Abstract
The aim of this work was to study the influence of phosphate concentrations on glucoamylase production by the fungus Aspergillus awamori. In culture media containing cassava flour, different levels of phosphates were tested and the fungal responses to increasing levels (whether or not coupled to pH adjustment throughout the run) were assessed in terms of the resulting glucoamylase production. Phosphate increments, associated with pH readjustments throughout the run, yielded around 1,200 U/L of quite stable glucoamylase activity in the broth, while under a conventional condition (low phosphate without pH readjustment), enzymatic activity was around 350 U/L, which decayed dramatically towards the end of the cultivation
Glucoamylase; Aspergillus awamori; phosphate influence
INFLUENCE OF PHOSPHATE CONCENTRATIONS ON GLUCOAMYLASE PRODUCTION BY Aspergillus awamori IN SUBMERGED CULTURE
J.M. Zaldivar-Aguero1, A.C. Badino Jr.2, P.R. Vilaça3, M.C.R. Facciotti4 and
W. Schmidell4
1Fellowship from FAPESP - 2Universidade Federal de São Carlos - 3Fellowship from CAPES
4Escola Politécnica da USP, Departamento de Engenharia Quimica, Caixa Postal 61548,
CEP: 05424-970 São Paulo-SP, Brazil. Phone: (55 11) 818-5382; Fax: (55 11) 211-3020;
E-mail: jaguero@usp.br
(Received: June 11, 1997; Accepted: October 30, 1997)
Abstract - The aim of this work was to study the influence of phosphate concentrations on glucoamylase production by the fungus Aspergillus awamori. In culture media containing cassava flour, different levels of phosphates were tested and the fungal responses to increasing levels (whether or not coupled to pH adjustment throughout the run) were assessed in terms of the resulting glucoamylase production.
Phosphate increments, associated with pH readjustments throughout the run, yielded around 1,200 U/L of quite stable glucoamylase activity in the broth, while under a conventional condition (low phosphate without pH readjustment), enzymatic activity was around 350 U/L, which decayed dramatically towards the end of the cultivation.
Keywords: Glucoamylase, Aspergillus awamori, phosphate influence.
INTRODUCTION
The culture medium is a key element in a successful fermentation process. This has also been proven to be true in the production of glucoamylase by Aspergillus. It is well known that the fungus needs dextrins, or specifically, maltose in order to synthesize large amounts of glucoamylase (Fowler et al., 1990). However, the role of phosphorous should not be underestimated, since it greatly contributes to cell survival. This element participates in synthesis (membrane and cell wall components, ATP, NADPH and nucleic acids), as well as in cellular physiology (pH homeostasis, cellular division, signal transduction and enzyme secretion). It has been demonstrated in fungi research that a high percentage of ATP is channeled into enzyme secretion (Caddick et al., 1986; Hondmann and Visser, 1994).
Usually culture media utilize dibasic and monobasic phosphates as phosphorous sources (Berry, 1975). However, there is no agreement on the amount to be employed for the optimal glucoamylase production by Aspergillus (Dworschack and Nelson, 1972; Hayashida, 1975; Metwally et al., 1991). In this work, a standard medium was modified in such a way that phosphate concentrations were increased while the levels of other nutrients were maintained according to the original formulation. Glucoamylase production was taken as the main parameter to assess fungal performance.
MATERIALS AND METHODS
Culture Methodology
Aspergillus awamori NRRL 3112 was cultivated in an NBS shaker (New Brunswick Sc. Co., USA) in 1 L flasks containing 230 mL of Aspergillus complete medium, ACM, composed (in g/L) of: total reducing sugars, TRS, from cassava flour and assayed as glucose, 20.0; (NH4)2SO4, 5.0; Na2HPO4.12H2O, 3.8; KH2PO4, 3.5; MgSO4.7H2O, 0.5 and yeast extract, 0.1. Initial pH was set at 5.0 in all the flasks. Fermentations were performed at 35 oC and 230 rpm during 63 hours. Experiments were designated R1, R2 and R3, respectively (Table 1). In R1, at the 36th hour of fermentation, one flask received a pulse containing all the components of the ACM (Aspergillus complete medium) and several others received a pulse containing each component of the ACM individually (only results of the phosphate-containing supplement will be presented). In the two remaining experiments (R2 and R3), phosphate concentrations were varied (Table 1), while other nutrient concentrations were maintained invariable in different experiments. In the last set of experiments (R3), the pH was periodically readjusted to the initial value throughout cultivation by adding NaOH 2N or HCl 2N.
Analytical Procedure
Fermentation kinetics was assessed by: a) cellular growth, measured by dry cell mass (DCM); b) substrate (TRS) consumption, by an enzymatic method in which the polysaccharyde in the sample had been previously converted into glucose (Schmidell and Fernandes, 1977) and the concentration of the latter was subsequently assayed by the glucose oxidase/peroxidase system, GOD/POD (Merck, Darmstadt); c) glucoamylase activity (Schmidell and Menezes, 1986). One unit of enzymatic activity was defined as the amount of enzyme that releases 1g of glucose, during a one-hour reaction, under standard conditions [60 oC, starch (Merck), 4% w/v, dissolved in an acetate buffer at pH = 4.2 as the substrate].
RESULTS AND DISCUSSION
Figure 1 illustrates a typical profile for glucoamylase production in a shaken flask (control). Between 24 and 29 hours, the enzyme reaches a peak of activity in the broth and during the following 6 hours its activity falls dramatically. In spite of the medium being buffered, a severe acidification (pH < 2.0) occurs. Since glucoamylase pH stability ranges from 3.0 to 7.0 (Saha and Zeikus, 1989), a low pH would cause enzyme instability similar to what was described for cellulase (Ghokale et al., 1992) and glucose-oxidase produced by A. niger (Kubicek and Röhr, 1986).
