Aspergillus niger. Development, Jerusalem, Israel. was adjusted manually. All fermentations were. carried out in duplicate.

Transcription

1 APPLIED MICROBIOLOGY, Nov., 1965 Copyright ( 1965 American Society for Microbiology Vol. 13, No. 6 Printed in U.S.A. Production of the Enzyme Naringinase by Aspergillus niger B. BRAM AND G. L. SOLOMONS Fermentation Unit, Hebrew University-Hadassah Medical School, and National Council for Research and Development, Jerusalem, Israel Received for publication 23 February 1965 ABSTRACT BRAM, B. (Hebrew University-Hadassah Medical School, Jerusalem, Israel), AND G. L. SOLOMONS. Production of the enzyme naringinase by Aspergillus niger. Appl. Microbiol. 13: The formation of naringinase, a glycolytic enzyme produced by Aspergillus niger, is repressed by glucose. Production of the enzyme is decreased below ph 4.0 and is stimulated by the presence of substrate. Fermentation conditions are described which cause the formation of the enzyme in approximately a fivefold greater concentration than that previously described. Naringinase is an enzyme which hydrolyzes the glycoside naringin, the bitter principle of grapefruit (Thomas, Smythe, and Labbee, 1958; Ting, 1958). Smythe and Thomas (1960), who described the production of this enzyme, obtained titers of approximately 100 units per ml. The purpose of the present study was to exae some factors which would increase enzyme production. MATERIALS AND METHODS Naringinase activity was measured by the method of Davis (1947), modified to develop the color produced by incubation in a water bath at 28 C. This was necessary because of the wide differences in ambient room temperature. Readings were taken in a Klett photometer with the use of the blue filter (420 m). A standard curve was prepared with pure naringin and with naringenin, which is the final hydrolysis product. An intermediate product, prunin, behaves very much like naringenin (Horowitz and Gentili, 1959). The method of preparing naringenin from naringin was that of Pulley and Loesecke (1939). Suitable blanks and controls were always included. Unit of naringinase. The unit adopted was that amount of enzyme which hydrolyzed 1 Amole of naringin in 1 at 45 C in ph 4.0 citrate buffer, when the substrate concentration was initially 1 mm and not more than 25% hydrolysis occurred. Shake-flask experiments were carried out at 30 C on a New Brunswick rotary shaker [describing a 1-inch (2.5-cm) circle] at 240. Flasks (1 liter) containing 200 ml of medium, 1 ml of antifoam, 20% Silicone RD Emulsion (Midland Silicone, London, England) were used. Spore suspensions were prepared as inoculum by use of 0.05% Lubrol W (I.C.I., London, England), a nontoxic wetting agent. Fermentations were carried out in 10-liter fermentors, full-baffled, with disc-turbine impellers, temperature control, and automatic antifoam control. Where indicated, ph was adjusted manually. All fermentations were carried out in duplicate. RESULTS In early shake-flask experiments with a number of strains grown on usual culture medium, e.g., malt extract, corn meal, etc., excellent growth was obtained, but no enzyme yield. With the salts of Czapek-Dox medium and the addition of 0.1% naringin, growth occurred with glucose, sucrose, lactate, or citrate as the carbon source, but no enzyme was produced. When peptone was made the carbon source, of 14 strains tested 7 gave no enzyme, 5 gave moderate titers, and 2 produced high titers. Using these two best enzyme-producing strains, we exaed a range of complex media for enzyme production. As shown in Table 1, the inclusion of starch as whole corn meal completely repressed enzyme formation by both strains of Aspergillus niger tested. As facilities were limited, all further experiments were carried out with A. niger NRRL To test for possible enzymatic adaptation during growth on substrate, the mold was subcultured four successive times on a modified Dox agar with 1% naringin replacing glucose as carbon source. When tested in shake flasks, enzyme titers were no higher than those given by cultures grown on malt-agar. Since malt-agar gave a heavier sporulation, this was adopted as the standard culture medium for producing spores for inoculum. 842 Downloaded from on July 13, 2018 by guest

