Invertase Production by Penicillium chrysogenum and Other Fungi in Submerged Fermentation

Transcription

1 APPLIED MICROBIOLOGY, Sept., American Society for Microbiology Vol. 13, No. 5 Printed in U.S.A. Invertase Production by Penicillium chrysogenum and Other Fungi in Submerged Fermentation F. M. POONAWALLA, K. L. PATEL, AND M. R. S. IYENGAR Microbiological Research Laboratory, Alembic Chemical Works, Baroda, India Received for publication 3 May 1965 ABSTRACT POONAWALLA, F. M. (Microbiological Research Laboratory, Alembic Chemical Works, Baroda, India), K. L. PATEL, AND M. R. S. IYENGAR. Invertase production by Penicillium chrysogenum and other fungi in submerged fermentation. Appl. Microbiol. 13: A study was made of Penicillium chrysogenum and some other fungi to determine the relative distribution of intra- and extracellular invertase produced by them in submerged fermentation. The proportion of each type of enzyme varied withthe organism and the period of fermentation. More of the enzyme initially bound to the mycelium was released into the medium with the progress of fermentation. Differences were observed in the effects of cultural conditions on enzyme production in P. chrysogenum and Saccharomyces cerevisiae. Considerably greater quantities of enzyme were produced by P. chrysogenum and the yeast in both laboratory and large-scale fermentors when sucrose was added continuously than when the same quantities of the sugar were added initially. In disagreement with previously accepted opinion, Wickerham (1958) demonstrated by a biological assay procedure that a few yeasts produce appreciable amounts of extracellular invertase. Dworschack and Wickerham (1958) determined some factors influencing the production of extracellular and bound invertase by Saccharomyces uvarum. The objective of the present work was to study Penicillium chrysogenum and a few other fungi to determine their capacity to produce invertase and the nature of the enzyme activity when cells were grown in submerged culture. Invertase production by P. chrysogenum in penicillin fermentation broth was studied by Damle, Singh, and Ghosh (1958). They reported that invertase in this mold appeared to be an exoenzyme. We have studied in detail the effects of various conditions contributing to the enhancement of extracellular invertase production by P. chrysogenum in submerged fermentation. The relative capacities of P. chrysogenum and S. cerevisiae to produce invertase were also studied. MATERIALS AND METHODS Organisms. The following organisms from our culture collection were used: P. chrysogenum 71, S. cerevisiae 8, Aspergillus oryzae 23, Colletotrichum falcatum 10, Neurospora crassa 27, N. sitophilia 28, and Alternaria tenuis 16. Media. Invertase production by fungi was studied in the following medium (grams per liter): sucrose, 40; corn steep liquor, 30; sodium nitrate, 3; potassium dihydrogen phosphate, 0.5; magnesium sulfate (heptahydrate), 0.05; calcium carbonate, 2.5; the ph was 5.5. For growth and sporulation of molds, and for growth of yeast, the following medium was used (grams per liter): malt extract (Difco), 40; yeast extract (Difco), 4; potassium dihydrogen phosphate, 1; agar (Difco), 20; the ph was 5.5. The organisms were grown at room temperature (28 C), and subcultures were maintained under refrigeration. Shaken flask fermentations were carried out on rotary shakers at 250 rev/min [1-inch (2.5 cm) stroke] with 100 ml of medium in 500-ml Erlenmeyer flasks. Laboratory fermentor runs were made in 25-gal (113.6-liter) stainless-steel laboratory fermentation tanks of conventional design. Reducing sugars. Reducing sugars were determined by Somogyi's (1945) method. Invertase. Invertase activity was determined by Sumner and Howell's (1935) method. The invertase activity was expressed as the milligrams of reducing sugar formed by the enzyme at ph 4.5 and 25 C in 1 hr in 1 ml of supernatant fluid or whole culture (Dworschack and Wickerham, 1958). Dry weight. To measure dry weight, 100 ml of unfiltered broth were centrifuged and the CaCO3 residue was decomposed by adding hydrochloric acid, washed thrice with distilled water, and dried at 8) C to constant weight. Extracellular invertase activity was determined on the supernatant fluids after centrifugation at 3,000 rev/min for 20 min and passage of the filtrates through bacteria-retaining sintered-glass funnels to remove small fragments of mycelia or cells. Intracellular activities were computed by 749

2 750 POONAWALLA, PATEL, AND IYENGAR APPL. MICROIBIOL. TABLE 1. Invertase production by fungi in shaken flask fermentations Invertase (units) Fungi 48 hr 72 hr 96 hr 120 hi T* I E T I E T I E T I E Saccharomyces cerevisiae Aspergillus oryzae Colletotrichum falcatum Penicillium chrysogenum Neurospora crassa N. sitophila Alternaria tenuis * T = total; I = intracellular; E = extracellular. difference from the total activities of whole broth samples. Corrections were made for reducing sugar present in the filtrate and whole broth samples. All analyses were carried out in duplicate. Data are uncorrected for temperature variation (±1 C). RESULTS Production of invertase by fungi in shaken flask cultures. Invertase production by P. chrysogenum and a few other fungi was studied in shaken flask cultures by inoculating 104 spores (or cells, in the case of S. cerevisiae) per milliliter of sucrosecorn steep liquor medium and assaying enzyme activity at various time intervals. Maximal titers of enzyme were reached between 72 and 96 hr with the yeast and fungi tested (Table 1). Among the filamentous fungi, the two species of Neurospora were the most active. A. tenuis was the least active. In A. oryzae and P. chrysogenum, the shift in the ratio of intracellular to extracellular enzyme, which occurred with time, indicated that the enzyme was initially bound to the mycelium and was released into the medium with increased time of fermentation. Though a similar trend was noticeable in N. crassa, the major part of the enzyme remained intracellular in that fungus. In N. sitophila and A. tenuis, also, the enzyme was mainly intracellular. It was interesting to note that in C. falcatum, which is a pathogen on sugar cane, the activity was almost wholly extracellular throughout the fermentation cycle. In S. cerevisiae, which was the largest producer of the enzyme under these conditions, there was good extracellular activity from the earliest stage examined. Invertase production by P. chrysogenum in shaken flask cultures. Extracellular invertase production by P. chrysogenum and S. cerevisiae was compared in sucrose-corn steep liquor and sucrose-peptone-yeast extract media (Wickerham, 1958) at 25 C (Fig. 1). The inoculum used was 104 spores per milliliter in the case of the mold, and 104 vegetative cells per milliliter in the case of yeast. Invertase titers reached the maximum at 120 hr with both the organisms in both the media. The titers in sucrose-peptone-yeast extract medium were lower than those in sucrose-corn steep liquor medium. In both the media, S. cerevisiae gave higher titers than P. chrysogenum. Also, the rate of production by the yeast was rapid until it reached the maximum at 120 hr, whereas with the mold the rate of production was gradual and, SCL: SUCROSE -CORNSTEEP LItSUOR MEDIUM TIME OF FERMENTATION,HR. FIG. 1. Invertase production by Penicillium chrysogenum and Saccharomyces cerevisiae in shaken flask fermentation.

3 VOL. 13, 1965 INVERTASE PRODUCTION BY FUNGI 751 TABLE 2. Effect of various factors on invertase production by Penicillium chrysogenum in shaken flask fermentations Condition* Extracellular invertase production (units) 24 hr 48 hr 72 hr 96 hr 120 hr Age of inoculum 24 hr hr hr Size of inoculum 1 % % % Aeration rate 50 mi/flask mi/flask mi/flask Sucrose 4 % % ph Temperature 25 C C Ct * Standard conditions used: age of inoculum, 48 hr; size of inoculum, 5% (v/v); aeration rate, corresponding to 100 ml of medium per flask; sucrose 4% (w/v), ph 5.5; and temperature, 25 C. Each condition was varied as indicated. t Comparative values for Saccharomyces cerevisiae. after reaching the maximum at 120 hr, there was a rapid decline in the activity. Under the conditions used, increase of dry cell weight of the yeast was relatively slower, reaching the maximum at 120 hr, together with invertase titers. In the case of the mold, the dry myceium weight increased rapidly until 48 hr, followed by a rapid decline. Most of the sugar was used by the organisms in about 72 hr in both media. Some factors affecting production of extracellular invertase by P. chrysogenum in shaken flask fermentation. Effects of age of vegetative inoculum, aeration rate, concentration of sucrose, size of inoculum, and temperature on the production of invertase were studied (Table 2). Age of inoculum had no appreciable effect except that very young inoculum gave less invertase. Quantity of inoculum had a definite effect on invertase titers. Increase in quantity of inoculum increased invertase titers. A 25%G inoculum gave the highest titer in 72 hr. Aeration had little effect except that, when it was very low, it depressed the enzyme titers. A 100-ml volume per flask gave optimal yields in 120 hr. Within the limits used, ph did not have any influence on invertase titers. Invertase titers were higher at the higher of the two temperatures used and reached their maximum in a much shorter time. The growth was faster at the higher temperature, but autolysis was more rapid too. The effect of sugar concentration was similar to that of temperature. Higher concentrations of sugar gave higher titers earlier, but after 48 hr the titers fell because of earlier autolysis. In sucrose-corn steep liquor medium at 25 C, the yeast produced slightly higher invertase titers than the mold (Fig. 1). On the other hand, at 28 C the mold produced slightly higher titers than the yeast (Table 2). Production of invertase in fermentors. Davey and Johnson (1953) and Soltero and Johnson (1953, 1954) clearly demonstrated that in penicillin-production media lactose could be completely replaced by sucrose without affecting antibiotic yields when sucrose was fed continuously in low concentrations. Using similar conditions, we first studied invertase production in 100-liter labo-

4 E~~~~~~~ 752 POONAWALLA, PATEL, AND IYENGAR APPL. MICROBIOL. TABLE 3. Effect of sugar addition on invertase production by Saccharomyces cerevisiae and Penicillium chrysogenum in 100-liter fermentations* Invertase (units/g of sugar consumed) Type of fermentation P. chrysogenum S. cerevisiae 18 hr 42 hr 18 hr 42 hr Sugar added initially only Continuous addition of sugar... 1,007 6,823 4,697 6,904 * Agitation with single impeller, 320 rev/min; aeration, 0.4 (v/v). D^ty WEIGHT gal capacity, where the continuous addition of 200. I sugar could be controlled better (Fig. 3). These 1.5 DL ERtTA9rE - results were consistently observed over a large, number of runs both in laboratory and production-scale fermentations. Since the production < 120- SUGAR o0.3 I E fermentors contained 3 % peanut cake in the corn 40- i steep liquor medium, the dry-weight measurements were made according to Maxon's (1959) o 0 Ao io O method. The correlation of dry weight and invertase activity showed that the enzyme was TIMJE Or FERMENTAzrION,NR. FIG. 2. Production of invertase by Penicillium extracellular under the conditions of large-scale chrysogenum on continuous addition of sugar in fermentation (Fig. 3). laboratory fermentor. DISCUSSION ratory fermentations at 25 C. A fermentor containing sucrose-corn steep liquor medium was fungi has been studied with the use of the Although invertase production in filamentous inoculated with 10%/ (v/v) of a 48-hr-old vegetative inoculum grown in the same medium in a cellular enzyme (Saksena and Bose, 1944; mycelium, on the assumption that it is an intra- 20-liter seed tank. The total concentration of Aritovskaya, 1948; Sainclinier, 1950; De Accadia, sugar, which was fed intermittently at 0.5-hr Russi, and Bellio, 1955), there is evidence that intervals during the fermentation cycle, amounted several filamentous fungi produce extracellular to 4.9%. Invertase production increased up to invertase (Gillespie, Jermyn, and Woods, 1952; 90 hr of fermentation, after which the rate Crewther and Lennox, 1953; Reese, Birzgalis, declined (Fig. 2). Total enzyme production was and Mandels, 1962; Damle et al., 1958). It could about five times more than the maximal titers obtained in shaken flask fermentations in the same medium in which sugar was added initially in the concentration range of 4 and 8% (Table 2). Thus, it was concluded that continuous addition of sucrose markedly increased the production of invertase. Also, since growth increased throughout the fermentation cycle, and conditions favoring autolysis were minimal, invertase production in the fermentor was considered mostly extracellular. On the basis of sugar consumed, P. chrysogenum produced about 25 times more invertase per gram of sugar when it was fed throughout the fermentation cycle than when all the sugar was added initially (Table 3). Under the same conditions, S. cerevisiae produced nearly 10 times more of this enzyme. Even higher titers-about three times more than in the laboratory fermentors-were produced by P. chrysogenum in production fermentors of 20, Seo z ui Z : TIME OF FERMtE.NTATION,HR. FIG. 3. Invertase production by Penicillium chrysogenum on continuous addition of sugar in production fermentor. - r,

