Factors regulating production of glucose oxidase by Aspergillus niger

D. i;. Hatziuikolaou aud B. J. Macris

Department of Chemical Engineering, National Technical University of Athens, Athens, Greece

Certain factors affecting production of extracellular and cell-bound glucose oxidase by Aspergillus niger were
investigated. The intention was to maximize total glucose oxidase activity of academic and potential commercial application by the selection of the appropriate strain and consecutive optimization of growth media and conditions. It was possible to identify combinations resulting in the utilization of molasses as the best carbon source and enhancing enzyme activity approximately 40-fold. Glucose oxiaase activities as high as 5.7 U ml- were produced, comparing favorably with those reported for other enzyme-producing microorganisms. These activity levels were obtained with molasses, indicating an economically attractive process for enzyme production. In aadition, our work identified CaCO, as a particularly strong inducer of glucose oxidase activity. Keywords: Aspergillus niger; glucose oxidase; production;

optimization

Introduction
Glucose oxidase (P-D-glucose:oxygen 1-oxidoreductase, (EC 1.1.3.4) catalyzes the oxidation of P-D-glucose to gluconic acid, utilizing oxygen as an electron acceptor with the simultaneous production of hydrogen peroxide. Glucose oxidase has found several commercial applications including glucose removal from dried egg and foods to improve color, flavor, and shelf life; oxygen removal from fruit juices and canned beverages, and from mayonnaise, to prevent rancidity; and in an automatic glucose assay in conjunction with catalase . ,* The most common microbial sources of the enzyme are
Aspergillus niger, Penicillium notatum, P. glaucum, P. amagasakiense, and P. purpurogenum, although the majority of its commercial preparations is obtained from A. niger. 2*3 The carbon sources employed in the production of glucose oxidase from A. nify were mainly glucoseh7 and

Materials aud methods

Microorganism
A number of A. niger ATCC strains and a wild-type strain, isolated in our laboratory from corn, and labeled as BTL, were used. The stock cultures were maintained on PDA at 4C.

Preculture
Precultures were grown in 250~ml Erlenmeyer flasks with a working volume of 70 ml. The medium composition was (in grams per liter): (NH&HPO,, 0.4; KH,PO,, 0.2; MgSO, . 7H,O, 0.2; peptone, 10; sucrose, 70. The pH was adjusted to 5.5, with 1 N NaOH, prior to sterilization. Following inoculation, the preculture was incubated for 24 h in a rotary shaker operating at 225 t-pm and 30C.

Growth media and conditions

The basal salt medium (BSM) was similar to that reported for glucose oxidase production and contained (in grams per liter): (NH,),HPO,, 0.4; KH,PO,, 0.2; MgSO, . 7H,O, 0.2. The BSM was supplemented with the appropriate amounts of carbon and nitrogen source as well as calcium carbonate. When beet molasses was used they were expressed as sucrose and contained 57% (w/w) sucrose and 3% (w/w) Kjeldahl nitrogen. All growth experiments were carried out in 2-l Erlenmeyer flasks with a 650~ml working volume. Sterilization was effected at 121C for 30 min. The flasks were inoculated with 40 ml of preculture and incubated in a rotary shaker operating at 175 rpm. Experiments were carried out at initial pH 5.5 and 30C unless CaCO, was added in the growth medium (resulting in an initial pH value in the range of pH 6.6 to 6.9). The culture pH was also

The use of cheaper carbon to a lesser extent sucrose. sources appears to be essential for the improvement of process economy. In this article an effort was made to optimize growth conditions toward glucose oxidase production from A. niger, using low-cost carbon sources.

Address reprint requests to Dr. Ma& at the Department of Chemical Engineering, National Technical University of Athens, Athens, Greece Received 26 April 1994; revised 23 September 1994; accepted 3 October 1994

Enzyme and Microbial Technology 17:530-534, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0141-0229/95/$10.00 SSDI 0141-0229(94)0069-4

Regulation

of A. niger glucose oxidase: D. G. Hatzinikolaou

and B. J. Macris

checked after sterilization and was never found to differ by more than 0.15 point from its initial value.

