the amino acid transport and protein-protein interaction properties of NBAT when expressed in Xenopusoocytes. Neutral amino acid (Ala, Phe)-cationic amino acid (Arg) heteroexchanges related to NBATmyc expression in oocytes are inactivated by treatment with the thiol-group reagent N-ethylmaleimide (NEM), although significant Arg-Arg and Ala-Ala homoexchanges persist. Inactivation of heteroexchange activity by NEM is accompanied by loss of > 85% of alanine and cystine uptake, with smaller (< 50%) inhibition of arginine and phenylalanine uptake. NEM-sensitive cystine uptake and arginine-alanine heteroexchange (system b0,+ activity) are not expressed by an NBAT truncation mutant (NBATmyc-Sph1) lacking the 13 C-terminal amino acid residues, but the mutant expresses NEM-resistant transport activity (system y+L-like) equivalent to that of full-length NBATmyc. The deleted region of NBATmyc-Sph1 contains two cysteine residues (671/683) which may be the targets of NEM action. The synthetic amino acid 2-trifluoromethylhistidine (TFMH) stimulated alanine efflux at pH 7.5 and arginine at pH 5.5, but not vice versa, establishing the existence of distinct pathways for cationic and neutral amino acid homoexchange (TFMH is zwitterionic at pH 7.5 and cationic at pH 5.5). We suggest that NBAT expresses a combination of system b0,+ and y+L-like activities, possibly by interacting with different light-chain subunits endogenous to oocytes (as does the homologous 4F2hc protein). The C-terminus of NBAT may also have an additional, direct role in the mechanism of System b0,+ transport (the major transport activity that is defective in Type 1 cystinuria).

Interactions between the thiol-group reagent N-ethylmaleimide and neutral and basic amino acid transporter-related amino acid transport

The neutral and basic amino acid transport protein (NBAT) expressed in renal and jejunal brush-border membranes is involved in amino acid and cystine absorption. NBAT mutations result in Type 1 cystinuria. A C-terminal myc-tagged NBAT (NBATmyc) retains the amino acid transport and protein-protein interaction properties of NBAT when expressed in Xenopus oocytes. Neutral amino acid (Ala, Phe)-cationic amino acid (Arg) heteroexchanges related to NBATmyc expression in oocytes are inactivated by treatment with the thiol-group reagent N-ethylmaleimide (NEM), although significant Arg-Arg and Ala-Ala homoexchanges persist. Inactivation of heteroexchange activity by NEM is accompanied by loss of >85% of alanine and cystine uptake, with smaller (<50%) inhibition of arginine and phenylalanine uptake. NEM-sensitive cystine uptake and arginine-alanine heteroexchange (system b(0,+) activity) are not expressed by an NBAT truncation mutant (NBATmyc-Sph1) lacking the 13 C-terminal amino acid residues, but the mutant expresses NEM-resistant transport activity (system y(+)L-like) equivalent to that of full-length NBATmyc. The deleted region of NBATmyc-Sph1 contains two cysteine residues (671/683) which may be the targets of NEM action. The synthetic amino acid 2-trifluoromethylhistidine (TFMH) stimulated alanine efflux at pH 7.5 and arginine at pH 5.5, but not vice versa, establishing the existence of distinct pathways for cationic and neutral amino acid homoexchange (TFMH is zwitterionic at pH 7.5 and cationic at pH 5.5). We suggest that NBAT expresses a combination of system b(0,+) and y(+)L-like activities, possibly by interacting with different light-chain subunits endogenous to oocytes (as does the homologous 4F2hc protein). The C-terminus of NBAT may also have an additional, direct role in the mechanism of System b(0,+) transport (the major transport activity that is defective in Type 1 cystinuria).