5 VOL. 13, 1965 INVERTASE PRODUCTION BY FUNGI be argued in some of these cases that the enzyme found in the external milieu resulted from autolysis. Our results indicate that in submerged fermentations, with the fungi studied, the enzyme was both intra- and extracellular, and that the proportion of such activity varied with the organism and the period of fermentation. The activity which was bound to the mycelium initially was released into the medium when the fermentation progressed. Such release was more in some cases and less in others, and was not always closely correlated with autolysis. A comparison of invertase production in shaken flask fermentation by P. chrysogenum and S. cerevisiae showed that, whereas the enzyme produced by the yeast cells was largely extracellular (because both the cell weight and extracellular invertase titers reached the maxima at the same time), in the mold the maximal titers of extracelluilar invertase were reached after autolysis had set in. These results from shake flask runs made it difficult to characterize the enzyme as truly extracellular. However, in the case of fermentor runs, where better correlation existed between growth and enzyme elaboration, evidence was less ambiguous. The major part of the enzyme produced during the growth phase was truly extracellular, and it could be clearly differentiated from the secondary increase of extracellular enzyme which occurred with the onset of autolysis. Since we have studied invertase production by P. chrysogenum under cultural conditions similar to those reported for the production of this enzyme by S. uvarum (Dworschack and Wickerham, 1958), it is interesting to compare the various conditions affecting the optimal yields of this enzyme in the two organisms. Age of the inoculum did not influence the yield of enzyme in both the organisms. Although variations in the size of inoculum did not have any effect on enzyme yield of the yeast, increased yields were obtained from the mold with increase of inoculum. In fermentor runs with P. chrysogenum and S. cerevisiae, when all the sugar was added at the start of fermentation, the growth of the organisms was rapid, but autolysis also set in early, resulting in less total growth and less enzyme production. In contrast, with continuous addition of sugar growth and extracellular enzyme elaboration were maintained longer and attained higher levels. Correlation between the rate of growth of molds with sucrose and the amount of invertase produced was previously demonstrated by Hawker and Chaudhuri (1946). Reese et al. (1962) reported on the production of sucrase by several fungi in 3-week shaken flask 753 runs. They found that yields of the enzyme were greatly enhanced when the fungi were grown on sucrose esters, particularly sucrose palmitate. The enhancement was explained as being due to the gradual liberation of the sugar from the ester by the action of a slowly acting esterase. In earlier work by Willstaetter, Lowry, and Schneider, cited by Neuberg and Roberts (1946), it was found that intermittent additions of small quantities of sucrose resulted in greater production of invertase by yeast. The very large increases in enzyme activity noted in our work show this to be true of a filamentous fungus in large-scale fennentations. Our results further extend the observation made by Nakai (1961) that filamentous fungi possess a marked potential for invertase production on a large scale. ACKNOWLED GMENT We are indebted to A. H. Amin for his keen interest and encouragement during this work. LITERATURE CITED ARITOVSKAYA, T. V Enzyme activity of northern strains of microorganisms. Microbiologia 17: CREWTHER, W. G., AND F. G. LENNOX Enzymes of Aspergillus oryzae. III. The sequence of appearance and some properties of the enzymes liberated during growth. Australian J. Biol. Sci. 6: DAMLE, S. P., K. SINGH, AND D. GHOSH Studies on soluble enzymes in penicillin fermentation broth. II. Invertase activity. Antibiotics Symposium, Council of Scientific and Industrial Research, New Delhi, p DAVEY, V. F., AND M. J. JOHNSON Penicillin production in cornsteep media with continuous carbohydrate addition. Appl. Microbiol 1: DE ACCADIA, F. D., S. RUSSI, AND A. BELLIO Action of Penicillium chrysogenum on sucrose. Rend. Ist. Super. Sanita 18: DWORSCHACK, R. G., AND L. J. WICKERHAM Production of extracellular invertase by the yeast, Saccharomyces uvarum NRRLY Y-972. Arch. Biochem. Biophys. 76: GILLESPIE, M. J., M. A. JERMYN, AND E. F. WOODS Multiple nature of the enzymes of Aspergillus oryzae and of horse-radish. Enzymes of Aspergillus oryzae. Nature 169: HAWKER, L. E., AND S. D. CHAUDHURI Growth and fruiting of certain ascomycetous fungi as influenced by the nature and concentration of carbohydrate in the medium. Ann. Botany (London) 10: MAXON, W. D Aeration-agitation studies on the novobiocin fermentation. J. Biochem. Microbiol. Technol. Eng. 1: NAKAI, T Concentration of sucrase obtained from filamentous fungi. Japanese- Patent 12,993.

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