Structural and catalytic properties of L-alanine dehydrogenase from Bacillus cereus

Alanine dehydrogenase from Bacillus cereus, a non-allosteric enzyme composed of six identical subunits, was purified to homogeneity by chromatography on blue-Sepharose and Sepharose 6B-CL. Like other pyridine-linked dehydrogenases, alanine dehydrogenase is inhibited by Cibacron blue, competitively with respect to NADH and noncompetitively with respect to pyruvate. The enzyme was inactivated by 0.1 M glycine/HCl (pH 2) and reactivated by 0.1 M phosphate (pH 8) supplemented with NAD+ or NADH. The reactivation was characterized by sigmoidal kinetics indicating a complex mechanism involving rate-limiting folding and association steps. Cibacron blue interfered with renaturation, presumably by competition with NADH. Chromatography on Sepharose 6B-CL of the partially renatured alanine dehydrogenase led to the separation of several intermediates, but only the hexamer was characterized by enzymatic activity. By immobilization on Sepharose 4B, alanine dehydrogenase from B. cereus retained 66% of the specific activity of the soluble enzyme. After denaturation of immobilized alanine dehydrogenase with 7 M urea, 37% of the initial protein was still bound to Sepharose, indicating that on the average the hexamer was attached to the matrix via, at most, two subunits. The ability of the denatured, immobilized subunits to pick up subunits from solution shows their capacity to fold back to the native conformation after urea treatment. The formation of "hybrids" between subunits of enzyme from B. cereus and Bacillus subtilis demonstrates the close resemblance of the tertiary and quaternary structures of alanine dehydrogenases from these species.     

Properties of water-insoluble matrix-bound lactate dehydrogenase.

Lactate dehydrogenase enzyme was immobilized by binding to a cyanogen bromideactivated Sepharose 4B-200 in 0.1 m phosphate buffer, pH 8.5. The immobilized enzyme was found to have lower K_m values for its substrates. K_m values for pyruvate and lactate were 8 × 10 ^?5m and 4 × 10^?3m, respectively, an order of magnitude less than the value for the native (free) enzyme. Chicken heart (H₄) lactate dehydrogenase was found to lose nearly all its substrate inhibition characteristics as a result of immobilization. The covalently bound muscle-type subunits of lactate dehydrogenase showed more favorable interaction with the muscle type than with the heart type subunits. An increase in thermal and acid stability of the dogfish muscle (M₄) lactate dehydrogenase as well as a decrease in the percentage of inhibition of enzyme activity by rabbit antisera and in the complement fixation was observed as a result of immobilization. The changes in the properties of the enzyme as a result of immobilization may be attributable to hindrance produced by the insoluble matrix as well as conformational changes in the enzyme molecules.     

Immobilization of horse liver alcohol dehydrogenase on copolymers of glycidyl methacrylate and ethylene dimethacrylate

Alcohol dehydrogenase has been immobilized to the basic copolymer and its several derivatives using various techniques. Enzyme coupling to the supports with amino groups by means of glutaraldehyde was found the most suitable. Activity of alcohol dehydrogenase coupled to these amino supports was comparable to that of the enzyme bound to Sepharose. Thermal and pH stability of alcohol dehydrogenase increased essentially upon immobilization. Kinetic properties of the immobilized enzyme differed from those of free alcohol dehydrogenase, pH optimum shifted to alkaline range, and apparent Michaelis constants for substrates and coenzymes increased. Curvatures observed in Lineweaver-Burk plots for coenzymes suggest an involvement of diffusion effects in the reaction catalyzed by alcohol dehydrogenase linked to these polymers.     

Preparation of 15N-labeled l-alanine by immobilized l-alanine dehydrogenase: Differential incorporation of 15N in bacterial proteins

This paper describes a system for continuous synthesis of ¹⁵N-labeled l-alanine from lactic acid, ¹⁵NH₄Cl and NADH, which uses immobilized alanine dehydrogenase and soluble lactate dehydrogenase as enzyme sources. Lactic acid acts both as hydrogen donor for the regeneration of NADH and as pyruvate source, thus providing the carbon skeleton of l-alanine. Citrobacter freundi grown on synthetic media containing 17 unlabeled amino acids and l-(¹⁵N)alanine as nitrogen source, incorporated 66% of ¹⁵N into alanine found in bacterial proteins. When ¹⁵N-labeled glutamic acid, aspartic acid or glycocol were added to the synthetic growth media, their ¹⁵N was “diluted” among different amino acids of bacterial proteins. Isotope enrichment of l-(¹⁵N)lysine found in newly synthesized proteins of C. freundi was practically unchanged as compared to the isotope content of free amino acid in the growth medium.     

