Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum

A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source, and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative molecular mass (M _r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K _m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C in the range pH 2.0–4.0 and at a pH above 7.0. Received: 18 November 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Pyranose 2-dehydrogenase, a novel sugar oxidoreductase from the basidiomycete fungus Agaricus bisporus

A novel C-2-specific sugar oxidoreductase, tentatively designated as pyranose 2-dehydrogenase, was purified 68-fold to apparent homogeneity (16.4 U/mg protein) from the mycelia of Agaricus bisporus, which expressed maximum activity of the enzyme during idiophasic growth in liquid media. Using 1,4-benzoquinone as an electron acceptor, pyranose 2-dehydrogenase oxidized d-glucose to d-arabino-2-hexosulose (2-dehydroglucose, 2-ketoglucose), which was identified spectroscopically through its N,N-diphenylhydrazone. The enzyme is highly nonspecific. d-,l-Arabinose, d-ribose, d-xylose, d-galactose, and several oligosaccharides and glycopyranosides were all converted to the corresponding 2-aldoketoses (aldosuloses) as indicated by TLC. d-Glucono-1,5-lactone, d-arabino-2-hexosulose, and l-sorbose were also oxidized at significant rates. UV/VIS spectrum of the native enzyme (λ_max 274, 362, and 465 nm) was consistent with a flavin prosthetic group. In contrast to oligomeric intracellular pyranose 2-oxidase (EC 1.1.3.10), pyranose 2-dehydrogenase is a monomeric glycoprotein (pI 4.2) incapable of reducing O₂ to H₂O₂ (> 5 × 10⁴-fold lower rate using a standard pyranose oxidase assay); pyranose 2-dehydrogenase is actively secreted into the extracellular fluid (up to 0.5 U/ml culture filtrate). The dehydrogenase has a native molecular mass of ∼79 kDa as determined by gel filtration; its subunit molecular mass is ∼75 kDa as estimated by SDS-PAGE. Two pH optima of the enzyme were found, one alkaline at pH 9 (phosphate buffer) and the other acidic at pH 4 (acetate buffer). Ag⁺, Hg²⁺, Cu²⁺, and CN^– (10 mM) were inhibitory, while 50 mM acetate had an activating effect. Received: 19 August 1996 / Accepted: 21 November 1996     

One-step purification and characterization of a β-1,4-glucosidase from a newly isolated strain of Stereum hirsutum

A highly efficient β-1,4-glucosidase (BGL) secreting strain, Stereum hirsutum SKU512, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. A BGL containing a carbohydrate moiety was purified to homogeneity from S. hirsutum culture supernatants using only a single chromatography step on a gel filtration column. The relative molecular weight of S. hirsutum BGL was determined as 98 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis or 780 kDa by size exclusion chromatography, indicating that the enzyme is an octamer. S. hirsutum BGL showed the highest activity toward p-nitrophenyl-β-D-glucopyranoside (V _max = 3,028 U mg-protein⁻¹, k _cat = 4,945 s⁻¹) ever reported. The enzyme also showed good stability at an acidic pH ranging from 3.0 to 5.5. The BGL was able to promote transglycosylation with an activity of 42.9 U mg-protein⁻¹ using methanol as an acceptor and glucose as a donor. The internal amino acid sequences of the isolated enzyme showed significant homology with hydrolases from glycoside hydrolase family 1 (GH1), indicating that the S. hirsutum BGL is a member of GH1 family. The characteristics of S. hirsutum BGL could prove to be of interest in several potential applications, especially in enhancing flavor release during the wine fermentation process.     

Purification and biochemical characterization of a transglucosilating β-glucosidase of <Emphasis Type="Italic">Stachybotrys</Emphasis> strain