Dry cell weight

The culture fluid was filtered through a Whatman (Clifton, NJ) No. 4 filter. If CaCO, supplemented the growth medium, the fluid was acidified to pH 2.5 with 4 N HCl, prior to filtration, in order to convert the insoluble CaCO, to soluble CaCl,. The separated mycelia were washed twice with deionized water and dried to constant weight at 75C.

extracellular) glucose oxidase activity. The results appear in Figure 1 and showed that the strain BTL, isolated in our laboratory from native corn crops, produced the highest glucose oxidase activity. The ATCC strains 364 and 567 also exhibited glucose oxidase activity but at significantly lower levels. Therefore, the strain A. niger BTL was employed for investigation in the subsequent experiments. Effect of carbon source A number of carbon sources was tested in order to determine their effect on growth and total glucose oxidase production by the selected strain. The rewlts are summarized in Table I and showed that the best activities were obtained with glucose, sucrose, and molasses. In fact, the microorganism was able to grow in all carbon sources tested, but glucose oxidase was produced at significant levels only in the three types of carbon source mehtioned above. This result indicates that glucose (either in pure form or as a product of sucrose hydrolysis by miaroorganismal invertase) is a principal inducer of the glucose oxidase gene. No correlation between biomass production and enzyme activity could be established, contradicting other relevant findings. Using the three best carbon sources listed in Table 1, the effect of cultivation time on glucose oxidase production was studied (Figure 2). Molasses yielded; the highest enzyme activity, closely followed by that obtained with sucrose. Because the former carbon source is a low-cost agroindustrial by-product, never reported previously for glucose oxidase production, it was decided to use hitas the main carbon source in the subsequent experiment& Sucrose was employed as a reference pure carbon souice in parallel experiments. Glucose has been exclusively used by other investigators to produce glucose oxidase from different microorganisms.4~1+2 Further optimization was carried out by studying the effect of concentration of molasses and sucrose on total glu-

Cell-bound

enzyme determination

Mycelia from the culture liquid were collected, washed on a sieve (160+m pore size), and suspended in 0.1 M citrate-phosphate buffer, pH 4. The mycelia were disrupted in a commercial blender, operating at maximum speed for 3 min, and the resulting suspension was subjected to sonication (Vibra-Cell sonicator; Sonits and Materials) in order to disrupt the cell membranes. Eight 1-min periods of sonication were used to avoid overheating. Cellbound glucose oxidase activity was determined in the fine particle suspension. Extracellular
rpm

enzyme determination

Aliquots of culture fluid were clarified by centrifugation at 5,200 for 15 min at room temperature and enzyme activities were measured in the clear supematant.

Enzyme assay Glucose oxidase activity was measured with the aid of a coupled o-dianisidine-peroxidase reaction. The reaction mixture contained in 0.1 M citrate-phosphate buffer, pH 5.0 (mg ml- ): CX-Dglucose, 37.5; o-dianisidine-HCl, 0.08; peroxidase, 0.008 (78 U mg- solid). The glucose solution was allowed to mutarotate overnight, prior to the addition of o-dianisidine and peroxidase. The linear rate of glucose consumption was determined from the rate of A,,, increase, measured in a Hitachi UV-VIS (ultravioletvisible) spectrophotometer, thermostatted at 30C. A calibration curve for the conversion of A,,, to H,O, concentration was used. One unit of glucose oxidase activity was defined as the amount of enzyme required to oxidize 1 pmol of glucose per minute under the above-described conditions.

Chemical analysis
Beet molasses were desulfutized by air stripping at 90C for 8 h and hydrolyzed prior to sucrose determination by the DNS method. lo All chemicals used were of analytical grade, and obtained through Sigma Chemical Co. (St. Louis, MO).

Reproducibility

of results

All fermentations were carried out in duplicate flasks and the experimental results represent the mean of two identical fermentations. Reproducibility was satisfactory. The standard deviation of the duplicate data never exceeded -C10% of the mean throughout this work.

