- Email: [email protected]
Enhancement of β-galactosidase production and secretion by high osmotic stress in Penicillium notatum
Microbiol. Res. (1998) 153,65-69
©
Gustav Fischer Verlag
Enhancement of ~-galactosidase production and secretion by high osmotic stress in Penicillium notatum 1. Fiedurek Department of Industrial Microbiology, Maria Curie-Sklodowska University, 20-033 Lublin, Poland Accepted: December 14, 1997
Abstract A strain of Penicillium notatum was cultured under various osmotic stresses. When the fungus was cultured under osmotic stress, total ~-galactosidase activity increased significantly (about 1.6-fold) with increasing concentration of NaCI. Mycelial growth was repressed with increased medium osmolarity. To release periplasmic ~-galactosidase, 72-h old mycelium was suspended on a concentrated solution of NaCI (0.2-1.6 M). The highest yield of ~-galactosidase activity was obtained at 0.4 M NaCI at pH 6.0, which improved the activity of this enzyme by about 1.9-fold in comparison with the control medium without this depressor. Key words: ~-galactosidase - osmotic stress - Penicillium notatum
Introduction ~- D-galactosidase [GAL (EC 3.2.1.23)] preparations are widely used for hydrolysis of lactose in milk, milk products and whey (Richmond et al. 1981; Gecas et aZ. 1985). Degradation of lactose into simple sugars increases availability of certain milk products and makes their consumption possible even by person with lactose intolerance. There are many reports proving that metabolism of microorganisms is strongly influenced by osmotic stress. This is also true for the production of secondary metabolites, such as microbial toxins (Troller 1980), enzymes (Mildenhal et al. 1981; Grajek and Gervais 1987; Pandey et af. 1994) and other compounds (Suzuki 1995). The amount of available water in the environment of microorganisms affects growth, respiration, enzyme synthesis, sporulation and other physiological
Corresponding author: 1. Fiedurek
functions (Troller 1980). The responses of cells to media with decreasing water activity (aw ) have been described (Hahn-Hagerdal 1986; Witter and Anderson 1987; Csonka 1989). It is important to know the optimal value of water activity for individual physiological phenomena such as microbial growth, sporulation and production of primary and secondary metabolites. By controlling water activity one can regulate fermentation and form conditions favoring the biosynthesis of desirable products. Maximum growth and metabolite overproduction are often observed at aw values between 0.9 and 1.0. Lactic acid bacteria show up to 1O-fold increase in a diacetyl production when aw is lowered to 0.95 (Troller and Stinson 1981). In ethanol fermentation by Saccharomyces cerevisiae, substrate uptake and glycerol production are greater at an aw of 0.971 than at 0.994. When aw is reduced below 0.990, a decreased growth rate and biomass concentration usually follow (Kenyon et af. 1986; OIz et af. 1993). Data on the influence of osmotic stress on the synthesis and excretion of enzymes into the medium are not uniform yet. To my knowledge no paper has mentioned the effect of water activity on production and secretion of ~-galactosidase. The aim of the present work was to examine the impact of osmotic stress on production and excretion of ~-galactosidase by Penicillium notatum.
Materials and methods Strain and media. In our studies a P. notatum mutant (strain 1) producing a high extracellular GAL activity in shaken cultures was used (Fiedurek et aZ. 1996). The fungus was grown on basal medium containing (gil): lactose - 10, peptone (type I, Sigma) - 1.5, yeast extract Microbial. Res. 153 (1998) 1
65
-1.0 (NH4hHP04 - 7.0, MgS0 4. 7Hp -1.0, K2HP0 4 - 1.0, CaCl2 - 1.0, with pH adjusted to 3.0. Basal medium of elevated osmotic strength were obtained by adding NaCI at the concentrations indicated below. Growth conditions. After autoclaving for 15 min at 121 DC medium was inoculated with 2 ml of conidia suspension (about 2 X 107 spores). The cultures were grown at 30°C and 200 rpm in 500 ml conical shaking flasks containing 100 ml of basal medium. After culti vation the mycelium of P. notatum 1 was separated from the medium by centrifugation at 6.000 xg for 10 min. GAL extraction by osmotic stress. Approximately 10 ml of the 72-h old culture grown on basal medium was centrifuged. A sample of the supernatant was assayed for GAL. For preparation of the enzyme extract by osmotic stress, the mycelium was washed with distilled water and pressed between filter paper to remove the excess of water. To the still wet mycelium (0.4 g) 10 ml of depressors (NaCI, KCI, Na2S04, glycerol, sorbitol) at 0.2-1.6 M concentration was added. After 1 h incubation in 100 ml conical flasks on rotary shake (220 revl min., shaking diameter 15 cm) at 30°C, the slurry was centrifuged and the supernatant tested for GAL activity. Analytical procedure. After centrifugation of the culture broth, GAL activity in the supernatant was measured by the previously described method (Fiedurek and Ilczuk 1990). One unit of the enzyme activity was defined as
40
E 2-
-
-
:§
E
30
9~
.~ QI
'0...