The filamentous fungus Stachybotrys sp has been shown to possess a rich β-glucosidase system composed of five β-glucosidases. One of them was already purified to homogeneity and characterized. In this work, a second β-glucosidase was purified and characterized. The filamentous fungal A19 strain was fed-batch cultivated on cellulose, and its extracellular cellulases (mainly β-glucosidases) were analyzed. The purified enzyme is a monomeric protein of 78 kDa molecular weight and exhibits optimal activity at pH 6.0 and at 50°C. The kinetic parameters, K _m and V _max, on para-nitro-phenyl-β-d-glucopyranosid (p-NPG) as a substrate were, respectively, 1.846 ± 0.11 mM and 211 ± 0.08 μmol min⁻¹ ml⁻¹. One interesting feature of this enzyme is its high stability in a wide range of pH from 4 to 10. Besides its aryl β-glucosidase activity towards salicin, methylumbellypheryl-β-d-glucoside (MU-Glc), and p-NPG, it showed a true β-glucosidase activity because it splits cellobiose into two glucose monomers. This enzyme has the capacity to synthesize short oligosaccharides from cellobiose as the substrate concentration reaches 30% with a recovery of 40%. We give evidences for the involvement of a transglucosylation to synthesize cellotetraose by a sequential addition of glucose to cellotriose.     

Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1

Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn²⁺ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure. Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

Chromatographic behaviour of glucose 1- and 2-oxidases from fungal strains on immobilized metal chelates

Glucose 2-oxidase (EC 1.1.3.10) from Coriolus versicolor and Phanerochaete chrysosporium and glucose 1-oxidase (EC 1.1.3.4) from Aspergillus niger bound to a CU(II)-IDA column in the pH range of 6–8. However, glucose 1-oxidase from Penicillium amagasakiense bound only partially to a CU(II)-IDA column at pH 8.0. Metal chelates containing either Ni(II) or Zn(II) were useful in the adsorption of glucose 2-oxidase from Phanerochaete chrysosporium. The binding of glucose 2-oxidase from P. chrysosporium to Ni(II) and Zn(II)-IDA agarose columns increases as a function of pH of the buffer system. The adsorption of glucose oxidases on metal(II)-IDA chelates was due to the available histidine residues on enzyme molecules since the addition of imidazole in the buffer system abolished the binding of glucose oxidases to these columns. Both glucose oxidases from C.versicolor, P. chrysosporium and A. niger were purified in one step by immobilized metal affinity chromatography on metal(II)-IDA agarose columns with a recovery of enzyme activity in the range of 80–91%. Purified preparations of glucose oxidases from fungal strains were apparently homogeneous on native PAGE and SDS-PAGE. Immobilized metal affinity chromatography was used to separate glucose 1-oxidase from the 2-oxidase on metal(II)-IDA agarose columns which was confirmed by analysis of the reaction products by HPLC. The different chromatographic behaviour of glucose oxidases on metal(II)-IDA chelates is apparently due to the number and spatial distribution of available histidine residues on these enzyme molecules. Received 12 May 1998/ Accepted in revised form 29 July 1998     

Pyranose 2-oxidase from Phanerochaete chrysosporium– further biochemical characterisation

Pyranose 2-oxidase (P2O) was purified 43-fold to apparent homogeneity from the basidiomycete Phanerochaete chrysosporium using liquid chromatography on phenyl Sepharose, Mono Q (twice) and phenyl Superose. The native enzyme has a molecular mass of about 250 kDa (based on native PAGE) and is composed of four identical subunits of 65 kDa. It contains three isoforms of isoelectric point (pI) 5.0, 5.05 and 5.15 and does not appear to be a glycoprotein. P2O is optimally stable at pH 8.0 and up to 60 °C. It is active over a broad pH range (5.0–9.0) with maximum activity at pH 8.0–8.5 and at 55 °C, and a broad substrate specificity. d-Glucose is the preferred substrate, but 1-β-aurothioglucose, 6-deoxy-d-glucose, l-sorbose, d-xylose, 5-thioglucose, d-glucono-1,5-lactone, maltose and 2-deoxy-d-glucose are also oxidised at relatively high rates. A Ping Pong Bi Bi mechanism was demonstrated for the P2O reaction at pH 8.0, with a catalytic constant (k _cat) of 111.0 s⁻¹ and an affinity constant (K _m) of 1.43 mM for d-glucose and 83.2 μM for oxygen. Whereas the steady-state kinetics for glucose oxidation were unaffected by the medium at pH ≥ 7.0, at low pH both pH and buffer composition affected the P2O kinetics with the k _cat/K _m value decreasing with decreasing pH. The greatest effect was observed in acetate buffer (0.1 M, pH 4.5), where the k _cat decreased to 60.9 s⁻¹ and the K _m increased to 240 mM. The activity of P2O was completely inhibited by 10 mM HgCl₂, AgNO₃ and ZnCl₂, and 50% by lead acetate, CuCl₂ and MnCl₂. Received: 28 August 1996 / Received revision: 25 November 1996 / Accepted: 29 November 1996     