~spergws ATCC56
Results and discussion Screening of strains Screening was carried out on a number of A. niger strains in order to isolate those with the highest total (cell-bound and
niger strain ATCC 699

CP-

Cultlvatlon the Wws)

Figure 1 Screening of different strains qf A. niger for total glucose oxidase production. The BSM was Supplemented with 5% glucose and 3% peptone

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Table 1 Effect of certain carbon sources on growth /us niger BTL and total glucose oxidase productiona
Biomass (mg ml-) 7.5 6.3 6.9 8.2 5.9 6.1 7.3

of Aspergil-

Carbon source Fructose Galactose Glucose Lactose Molasses Sucrose Starch

Total enzyme activity (U ml-) 0.15 0.00

f:;~:fl
15
b
0,s

1.74
0.00 1.83 2.17 0.05

O0 2 4 6 8 10 12 Carbon source concentretlon (%)

The BSM was supplemented with 8% carbon source, 1% peptone, and 4% CaCO,. The cultivation time was 5 days. With beet molasses as a carbon source the CaCO, concentration was 5%

Effect of carbon source concentration on total glucose oxidase production by A. niger BTL, grown for 4 days on BSM, supplemented with 1% peptone and 4 and 5% CaCO, when sucrose (A) and molasses (0) were used, respectively

Figure 3

case oxidase production (Figure 3). With sucrose as a carbon source, the enzyme activity was increased with increasing concentration and remained practically constant at levels above 10% (w/v). With molasses, glucose oxidase activities leveled off at a considerably lower concentration, namely 4% (w/v), and decreased slightly thereafter, indicating the possible existence of inhibitory factors (e.g., SO*, heavy metals). The pattern of enzyme production with both carbon sources was completely different from that reported for other fungi producing glucose oxidase. Effect of calcium carbonate Calcium carbonate is an insoluble salt frequently used in gluconic acid13,14and glucose oxidase production.5 Its role in gluconate production is not clear. It was suggested as a mechanical support of fungal growth.i4 The results of Figure 4 clearly demonstrated that the addition of CaCO, in the growth medium substantially induced glucose oxidase activity. More importantly, this salt was found to be essential for the production of elevated levels of the enzyme from both sucrose and molasses. Although highly insoluble in water, CaCO, proved to work better at higher concentrations, leaving no other explanation than the direct effect of solid salt phase on the mechanism of enzyme production. The observed optimum CaCO, concentration of 4 and 5% for sucrose and molasses, respectively, was used in the subsequent experiments.

Effect of nitrogen source Different nitrogen sources were tested for their effect on growth and total glucose oxidase production. The results are summarized in Table 2 and showed that peptone was the best nitrogen source for both types of carbon source used. With sucrose, poor growth and low enzyme activities were observed on inorganic nitrogen. Better growth and higher enzyme activities were obtained with molasses on all types of nitrogen source used. Peptone and NaN035*6T15 or steep liquor **were the nitrogen sources used previously for glucose oxidase production by different microorganisms. Peptone was chosen as the nitrogen source in the subsequent experiments. The concentration of peptone markedly affected total glucose oxidase production (Figure 5). Similar results were reported previously for other nitrogen sources. l1 With sucrose as a carbon source maximum enzyme activity was observed at a peptone concentration of l-2 (%) and with molasses a sharp peak of enzyme activity was observed at 0.2-0.3 (%). The latter low concentration could be explained on the basis that the microorganism was capable of utilizing nitrogen sources available in molasses and any further supplementation had an inhibitory effect on enzyme production. Obviously, the low concentration of peptone has a positive effect on process economy

2 E2

Il. : i? 5 0 0 2 4 6 0

Lil OlY
0 1 2 3 4 5 6 7

I
Cultivation tlme (days)

<~C

Celolum carbonate oonoentmtlon (%)