Characterization of the nitrate transporter in root plasma membranes of Cucumis sativus L

Nitrate uptake in right-side out plasma membrane vesicles isolated from cucumber roots was characterized. Nitrate uptake into vesicles was driven by an artificially imposed pH gradient. The uptake was strongly inhibited by phenylglyoxal, an arginyl residue modificator. Only a slight repression of NO 3 − transport in vesicles was observed in the presence of NEM, a thiol group reagent. pCMBS, an other thiol reagent and DEPC, an effector of histidine residue, had no effect on the nitrate transport in plasma membranes. ATP-driven proton transport in vesicles was not significantly affected in the presence of both, phenylglyoxal and DEPC, whereas pCMBS and NEM abolished it almost completely. The possible role of the particular amino acids residues in the active nitrate transport is discussed. NO 3 − uptake into vesicles isolated from both, nitrate-induced and nitrate-depleted plant material was higher than that observed in the vesicles obtained from uninduced plants. Thus, isolated vesicles reflect the well-known in vivo response of intact plants on the exogenous nitrogen regime.

Chemical inactivation of bacterial GABA aminotransferase.

The effects of three potential irreversible inhibitors of gamma-aminobutyrate aminotransferase from Pseudomonas fluorescens were studied in order to throw more light on the nature of the active site of the enzyme. The thiol group reagent mercuric chloride inactivated the enzyme in a concentration-dependent manner. Inhibition kinetics were consistent with a simple bimolecular reaction. The second-order rate constant was 4.2 x 10(3) +/- 0.61 M-1 sec-1. In contrast to either of the substrates, the cofactor pyridoxal 5'-phosphate could protect the enzyme from the inhibition, suggesting cysteinyl residues are important for cofactor binding at the active site. p-Chloromercuribenzoic acid produced a similar inactivation of the enzyme. 4-Amino-2-fluorobutanoic acid also inhibited enzymic activity but in this case the inhibition was reversible and competitive with respect to gamma-aminobutyric acid (GABA). The inhibitor constant (Ki) was 0.83 +/- 0.44 mM. We found no evidence that this fluorinated analogue of GABA could act as a substrate for the enzyme.

Purification and Characterization of a Tripeptidase from Lactobacillus sake.

A tripeptidase was purified to homogeneity from the cell extract of Lactobacillus sake by ammonium sulfate precipitation, hydrophobic interaction chromatography, gel filtration chromatography, and two steps of anion exchange chromatography. After SDS-PAGE a single band of protein was detected of approximately 55 kDa. A similar molecular mass was estimated by gel filtration. The tripeptidase activity was optimal at pH 7.0 and at 40 degrees C. The enzyme was strongly inhibited by metal chelators, reducing agents, and bestatin while thiol group reagents, serine proteinase inhibitors, and aspartic proteinase inhibitors had no effect on the activity. The enzyme was activated by Mn(2+) and almost totally inhibited by Zn(2+) and to a lesser extent by Sn(2+). The enzyme only exhibited activity against tripeptides, and those hydrolyzed at higher rates were Ala-Ala-Ala, Ser-Ser-Ser, and Leu-Gly-Gly.

Molecular and kinetic characterization of glutamate synthase from the phototrophic bacterium Rhodobacter capsulatus E1F1

Glutamate synthase (GOGAT) from the phototrophic non-sulphur purple bacterium Rhodobacter capsulatus ElFl has been purified to electrophoretic homogeneity by affinity chromatography. The native protein consisted of two different subunits of 175 and 53 kDa and contained 4 mol FAD, 4 mol iron and 4 mol labile sulphide per mol of dimer enzyme. The enzyme used NADPH as the electron donor and was inhibited by iron-chelating and thiol group reagents. GOGAT exhibited NAD(P)H-diaphorase activity which used sodium ferricyanide, cytochrome c and dichlorophenol indophenol as alternative electron acceptors. By contrast, glutaminase activity was not detected in purified GOGAT. The amino acid composition was quite different from that of other bacterial GOGATs, and the protein presented different aggregation states depending on the ionic strength. Two major multimeric active species with Stokes’ radii of 6·18 and 7·32 nm could be separated by gel-filtration of protein solutions made in 0·5 m-KCl, whereas in the absence of salt, the maximal GOGAT activity corresponded to an oligomer with Stokes radius of 6·80 nm. The enzyme exhibited apparent negative cooperativity for glutamine, and was competitively inhibited by l-glutamate and NADP+.