Purification and characteristics of fucoidanase obtained from <Emphasis Type="Italic">Dendryphiella arenaria</Emphasis> TM94

Dendryphiella arenaria TM94 is an obligate marine fungus. Fucoidanase expressed by TM94 by solid state fermentation was purified. The fermented solid medium was extracted with citric acid buffer, and the extracts were precipitated by acetone and separated on Sephadex G-100 chromatography. The specific fucoidanase activity of purified enzyme was 27-fold than that of the crude enzyme. The recovery of the enzyme was 17.69%. SDS-PAGE was used to identify the purity and the molecular weight of the fucoidanase. A single band appeared on SDS-PAGE gel which suggested that relatively pure fucoidanase has been obtained. The molecular weight of fucoidanase is 180 kDa and the isoelectric point was about pH 4.4. The purified fucoidanase appeared to have the maximum enzymatic activity at pH 6.0. K_M and the maximum velocity of the enzyme was 6.56 mg·mL⁻¹ and 6.55 mg·mL⁻¹·min⁻¹ by using fucoidan from Fucus vesiculosus as substrate. The enzyme may be a type of endo-fucoidanase which could hydrolyze high molecular weight fucoidan to low molecular weight fucoidan rather than to fucose.     

Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts – partial purification and biochemical properties

 Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersicon esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa, with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn²⁺ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with K _m values for Mn²⁺, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 μM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including β-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences. Received: 20 January 2000 / Accepted: 16 February 2000     

Purification and properties of a thermoactive and thermostable pullulanase from Thermococcushydrothermalis, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent

The extremely thermophilic archaeon Thermococcus hydrothermalis, isolated from a deep-sea hydrothermal vent in the East Pacific Rise at 21°N, produced an extracellular pullulanase. This enzyme was purified 97-fold to homogeneity from cell-free culture supernatant. The purified pullulanase was composed of a single polypeptide chain having an estimated molecular mass of 110 kDa (gel filtration) or 128 kDa (sodium dodecyl sulfate/polyacryl amide gel electrophoresis). The enzyme showed optimum activity at pH 5.5 and 95 °C. The thermostability and the thermoactivity were considerably increased in the presence of Ca²⁺. The enzyme was activated by 2-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and α-cyclodextrin were inhibitors. This enzyme was able to hydrolyze, in addition to the α-1,6-glucosidic linkages in pullulan, α-1,4-glucosidic linkages in amylose and soluble starch, and can therefore be classified as a type II pullulanase or an amylopullulanase. The purified enzyme displayed Michaelis constant (K _m) values of 0.95 mg/ml for pullulan and 3.55 mg/ml for soluble starch without calcium and, in the presence of Ca²⁺, 0.25 mg/ml for pullulan and 1.45 mg/ml for soluble starch. Received: 19 November 1997 / Received revision: 9 March 1998 / Accepted: 14 March 1998     

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin was 55°C and the optimal pH for sucrose was 4.75. The apparent K _m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides. The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69. Received 17 November 1999/ Accepted in revised form 30 May 2000     

Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2

Rhodococcus sp. R_14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH₂-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a rather wide pH range of 4.0–10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly oxidizes 3β-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3α-hydroxysteroids. Thus, the presence of a 3β-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated at 55 μM; the COX activity was markedly inhibited by metal ions such as Hg²⁺ and Fe³⁺ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione. These suggests that -SH groups may be involved in the catalytic activity of the present COX.     

