Glucose Oxidase Pellets

Transcription

1 BIOTECHNOLOGY AND BIOENGINEERING VOL. XIX (1977) Glucose Oxidase Pellets INTRODUCTION Considerable world-wide interest has arisen in the use of immobilized enzymes as catalysts in industrial process and analytical chemistry.1-3 Recently, aminoacylase was immobilized on the mycelium pellets of Aspergillus ochraceus by using albumin and glutaraldehyde.4 Mycelium pellets could be used for the carrier of the aminoacylase immobilization. A glucose oxidase producing microorganism, Aspergillus sp., also became pellets through a fermentation process. Glucose oxidase has a potential application in the production of gluconic acid. Glucose oxidase was immobilized on the mycelium pellets of f!spergillus sp. by using albumin and glutaraldehyde. In this paper, properties of glucose oxidase pellets (immobilized glucose oxidase) were described. MATERIALS AND METHODS Materials Glutaraldehyde (26y0 solution), ovalbumin, and o-dianisidine were obtained from Tokyo Kasei Kogyo Co. Glucose oxidase was obtained from culture broth (2 units/mg protein). Peroxidase (horseradish) was obtained from Miles Laboratory Ltd. Glucose was purchased from Wako Pure Chemicals Co. Other reagents were commercially available analytical reagents or laboratory grade materials. Culture of Strain Aspergillus sp. No. 319 was used for the experiments. Fermentation was carried out at 30 C for 48 hr in a 500 ml Sakaguchi flask containing 75 ml of the Czapek-Dox medium which contained 0.5% yeast extract. After cultivation, Aspergillus sp. pellets were harvest,ed by filtration and washed with deionized water. Immobilization of Glucose Oxidase Immobilization of glucose oxidase on the mycelium pellets was carried out as described previ~usly.~ After reaction, pellets were separated by filtration and washed with deionized water. Enzyme assay Fifty to 100 rg of glucose oxidase or mg glucose oxidase pellets (1-3 mg, dry basis), 1 ml of 0.35% o-dianisidine ethanol solution, 5 ml of 0.004y0 peroxidase solution, 10 ml of water, and 30 ml of 3% glucose solution (in 0.1M acetate buffer ph 5.0 or 0.1M phosphate buffer) were added into 100 ml flask and incubated for 15 min at 40 C. The reaction was stopped with 10 ml of 2N hydrochloride solution and allowed to stand for 15 min at room temperature. The 1977 by John Wiley & Sons, Inc. 1233

2 1234 BIOTECHNOLOGY AND BIOENGINEERING VOL. XIX (1977) at 402 nm was measured with a spectrophotometer (Shimadzu model UV-200). Ten pg of glucose oxidized by glucose oxidase in 1 min were defined as the activity of one unit. Batch reaction One g (wet) of glucose oxidase pellets was added to 50 ml of various concentrations of glucose solution in 0.1M acetate buffer (ph 5.0) and incubated at 40 C. The glucose solution was saturated with dissolved oxygen during the experiments. Column reaction The apparatus used for the experiments consists of a water-jacketted reactor (0.8 cm diam, 14 cm height), a peristaltic pump (Mitumi Science Co., Model SJ-1210), and a fluid reservoir. Eleven g of wet glucose oxidase pellets (11 cm3) were placed in the reactor. Various concentrations of glucose solution in 0.1M acetate buffer (ph 5.0) were employed for the experiments. RESULTS The glucose oxidase activity of mycelium pellets of Aspergillus sp. was 21 units/g wet cell. Glucose oxidase was immobilized on mycelium pellets by using glutaraldehyde and ovalhumin. The activity yield of the glucose oxidase pellets was 95%. The glucose oxidase pellets were incubated in O.1M acetate buffer (ph 5.0) for four days. However, no leakage of aminoacylase was observed from the pellets. The activity of the glucose oxidase pellets was 200 units/g wet cell. Figure 1 shows the ph-activity profiles of glucose oxidase pellets and native glucose oxidase. The optimum ph of native glucose oxidase was ph 6.0 and shifted to ph 5.0 by immobilization. The activity of glucose oxidase pellets was h ' Fig. 1. ph-activity profiles of glucose oxidase pellets and native glucose oxidase. 1 g glucose oxidase pellets (wet) and 50 pg of native glucose oxidase were used and the enzyme assay was carried out under standard conditions at various ph's. 0.1M acetate buffer (ph 3-5), 0.1M phosphate buffer (ph 6-8), and 0.1M Verona1 buffer (ph ) were employed. (0-0) Glucose oxidase pellets; (0-0) native glucose oxidase. PH