2 VOL. 13, 1965 NARINGINASE PRODUCTION BY A. NIGER 843 TABLE 1. Effect of medium containing starch on enzyme production by-aspergillus niger* Medium constituent (NO) Medium constituent ~~~~~~O/~ Enzymeatiter 72 (units/mi) CSL YE SBM NaNOa WCM KH2PO4 Naringin Ca COs A. niger A. niger NRRL 330 NRRL _ * The ph was adjusted to 5.0 to 5.5; CaCO3 was added after sterilization. CSL = corn steep liquor; YE = yeast extract; SBM = soya bean meal; WCM = whole corn meal. TABLE 2. Effect of soya bean meal (SBM) and yeast extract (YE) on enzyme formation by Aspergillus niger NRRL 72-4 Medium constituent (%) ph Enzyme titer SBM YE O Narin- CaCO: gin As soya bean meal-yeast extract medium and corn steep liquor-yeast extract medium had produced high enzyme titers, two experiments were done in which the relative amounts of the constituents were varied (Tables 2 and 3). The highest enzyme titers were produced by the corn steep liquor-yeast extract media, with 4% corn steep liquor4% yeast extract medium giving the best results; this medium was adopted as a standard basal medium for further work. The optimal concentration of calcium carbonate in the medium was then detered (Table 4). The ph decreased with increasing concentrations of carbonate up to 1%, but no appreciable differences in enzyme titers were apparent. The final shake-flask experiment was designed to investigate the effect of naringin addition on enzyme production. As shown in Table 5, the stepwise addition of a smaller concentration of naringin was more effective than a higher concentration added at the beginning of the fermentation. TABLE 3. Effect of varying the concentration of corn steep liquor (CSL) and yeast extract (YE) on enzyme production Medium constituent (%) ph Enzyme titer (units/mi) CSL YE KH2PO4 Narin- CaCOs gin TABLE 4. Effect of calcium carbonate concentration on enzyme production after 120 of growth* CaCOs ph Enzyme titer 5%o units/ml Control * CaCOa was added as indicated to a basal medium containing 4% corn steep liquor, 4% yeast extract, 0.2% KH2PO4 and 0.5% naringin. During the course of development, enzyme titers in shake-flask experiments had risen from 30 to 400 units per ml. At this point, work was directed to the production of enzyme in 10-liter laboratory fermentors. These were fully baffled systems (four baffles, each 0.1 diameter of the vessel) with a centrally mounted disc turbine (0.5 diameter of the vessel). The depth of the unsparged liquid equalled the diameter of the vessel. Air was supplied at the rate of 1 volume per volume of medium per, and the fermentor was maintained at 15 psi of pressure. Incubation temperature was 30 i 1 C. The oxygen transfer rates (OTR) of the

3 844 BRAM AND SOLOMONS APPL. MICROBIOL. TABLE 5. Effect of naringin concentration on enzyme production after 120 of growth* Naringin ph Enzyme titer % units/ml Control t * Naringin was added as indicated to a basal medium containing 4% corn steep liquor, 4% yeast extract, 0.2% KH2PO4, and 0.5% CaCO3. t Added as four lots of 0.1% each at 24- intervals. TABLE 6. Effect of stirrer speed on enzyme production in 10-liter fermentors* ph Enzyme titer (units/ml) Time * Pasal medium contained 4% corn steep liquor, 4% yeast extract, 0.2% KH2PO4, 0.1% naringin, and 1% CaCO3. fermentors were measured by the sulfite oxidation technique. (Cooper, Fernstrom, and Miller, 1944) for stirrer speeds of 500, 700, and 900. For the respective speeds, the OTR values were 170, 256, and 350 mmoles of 02 per liter per. A standard spore suspension was used so that the fermentor contents were inoculated with 105 spores per milliliter of medium. All fermentations were carried out in duplicate. Table 6 shows the effect of stirring rate on enzyme production in 10-liter fermentors. Since the slowest stirrer speed gave the highest enzyme titer, 500 was adopted for further experiments. As the incremental addition of naringin had proved so effective in shake-flask experiments (Table 5), a similar experiment was tried in the fermentors (Table 7). Continuous addition of naringin produced the highest enzyme yield, but as this was technically more difficult the method of adding the naringin in six divided doses was adopted in further experiments. The ph values given in Table 7 show that even the presence of 1 % calcium carbonate was insufficient to hold the ph of the fermentation above 4.0. TABLE 7. Effect of naringin addition on enzyme nroduction in 10-liter fermentors im ph Enzyme titer (units/ml) I* II III I II III t t Ot * Basal medium contained 4% corn steep liquor, 4% yeast extract, 0.2% KH2PO4, and 1% CaCO3. The stirrer speed was 500, and the aeration rate was 1 volume per volume of medium per ute. Medium I was a control containing no naringin. Medium II contained 0.1% naringin added as a continuous feed toughout the period of fermentation. Medium III contained 0.1% naringin added as six divided doses at 24- intervals. t At these times, 50% sodium hydroxide was added to adjust the ph to values shown. TABLE 8. Time Effect of 12 hourly adjustment of ph on enzyme production Unadjusted pih Adjusted Enzyme titer units/ml * Basal medium contained 4% corn steep liquor, 4% yeast extract, 0.2% KH2PO4, and 0.1% naringin added in divided doses at 24- intervals. The stirrer speed was 500, and the aeration rate was 1 volume per volume of medium per ute. At times shown, the ph was adjusted by addition of 50% sodium hydroxide.

