1 •k J. Appl. Glycosci., Vol.46, No.1, p.1-7 (1999)•l Oxidation of Uronic Acids by a Large Excess of Glucose Oxidase Preparations Mikihiko Kobayashi,* Hirofumi Nishihara1 and Shoichi Kobayashi National Food Research Institute (2-1-2, Kannondai, Tsukuba 305-8642, Japan)1 Department of Applied Bioresource Science, School of Agriculture, Ibaraki University (3998 Ami-machi, Ibaraki 300-0393, Japan) Three commercial enzyme preparations of glucose oxidase (GOD) showed an oxidizing ability of glucuronic acid and galacturonic acid to form sugar acids. The optimum conditions of GOD reaction were at pH 8.0 and 50•Ž, whereass those of uronic acid oxidase (UOD) reaction were at pH 3.5 and 40•Ž. In spite of extensive attempts to separate GOD from UOD, the isolation of each activity was unsuccessful, and UOD reaction resulted from the wide substrate specificity of GOD reaction. More than 6-fold higher Km values for glucuronic acid and galacturonic acid than for glucose might suggest that UOD reaction was the alternative reaction of GOD enzyme. However, a more definite conclusion on UOD activity should be drawn from further studies. The reaction products from uronic acids were analyzed by HPLC and paper chromatography, and some products were shown to be identical with sugar acids. Beet pulp contained a large amount of uronic oxidation of uronic acids with glucose oxidase acids, which were found not only in the pectic (GOD) preparation from commercial sources. fraction, but also in the hemicellulose and cellulose, fractions.1) Although the effective MATERIALS AND METHODS saccharification of beet pulp might increase biomass content about 1.4-fold for the ethanol Materials. Three preparations of glucose production by yeast fermentation, uronic acids oxidase were obtained from Toyobo (GODT:165 could not be used as carbon sources. The units/mg, isolated from Aspergillus species), chemical conversion of uronic acids by water Wako Pure Chemical Industries Ltd. (150-250 soluble carbodiimide increased the yield of units/mg, isolated from Aspergillus niger), and glucose and galactosee in beet pulp . fraction on Oriental Yeast Co. (348 units/mg, isolated from enzymic hydrolysis with pectinases and hemi microorganism). The glucose was measured cellulases.2) However, no papers were found by the glucose oxidase kit (Glucose B-Test concerning the enzymic reduction of uronic Wako). Horseradish peroxidase (100 units/ acids to neutral sugars. We have been screened mg) was purchased from Wako Pure Chemical the enzyme that converted uronic acids to neu Industries Ltd. Most sugars, including sugar tral sugar. In contrast to our object, no enzyme acids, were commercial preparations purchased concerning the production of neutral sugar from from Wako. acidic sugar was obtained, but reaction catalyz Assay of uronic acid, oxidizing activity. The ing a further oxidation of uronic acids was reaction mixture contained enzyme solution observed. In the present paper, we describe the (20 ,u L) , substrate solution (10 p L) , and buffer solution including additional components (20 * To whom correspondence should be addressed . Present address: Akita Research Institute for Food p L) . After incubation at 30•Ž for 20 min, the and Brewing (4-26, Sanuki, Araya-machi, Akita reaction was stopped by the addition of 1 M 010-1623, Japan) NaOH (25 uL). The mixture was neutralized 2 J. Appl. Glycosci., Vol. 46, No. l (1999) with 1 M HCl (25 ,u L) and subjected to a UOD assay, which was essentially based on the GOD RESULTS assay kit of B-Test Wako. For the UOD assay, a cocktail from the GOD assay kit was treated Screening of UOD activity. at 100•Ž for 5 min for the inactivation of GOD; About 40 commercial enzyme preparations, that is, phenol and 4-aminophenazone in the including some oxidases, were tested for the GOD cocktail were used. The peroxidase oxidase activity of glucuronic acid and galact of horseradish (0.2 mg/mL 67 mM phosphate uronic acid conversion. UOD activity was buffer, pH 7.6) was supplemented for the UOD assayed for the production of H2o2 with the assay. Inactivated GOI) cocktail (100 uL) and color development reaction coupled with per peroxidase solution (25 ,u L) were added to the oxidase, phenol, and 4-aminophenazone. As above reaction mixture and incubated at 30•Ž shown in Table 1, three GOD preparations gave for 20 min. After dilution with water (375 ,u L) , UOD activity. No remarkable UOD activity absorbance was measured at 505 nm. One unit was detected from the remaining 37 enzyme each of GOD and UOD activity was defined as