3 COMMUNICATIONS TO THE EDITOR 1235 lower than that of native glucose oxidase between ph 5 and 8. However, the activity of glucose oxidase pellet was higher than that of native glucose oxidase in acidic and alkaline conditions. No difference in the temperature-activity profile was observed between glucose oxidase pellets and native glucose oxidase. Figure 2 shows the ph stability of glucose oxidase pellets and native glucose oxidase. Glucose oxidase pellets and native glucose oxidase were incubated in various buffer solutions for 15 min at 50 C. No difference of the stability was observed in the ph range from 5 to 7. However, glucose oxidase pellets were more stable than native glucose oxidase in alkaline conditions and less stable than native glucose oxidase in acidic conditions. The thermostability of the glucose oxidase pellets and native glucose oxidase is shown in Figure 3. The glucose oxidase pellets and native glucose oxidase PH Fig. 2. ph-stability of glucose oxidase pellets and native glucose oxidase. 1 g of glucose oxidase pellets (wet) and 50 pg of native glucose oxidase were used and the enzyme assay was carried out under standard conditions. ( O- 0) Glucose oxidase pellets; (0-0) native glucose oxidase. x c.-.- > c U rd - c;n I- \ \ 2-.- '0 m aj U Tempera t u re ( 'c) Fig. 3. Thermostability of glucose oxidase pellets and native glucose oxidase. 1 g of pellets (wet) in 10 ml of 0.1M acetate buffer (ph 5.0) were incubated at.io"c. Glucose oxidase assay was carried out under standard conditions. (0-- 0) Glucose oxidase pellets; (0-0) native glucose oxidase.

4 1236 BIOTECHNOLOGY AND BIOENGINEERING VOL. XIX (1977) were incubated for 15 min at various temperatures. The activity of native glucose oxidase was decreased with increasing temperature. The complete inactivation of native glucose oxidase was observed at 65 C. On the other hand, the glucose oxidase pellets retained 85% of the initial activity at 6.5OC. The effect of flow rates on the conversion of glucose to gluconic acid is shown in Figure 4. Various concentrations of glucose solution were flowed upward through the column. Conversion of glucose to gluconic acid was dependent on flow rate and increased with increasing flow rate. The complete oxidat.ion of glucose to gluconic acid was examined by the recycle reactor system (Fig. 5). Fifty ml of 0.188% glucose solution (ph 5.0) were flowed upward through the column at a space velocity (SV) of 48 hr-l. The complete conversion was attained after 4 hr. The glucose oxidase pellet column maintained the original activity for 15 days. Figure 6 shows the complete oxidation of glucose by the glucose oxidase pellets with use of the batch system. One g of the wet glucose oxidase pellets was added to 50 ml of various concentrations of glucose solution (ph 5.0). The complete oxidation of glucose occurred when the glucose solution was saturated with 1 I 1 I I I Flow rate SV (h- ) Fig. 4. Effect of flow rates on conversion. Experimental details are given in the text. Enzyme assay was carried out under standard conditions. Time (hr) Fig. 5. Complete oxidation of glucose by the recycle reactor system. Experimental details are given in the text. Glucose oxidase assay was carried out under standard condition.