Immobilization of nucleoside diphosphatase at its allosteric site using immobilized derivatives of pyridoxal 5'-phosphate

3-O-Immobilized and 6-immobilized pyridoxal 5′-phosphate analogs of Sepharose were bound to the allosteric site of nucleoside diphosphatase with very high affinity. Active immobilized nucleoside diphosphatase was prepared by reduction of the Schiff base linkage between the enzyme and pyridoxal 5′-phosphate bound to Sepharose with NaBH₄. 3-O-Immobilized pyridoxal 5′-phosphate analog gave more active immobilized enzyme than the 6-analog; the immobilized enzyme on the 3-O-immobilized pyridoxal 5′-phosphate analog showed about 90% of activity of free enzyme. The immobilized enzyme thus prepared was less sensitive to ATP, an allosteric effector, and showed a higher heat stability than the free enzyme. When an assay mixture containing inosine diphosphate and MgCl₂ was passed through a column of the immobilized enzyme at 37 °C, inosine diphosphate liberated inorganic phosphate almost quantitatively. Properties of the immobilized enzyme on the pyridoxal 5′-phosphate analog were compared with those of the immobilized enzyme on CNBr-activated Sepharose.     

Immobilized flounder muscle glyceraldehyde 3-phosphate dehydrogenase

Summary Partially purified flounder muscle (Pseudopleuronectus americanus) glyceraldehyde 3-phosphate dehydrogenase was immobilized on cyanogen bromide-activated Sepharose. The catalytic properties of the immobilized preparation were studied to determine if immobilization alters the kinetic properties of the native holoenzyme. The results indicate that the pH activity profile of immobilized glyceraldehyde 3-phosphate dehydrogenase did not differ from that of the native enzyme. The Michaelis constants (Km) for NAD and glyceraldehyde 3-phosphate were somewhat altered. The enzyme stability toward various inactivation treatments in the presence and absence of NAD was characterized and compared to that of he native enzyme. When either form of the enzyme was incubated with urea at concentrations greater than 2m, inactivation occurred very rapidly. Incubation in 0.1% trypsin for 60 minutes decreased the activity of immobilized glyceraldehyde 3-phosphate dehydrogenase by 45% and of the native soluble enzyme by 70%. The immobilized enzyme also exhibited considerably more stability than the native soluble enzyme when exposed to a temperature of 50° or to 20 mm ATP. In all cases NAD either greatly reduced the rate of inactivation or completely protected the enzyme from inactivation.     

Renaturation and urea-induced denaturation of 20 beta-hydroxysteroid dehydrogenase studied in solution and in the immobilized state

Tetrameric 20 beta-hydroxysteroid dehydrogenase (17,20 beta,21-trihydroxysteroid:NAD+ oxidoreductase, EC 1.1.1.53) from Streptomyces hydrogenans was reactivated after inactivation, dissociation and denaturation with urea. The effect of several factors such as NAD+, NADH, substrate, sulphydryl reducing agents, extraneous proteins, pH and enzyme concentration on reactivation was investigated. The coenzymes were found to be essential for obtaining a high reactivation yield (about 90%), since in their absence the reactivation was less than 10%. NADH was effective at lower concentrations than NAD+. The reactivated enzyme, after the removal of inactive aggregates, showed physical and catalytic properties coincident with those of the native enzyme. The mechanism by which NADH affects the reconstitution of 20 beta-hydroxysteroid dehydrogenase was investigated using both soluble enzyme and enzyme immobilized on Sepharose 4B. The immobilization demonstrates that isolated subunits are inactive and incapable of binding NADH and suggests that subunit association to the tetramer is essential for enzymatic activity. NADH appears to act, after subunit assembly has taken place, by stabilizing tetramers and preventing their aggregation with monomers that would give rise to inactive polymers.     