~
Q.
CIS
.s::.
C
U
:;
C)
Osmotic stress was expected to affect the enzyme biosynthesis by microorganisms (Mildenhal et al. 1981; Grajek and Gervais 1987; Davis and Baudoin 1987). To evaluate this effect, the cultures of P. notatum 1 were carried out in mineral media containing NaCI in various concentration up to 1.6 M (0.945 aw ). ~-galactosidase accumulation increased significantly (about 1.6-fold) until 0.4 M NaCI (0.985 ~), but decreased sharply at 0.6 M (0.98 aw) or higher. At 1.6 M NaCI (0.945 aw) synthesis of the enzyme dropped to zero. On the other hand, the dry weight was still at 18% of the control at 0.8 M NaCI and dropped to 4.0% at 1.2 M (Fig. 1). Si-
8
·ii
c(
Results and discussion
12
~ .:; ..J
the amount producing 1 !lmol glucose per min at 60 DC. The influence of osmotic stress on enzyme activities was measured by adding adequate quantities of depressors to the enzyme-substrate solutions. The protein contents in the medium and post-culture liquid was determined by the method of Schacterle and Pollack (1973). After cultivation, the mycelium dry weight was determined by washing and drying it at 105°C. Fermentations were performed in 3 replicate culture flasks, and analyses carried out in duplicate. The data given are the means of the measurements. The means standard error of GAL estimate was ±0.19 and ranged from ±0.004 to ±0.25 U/ml.
6
~
"C
20
6
4
10
3
2
o~--------~--~----~---------+--------~~~~--~
o
0,4
0,8 NaCI concentration (M)
66
Microbiol. Res. 153 (1998) 1
1,2
1,6
Fig. 1. The effect of various NaCL concentration on ~-galac tosidase production by Penicillium notatum 1: ... - GAL activity; • - Protein; D - dry weight
milar results were obtained for production of cellulase activity by Trichoderma reesei and Sporotrichum cellulophilum growing on wheat bran (Kim et al. 1985). Cell growth and glucoamylase production by A. niger seem to be similarly correlated to osmotic stress (Pandey et at. 1994). A number of reports are published on effects of osmotic potential on microbial enzyme production, which allow a few general conclusions on the nature of these effects. Enzyme levels at lower osmotic potentials may be increased, non-effected (Edgley and Brown 1983) or decreased (Mildenhal el al. 1981). Increases and decreases have been reported both for fungal and for bacterial enzymes, extracellular as well as intracellular ones. Hypo-osmotic shock was reported to cause a rapid increase in cAMP content in the halotoerant green alga Dunaliella viridis (Ohsawa et at. 1992), while in the yeast Zygosaccharomyces rouxii the role of cAMP in osmoregulation was described during the initial stages of salt stress (Nishi and Yagi 1993). cAMP, know to play an important role as a second mesenger in signal transduction and to control various aspects of intracellular metabolism in yeast (Matsumoto et al. 1985; Thevelein 1994), apparently has a significant role in P. notatum cells. Our experiments tested the effect of hypoosmotic shock on ~-galactosidase production by P. notatum 1. To induce enzyme synthesis a high lactose concentration (20%) was used for the germination of spores during
36 h; the substrate was suddenly diluted to I % and cultures were continued for the next 36 h. In these conditions it was possible to substantially elevate (from 22.7 to 55.6 Vlml) the ~-galactosidase activity. The significant part of GAL produced by P. notatum 1 is bound to intracellular structures of the fungus. Therefore, it seems important from the technological point of view to elaborate a method for effective extraction of the intracellular GAL fraction which plays a significant role in the overall synthesis of the enzyme (Fiedurek et al. 1996). In subsequent studies, the 72-h old mycelium was suspended in concentrated solutions of NaCl (0.2-1.6 Mil) to release periplasmic GAL. Fig. 2 shows the effect of GAL secretion after adding different concentration ofNaCl in pH from 3.0 to 7.0. The highest yield of GAL activity was obtained at 0.4 M concentration of NaCI at pH 6.0, which improved the activity of the enzyme by about 1.9-fold as compared to the control medium without this depressor. The lowest activities of enzyme were obtained in pH 3.0 (at 1.6 M NaCl). Different depressors were also used to discount, at the same osmotic potential, their specific effect on the enzyme extraction, and to draw an objective conclusion on optimal conditions for enzyme secretion. The results of GAL extraction in the media with different concentration of depressors are shown in Fig. 3. All results presented in the figures were corrected with respect to the influence of osmotic stress on enzyme