Figure 2
grown course source, source

Total glucose oxidase production by A. niger BTL on glucose (0). sucrose (A), and molasses (0) in the of time. The BSM was supplemented with 8% carbon 1% peptone, and 4% CaCO,. With molasses as a carbon the CaCO, concentration was 5%

Figure 4 Effect of calcium carbonate cose oxidase production by A. niger BSM, supplemented with 1% peptone trations of molasses (0) and sucrose

concentration on total gluBTL, grown for 4 days on and the optimum concen(A) found above

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and 8. J. Macris

Table 2 Effect of different nitrogen sources on growth of Aspergillus niger BTL and total glucose oxidase production Sucrose Enzyme activity (U ml-) 0.32 2.55 2.21 0.50 0.18 0.02 Molasses Enzyme activity (U ml-) 0.49 2.45 0.20 1.85 2.16 0.05 0 sucrose, moThe nitrogen liquor, which was 4 days

Nitrogen source Yeast extract Peptone Urea Corn steep liquor NaNO, NH,NO,

Biomass (mg ml-) 6.4 12.7 14.1 10.9 2.4 1.2

Biomass (mg ml-) 4.7 5.6 6.3

Cultlvatlon time (h) Figure 6 Effect of cultivation time on biomass (+ 1 and extracellular (0). cell-bound (A), and total glukose oxidase (0) production by A. niger BTL grown on molasses under the optimum conditions found above

PJH,),SO,

0.8

Z:Z 4.2 1.4

The BSM was supplemented with the optimum lasses, and CaCO, concentrations found above. source concentration was l%, except for steep was 3%. The pH was 6.6-6.9. The cultivation time

the enzyme proposed by other investigators*,16 and supporting the assumption of controlled secretion.77 In general, the level of extracellular glucose oxidase activity varied from about 10% of the total at the initial stages of the fermentation to about 25% at the final stages.
Comparison of enzyme activity

Effect of growth temperature The effect of growth temperature on total glucose oxidase

production was examined at the optimum medium composition described above. Five fermentation temperatures were tested, namely 22.5, 25, 21.5, 30, and 32.K. Optimum enzyme activities were obtained at 27.5C. Slightly higher growth temperatures were used by other investigators to maximise glucose oxidase production.5~7-9*11
Effect of growth time

Finally, the effect of cultivation time on glucose oxidase production under the optimum conditions found above is depicted in Figure 6 and showed an optimum growth time of 70 h for maximum total enzyme production. The cellbound and extracellular glucose oxidase activity exhibited different patterns in the course of time. It is important to note that these patterns were conserved in all fermentations carried out in this work. While the cell-bound enzyme activity followed, more or less, the pattern of biomass production the extracellular enzyme activity was continuously excreted, contradicting the exclusive cell-bound nature of

The results of consecutive optimization of growth medium and conditions with respect to total glucose oxidase production by A. niger BTL were compared with the maximum activities reported in the literature for other microorganisms overproducing glucose oxidase (Figure 7). It is evident from this comparison that our system produced considerably higher total glucose oxidase activity. In conclusion, within the experimental limits of this work, it was possible to optimize consecutively glucose oxidase production by A. niger on molasses as carbon source and increase the activity of the enzyme to levels competing favorably with those reported in the literature.4*5.7-9*11*12 These glucose oxidase activity levels were obtained at low nitrogen source (peptone) requirements,

09

Peptone concentration (%) Figure 5 Effect of peptone concentration on total glucose production by A. niger BTL, grown for 4 days on BSM, supplemented with the optimum molasses (O), sucrose (A), and calcium carbonate concentration found above

Figure 7 Comparison of maximum glucose oxidase activity produced by A. niger BTL and other overproducing microorganisms reported in the literature

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suggesting an economically attractive growth medium for the production of glucose oxidase. In addition, our work revealed the inductive role of calcium carbonate in glucose oxidase production by A. n&r BTL. This effect is currently under investigation in our laboratory at the biochemical level.

Acknowledgments
This work was supported in part by Grant No. 89012, AGRF-0032-C from the European Economic Community.

8 9 10 11 12 13 14 15 16 17

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