IMMOBILIZATION OF OXALATE DECARBOXYLASE TO EUPERGIT AND PROPERTIES OF THE IMMOBILIZED ENZYME

Oxalate decarboxylase, an oxalate degradation enzyme used for medical diagnosis and decreasing the oxalate level in the food or paper industry, was covalently immobilized to Eupergit C. Different immobilization parameters, including ratio of enzyme to support, ammonia sulfate concentration, pH, and incubation time, were optimized. Under the condition of enzyme/support ratio at 1:20, pH 9, with 1.5 mol/L (NH₄)₂SO₄, room temperature, and shaking at 30 rpm for 24 hr, activity recovery of immobilized Oxdc reached 90% with an apparent specific activity of 0.44 U/mg support. The enzymatic properties of immobilized Oxdc were investigated and compared with those of the soluble enzyme. Both shared a similar profile of optimum conditions; the optimum pH and temperature for soluble and immobilized Oxdc were 3.5 and 50°C, respectively. The immobilized enzyme was more stable at lower pH and higher temperatures. The kinetic parameters for soluble and immobilized enzyme were also determined.     

Purification, Immobilization, and Some Properties of Glucose Isomerase from Streptomyces flavogriseus

Glucose isomerase (EC 5.3.1.5) produced from Streptomyces flavogriseus was purified by fractionation with (NH₄)₂SO₄ and chromatography on diethylaminoethyl (DEAE)-cellulose and DEAE-Sephadex A-50 columns. The purified enzyme was homogeneous as shown by ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Benzyl DEAE-cellulose, triethylaminoethyl-cellulose, and DEAE-cellulose were effective in the immobilization of partially purified glucose isomerase. Several differences in properties were found between purified soluble enzyme, immobilized enzyme (DEAE-cellulose-glucose isomerase), and heat-treated whole cells. Glucose and xylose served as substrate for the enzyme. Whole cells had the highest K_m values for glucose and xylose; the soluble enzyme had the lowest values. The optimum temperature for activity of the soluble and immobilized enzymes was 70°C; that for whole cells was 75°C. The pH optimum for the three enzyme preparations was 7.5. Magnesium ion or Co²⁺ was required for enzyme activity; an addition effect resulted from the presence of both Mg²⁺ and Co²⁺. The enzyme activity was inhibited by Hg²⁺, Ag⁺, or Cu²⁺. The conversion ratio of the enzyme for isomerization was about 50%. The soluble and immobilized enzymes showed a greater heat stability than whole cells. The soluble enzyme was stable over a slightly wider pH (5.0 to 9.0) range than the immobilized enzyme and whole cells (pH 5.5 to 9.0). The molecular weight of the enzyme determined by the sedimentation equilibrium method was 171,000. A tetrameric structure for the enzyme was also indicated. After operating at 70°C for 5 days, the remaining enzyme activity of the immobilized enzyme and whole cells, which were used for the continuous isomerization of glucose in a plug-flow type of column in the presence of Mg²⁺ and Co²⁺, was 75 and 55%, respectively. Elimination of Co²⁺ decreased operational stability.     

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.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

Affinity chromatography of Pseudomonas salicylate hydroxylase

In order to facilitate the purification of salicylate hydroxylase (salicylate 1-monooxygenase, EC 1.14.13.1) from Pseudomonas sp. RPP (ATCC 29351), an affinity chromatography procedure was developed employing immobilized salicylate as the affinity ligand. The immobilization was achieved by reacting p-aminosalicylate with the N-hydroxysuccinimide ester of Sepharose 4B-6-aminohexanoic acid. When the bacterial crude extract was chromatographed with this affinity column, salicylate hydroxylase was absorbed to the gel while the bulk of protein freely passed through. The absorbed enzyme was subsequently eluted from the affinity column by applying a 0–60 mm sodium salicylate gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzymatically most active fraction of the affinity effluent revealed salicylate hydroxylase was by far the most predominant protein but there were also small amounts of contaminating proteins. However, a virtually homogeneous enzyme preparation was obtained when the crude extract was first fractionated with a DE-52 anion-exchange column followed by the affinity step. The enzyme preparation obtained by this two-step procedure showed a specific activity of 14.9 units/mg and an A₄₅₀:A₃₇₂:A₂₈₀ of 1.01:1:10.23. Because most of the enzymes belonging to the class of external flavoprotein monooxygenase utilize salicylate analogs as substrates and share many other common properties, there is a strong possibility that the salicylate column may be useful for the purification of other member monooxygenases.     