40
-.-.. E 2>-
:;
30
:; U
III
..J c(
C)
20
10
o+---~---+--~----+---~--~--~----~--~--~
o
0,4
0,8 NaCI concentration (M)
1,2
1,6
Fig. 2. The effect of various pH levels on ~-gal actosidase extraction from mycelium of Penicillium notatum I under osmotic stress (0.2-\.6 M NaCI) : 0- pH 3.0 ; Ii - pH 4.0; D - pH 5.0; .-pH6.0; .-pH7.0 Microbiol. Res. 153 (1998) 1
67
40
E
2-
30
~ .:; :0:: u
III
.J
20 < C)
10
O~--~----~----r----+--------~~---+----+---~--~
o
0,4
0,8
depressor concentration (M)
Table 1. Cultural factors affeting ~-galactosidase extraction by osmotic stress (OA M NaCl, 1 h) from mycelium Penicillium notatum 1
Factor varied
GAL activity (Ulml)
Protein (mg/ml)
Temperature (0C) 4 20 30 40 50 60
3.6 21.4 40.5 32.0 18.1
0.36 4.10 6.50 SAO
3.10
3.7
0.48
Agitation speed (rpm) 50 21.6 100 22.9 200 33.8 300 41.0
4.10 4.20 5.30 6.50
Age of mycelium (h) 48 18.8 72 41.0 96 30A
3.60 6.60 6.10
assays in the dilutions used in extraction and analytical procedures. The results obtained with GAL demonstrated strong inducing effect of all ionic (NaCI, KCI, NazS04) and nonionic depressors on enzyme extraction. The highest yield of GAL activity was obtained at 0.4 M or 0.8 M NaCl or glycerol (pH 6.0), which improved the activity of this enzyme about 1.9-fold 68
Microbial. Res. 153 (1998) I
1,2
1,6
Fig. 3. The effect of different depressors on ~-galactosidase extraction from mycelium of Penicillium notatum 1 (pH 6.0) : e-NaCl; O-KCl; 6-Na2S04 ; D - sorbitol; • - glycerol
as compared with the control medium without depressors. The next stage of experiments was to maximize GAL extraction by altering the cultural factors under osmotic stress. Maximal enzyme extraction occured at a temperature of 30°C. The effect of increasing the shaking speed from 50 to 300 rpm on GAL extraction during osmotic stress (0.4 M NaCl, pH 6.0) showed the highest enzyme value at 300 rpm. Increasing agitation speed - from 50 to 300 rpm - resulted in significant increase in GAL activity (from 21.6 to 41.0U/ml). The highest enzyme activities were obtained when using 72-h old mycelium (Table 1). To study the effect of osmotic stress on the enzymatic hydrolysis of lactose used in assay methods, the activity determinations were performed using reaction mixtures with 0.2-2.6 M NaCl. It was noticed that the enzymatic hydrolysis was insignificantly affected by concentration of the depressor. The highest increase of GAL activity (about 7%) was observed at 0.4 M NaCl. Significant decrease of GAL activity (46-60%) was found at 2-2.6 M NaCI concentration (data not shown). Among various selective extraction procedures, osmotic pressure treatment is technically feasible, as it may be applied on different scales. There are no costly chemicals involved and the equipment required is the same as that used for production purposes. The fermenter used as an extraction vessel and the centrifuge
to recover the cells may be the same as the one employed to process the fermentation broth. Since the influence of osmotic potential on enzyme levels has been studied in only a few microbial systems, and significant albeit variable effects have been reported for several of those systems, such effects appear to be common. Therefore, osmotic potential should be considered as a possible regulating factor in studies on the synthesis and secretion of microbial enzymes. The results reported here show the possibility of significant improving the yield of GAL extraction from mycelium P. notatum by osmotic stress.