Reactivity of Sulfhydryl Groups of the Catalytic Subunits of Rabbit Skeletal Muscle Protein Phosphatases 1 and 2A

Chemical modification of the sulfhydryl residues of the catalytic subunits of protein phosphatases 1 and 2A was studied. Both enzymes were inactivated by a variety of thiol group reagents. Mercurial compounds were the most effective inactivators. Of the alkylating agents the maleimides were more effective than iodoacetate or iodoacetamide which were relatively slow reacting. Both enzymes were also inactivated by disulfides, including glutathione disulfide, 5,5′-dithiobis(2-nitrobenzoic acid), and 4,4′-dipyridyl disulfide. The latter two were much more reactive than glutathione disulfide and, in addition, displayed a significant differential reactivity toward phosphatase 1. The apparent second-order rate constants for the inactivation of phosphatase 1 were 20- to 70-fold higher than for phosphatase 2A with 5,5′-dithiobis(2-ni-trobenzoic acid) and 4,4′-dipyridyl disulfide. Kinetic analysis indicated that inactivation of both enzymes could be correlated with the modification of one cysteine per one mole of enzyme.

A quantitative investigation of proton— and lead(II)—dithioerythritol complex equilibria under physiological conditions

Dithioerythritol (DTE) is frequently employed as a reducing agent or a protective reagent for thiol groups in biological assays. Owing to its known inhibiting properties in enzyme catalysis reactions, lead is also commonly used in such experiments, and often simultaneously with DTE. Given the potential affinity of these two reactants, their measured individual effects may well depend on their interactions in the medium used. Any quantitative assessment of these interactions necessitates, however, that the complex equilibria between lead and DTE be investigated beforehand. To test this hypothesis, the formation constants of lead(II) complexes with DTE under physiological conditions (37°, NaCl 0.15M) have been calculated from the results of glass electrode potentiometry, with the help of the MINIQUAD and ESTA computer programs. The p K values for dissociation of DTE have been found equal to 8.926 ± 0.003 and 9.840 ± 0.003. The following lead—DTE species have been characterized: ML (12.774 ± 0.037), MLH −1 (2.858 ± 0.037), M 2LH −1 (13.349 ± 0.025) and M 6L 5 (86.586 ± 0.099); the log *β-values are given in the parentheses. Appropriate computer simulations effectively show that the interactions of the two reactants are indeed quite significant within the concentration ranges commonly used in in vitro biological assays. They should thus be taken into account in interpretation of the effects observed.

Importance of thiol groups in ligand binding to D2 dopamine receptors from brain and anterior pituitary gland

The effects of the thiol group reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) on D2 dopamine receptors have been examined in three brain regions (caudate nucleus, putamen and olfactory tubercle), and in the anterior and neurointermediate lobes of the pituitary gland. Whereas the receptors in brain were insensitive to DTNB, a dose-dependent inhibition of [3H]spiperone binding to D2 receptors in both lobes of the pituitary gland was observed. The effects in the pituitary could be reversed by subsequent treatment with dithiothreitol and could be prevented by prior occupancy of the receptor binding site with a ligand. The effect of DTNB was to decrease the number of ligand-binding sites without altering the affinity of those remaining. The results show that modification of a thiol group of the D2 dopamine receptor in the two lobes of the pituitary gland tested here significantly affects ligand binding. There is therefore a difference between the D2 dopamine receptor populations in brain and pituitary in their sensitivity to modification by DTNB, and this may imply the existence of different receptor isoforms in the two issues.

