Applied and Environmental Microbiology, May 2003, p . 2603-2607, Vol . 69, No . 5

A Novel NAD-Dependent Dehydrogenase, Highly Specific for 1,5-Anhydro-D-Glucitol, from Trichoderma longibrachiatum Strain 11-3

Nobuyuki Yoshida,^* Etsuko Uchida, Tohoru Katsuragi, and Yoshiki Tani

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

Received 31 October 2002/ Accepted 28 January 2003

The 1,5-AG concentration in serum has been determined by high-performance liquid chromatography (9, 13, 14) and gas-liquid chromatography (10, 17, 21), but the specificities for 1,5-AG of these methods are relatively low and the procedure, which involves assaying large number of samples, is too tedious . There have been some reports about the enzymes which exhibit activity with 1,5-AG . Pyranose oxidase from Polyporus obtusus is used for enzymatic determination of 1,5-AG . This method requires removal of glucose from samples with a column (16) or conversion of glucose in samples to glucose 6-phosphate, which is not the substrate for pyranose oxidase (4) . Other oxidases and dehydrogenases that oxidize 1,5-AG have been found in several microorganisms (3, 7, 8, 15) . Oxidases derived from Pseudomonas sp . strain NK-8500, Pycnoporus coccineus, and Coriolus consors exhibit activity with some other sugars and sugar alcohols, such as glucose, galactose, mannitol, and xylitol, in addition to 1,5-AG (7) . Dehydrogenases that react with 1,5-AG have been found in Agrobacterium sp . (3), Eupenicillium crustaceum, Hansenula california (8), and Cytophaga marinoflava (15) . These enzymes, however, oxidize some other sugars and sugar alcohols, such as fructose, galactose, mannitol, and xylitol . The specificities are crucial for diagnostic use, because sugars and sugar alcohols which are substrates for the enzymes described above are present in abundance in blood .

In this study, we screened microorganisms from the soil for 1,5-AG-degrading activity that is highly specific for 1,5-AG to isolate a fungus, Trichoderma longibrachiatum strain 11-3 .

Screening for 1,5-AG-utilizing microorganisms.
The enrichment culture technique was used with a medium composed of 3 g of (NH₄)₂SO₄, 1 g of K₂HPO₄, 1 g of NaH₂PO₄, 0.5 g of MgSO₄ · 7H₂O, 0.1 g of CaCl₂ · 2H₂O, 5 g of 1,5-AG, 1 ml of a vitamin mixture (see below), and 10 ml of a metal solution (see below) in 1,000 ml of distilled water (pH 5.5) . The vitamin mixture contained 1 mg of thiamine-HCl, 2 mg of riboflavin, 2 mg of Ca pantothenate, 2 mg of pyridoxine-HCl, 0.1 mg of biotin, 1 mg of p-aminobenzoic acid, 2 mg of nicotinic acid, and 0.1 mg of folic acid in 100 ml of distilled water . The metal solution contained 11.7 g of MnSO₄ · 3H₂O, 2.2 g of ZnSO₄ · 7H₂O, 0.4 g of CuSO₄ · 5H₂O, 0.28 g of CoCl₂ · 2H₂O, 0.26 g of NaMoO₄ · 2H₂O, 0.4 g of H₃BO₃, and 0.06 g of KI in 1,000 ml of distilled water . A soil sample was added to 5 ml of the medium in a test tube and shaken (300 strokes per min) at 30°C for 2 days . Then 0.01 ml of the culture was transferred to fresh medium, and cultivation was continued under the same conditions . Pure cultures were obtained by the streak-plate technique with agar plates containing the same medium .

Detection of glucose in culture filtrate.
Glucose in the culture filtrate was detected by the glucose oxidase method . The assay mixture (total volume, 3 ml) contained 100 µmol of 2-(N-morpholino)ethanesulfonic acid, 4.5 µmol of 4-aminoantipyrine, 6.0 µmol of phenol, 6.0 U of horseradish peroxidase, 100 U of glucose oxidase, and 50 µl of sample (pH 5.7) . The reaction mixture was incubated at 30°C for 30 min, and the absorbance was measured at 505 nm .