Acknowledgement This work was financially supported by research program BWIIWBiNoZII. Microb. Biotechnol.
References Csonka, L. N. (1989): Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev. 53, 121-147. Davis, L. L., Baudoin, A. B. M. (1987): Effect of osmotic potential on synthesis and secretion of polygalacturonase and cellulase by Geotrichum candidum. Can. 1. Microbiol. 33, 138-141,1987. Edgley, M., Brown, A. D. (1983): Yeast water relations: physiological changes induced by solute stress in Saccharomyces cerevisiae and Saccharomyces rouxii. 1. Gen. MicrobioI. 129,3453-3463. Fiedurek, 1., Ilczuk, Z. (1990): Screening of microorganisms for improvement of ~-galactosidase production. Acta Microbiol. Polon., 39, 37-42. Fiedurek, 1., Gromada, A., Jamroz, 1. (1996): Effect of medium components and metabolic inhibitors on ~-galacto sidase production and secretion by Penicillium notatum 1. J. Basic Microbiol. 36,27 -32. Gecas, v., Lopez-Leiva, M. (1985): Hydrolysis of lactose: a literature review. Process Biochem. 20, 2-11. Grajek, w., Gervais. P. (1987): Influence of water activity on the enzyme biosynthesis and enzyme activities produced by Trichoderma viride TS in solid-state fermentation. Enzyme Microb. Technol. 9,658-662. Hahn-Hagerdal, B. (1986): Water activity: a possible external regulator of biotechnological process. Enzyme Microb. Technol. 8, 322-327.
Kim, H. K., Mosobuchi, M., Kishimoto, M. M, Seki, T., Ryu, D. D. Y. (1985): Cellulase production by a solid state culture system. Biotechnol. Bioeng. 27, 1445-1450. Kenyon, C. P., Prior, B. A., Vuuren, H. 1. 1., (1986): Water relations of etanol fermentation by Saccharomyces cerevisiae.' glycerol production under solute stress. Enzyme Microb. Technol. 8, 461-464. Matsumoto, K., Uno, T., Ishikawa, T. (1985): Genetic analysis of the role of cAMP in yeast. Yeast 1,15-24. Mildenthal,1. P., Prior, B. A., Trollope, L. A. (1981): Water relations of Erwinia chrysanthemi,' growth and extracellular pectic acid lyase production. 1. Gen. Microbiol.l27, 27 -34. Nishi, T., Yagi T. (1993): Participation of cAMP in the induction of the synthesis of glycerol for the osmoregulatory response in the salt-tolerant yeast Zygosaccharomyces rouxii. 1. Gen. Appl. Microbiol. 39, 493-503. Ohsawa, H., Nakayama, K., Okada, M. (1992): Adenylate cyclase and phosphodiesterase activities in relation to a rapid increase in cellular cAMP concentration by hypoosmotic shock in Dunaliella viridis. Bot. Mag. Tokyo 105,393-404. Olz, R., Larsson, K., Adler, L., Gustavsson, L. (1993): Energy flux and osmoregulation of Saccharomyces cerevisiae grown in chemostat under NaCI stress. 1. Bacteriol. 175, 2205-2213. Pandey, A., Ashakumary, L., Selvakumar, P., Vijayalakshrni, K. S. (1994): Influence of water activity on growth and activity of Aspergillus niger for glucoamylase production in solid-state fermentation. World 1. Microbiol. Biotechnol. 10,485-486. Richmond, M. L., Grey, 1. I., Stine, C. M. (1981): ~-galacto sidase: Review of recent research related to technological applications, nutritional concerns, and immobilization. J. Dairy Sci. 64,1759-1771. Schacterle, G. R., Pollack, R. L. (1973): A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal. Biochem. 51, 654-655. Suzuki, M. (1995): Enhancements of antocyanin accumulation by high osmotic stress ad low pH in grape cells (Vitis hybrids). 1. Plant Physiol.l47, 152-155. Thevelein,1. M. (1994): Signal transduction in yeast. Yeast 10, 1753-1790. Troller, 1. A. (1980): Influence of water activity on microorganisms in food. Food Technology 5, 76-82. Troller, J. A., Stinson, J. V. (1981): Moisture requirements for growth and metabolite production by lactic acid bacteria. Appl. Environm. Microbial. 42, 682-687. Witter, L. D., Anderson, C. B. (1987): Osmoregulation by microorganisms at reduced water activity. In "Food Microbiology" Ed: Montville, T. 1. 1, 1-34, Boca Raton: CRC Press Inc.