Group-selective reagent modification of the sodium- and chloride-coupled glycine transporter under native and reconstituted conditions

Glycine transporter from rat brain stem and spinal cord is inactivated by specific sulfhydryl reagents. Modification of lysine residues also promotes a decrease of the transporter activity but in a lesser extent than that promoted by thiol group reagents. Mercurials showed a more marked inhibitory effect than maleimide derivatives. SH groups display a similar reactivity for p-chloromercuribenzenesulfonate (pCMBS) and mersalyl in synaptosomal membrane vesicles and proteoliposomes reconstituted with the solubilized transporter. However, different reactivity is observed with N-ethylmaleimide (MalNEt), the greatest effect being attained in membrane vesicles. The rate of inactivation by pCMBS and MalNEt is pseudo-first-order showing time- and concentration-dependence. pCMBS and MalNEt decrease the V max for glycine transport and to a lesser extent act on the apparent K m. Treatment with dithiothreitol (DTT) of the transporter modified by pCMBS results in a complete restoration of transporter activity indicating that the effect exercised by the reagent is specific for cysteine residues on the protein. It is concluded that SH groups are involved in the glycine transporter function and that these critical residues are mostly located in a relatively hydrophilic environment of the protein.

Directed evolution of amidase in Methylophilus methylotrophus; purification and properties of amidases from wild-type and mutant strains

The obligately methylotrophic bacterium Methylophilusmethylotrophus hydrolyses acetamide and acrylamide using a cytoplasmic amidase. In previous work, continuous culture was used to isolate spontaneous mutants which overexpressed either the wild-type amidase (strain MM6) or a mutant amidase with an apparently higher K cat (strain MM8). We now report that NTG mutagenesis of strain MM8 followed by acrylamide-limited growth at low dilution rate (D 0·025 h-1; 37 °C) led to the selection of a strain which continued to overexpress the amidase, but which exhibited an unexpectedly low amidase activity and a greatly decreased K m for acrylamide (strain MM15). Amidases from the wild-type and mutant strains were purified and shown to be homotetramers (subunit M r 38000, pI 4·1). The N-terminal amino acid sequence of the wild-type enzyme was 90% homologous with the aliphatic amidase from Pseudomonas aeruginosa, and Southern blotting using an oligonucleotide probe for this region showed that overexpression of the enzyme in the mutant strains was not due to gene amplification. Compared with the wild-type and MM6 enzymes, the MM8 enzyme exhibited a threefold higher K m and a slightly lower K m for acrylamide, whereas the MM15 enzyme exhibited a similar K cat and an eightfold lower K m for acrylamide. The MM15 enzyme also reacted more extensively with the thiol group reagent DTNB, had a significantly lower sedimentation coefficient and exhibited a more relaxed substrate specificity, all of which were compatible with a looser tetrameric structure. It was also much more susceptible than the other three enzymes to inactivation by high temperature or by freezing and thawing (MM15»MM8&gt;MM6/wild-type), both of which variably dissociated the enzyme into inactive dimers and monomers. The amidase activity of strain MM15 was almost 15-fold higher following growth at 25 °C than at 37 °C, since at this lower temperature the enzyme exhibited a similar K cat to the MM8 enzyme and was not significantly dissociated. However, as strain MM15 readily outgrew the organism from which it was derived (strain MM8) during acrylamide-limited continuous culture at 37 °C, it is clear that under these conditions a low K m was a greater selective advantage than a high K cat.

Properties of cellobiase from Aspergillus niger

Cellobiase enzyme was partially purified from the culture filtrate of Aspergillus niger AS-101 and the general and kinetic properties of the enzyme were examined. The enzyme was unstable on storage. However, it was protected by the addition of BSA, glycerol or sodium azide. Addition of glycerol also protected the enzyme from denaturation due to freezing and thawing. Effect of thiol group reagents revealed the presence of — SH groups at the active site of the enzyme. Different modulators such as metal ions and macroionic compounds illustrated varying effects on the purified cellobiase.