4 VOL. 13, 1965 This compares with a ph value of 4.6 produced by the same culture medium in shake-flask experiments. The lowering of the ph to values below 4.0 caused losses in enzyme titer, so fermentations were carried out without carbonate in the medium, but the ph was adjusted every 12 with 50% sodium hydroxide. The resulting ph values and enzyme production are given in Table 8. In all the experiments in 10-liter fermentors, 3% octadecanol in a metabolizable oil was used as antifoam. When 10% Hodag KG1 in liquid paraffin was substituted as the antifoam, the ph of the fermentations remained constant at 6.5 after the first 45. The enzyme titers, however, only reached 400 units per ml at 113, compared with 540 units per ml in the same time when octadecanol was used. DISCUSSION The production of naringinase by A. niger NRRL 724 when grown on a synthetic medium is repressed in the presence of glucose, lactate, or citrate (Clarke and Brammer, 1964). Sucrose and starch similarly suppress enzyme formation, although they support excellent growth. Soya bean meal-yeast extract and corn steep liquoryeast extract media both produced high enzyme titers, probably because both of these media have a low carbohydrate content. Of the synthetic media exaed, only the one with peptone as carbon source produced measurable enzyme titers, although all supported good growth. Added growth factors are not required for growth, and the high naringinase titers obtained on corn steep liquor-yeast extract medium are attributed to low carbohydrate content. This is supported by the fact that malt extract medium, which is an excellent source of growth factors but has a high carbohydrate content, produces no naringinase. The corn steep liquor medium was preferred, since it not only gave higher enzyme production, but a nonparticular culture medium was easier to handle and reduced sterilization difficulties, especially on a large scale. Although naringin was not essential for enzyme formation, its presence increased the yields in shake-flask experiments by teefold; in the fermentors, the increase was only 50%. It is possible that corn steep liquor contains an induction factor; in a recent review of some Japanese work, Mateles et al. (1965) reported that rhamnose or plant meal containing rhamnose glucosides increases naringinase production. The stimulation by naringin was much greater in shake flasks where less oxygen was available, suggesting that the metabolic rate may be important. This seems to be supported by the fact NARINGINASE PRODUCTION BY A. A;IGER that, in 10-liter fermentors, the lowest stirrer speed used gave the highest enzyme titer. Citric acid production by this organism is also inhibited by vigorous aeration. Enzyme production was little affected by ph in the range of 4.5 to 7.5, but yields fell markedly at ph values below 4. Since the strain of Aspergillus used normally produces citric acid, the natural tendency of the culture was to become very acid. In shake-flask experiments where available oxygen was limited, this tendency could be controlled by the use of 1% calcium carbonate. By contrast, in 10-liter fermentors, because of the great increase in available oxygen, the ph even in the presence of 1 % calcium carbonate fell below 4, and control had to be effected by use of sodium hydroxide. The effect of the antifoam added was of interest, the organism being able to metabolize the carrier oil used with octadecanol. A similar utilization of the carrier oil by Pencillium was reported by Rolinson and Lumb (1953). This lipid nutrient did not interfere with enzyme production but rather produced higher titers, since the nonmetabolizable liquid paraffin antifoam only produced 70% of the enzyme titer when grown under comparable conditions. LITERATURE CITED CLARKE, P. M., AND W. J. BRAMMER Regulation of bacterial enzyme synthesis by induction and repression. Nature 203: COOPER, C. M., G. A. FERNSTROM, AND S. A. MILLER Performance of agitated gasliquid contactors. Ind. Eng. Chem. 36: DAVIS, W. B Deteration of flavanones in citrus fruits. Anal. Chem. 19: HOROWITZ, R. M., AND B. GENTILI Use of the Davis method to estimate flavanones. Food Res. 24: MATELES, R. I., D. PERLMAN, A. E. HUMPHREY, AND F. H. DEINDORFER Fermentation review. Biotech. Bioeng. 7: PULLEY, G. N., AND H. M. V. LOESECKE Preparation of rhamnose from naringin. J. Am. Chem. Soc. 61: RoLINSON, L. N., AND M. LUMB The effect of aeration on the utilisation of respiratory substrates by Penicillin cysogenum in submerged culture. J. Gen. Microbiol. 8: SMYTHE, C. W., AND D. W. THOMAS Conversion of flavanoid glycosides. U. S. Patent. 2,950,974. THOMAS, D. W., C. V. SMYTHE, AND M. D. LABBEE Enzymatic hydrolysis of naringin: the bitter principle of grapefruit. Food Res. 23: TING, F. V Fruit glycoside hydrolysisenzymatic hydrolysis of naringin in grapefruit. J. Agr. Food Chem. 6:

5 APPLED MICROBIOLOGY, May, 1966 Vol. 14, No American Society for Microbiology Printed in U.S.A. ERRATUM Production of the Enzyme Naringinase by Aspergillus niger B. BRAM AND G. L. SOLOMONS Fermentation Unit, Hebrew University-Hadassah Medical School, and National Council for Research and Development, Jerusalem, Israel Vol. 13, no. 6, p. 842, col. 1, lines 18 and 19 of MATERIALS AND METHODS: Change "which hydrolyzed 1 j,mole of naringin" to "which hydrolyzed 1 m,umole of naringin." 477