Microbiol. Res. 153 (1998) 1
69
©
Gustav Fischer Verlag
Enhancement of ~-galactosidase production and secretion by high osmotic stress in Penicillium notatum 1. Fiedurek Department of Industrial Microbiology, Maria Curie-Sklodowska University, 20-033 Lublin, Poland Accepted: December 14, 1997
Abstract A strain of Penicillium notatum was cultured under various osmotic stresses. When the fungus was cultured under osmotic stress, total ~-galactosidase activity increased significantly (about 1.6-fold) with increasing concentration of NaCI. Mycelial growth was repressed with increased medium osmolarity. To release periplasmic ~-galactosidase, 72-h old mycelium was suspended on a concentrated solution of NaCI (0.2-1.6 M). The highest yield of ~-galactosidase activity was obtained at 0.4 M NaCI at pH 6.0, which improved the activity of this enzyme by about 1.9-fold in comparison with the control medium without this depressor. Key words: ~-galactosidase - osmotic stress - Penicillium notatum
Introduction ~- D-galactosidase [GAL (EC 3.2.1.23)] preparations are widely used for hydrolysis of lactose in milk, milk products and whey (Richmond et al. 1981; Gecas et aZ. 1985). Degradation of lactose into simple sugars increases availability of certain milk products and makes their consumption possible even by person with lactose intolerance. There are many reports proving that metabolism of microorganisms is strongly influenced by osmotic stress. This is also true for the production of secondary metabolites, such as microbial toxins (Troller 1980), enzymes (Mildenhal et al. 1981; Grajek and Gervais 1987; Pandey et af. 1994) and other compounds (Suzuki 1995). The amount of available water in the environment of microorganisms affects growth, respiration, enzyme synthesis, sporulation and other physiological
Corresponding author: 1. Fiedurek
functions (Troller 1980). The responses of cells to media with decreasing water activity (aw ) have been described (Hahn-Hagerdal 1986; Witter and Anderson 1987; Csonka 1989). It is important to know the optimal value of water activity for individual physiological phenomena such as microbial growth, sporulation and production of primary and secondary metabolites. By controlling water activity one can regulate fermentation and form conditions favoring the biosynthesis of desirable products. Maximum growth and metabolite overproduction are often observed at aw values between 0.9 and 1.0. Lactic acid bacteria show up to 1O-fold increase in a diacetyl production when aw is lowered to 0.95 (Troller and Stinson 1981). In ethanol fermentation by Saccharomyces cerevisiae, substrate uptake and glycerol production are greater at an aw of 0.971 than at 0.994. When aw is reduced below 0.990, a decreased growth rate and biomass concentration usually follow (Kenyon et af. 1986; OIz et af. 1993). Data on the influence of osmotic stress on the synthesis and excretion of enzymes into the medium are not uniform yet. To my knowledge no paper has mentioned the effect of water activity on production and secretion of ~-galactosidase. The aim of the present work was to examine the impact of osmotic stress on production and excretion of ~-galactosidase by Penicillium notatum.
Materials and methods Strain and media. In our studies a P. notatum mutant (strain 1) producing a high extracellular GAL activity in shaken cultures was used (Fiedurek et aZ. 1996). The fungus was grown on basal medium containing (gil): lactose - 10, peptone (type I, Sigma) - 1.5, yeast extract Microbial. Res. 153 (1998) 1
65
-1.0 (NH4hHP04 - 7.0, MgS0 4. 7Hp -1.0, K2HP0 4 - 1.0, CaCl2 - 1.0, with pH adjusted to 3.0. Basal medium of elevated osmotic strength were obtained by adding NaCI at the concentrations indicated below. Growth conditions. After autoclaving for 15 min at 121 DC medium was inoculated with 2 ml of conidia suspension (about 2 X 107 spores). The cultures were grown at 30°C and 200 rpm in 500 ml conical shaking flasks containing 100 ml of basal medium. After culti vation the mycelium of P. notatum 1 was separated from the medium by centrifugation at 6.000 xg for 10 min. GAL extraction by osmotic stress. Approximately 10 ml of the 72-h old culture grown on basal medium was centrifuged. A sample of the supernatant was assayed for GAL. For preparation of the enzyme extract by osmotic stress, the mycelium was washed with distilled water and pressed between filter paper to remove the excess of water. To the still wet mycelium (0.4 g) 10 ml of depressors (NaCI, KCI, Na2S04, glycerol, sorbitol) at 0.2-1.6 M concentration was added. After 1 h incubation in 100 ml conical flasks on rotary shake (220 revl min., shaking diameter 15 cm) at 30°C, the slurry was centrifuged and the supernatant tested for GAL activity. Analytical procedure. After centrifugation of the culture broth, GAL activity in the supernatant was measured by the previously described method (Fiedurek and Ilczuk 1990). One unit of the enzyme activity was defined as
40
E 2-
-
-
:§
E
30
9~
.~ QI
'0...