preparations. Since GOD of Toyobo (GODT) the amount of enzyme, which converted 11umol had the highest activity for galacturonic acid glucose/min at pH 7.5 (50 mM TEMED-HC1 and was available in a large quantity at a low buffer) and 1 nmol glucuronic acid/min at pH price, further experiments were done with this 3.5 (50 mM Tris-acetate buffer), respectively, enzyme. at 30•Ž incubations. Calibration curves with standard sugars were obtained by the above Characteristic of UOD reaction. methods, including alkaline inactivation and The general properties of the GODT prepara subsequent neutralization steps that required 2 tion in the reaction with galacturonic acid or 3 min. (UOD activity) were compared with those in HPLC analysis. HPLC gel filtration analysis the reaction with glucose (GOD activity). As was done with Tosoh G3000SW column (7.8 x shown in Fig. la, pH optimum for GOD activity 300 mm), which was equilibrated with 17 mM was at pH 8.0, and higher activity was obtained phosphate buffer (pH 7.6) -0.2 M NaCI-0.02% at neutral to alkaline pH ranges. In contrast, NaN3. GOTT was eluted from the column at the maximum activity of UOD reaction was 0.6 mL/min, and fractions,were collected every obtained at pH 3.5; higher activity occurred at 30 s to measure the enzyme activity and protein the acidic pH regions. A comparison of opti by a UV detector (Jasco UV-870). The product mum temperatures also revealed a marked sugars of UOD reaction were separated by the difference between the GOD and UOD activities. column of Aminex carbohydrate HPX-87C (7.8 The former activity was maximum , at 40 to X 300 mm). The reaction mixture was eluted 60•Ž, and the UOD reaction was maximum at 30 from the column with water (0.6 mL/min), and to 50 •Ž ; about 60% of the UOD activities were sugars were detected by an RI detector (Jasco detected at the low temperature of 10•Ž, at RI-830). Paper chromatography (PPC). A product Table 1. The comparison of uronate oxidizing analysis of the UOD reaction was done with activity among the GOD preparations. filter paper (20 x 20 cm, Advantec Toyo, No. 50). The development was done at room tem perature overnight with 65% 1-propanol, and sugars were detected by the silver nitrate dip procedure. a The reaction mixture (300 ,u L) contained enzyme (10 mg/mL,100 ,u L) ,10 % substrate sugar (100 ,u L) , and 0.2 M TEMED-HCl buffer (pH 7.5, 100 uL). After incubation at 30•Ž for 20 h, the enzyme activ ity was measured as described in the text. Oxidation of Uronic Acids by Glucose Oxidase 3 Table 2. The carbohydrate oxidizing activity of commercial GODT preparation. a The reaction mixture (80 p L) contained enzyme Fig. 1. Optimum conditions of GOD and UOD reac (10 mg/mL, 10 ,u L) , 1% substrate sugar (20 p L) , and 50 mM HCl-acetate buffer (pH 3.5, 50 ,u L). tions. After incubation at 30•Ž for 20 h, the enzyme (a) Optimum pH. (b) Optimum temperature. The activity was measured as described in the text. reaction mixture (1001uL) for GOD activity (0) con tained GODT (10 a g/mL, 25 ,u L) , 0.1 M glucose (25 as shown in Figs. 2a and b. The GODT prepara uL), and 0.2 M TEMED-HC1 buffer (pH 3.75-11.0, 50 tion gave biphasic lines for glucuronic acid and ,u L). After incubation at 30•Ž for 10 min, the enzyme galacturonic acid; two extrapolated Km values, activity was measured as described in the text. The 99 and 329 mM for glucuronic acid; and 86 and optimum temperature was measured at pH 8.0. The reaction mixture (100 pL) for UOD activity (•œ 235 mM for galacturonic acid. In contrast, contained GODT (10 mg/ml, 25 ,u L) , 10% galacturonic the Km value for glucose was 13.2 mM at pH 7.5, acid (25 uL), and 0.2 M Tris-acetate buffer (pH 3.1-8.5, whereas the Km value was 25.5 mM at pH 3.5. 50 uL). After incubation at 30•Ž for 20 min, the enzyme A comparison of the ko values showed a large activity was measured as above. The optimum tem difference in the UOD and GOD reactions. The perature was measured at pH 3.5. ko values for galacturonic acid (1.2-2.7 min-1) were about 1000-fold smaller . than those for which GOD activity was less than 10% (Fig. glucose (1.2 and 2.9 x 103 min-1 at pH 7.5 and pH 1b). 3.5, respectively). The carbohydrate oxidizing activity of com mercial GODT preparation was examined with Separation of UOD from GOD activity. neutral and acidic sugars (Table 2). Although To determine whether UOD activity was iden glucose and xylose gave significantly high activ tical with GOD activity, a separation of UOD ity, galactose and mannose also served as good and GOD activities was examined by various substrates.