Resting cell reaction with 1,5-AG.
The reaction mixture for the resting cell reaction (total volume, 1 ml) contained 100 µmol of Tris-HCl (pH 8.0), 2 µmol of 1,5-AG, and 0.1 g (wet weight) of resting cells . The reaction was carried out at 30°C for 1 to 4 h with reciprocal shaking . The reaction mixture was put on a silica gel plate (20 by 20 cm; Merck KGaA, Darmstadt, Germany), and thin-layer chromatography (TLC) was carried out by using phenol-water (4:1, vol/vol) as the developing solvent . The spots were detected by spraying with 20% sulfuric acid .

Cultivation of the isolate.
The isolated fungus was cultivated at 28°C for 2 days in 7 liters of medium (pH 5.5) containing 40 g of glucose, 40 g of yeast extract, 24 g of NaNO₃, 8 g of K₂HPO₄, 4 g of MgSO₄ · 7H₂O, and 0.8 g of CaCl₂ · 2H₂O (pH 5.5) by using a 10-liter jar fermentor (agitation, 500 rpm; aeration, 5.0 liters/min) . Growth of the fungus was monitored by measuring wet mycelial weight after filtration on a glass filter .

Enzyme purification.
Of the SH reagents tested, dithiothreitol was the most effective in preventing enzyme inactivation and was always used in the buffer (50 mM Tris-HCl buffer [pH 8.0]) at a concentration of 2 mM unless otherwise stated . All purification procedures were carried out at 4°C .

(i) Step 1: preparation of cell extract.
Washed mycelia (100 g, wet weight) were suspended in 130 ml of 0.1 M Tris-HCl (pH 8.0) and disrupted in a glass bead mill (model 11079-S Biospec bead beater; Japan Lambda) . The homogenate was centrifuged at 9,000 x g for 30 min at 4°C to remove unbroken cells and debris . The supernatant solution was used as the cell extract .

(ii) Step 2: ammonium sulfate fractionation.
To the cell extract, solid (NH₄)₂SO₄ was added to 35% saturation with stirring, and the pH was adjusted to 7.0 with a 35% NH₄OH solution . After the preparation stood for 1 h, the precipitate was separated by centrifugation at 16,000 x g for 30 min and discarded . Further (NH₄)₂SO₄ fractionation was carried out in the similar way, and the precipitate obtained at 55% (NH₄)₂SO₄ saturation was dissolved in the buffer . The enzyme solution was then dialyzed with the buffer for 18 h .

(iii) Step 3: DEAE-Toyopearl column chromatography.
The dialyzed solution was applied to a DEAE-Toyopearl column (diameter, 2.2 cm; length, 20 cm) equilibrated with the buffer . The absorbed protein was eluted with a linear gradient of KCl (0 to 0.5 M) . Active fractions were collected and dialyzed with the buffer containing (NH₄)₂SO₄ at 55% saturation .

(iv) Step 4: phenyl-Sepharose column chromatography.
The dialyzed solution was loaded on a phenyl-Sepharose column (diameter, 1.0 cm; length, 10 cm) equilibrated with the same buffer . After washing with the same buffer, the enzyme solution was eluted with a linear gradient of (NH₄)₂SO₄ (55 to 0% saturation) at a flow rate of 1 ml/min .

Protein assay.
The molecular mass of the purified enzyme was determined by gel filtration on a HiLoad 16/60 Superdex 200 pg column equilibrated with 0.1 M Tris-HCl buffer (pH 8.0) containing 2 mM dithiothreitol and 0.1 M NaCl . The molecular mass of the subunits was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) by the method of Laemmli (6) with a 10% polyacrylamide gel (8 by 9 cm; thickness, 1 mm) . Protein was stained with Coomassie brilliant blue R-250 . The amount of protein was determined by the method of Bradford (2) with a protein assay kit (Bio-Rad Laboratories, Tokyo, Japan) by using bovine serum albumin as the standard .