~
Q.
CIS
.s::.
C
U
:;
C)
Osmotic stress was expected to affect the enzyme biosynthesis by microorganisms (Mildenhal et al. 1981; Grajek and Gervais 1987; Davis and Baudoin 1987). To evaluate this effect, the cultures of P. notatum 1 were carried out in mineral media containing NaCI in various concentration up to 1.6 M (0.945 aw ). ~-galactosidase accumulation increased significantly (about 1.6-fold) until 0.4 M NaCI (0.985 ~), but decreased sharply at 0.6 M (0.98 aw) or higher. At 1.6 M NaCI (0.945 aw) synthesis of the enzyme dropped to zero. On the other hand, the dry weight was still at 18% of the control at 0.8 M NaCI and dropped to 4.0% at 1.2 M (Fig. 1). Si-
8
·ii
c(
Results and discussion
12
~ .:; ..J
the amount producing 1 !lmol glucose per min at 60 DC. The influence of osmotic stress on enzyme activities was measured by adding adequate quantities of depressors to the enzyme-substrate solutions. The protein contents in the medium and post-culture liquid was determined by the method of Schacterle and Pollack (1973). After cultivation, the mycelium dry weight was determined by washing and drying it at 105°C. Fermentations were performed in 3 replicate culture flasks, and analyses carried out in duplicate. The data given are the means of the measurements. The means standard error of GAL estimate was ±0.19 and ranged from ±0.004 to ±0.25 U/ml.
6
~
"C
20
6
4
10
3
2
o~--------~--~----~---------+--------~~~~--~
o
0,4
0,8 NaCI concentration (M)
66
Microbiol. Res. 153 (1998) 1
1,2
1,6
Fig. 1. The effect of various NaCL concentration on ~-galac tosidase production by Penicillium notatum 1: ... - GAL activity; • - Protein; D - dry weight
milar results were obtained for production of cellulase activity by Trichoderma reesei and Sporotrichum cellulophilum growing on wheat bran (Kim et al. 1985). Cell growth and glucoamylase production by A. niger seem to be similarly correlated to osmotic stress (Pandey et at. 1994). A number of reports are published on effects of osmotic potential on microbial enzyme production, which allow a few general conclusions on the nature of these effects. Enzyme levels at lower osmotic potentials may be increased, non-effected (Edgley and Brown 1983) or decreased (Mildenhal el al. 1981). Increases and decreases have been reported both for fungal and for bacterial enzymes, extracellular as well as intracellular ones. Hypo-osmotic shock was reported to cause a rapid increase in cAMP content in the halotoerant green alga Dunaliella viridis (Ohsawa et at. 1992), while in the yeast Zygosaccharomyces rouxii the role of cAMP in osmoregulation was described during the initial stages of salt stress (Nishi and Yagi 1993). cAMP, know to play an important role as a second mesenger in signal transduction and to control various aspects of intracellular metabolism in yeast (Matsumoto et al. 1985; Thevelein 1994), apparently has a significant role in P. notatum cells. Our experiments tested the effect of hypoosmotic shock on ~-galactosidase production by P. notatum 1. To induce enzyme synthesis a high lactose concentration (20%) was used for the germination of spores during
36 h; the substrate was suddenly diluted to I % and cultures were continued for the next 36 h. In these conditions it was possible to substantially elevate (from 22.7 to 55.6 Vlml) the ~-galactosidase activity. The significant part of GAL produced by P. notatum 1 is bound to intracellular structures of the fungus. Therefore, it seems important from the technological point of view to elaborate a method for effective extraction of the intracellular GAL fraction which plays a significant role in the overall synthesis of the enzyme (Fiedurek et al. 1996). In subsequent studies, the 72-h old mycelium was suspended in concentrated solutions of NaCl (0.2-1.6 Mil) to release periplasmic GAL. Fig. 2 shows the effect of GAL secretion after adding different concentration ofNaCl in pH from 3.0 to 7.0. The highest yield of GAL activity was obtained at 0.4 M concentration of NaCI at pH 6.0, which improved the activity of the enzyme by about 1.9-fold as compared to the control medium without this depressor. The lowest activities of enzyme were obtained in pH 3.0 (at 1.6 M NaCl). Different depressors were also used to discount, at the same osmotic potential, their specific effect on the enzyme extraction, and to draw an objective conclusion on optimal conditions for enzyme secretion. The results of GAL extraction in the media with different concentration of depressors are shown in Fig. 3. All results presented in the figures were corrected with respect to the influence of osmotic stress on enzyme