5 COMMUNICATIONS TO THE EDITOR 1237 Ti me (hr) Fig. 6. Complete oxidation of glucose by glucose oxidase pellets wit,h batch system. Experimental details are given in the text. Enzyme assay was carried out under standard conditions. (O- 0) With aeration, (0-0) without aeration. dissolved oxygen by bubbling air through the system. On the other hand, the conversion of glucose was limited without bubbling air. The storage stability of glucose oxidase pellets was examined. The glucose oxidase pellets showed no detectable decrease in their activity after three months storage at 4 C. DISCUSSION Glucose oxidase of Aspergillus sp. is believed to be a cell bound enzyme similar to the aminoacylase of Aspergillus ochraceus and the 0-amylase of Asp. ory~ae.~ In the case of aminoacylase on the Asp. ochraceus, the most enzyme was easily liberated from the mycelium during incubation.' However, 85% of the initial glucose oxidase activity remained on the mycelium after a four day incubation at 20 C. Therefore, glucose oxidase was more tightly bound to proteins and amino sugars which exist on the surface of the cell wall than aminoacylase. Glucose oxidase was immobilized on the mycelium pellets of Aspergillus sp. by using glutaraldehyde and ovalbumin. No leakage of glucose oxidase was observed from the mycelium pellets after treatment. Most of the glucose oxidase may be covalently bound to the mycelium. The activity yield of the immobilization was 95%. The activity yield of aminoacylase immobilized on the mycelium pellets of Asp. ochraceus was 33%.' Therefore, glucose oxidase is stable against glutaraldehyde treatment. The optimum ph of the glucose oxidase pellets was shifted to a lower ph. It is well known that glutaraldehyde reacts with the free amino groups of proteins. A part of the free amino groups of glucose oxidase is modified during glutaraldehyde treatment. The modification may result in a shift of the optimum ph of the glucose oxidase pellets. Glucose oxidase pellets were unstable in acidic conditions. However, they showed a high stability in alkaline conditions. Native glucose oxidase was completely inactivated at ph 10. In the case of glucose oxidase pellets, 50y0 of the original activity still remained in the same condition. Therefore, the optimum condition for glucose oxidase pellets was shifted to higher ph. This result is quite different from that obtained from the aminoacylase pellets.' The thermostability of the glucose oxidase pellets was markedly increased with modification. This may be

6 1238 BIOTECHNOLOGY AND BIOENGINEERING VOL. XIX (1977) caused by the modification of glucose oxidase with glutaraldehyde. It is suggested that some minor conformational changes are induced by the chemical modification. In the case of the aminoacylase pellets, the thermostability was decreased after glutaraldehyde treatment. Conversion of glucose to gluconic acid was increased with increasing flow rate. In general, a rate of the reaction of immobilized enzymes was decreased with increasing flow rate. It was assumed that low conversion at a low flow rate is caused by production inhibition. However, no difference of conversion was observed when the substrate containing 0.094% gluconic acid or 0.094y0 hydrogen peroxide was flowed through the column. One mol of oxygen is required for the oxidation of glucose by glucose oxidase. Therefore, this effect may be a result of the lack of oxygen in the column at low flow rates. The complete oxidation of glucose was attained with a recycle reactor system and a batch system when the oxygen was supplied by bubbling air. On the other hand, the conversion is low when the air supply was limited. It was previously noted by Messing6 that glucose substrates devoid of hydrogen peroxide could not be utilized to attain reproducible results. The glucose oxidase pellets showed the catalase activity. Therefore, the employment of the substrate containing hydrogen peroxide would increase the activity of the glucose oxidase pellets. The least stable glucose oxidase pellets exhibited no detectable decrease in their activity after three months of storage at 4 C. This compares very favorably with the best system of Herring et al. which exhibited a.ioo/b activity loss in 25 days of storage at 5 C. References 1. I. Chibata, T. Tosa, T. Sato, T. Mori, and Y. Matsuo, Proc. ZV ZFS: Ferment. Technol. Today, 4, 383 (1972). 2. S. Suzuki, I. Karube, and I. Satoh, in Biochemical Application of Immobilized Enzymes and Proteins, Plenum, New York, 1977, p I. Satoh, I. Karube, and S. Suzuki, Biotechnol. Bioeng., 18, 269 (1976). 4. K. Hirano, I. Karube, and S. Suzuki, Biotechnol. Bioeng., 19, 311 (1977). 5. K. Tonomura, F. Futai, and 0. Tanabe, Biochim. Biophys. Acta, 78, 802 (1963). 6. R. A. Messing, Biotechnol. Bioeng., 16, 897 (1974). 7. W. M. Herring, R. L. Laurance, and J. R. Kittrell, Biotechnol. Bioeng., 14, 975 (1972). Research Laboratory of Resources Utilization Tokyo Institute of Technology Ookayama, Meguro-ku, Tokyo, Japan Accepted for Publication February 28, 1977 ISAO KARUBE KEN-ICHI HIRANO* SHUICHI SUZUKI * Alternate address: Amano Pharmaceutical Co., Nishiharumachi, Nishikasugaigun, Aichiken, Japan.