Enzyme assay.
NAD-dependent dehydrogenase activity was measured at 30°C by determining the increase in absorbance at 340 nm with a spectrophotometer (model U-3300; Hitachi Ltd., Tokyo, Japan) . The reaction mixture (total volume, 3 ml) contained 100 µmol of Tris-HCl (pH 8.0), 15 µmol of substrate, and 5.0 µmol of NAD⁺ . The activity was calculated by using an extinction coefficient of 6,220 M^-1 · cm^-1 for NADH . One unit of enzyme activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol of NADH per min . Specific activity was expressed in units per milligram of protein . To examine the optimum temperature and pH of the enzyme reaction, the activities were measured at various temperatures and with various buffers at 30°C, respectively . To examine enzyme stability, 0.5 U of purified 1,5-AG dehydrogenase (AGH) was incubated with 0.1 M Tris-HCl (pH 8.0) at various temperatures or with various buffers, and the residual activities were measured under the standard conditions . The enzyme activities were also measured in the standard reaction mixture containing various metal ions or chemical inhibitors at a concentration of 1 mM; the only exception was p-chloromercuribenzoic acid, which was used at a concentration of 0.1 mM . Kinetic constants were calculated by constructing double-reciprocal plots of the initial velocity of the enzyme versus various substrate concentrations in the standard reaction mixture by using 0.5 U of the enzyme .

 
Furthermore, enhanced reduction of NAD was observed when the cell extract was added to the reaction mixture with 1,5-AG, although conversion of 1,5-AG into glucose was not detected in the cell extract or in the membrane fraction of the fungus . This activity with 1,5-AG seemed to be an AGH activity and to be dependent on NAD . NADP was not effective under the same conditions .

Culture conditions for strain 11-3.
To facilitate large-scale production of AGH, glucose and glycerol, as well as 1,5-AG, were tested for enzyme productivity . After strain 11-3 was cultivated at 30°C for 2 days, the enzyme activity and the growth were measured . As shown in Table 1, the highest enzyme activity and the best growth were obtained when glucose was used . Strain 11-3 was cultivated at 28°C in 7 liters of medium which contained glucose and NaNO₃ as carbon and nitrogen sources, respectively (Fig . 2) . The growth and the enzyme activity increased until 40 h in the early stationary phase, when they reached the maximum values .

 
Identification of strain 11-3.
Strain 11-3 was a filamentous fungus, and the colonies of this fungus were green after culture . Many phialoconidia were observed at the tops of branched phialides by microscopy, and the conidia formed lumps but not chains . From these observations, we speculated that strain 11-3 belongs to the genus Trichoderma . This fungus was definitively identified as Trichoderma longibrachiatum by Centraalbureau voor Shimmelcultures (Delft, The Netherlands) .

Properties of AGH.
AGH was purified 18-fold from the cell extract (Table 2) . The purified AGH had a specific activity of 3.3 U/mg of protein . The purified preparation produced a single band on SDS-PAGE gels, indicating the apparent homogeneity of the protein (Fig . 3) .

 
The relative molecular mass of the enzyme subunit was estimated to be 36 kDa by SDS-PAGE (Fig . 3) . As gel filtration showed that the molecular mass of AGH was 141 kDa, the enzyme seemed to be a homotetramer . The optimum temperature and optimum pH of the enzyme were 40°C and 9.0, respectively (Fig . 4A and B) . The enzyme was stable at temperatures up to 60°C, and 60% of the enzyme activity remained at 70°C . The enzyme activity was lost completely after incubation for 10 min at 80°C (Fig . 4C) . The enzyme was stable at pH values ranging from 6 to 11 (Fig . 4D) . The enzyme activity was completely inhibited by Mn²⁺, Fe³⁺, Ni²⁺, Ca²⁺, Ba²⁺, and Li⁺ and was partially inhibited by Mg²⁺, Cu²⁺, Zn²⁺, and Hg²⁺ . Fe²⁺ activated the enzyme activity (about 2.7-fold) . The enzyme was strongly inhibited by p-chloromercuribenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid), NaN₃, and o-phenanthroline . These data suggested that the SH group and iron ion are involved in the enzyme reaction . AGH did not exhibit activity with glucose, sorbitol, xylitol, mannitol, glyceraldehyde 3-phosphate, and acetaldehyde (Table 3) . There was a trace of activity with fructose, but it could not be measured definitely even if the amount of the enzyme and/or the amount of the substrate was increased . The enzyme activity with glyceraldehyde was 52% of that with 1,5-AG . The apparent Michaelis constants for 1,5-AG and NAD were determined to be 0.67 and 0.50 mM, respectively .

 
Enzymatic measurement of 1,5-AG.
Enzymatic determination of the amount of 1,5-AG in samples was examined by using the purified AGH . The mean concentration of 1,5-AG in normal subjects is 2.5 ± 0.7 mg/dl, and the cutoff value for diabetic diagnosis has been proposed to be 1.4 mg/dl . AGH activities were measured with 1,5-AG at various concentrations around the cutoff value . As shown in Fig . 5A, a linear relationship between the concentration of 1,5-AG and the enzyme activity was observed . The relationship was unchanged when glucose or fructose was present (Fig . 5B and C) .

 
Enzymes which exhibit activity with 1,5-AG have been purified from various microorganisms and characterized (3, 7, 8, 15) . However, almost all of these enzymes also exhibit activity with glucose . For example, the level of activity of pyranose oxidase from P . obtusus (7), which is the only commercial enzyme, with glucose is the same as the level of activity of this enzyme with 1,5-AG . Because 1,5-AG is structurally similar to glucose, selection for enzyme activity specific for 1,5-AG seems to be difficult . Thus, we tried to obtain microorganisms which transform 1,5-AG into glucose without further consumption of glucose .

AGH from T . longibrachiatum 11-3 was unique in its substrate specificity . This enzyme did not exhibit activity with any sugar other than 1,5-AG or with any sugar alcohol . The enzyme gained prominence because of its substrate specificity compared with the specificities of the other enzymes described so far . This characteristic of AGH is adequate for diagnostic application of the enzyme . In this study, a preliminary examination of enzymatic measurement of 1,5-AG was carried out . A linear relationship between the concentration of 1,5-AG and the enzyme activity was obtained by using reaction mixtures with an excess amount of purified AGH . It was possible that AGH exhibited activity with glucose in the serum sample because large amounts of glucose (50 times the amount of 1,5-AG) are present in blood serum . Thus, 1,5-AG samples containing 110 mg of glucose per dl, which is a border value for the serum glucose concentration for diabetes diagnosis, were used for the AGH assay . As a result, we found that the relationship did not change with or without glucose (Fig . 5B) . The purified AGH also exhibited trace activity with fructose . Fructose is produced from ingested sucrose and then inverts to glucose, but the amount of fructose is not negligible (0.56 mg/dl in normal subjects) . As shown in Fig . 5C, the presence of fructose at a physiological level did not affect the 1,5-AG determination with AGH (Fig . 5C) . The activity with glyceraldehyde may not be an obstacle to practical use because glyceraldehyde is thought not to be present in serum . Furthermore, the enzyme activity remained after incubation at 70°C for 10 min and was stable after storage at 4°C for at least 6 months . These results suggested that AGH is a novel enzyme and is adequate for diagnosing diabetes mellitus .

1,5-AG is generally found in animals and plants . 1,5-AG in animals is produced from glucose and is ingested with food (20) . However, its physiological role is unknown . Shiga et al . have reported that 1,5-AG is synthesized and phosphorylated in Escherichia coli C600 . They have also shown that E . coli C600 synthesizes 1,5-AG when glucose is exhausted in the medium (12) and that E . coli takes 1,5-AG back from the medium and phosphorylates it to 1,5-AG 6-phosphate . These authors suggested that 1,5-AG and/or its phosphate may be a signal substance in cell-to-cell communication for bacterial growth (11) . T . longibrachiatum strain 11-3 mycelia converted 1,5-AG into glucose in the resting cell reaction . AGH activity was detected after the fungus was cultivated on medium containing glucose as a carbon source and then immediately decreased in the stationary phase of the growth . These results suggest that AGH may also be an engaged signal for starvation for carbon sources in strain 11-3, as it is in E . coli C600 .

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