Development of Bed Reactor Using Brick Dust Immobilized CIVI-Cellulase from Seeds of Cowpea (Vigna sinensis L)

Cellulase extracted from seeds of Cowpea (Vigna sinensis L var VITA-4) was partially purified and immobilized on brick dust as solid support via glutaraldehyde. The percentage retention of the enzyme activity on brick dust was nearly 85%. After immobilization specific activity of the enzyme increased from 0.275 to 0.557 U mg^?1 protein with about 2 fold enrichment. The optimum pH and temperature of soluble enzyme were determined as pH 4.6 and WC, respectively whereas immobilized enzyme showed at pH 5.0 and 37°C, respectively. The V_max values for soluble and immobilized enzyme were determined as 6.67 and 1.25 mg min^?1, respectively whereas K_m values were 4.35 and 4.76 mg ml^?1, respectively. The immobilized enzyme displayed higher thermal stability than soluble enzyme and retained about 50% of its initial activity after 12 reuses. Immobilized enzyme was packed in an indigenously designed double walled glass bed reactor for continuous production of reducing sugars.     

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K_m = 12 micromolar) and coniferaldehyde (K_m = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.     

A novel method for the immobilization of glucoamylase onto polyglutaraldehyde-activated gelatin

A novel method was developed for the immobilization of glucoamylase from Aspergillus niger. The enzyme was immobilized onto polyglutaraldehyde-activated gelatin particles in the presence of polyethylene glycol and soluble gelatin, resulting in 85% immobilization yield. The immobilized enzyme has been fully active for 30 days. In addition, the immobilized enzyme retained 90 and 75% of its activity in 60 and 90 days, respectively. The enzyme optimum conditions were not affected by immobilization and the optimum pH and temperature for free and immobilized enzyme were 4 and 65 °C, respectively. The kinetic parameters for the hydrolysis of maltodextrin by free and immobilized glucoamylase were also determined. The K_m values for free and immobilized enzyme were 7.5 and 10.1 g maltodextrin/l, respectively. The V_max values for free and immobilized enzyme were estimated as 20 and 16 μmol glucose/(min μl enzyme), respectively. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes.     

Bioaffinity Based Oriented Immobilization of Stem Bromelain

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K _m values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V _max values remained unaffected on immobilization.     

Novel and efficient method for immobilization and stabilization of β-d-galactosidase by covalent attachment onto magnetic Fe3O4–chitosan nanoparticles

A novel and efficient immobilization of β-d-galactosidase from Aspergillus oryzae has been developed by using magnetic Fe₃O₄–chitosan (Fe₃O₄–CS) nanoparticles as support. The magnetic Fe₃O₄–CS nanoparticles were prepared by electrostatic adsorption of chitosan onto the surface of Fe₃O₄ nanoparticles made through co-precipitation of Fe²⁺ and Fe³⁺. The resultant material was characterized by transmission electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, vibrating sample magnetometry and thermogravimetric analysis. β-d-Galactosidase was covalently immobilized onto the nanocomposites using glutaraldehyde as activating agent. The immobilization process was optimized by examining immobilized time, cross-linking time, enzyme concentration, glutaraldehyde concentration, the initial pH values of glutaraldehyde and the enzyme solution. As a result, the immobilized enzyme presented a higher storage, pH and thermal stability than the soluble enzyme. Galactooligosaccharide was formed with lactose as substrate by using the immobilized enzyme as biocatalyst, and a maximum yield of 15.5% (w/v) was achieved when about 50% lactose was hydrolyzed. Hence, the magnetic Fe₃O₄–chitosan nanoparticles are proved to be an effective support for the immobilization of β-d-galactosidase.     

Characterization of functionally active immobilized carbonic anhydrase purified from sheep blood lysates

Carbonic anhydrase (CA) catalyzes the reversible reaction of hydration of CO₂ to bicarbonate and the dehydration of bicarbonate back to CO₂. Sequestration of CO₂ from industrial processes or breathing air may require a large amount of highly active and stable CA. Therefore, the objectives of the present study were to purify large amounts of CA from a cheap and easily accessible source of the enzyme and to characterize the enzymatic and kinetic properties of soluble and immobilized enzyme. We recovered 80% of pure enzyme with a specific activity of 4870 EU/mg protein in a single step using sheep blood lysates from slaughter house waste products and CA specific inhibitor affinity chromatography. Since affinity pure CA showed both anhydrase and esterase activities, we measured the esterase activities for enzymology. The Michaelis–Menten constant, K_M, pH optimum, activation energy, and thermal stability of soluble enzymes were 8 × 10^?2 M, 7.3 pH, 7.3 kcal/mol and 70 °C, respectively.The immobilization of the enzyme to Affigel-10 was very efficient and 83% of purified enzyme was immobilized. The immobilized enzyme showed a K_M of 5 × 10^?2 M and activation energy of 8.9 kcal/mol, suggesting a better preference of substrate for immobilized enzyme in comparison to soluble enzyme. In contrast to soluble enzyme, immobilized enzyme showed relatively higher activity at pH 6–8. From these results, we concluded that a shift in pH profile toward acidic pH is due to modification of lysine residues involved in the immobilization process. The immobilized enzyme was stable at higher temperatures and showed highest activity at 80 °C. The activity of immobilized enzyme in a flow reactor at 0.5–2.2 ml/min flow rate was unaffected. Collectively, results from the present study suggested the application of blood lysate waste from animal slaughterhouses for purification of homogeneous enzyme for CO₂ capture in a flow reactor.     

Covalent Immobilization of Alcohol Dehydrogenase (ADH2) from Haloferax volcanii: How to Maximize Activity and Optimize Performance of Halophilic Enzymes

Alcohol dehydrogenase from halophilic archaeon Haloferax volcanii (HvADH2) was successfully covalently immobilized on metal-derivatized epoxy Sepabeads. The immobilization conditions were optimized by investigating several parameters that affect the halophilic enzyme–support interaction. The highest immobilization efficiency (100 %) and retention activity (60 %) were achieved after 48 h of incubation of the enzyme with Ni-epoxy Sepabeads support in 100 mM Tris–HCl buffer, pH 8, containing 3 M KCl at 5 °C. No significant stabilization was observed after blocking the unreacted epoxy groups with commonly used hydrophilic agents. A significant increase in the stability of the immobilized enzyme was achieved by blocking the unreacted epoxy groups with ethylamine. The immobilization process increased the enzyme stability, thermal activity, and organic solvents tolerance when compared to its soluble counterpart, indicating that the immobilization enhances the structural and conformational stability. One step purification–immobilization of this enzyme has been carried out on metal chelate-epoxy Sepabeads, as an efficient method to obtain immobilized biocatalyst directly from bacterial extracts.     

β-d-Galactosidases immobilized on soluble matrices: Kinetics and stability

Three β-d-galactosidases (β-d-galactoside galactohydrolase, EC 3.2.1.23) from different origins have been immobilized on sucrose-polyacrolein and sucrose sulphate-polyacrolein. This gave enzyme conjugates insoluble in the immobilization medium but which could be made soluble by reduction with sodium borohydride before use. The optimum conditions for both copolymer synthesis and the immobilization reaction were investigated. I.r. and ¹³C n.m.r. spectroscopy were used to follow the sulphation and the copolymerization reaction. The characteristics of the enzyme conjugates were compared with those of the free enzymes: the V_max values of the enzyme conjugates were lower than those of the corresponding free enzymes, whilst the K_m values were similar. The thermal stability of the enzyme conjugates depended on the enzyme origin, while their pH stability was in all cases higher than that of the free enzymes. These data suggest some advantages in using enzyme immobilization supports which can be made soluble after separation of the immobilized enzyme without altering the enzyme characteristics.