The initial approach in carrying out this work was to verify a nutrient limitation likely to occur towards the end of the cultivation. Thus, in cultures aging for 36 hours, in which glucoamylase levels were 80% lower than the peak value, one pulse of nutrients was added to several flasks, as indicated in Table 1. Subsequent analysis revealed the presence of residual sugar (approximately 4 g/L) in each one of the flasks at the 36th hour. The addition of either ACM (containing phosphorous, carbon and nitrogen sources) or phosphates ("0.5P" and "2P") increased glucoamylase activity and this suggested that phosphate would be the limiting factor (Figure 1A). Furthermore, the concentrated phosphate pulse ("2P") promoted a significantly higher glucoamylase activity (550 U/L). It could be speculated that additional phosphates exerted their influence on both fungal metabolism and glucoamylase stability. Regarding the former, the supplement apparently contributed to the synthesis of needed compounds, such as ATP and nucleotides (Hondmann and Visser, 1994), which allowed for glucoamylase resynthesis and/or secretion, thus enhancing the carbohydrase extracellular levels. On the other hand, enzyme stability could be explained by two factors. One of them was the less acidic pH. Indeed, it was verified that a more concentrated phosphate pulse ("2P," Figure 1A) shifted the pH from 1.6 to 2.8, with less degradation occurring thereafter. The second was the ionic strength (and/or molarity), whose change by phosphate supplementation could modify the stability of the glucoamylase (Archer et al., 1990).
Based upon these results, the next step was directed towards increasing the initial phosphate levels, while maintaining the levels of other nutrients. Run R2 clearly resulted in better production, as illustrated in Figure 1B. In the control flask, enzyme activity reached 350 U/L; by doubling or quadruplicating the phosphate levels utilized in the control, enzyme production was 450 U/L and 600 U/L, respectively, and thus higher than in the control. In those cases, the glucoamylase was more stable than the control, but it degraded towards the end of the run, a fact attributed to the acidic broth (the final pH in the control was 1.6, while in flasks "2P" and" 4P" it was approximately 2.4).
In the last set of experiments (run R3), in order to minimize the adverse effects of the pH, flasks containing increased levels of phosphates were periodically pH-readjusted (Figure 1C). This strategy allowed for a superior enzyme production: 560 U/L in flask "1P 5," 1,050 U/L in "2P 5," 1,200 U/L in "3P 5" and approximately 850 U/L in" 4P 5." Therefore, there is an ideal range of phosphate concentrations (7.6-11.4 g/L Na2HPO4.12H2O and 7.0-10.5 g/L KH2PO4) within which glucoamylase production is higher, and below or above this range, fungal performance may be less satisfactory. Taking into account that runs R2 and R3 were sugar depleted almost at the same time, it is clear that higher phosphate levels, associated with pH readjustment, promoted higher enzyme production, a highly desirable feature in the enzyme industry. For instance, focusing on the 36th hour, it can be observed (Figures 1B and 1C) that under pH readjustment 800 U/L was obtained, while about 600 U/L was obtained without readjustment. Therefore, maintaining the broth pH at around 5.0 during cultivation would be advantageous in two ways: it renders higher levels of glucoamylase, which is in agreement with the literature (Dworschack 1972; Zaldivar-Aguero et al., 1990) and the enzyme displays a higher stability.
Table 2 summarizes the results obtained in runs R2 and R3. Additionally, specific activities, SA (glucoamylase activity normalized with biomass) and productivities, PrGLA (enzyme production divided by the time required to attain it) were calculated and appear in that table. It can be observed that higher levels of phosphate, without pH-readjustment, promoted specific activities of approximately 70 U/g, while under periodical pH readjustments, biomass was more productive, rendering specific activities about twice as high (120.2 U/g and 143.3 U/g in flasks "2P 5" and "3P5," respectively). Similar behavior was verified in productivity values. Thus, while in flasks "2P" and" 4P" productivities were 15.2 and 15.9 U/L.h, respectively, in flasks "2P 5" and "3P 5" they were 22.0 and 25.1 U/L.h , respectively.
Finally, it should be mentioned that we are presently studying the influence of phosphates on glucoamylase production by Aspergillus in bench reactors.
*GLAmax: maximal glucoamylase activity; tGLAmax: time required to attain GLAmax; XtGLAmax: biomass at tGLAmáx; SA: specific glucoamylase activity; PrGLA: glucoamylase productivity.
th hour of fermentation (A); increased phosphate levels without (B) and with (C) pH readjustment throughout the run.
CONCLUSIONS
-
Increased concentrations of phosphates (11.4 g/L of Na
2HPO
4.12H
2O and 10.5 g/L of KH
2PO
4) allow for higher glucoamylase production in
Aspergillus.
-
In shaken flasks, high levels of phosphates associated with pH readjustment further improve glucoamylase production and the enzyme displays enhanced stability.
-
The presence of glucoamylase in a very acidic environment (pH < 2.0) for an extended period contributes to enzyme degradation.
NOMENCLATURE
ACM Aspergillus complete medium
ATP Adenosine triphosphate
GLAmax Glucoamylase maximal activity
NADPH Nicotinamide adenine dinucleotide phosphate
PrGLA Glucoamylase productivity
SA Specific glucoamylase activity
TRS Total reducing sugars (assayed as glucose)
tGLAmax Time for GLAmax
XtGLAmax Biomass at tGLAmáx
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Publication Dates
-
Publication in this collection
06 Oct 1998 -
Date of issue
Dec 1997
History
-
Accepted
30 Oct 1997 -
Received
11 June 1997