40
-.-.. E 2>-
:;
30
:; U
III
..J c(
C)
20
10
o+---~---+--~----+---~--~--~----~--~--~
o
0,4
0,8 NaCI concentration (M)
1,2
1,6
Fig. 2. The effect of various pH levels on ~-gal actosidase extraction from mycelium of Penicillium notatum I under osmotic stress (0.2-\.6 M NaCI) : 0- pH 3.0 ; Ii - pH 4.0; D - pH 5.0; .-pH6.0; .-pH7.0 Microbiol. Res. 153 (1998) 1
67
40
E
2-
30
~ .:; :0:: u
III
.J
20 < C)
10
O~--~----~----r----+--------~~---+----+---~--~
o
0,4
0,8
depressor concentration (M)
Table 1. Cultural factors affeting ~-galactosidase extraction by osmotic stress (OA M NaCl, 1 h) from mycelium Penicillium notatum 1
Factor varied
GAL activity (Ulml)
Protein (mg/ml)
Temperature (0C) 4 20 30 40 50 60
3.6 21.4 40.5 32.0 18.1
0.36 4.10 6.50 SAO
3.10
3.7
0.48
Agitation speed (rpm) 50 21.6 100 22.9 200 33.8 300 41.0
4.10 4.20 5.30 6.50
Age of mycelium (h) 48 18.8 72 41.0 96 30A
3.60 6.60 6.10
assays in the dilutions used in extraction and analytical procedures. The results obtained with GAL demonstrated strong inducing effect of all ionic (NaCI, KCI, NazS04) and nonionic depressors on enzyme extraction. The highest yield of GAL activity was obtained at 0.4 M or 0.8 M NaCl or glycerol (pH 6.0), which improved the activity of this enzyme about 1.9-fold 68
Microbial. Res. 153 (1998) I
1,2
1,6
Fig. 3. The effect of different depressors on ~-galactosidase extraction from mycelium of Penicillium notatum 1 (pH 6.0) : e-NaCl; O-KCl; 6-Na2S04 ; D - sorbitol; • - glycerol
as compared with the control medium without depressors. The next stage of experiments was to maximize GAL extraction by altering the cultural factors under osmotic stress. Maximal enzyme extraction occured at a temperature of 30°C. The effect of increasing the shaking speed from 50 to 300 rpm on GAL extraction during osmotic stress (0.4 M NaCl, pH 6.0) showed the highest enzyme value at 300 rpm. Increasing agitation speed - from 50 to 300 rpm - resulted in significant increase in GAL activity (from 21.6 to 41.0U/ml). The highest enzyme activities were obtained when using 72-h old mycelium (Table 1). To study the effect of osmotic stress on the enzymatic hydrolysis of lactose used in assay methods, the activity determinations were performed using reaction mixtures with 0.2-2.6 M NaCl. It was noticed that the enzymatic hydrolysis was insignificantly affected by concentration of the depressor. The highest increase of GAL activity (about 7%) was observed at 0.4 M NaCl. Significant decrease of GAL activity (46-60%) was found at 2-2.6 M NaCI concentration (data not shown). Among various selective extraction procedures, osmotic pressure treatment is technically feasible, as it may be applied on different scales. There are no costly chemicals involved and the equipment required is the same as that used for production purposes. The fermenter used as an extraction vessel and the centrifuge
to recover the cells may be the same as the one employed to process the fermentation broth. Since the influence of osmotic potential on enzyme levels has been studied in only a few microbial systems, and significant albeit variable effects have been reported for several of those systems, such effects appear to be common. Therefore, osmotic potential should be considered as a possible regulating factor in studies on the synthesis and secretion of microbial enzymes. The results reported here show the possibility of significant improving the yield of GAL extraction from mycelium P. notatum by osmotic stress.
Acknowledgement This work was financially supported by research program BWIIWBiNoZII. Microb. Biotechnol.
References Csonka, L. N. (1989): Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev. 53, 121-147. Davis, L. L., Baudoin, A. B. M. (1987): Effect of osmotic potential on synthesis and secretion of polygalacturonase and cellulase by Geotrichum candidum. Can. 1. Microbiol. 33, 138-141,1987. Edgley, M., Brown, A. D. (1983): Yeast water relations: physiological changes induced by solute stress in Saccharomyces cerevisiae and Saccharomyces rouxii. 1. Gen. MicrobioI. 129,3453-3463. Fiedurek, 1., Ilczuk, Z. (1990): Screening of microorganisms for improvement of ~-galactosidase production. Acta Microbiol. Polon., 39, 37-42. Fiedurek, 1., Gromada, A., Jamroz, 1. (1996): Effect of medium components and metabolic inhibitors on ~-galacto sidase production and secretion by Penicillium notatum 1. J. Basic Microbiol. 36,27 -32. Gecas, v., Lopez-Leiva, M. (1985): Hydrolysis of lactose: a literature review. Process Biochem. 20, 2-11. Grajek, w., Gervais. P. (1987): Influence of water activity on the enzyme biosynthesis and enzyme activities produced by Trichoderma viride TS in solid-state fermentation. Enzyme Microb. Technol. 9,658-662. Hahn-Hagerdal, B. (1986): Water activity: a possible external regulator of biotechnological process. Enzyme Microb. Technol. 8, 322-327.
Kim, H. K., Mosobuchi, M., Kishimoto, M. M, Seki, T., Ryu, D. D. Y. (1985): Cellulase production by a solid state culture system. Biotechnol. Bioeng. 27, 1445-1450. Kenyon, C. P., Prior, B. A., Vuuren, H. 1. 1., (1986): Water relations of etanol fermentation by Saccharomyces cerevisiae.' glycerol production under solute stress. Enzyme Microb. Technol. 8, 461-464. Matsumoto, K., Uno, T., Ishikawa, T. (1985): Genetic analysis of the role of cAMP in yeast. Yeast 1,15-24. Mildenthal,1. P., Prior, B. A., Trollope, L. A. (1981): Water relations of Erwinia chrysanthemi,' growth and extracellular pectic acid lyase production. 1. Gen. Microbiol.l27, 27 -34. Nishi, T., Yagi T. (1993): Participation of cAMP in the induction of the synthesis of glycerol for the osmoregulatory response in the salt-tolerant yeast Zygosaccharomyces rouxii. 1. Gen. Appl. Microbiol. 39, 493-503. Ohsawa, H., Nakayama, K., Okada, M. (1992): Adenylate cyclase and phosphodiesterase activities in relation to a rapid increase in cellular cAMP concentration by hypoosmotic shock in Dunaliella viridis. Bot. Mag. Tokyo 105,393-404. Olz, R., Larsson, K., Adler, L., Gustavsson, L. (1993): Energy flux and osmoregulation of Saccharomyces cerevisiae grown in chemostat under NaCI stress. 1. Bacteriol. 175, 2205-2213. Pandey, A., Ashakumary, L., Selvakumar, P., Vijayalakshrni, K. S. (1994): Influence of water activity on growth and activity of Aspergillus niger for glucoamylase production in solid-state fermentation. World 1. Microbiol. Biotechnol. 10,485-486. Richmond, M. L., Grey, 1. I., Stine, C. M. (1981): ~-galacto sidase: Review of recent research related to technological applications, nutritional concerns, and immobilization. J. Dairy Sci. 64,1759-1771. Schacterle, G. R., Pollack, R. L. (1973): A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal. Biochem. 51, 654-655. Suzuki, M. (1995): Enhancements of antocyanin accumulation by high osmotic stress ad low pH in grape cells (Vitis hybrids). 1. Plant Physiol.l47, 152-155. Thevelein,1. M. (1994): Signal transduction in yeast. Yeast 10, 1753-1790. Troller, 1. A. (1980): Influence of water activity on microorganisms in food. Food Technology 5, 76-82. Troller, J. A., Stinson, J. V. (1981): Moisture requirements for growth and metabolite production by lactic acid bacteria. Appl. