Purification and biochemical properties of SDS-stable low molecular weight alkaline serine protease from Citrullus colocynthis

A low molecular weight serine protease from seeds of Citrullus colocynthis was purified to electrophoretic homogeneity with high level of catalytic efficiency (22,945 M^?1 S^?1). The enzyme was a monomer with molecular mass of 25 kDa estimated by SDS–PAGE. The enzyme was highly active over a pH range of 6.5–9.0 and temperature range of 20–80 °C, with maximum activity at pH 7.5 and at 50 °C. The K_m and K_cat were 73 μg/mL and 67/s, respectively. The enzyme was strongly inhibited by PMSF, moderately by soybean trypsin inhibitor, indicating that the enzyme was a serine protease. The enzyme retained 86 and 73% of its activity in the presence of urea and DTT, respectively, and its activity was slightly enhanced in the presence of anionic detergent (SDS). Thus, the enzyme is a novel SDS-stable protease with high catalytic efficiency over wide ranges of pH and temperature which is commercially promising for various industrial applications.     

Resveratrol and arctigenin production from polydatin and arctiin respectively by a thermostable β-glucosidase from Thermotoga maritima

Abstract

For preparation of resveratrol and arctigenin from peanut hulls and arctium lappa fruits, respectively, a recombinant β-glucosidase (TmBglA) from hyperthermophile Thermotoga maritima was purified and characterized. The hydrolytic activity was the highest at 90?°C and pH 6.2 for arctiin with K_m of 1.61?mM and k_cat of 197.4?s^?1, and 90?°C and 5.8 for polydatin with K_m of 0.38?mM and k_cat of 47.6?s^?1. The enzyme produced 215.4?mg L^?1 resveratrol and 355.7?mg L^?1 arctigenin from 400?mg L^?1 polydatin and 540?mg L^?1 arctiin after 60?min of incubation at 80?°C, with capable of hydrolyzing up to 92.1 and 94.9% of polydatin and arctiin, respectively. The enzymatic hydrolysis of peanut hulls and fructus arctii displayed a conversion yield of 3.8 and 0.33?mg resveratrol and arctigenin per gram of substrate material flour, respectively. Of the reported β-glucosidase, TmBglA exhibited the highest thermostability, k_cat, k_cat/K_m, and conversion productivity for hydrolyzing polydatin and arctiin, and has great potential applications in functional food and medicine production.     

Erratum to: Catalytic Properties of Two Rhizopus oryzae 99-880 Glucoamylase Enzymes Without Starch Binding Domains Expressed in Pichia pastoris

Catalytic properties of two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 are determined using heterologously expressed enzyme purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly one unit higher than the Rhizopus AmyA glucoamylase enzyme. Optimal initial activities are at 60 and 50 °C for AmyC and AmyD, respectively. Inactivation of both enzymes occurs at 50 °C following 30 min pre-incubation. The two enzymes demonstrate substantially slower catalytic rates toward soluble starch relative to AmyA. AmyC has similar k _cat and K _m for oligosaccharides to other Rhizopus and Aspergillus glucoamylases; however, the enzyme has a 2-fold lower K _m^maltose. AmyD has a 3-fold higher K _m and lower k _cat for maltooligosaccharides than AmyC and other glucoamylases. AmyC (but not AmyD) exhibits substrate inhibition. K _i for substrate inhibition decreases with increasing length of the oligosaccharides. Data from pre-steady-state binding of AmyC to maltose and maltotriose and pre-steady-state to steady-state catalytic turnover experiments of AmyC acting on maltotriose were used to interrogate models of substrate inhibition. In the preferred model, AmyC accumulates an enzyme-maltose-maltotriose dead-end complex in the steady state.     

Purification and Characterization of Pectin Lyase Produced by Aspergillus terricola and its Application in Retting of Natural Fibers

An indigenously isolated fungal strain identified as Aspergillus terricola with assigned fungal strain number MTCC 7588 has been used as source for pectin lyase production. The extracellular pectin lyase was purified to homogeneity from the culture filtrate of A. terricola by ion exchange and gel filtration chromatography. The determined molecular weight was 35 ± 01 kDa. The K _m and k _cat (turnover) values of the purified enzyme at 37 °C using citrus pectin as the substrate were found to be 1.0 mg/ml and 110.0 s⁻¹, respectively. The pH and temperature optima of the enzyme were 8.0 and 50 °C, respectively. The retting ability of the purified pectin lyase for natural fibers viz. Cannabis sativa and Linum usitatissimum has been demonstrated for the first time.     

Cloning of a Heat-Stable Chitin Deacetylase Gene from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and its Functional Expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A gene encoding chitin deacetylase was cloned by polymerase chain reaction from Aspergillus nidulans. Sequencing result showed 40% homology to the corresponding gene from Colletotrichum lindemuthianum. The complete gene contains an open reading frame of 747 nucleotides encoding a sequence of 249 amino acid residues. The chitin deacetylase gene was subcloned into a pET28a expression vector and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a His-bind column. The purified chitin deacetylase demonstrated an activity of 0.77 U ml⁻¹ for the glycol chitin substrates, and its specific activity was 4.17 U mg⁻¹ for it. The optimal temperature and pH of the purified enzyme were 50 °C and 8.0, respectively. When glycol chitin was used as the substrate, K _m was 4.92 mg ml⁻¹, and K _cat showed 6.25 s⁻¹, thus the ratio of K _cat and K _m was 1.27 ml s⁻¹ mg⁻¹. The activity of chitin deacetylase was affected by a range of metal ions and ethylenediaminetetraacetic acid.     

A Laccase of <Emphasis Type="Italic">Fomes durissimus</Emphasis> MTCC-1173 and Its Role in the Conversion of Methylbenzene to Benzaldehyde

A laccase has been purified from the liquid culture growth medium containing bagasse particles of Fomes durissimus. The method involved concentration of the culture filtrate by ultrafiltration and anion exchange chromatography on diethyl aminoethyl cellulose. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis both gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the purified laccase determined from SDS-PAGE analysis was 75 kDa. Using 2,6-dimethoxyphenol as the substrate, the determined K _m and k _cat values of the laccase are 182 μM and 0.35 s⁻¹, respectively, giving a k _cat/K _m value of 1.92 × 10³ M⁻¹ s⁻¹. The pH and temperature optimum were 4.0 and 35 °C, respectively. The purified laccase has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover, it transformed methylbenzene to benzaldehyde in the absence of mediator molecules, property exhibited by yellow laccases.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

Recombination System Based on Cre α Complementation and Leucine Zipper Fusions

A gene encoding β-1,3-1,4-glucanase was cloned by polymerase chain reaction (PCR) from Bacillus subtilis MA139. Sequencing result showed 97% homology to the corresponding gene from Bacillus licheniformis. The open reading frame (ORF) of the gene contained 690 bp coding for a 226 amino-acid matured protein with the estimated molecular weight of 24.44 kDa. The β-1,3-1,4-glucanase gene was subcloned into an expression vector of pET28a and expressed in Escherichia coli BL21 and then purified by metal affinity chromatography using a nickel–nitrilotriacetic acid (Ni–NTA) column. The purified β-1,3-1,4-glucanase demonstrated 24.05 and 12.52 U ml^-1 activities for the substrates of barley β-glucan and lichenan, respectively, and the specific activities were 728.79 and 379.1 U mg^-1 for them, respectively. The optimal temperature and pH of the purified enzyme were 40°C and 6.4, respectively. When barley β-glucan was used as the substrate, K _m was 5.34 mg ml^-1, and K _cat showed 7,206.71 S^-1, thus the ratio of K _cat and K _m was 1,349.67 ml s^-1 mg^-1. The activity of β-1,3-1,4-glucanase was affected by a range of metal ions or ethylenediaminetetraacetic acid (EDTA).     

Cloning, heterologous expression, and characterization of Thielavia terrestris glucoamylase

Thielavia terrestris is a soil-borne thermophilic fungus whose molecular/cellular biology is poorly understood. Only a few genes have been cloned from the Thielavia genus. We detected an extracellular glucoamylase in culture filtrates of T. terrestris and cloned the corresponding glaA gene. The coding region contains five introns. Based on the amino acid sequence, the glucoamylase was 65% identical to Neurospora crassa glucoamylase. Sequence comparisons suggested that the enzyme belongs to the glycosyl hydrolase family 15. The T. terrestris glaA gene was expressed in Aspergillus oryzae under the control of an A. oryzae α-amylase promoter and an Aspergillus niger glucoamylase terminator. The 75-kDa recombinant glucoamylase showed a specific activity of 2.8 μmol/(min·mg) with maltose as substrate. With maltotriose as a substrate, the enzyme had an optimum pH of 4.0 and an optimum temperature of 60°C. The enzyme was stable at 60°C for 30 min. The K _m and k _cat of the enzyme for maltotriose were determined at various pHs and temperatures. At 20°C and pH 4.0, the enzyme had a K _m of 0.33±0.07 mM and a k _cat of (5.5±0.5)×10³ min⁻¹ for maltotriose. The temperature dependence of k _cat /K _m indicated an activation free energy of 2.8 kJ/mol across the range of 20–70°C. Overall, the enzyme derived from the thermophilic fungus exhibited properties comparable with that of its homolog derived from mesophilic fungi.     

Catalytic Properties of Two <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> 99-880 Glucoamylase Enzymes Without Starch Binding Domains Expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Catalytic properties of two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 are determined using heterologously expressed enzyme purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly one unit higher than the Rhizopus AmyA glucoamylase enzyme. Optimal initial activities are at 60 and 50 °C for AmyC and AmyD, respectively. Inactivation of both enzymes occurs at 50 °C following 30 min pre-incubation. The two enzymes demonstrate substantially slower catalytic rates toward soluble starch relative to AmyA. AmyC has similar k _cat and K _m for oligosaccharides to other Rhizopus and Aspergillus glucoamylases; however, the enzyme has a 2-fold lower K _m ^maltose . AmyD has a 3-fold higher K _m and lower k _cat for maltooligosaccharides than AmyC and other glucoamylases. AmyC (but not AmyD) exhibits substrate inhibition. K _i for substrate inhibition decreases with increasing length of the oligosaccharides. Data from pre-steady-state binding of AmyC to maltose and maltotriose and pre-steady-state to steady-state catalytic turnover experiments of AmyC acting on maltotriose were used to interrogate models of substrate inhibition. In the preferred model, AmyC accumulates an enzyme-maltose-maltotriose dead-end complex in the steady state.     

Purification and characterization of organic solvent stable serine alkaline protease from newly isolated Bacillus circulans M34

A protease from newly isolated Bacillus circulans M34 was purified by Q‐Sepharose anion exchange chromatography and Sepharose–bacitracin affinity chromatography followed by (NH₄)₂SO₄ precipitation. The molecular mass of the purified enzyme was determined using SDS–PAGE. The optimum pH and temperature for protease activity were 11 and 50°C, respectively. The effect of various metal ions on protease activity was investigated. Alkaline protease from Bacillus circulans M34 wase activated by Zn²⁺, Cu²⁺ and Co²⁺ up to 31%. The purified protease was found to be stable in the organic solvents, surfactants and oxidizing agent. The substrate specificity of purified protease was investigated towards different substrates. The protease was almost completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The kinetic parameters of the purified protease, maximum rate (V_max) and Michaelis constant (K_m), were determined using a Lineweaver–Burk plot. Copyright © 2015 John Wiley & Sons, Ltd.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

Extracellular l-Asparaginase from a Protease-Deficient Bacillus aryabhattai ITBHU02: Purification, Biochemical Characterization, and Evaluation of Antineoplastic Activity In Vitro

An extracellular l-asparaginase produced by a protease-deficient isolate, Bacillus aryabhattai ITBHU02, was purified to homogeneity using ammonium sulfate fractionation and subsequent column chromatography on diethylaminoethyl-Sepharose fast flow and Seralose CL-6B. The enzyme was purified 68.9-fold with specific activity of 680.47 U mg^?1. The molecular weight of the purified enzyme was approximately 38.8 kDa on SDS-PAGE and 155 kDa on native PAGE gel as well as gel filtration column revealing that the enzyme was a homotetramer. The optimum activity of purified l-asparaginase was achieved at pH 8.5 and temperature 40 °C. Kinetic studies depicted that the K _m, V _max, and k _cat values of the enzyme were 0.257 mM, 1.537 U μg^?1, and 993.93 s^?1, respectively. Circular dichroism spectroscopy has showed that the enzyme belonged to α?+?β class of proteins with approximately 74 % α-helices and 12 % β-sheets. BLASTP analysis of N-terminal sequence K-T-I-I-E-A-V-P-E-L-K-K-I-A of purified l-asparaginase had shown maximum similarity with Bacillus megaterium DSM 319. In vitro cytotoxicity assays with HL60 and MOLT-4 cell lines indicated that the l-asparaginase has significant antineoplastic properties.     

Purification and Characterization of a Low Molecular Weight of β-Mannanase from Penicillium occitanis Pol6

The highest β-mannanase activity was produced by Penicillium occitanis Pol6 on flour of carob seed, whereas starch-containing medium gave lower enzymes titles. The low molecular weight enzyme was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography procedures. The purified β-mannanase (ManIII) has been identified as a glycoprotein (carbohydrate content 5%) with an apparent molecular mass of 18 kDa. It was active at 40 °C and pH 4.0. It was stable for 30 min at 70 °C and has a broad pH stability (2.0–12.0). ManIII showed K _m, V _max, and K _cat values of 17.94 mg/ml, 93.52 U/mg, and 28.13 s⁻¹ with locust bean gum as substrate, respectively. It was inhibited by mannose with a K _I of 0.610⁻³ mg/ml. ManIII was activated by CuSO₄ and CaCl₂ (2.5 mM). However, in presence of 2.5 mM Co²⁺, its activity dropped to 60% of the initial activity. Both N-terminal and internal amino acid sequences of ManIII presented no homology with mannanases of glycosides hydrolases. During incubation with locust bean gum and Ivory nut mannan, the enzyme released mainly mannotetraose, mannotriose, and mannobiose.     

Expression,Purification and Characterization of Human α‐l‐Fucosidase

α‐l ‐Fucosidases (EC 3.2.1.51) are exo‐glycosidases. On the basis of the multi‐alignment of amino acid sequence, α‐l ‐fucosidases were classified into two families of glycoside hydrolases, GH‐29 and GH‐95. They are responsible for the removal of l ‐fucosyl residues from the non‐reducing end of glycoconjugates. Deficiency of α‐l ‐fucosidase results in Fucosidosis due to the accumulation of fucose‐containing glycolipids, glycoproteins and oligosaccharides in various tissues. Recent studies discovered that the fucosylation levels are increased on the membrane surfaces of many carcinomas, indicating the biological function of α‐l ‐fucosidases may relate to this abnormal cell physiology. Although the gene of human α‐l ‐fucosidase (h‐fuc) was cloned, the recombinant enzyme has rarely been overexpressed as a soluble and active from. We report herein that, with carefully control on the growing condition, an active human α‐l ‐fucosidases (h‐Fuc) was successfully expressed in Escherichia coli for the first time. After a series steps of ion‐exchange and gel‐filtration chromatographic purification, the recombinant h‐Fuc with 95% homogeneity was obtained. The molecular weight of the enzyme was analyzed by SDS‐PAGE (～50 kDa) and confirmed by ESI mass (50895 Da). The recombinant h‐Fuc was stable up to 55 °C with incubation at pH 6.8 for 2 h; the optimum temperature for h‐Fuc is approximately 55 °C. The enzyme was stable at pH 2.5–7.0 for 2 h; the enzyme activity decreased greatly for pH greater than 8.0 or less than 2.0. The K_m and k_cat values of the recombinant h‐Fuc (at pH 6.8) were determined to be 0.28 mM and 17.1 s^?1, respectively. The study of pH‐dependent activity showed that the recombinant enzyme exhibited optimum activity at two regions near at pH 4.5 and pH 6.5. These features of the recombinant h‐Fuc are comparable to the native enzyme purified directly from human liver. Studies on the transfucosylation and common intermediate of the enzymatic reaction by NMR support that h‐Fuc functions as a retaining enzyme catalyzing the hydrolysis of substrate via a two‐step, double displacement mechanism.     

Kinetic Study of Peroxidase‐Catalyzed Coupling of Benzene‐1,4‐diamine and N‐(2‐Aminoethyl)naphthalen‐1‐amine: Development of Micromolar Hydrogen Peroxide Reaction System

A novel chromogenic method to measure the peroxidase activity using para‐phenylenediamine dihydrochloride (=benzene‐1,4‐diamine hydrochloride; PPDD) and N‐(1‐naphthyl)ethylenediamine dihydrochloride (=N‐(2‐aminoethyl)naphthalen‐1‐amine; NEDA) is presented. The PPDD entraps the free radical and gets oxidized to electrophilic diimine, which couples with NEDA to give an intense red‐colored chromogenic species with maximum absorbance at 490 nm. This assay was adopted for the quantification of H₂O₂ between 20 and 160 μM . Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 4.47×10⁴ M ^?1 min^?1 and 3.38×10^?4 min^?1, respectively. The catalytic constant (k_cat) and specificity constant (k_cat/K_m) at saturated concentration of the co‐substrates were 0.0245×10³ min^?1 and 0.0445 μM ^?1 min^?1, respectively. The chromogenic coupling reaction has a minimum interference from the reducing substances such as ascorbic acid, L ‐cystein, citric acid, and oxalic acid. The method being simple, rapid, precise, and sensitive, its applicability has been tested in the crude vegetable extracts that showed peroxidase activity.     

Structure-function relationships of a catalytically efficient β-<Emphasis Type="SmallCaps">D</Emphasis>-xylosidase

β-d-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k _cat, k _cat/K _m) for promoting hydrolysis of 1,4-β-d-xylooligosaccharides. ¹H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 1,4-β-d-xylobiose (X2). Progress curves of 4-nitrophenyl-β-d-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k _cat and k _cat/K _m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites −1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites bey ond−1 are blocked by protein and subsites bey ond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k _cat and k _cat/K _m by <10³ and to asimilar extent for substrates 4NPX and 4-nitrophenyl-α-l-arabinofuranoside (4NPA), indicating that the two substrates share the same active site. With 4NPX and 4NPA, pH governs k _cat/K _m with pK _a values of 5.0 and 7.0 assigned to D14 and E186, respectively. k _cat (4NPX) has a pK _a value of 7.0 and k _cat (4NPA) is pH independent above pH 4.0, suggesting that the catalytically inactive, “dianionic” enzyme form (D14-E187-) binds 4NPX but not 4NPA. The mention of firm names or trade products does not imply that they are end orsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Characterization of xylitol dehydrogenase from debaryomyces hansenii

The xylitol dehydrogenase (EC 1.1.1.9) from xylose-grown cells ofDebaryomyces hansenii was partially purified in two Chromatographic steps, and characterization studies were carried out in order to inves tigate the role of the xylitol dehydrogenase-catalyzed step in the regu lation of D-xylose metabolism. The enzyme was most active at pH 9.0–9.5, and exhibited a broad polyol specificity. The Michaelis con stants for xylitol and NAD⁺ were 16.5 and 0.55 mM, respectively. Ca²⁺, Mg²⁺, and Mn²⁺ did not affect the enzyme activity. Conversely, Zn²⁺, Cd²⁺, and Co²⁺ strongly inhibited the enzyme activity. It was concluded that NAD⁺-xylitol dehydrogenase from D.hansenii has similarities with other xylose-fermenting yeasts in respect to optimal pH, substrate specificity, and K_m value for xylitol, and therefore should be named L-iditol:NAD⁺-5-oxidoreductase (EC 1.1.1.14). The reason D.hansenii is a good xylitol producer is not because of its value of K_m for xylitol, which is low enough to assure its fast oxidation by NAD⁺ xylitol dehydrogenase. However, a higher K_m value of xylitol dehydro genase for NAD⁺ compared to theK _m values of other xylose-ferment ing yeasts may be responsible for the higher xylitol yields.     

Preparation and properties of glucose isomerase immobilized on Indion 48-R

Partially purified glucose isomerase fromStreptomyces thermonitrificans when coupled to glutaraldehyde-activated Indion 48-R, retained 30–40% activity of the soluble enzyme. However, an approximately twofold increase in the activity could be achieved by binding the enzyme in the presence of glucose. Binding the enzyme to matrices presaturated with either glucose or fructose and influence of lysine modification on the activity of the soluble enzyme revealed that the comparatively low activity observed in case of the enzyme bound in the absence of substrate is the result of the nonspecific binding of either substrate or product to the matrix. Immobilization did not affect the pH and temperature optima of the enzyme, but it lowered the temperature stability. Immobilization resulted in a marginal increase in theK _m and a threefold decrease in theV _max . Substrate concentrations as high as 36% glucose could be converted to 18.5% fructose in 5 h, at pH 7.0 and 70‡C. The bound enzyme, however, showed inferior stability to repeated use and lost approx 40% of its initial activity after five cycles of use. Indion 48-R bound glucose isomerase could be stored, in wet state, for 30 d without any apparent loss in its initial activity.