Biochemical Characterization of a β-1,3-Glucanase from Trichoderma koningii Induced by Cell Wall of Rhizoctonia solani

Trichoderma species are readily isolated from Brazilian cerrado soil by conventional methods and some of them were characterized as Trichoderma koningii. The effect of carbon source on the production of β-1,3-glucanases in the culture filtrates of a specific Trichoderma koningii strain (ALL 13) was investigated. Enzyme activity was detected in all carbon sources tested and only one band of β-1,3-glucanase was detected in non-denaturing PAGE. This enzyme was purified by Sephacryl S-200 gel filtration and Phenyl Sepharose CL 4B chromatography. A typical procedure provided 105-fold purification with 13.4% yield. The molecular weight of the purified enzyme was 75 kDa as estimated by SDS-PAGE. The enzyme hydrolyzed laminarin in an endo-like fashion to form small oligosaccharides and glucose. The K_m and V_max values for β-1,3-glucanase, using laminarin as substrate, were 0.148 mg.mL⁻¹ and 0.159 U.min⁻¹, respectively. The pH optimum for the enzyme was pH 4.6 and maximum activity was obtained at 50°C. Hg²⁺ inhibited the purified enzyme.     

α-Galactosidase of Bifidobacterium adolescentis DSM 20083

Bifidobacterium adolescentis was grown anaerobically in medium enriched with α-D-galactosides. α-Galactosidase (EC 3.2.1.22) was released from the cells by ultrasonic treatment and purified 36-fold by ultrafiltration, ammonium-sulphate precipitation, anion-exchange chromatography, and size-exclusion chromatography. Two protein bands were consistantly observed after sodium-dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretically homogeneous α-galactosidase was only obtained by electroelution. The enzyme had an apparent molecular mass of 344 kDa and 79 kDa as judged by size-exclusion chromatography and SDS-PAGE, respectively. Activity-staining after nondenaturing SDS-PAGE indicated an apparent molecular mass of 145 kDa. Thus, a tetrameric structure of the protein is suggested. The α-galactosidase showed optimal activity at pH 5.5 and 55°C. Lower pH values and higher temperatures rapidly inactivated α-galactosidase. The enzyme hydrolyzed specifically α-galactosidic linkages, and α-(1-3)-linkages were hydrolyzed at a higher rate compared to α-(1-6)-linkages. Hydrolysis of galactosides followed normal saturation kinetics; K_M-values for p-nitrophenyl-α-galactopyranoside (p-NPG) and raffinose were calculated with 0.957 mM and 4.12 mM, respectively. Received: 7 August 1998 / Accepted: 9 September 1998     

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products

A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin. The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and V _max values toward phosphoric-acid-swollen cellulose as substrate, except that their K _m values were about fourfold higher than that of the native enzyme. Received: 4 August 1997 / Received revision: 13 October 1997 / Accepted: 31 October 1997     

Keratinase of Doratomyces microsporus

 The fungus Doratomyces microsporus produced an extracellular keratinase during submerged aerobic cultivation in a medium containing a protein inducer for enzyme synthesis. The keratinase was purified to homogeneity using hydrophobic interaction chromatography followed by gel chromatography. The molecular weight was estimated to be 33 kDa (from SDS-PAGE analysis) or 30 kDa (by gel chromatography), suggesting a monomeric structure. The isoelectric point of the enzyme was determined to be around 9. The optimal pH and temperature for keratinolytic activity were pH 8–9 and 50 °C, respectively. The serine protease inhibitor PMSF totally inhibited the keratinase. The enzyme was not glycosylated. It was capable of hydrolysing different keratinous materials as well as some non-keratinous proteins. Hydrolysis of some synthetic substrates, specific for known proteinases, suggested that the keratinase of D. microsporus is close to proteinase K. Received: 9 July 1999 / Received revision: 13 September 1999 / Accepted: 17 September 1999     

Biochemical properties of an extracellular trehalase from <Emphasis Type="Italic">Malbranchea pulchella</Emphasis> var. Sulfurea

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0–5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K_M values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70±0.29 and 2.58±0.13 mM, respectively. Manganese ions affected only the apparent V_max, increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.     

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW_XL and G3000SW_XL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SW_XL and G4000SW_XL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl₂ and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type. Received: 29 October 1998 / Accepted: 10 December 1998     

Discovery and characterization of a putrescine oxidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> NCIMB 11540

A gene encoding a putrescine oxidase (PuO_Rh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K _M = 8.2 μM, k _cat = 26 s⁻¹). PuO_Rh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuO_Rh is a reasonably thermostable enzyme with t _1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuO_Rh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuO_Rh. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.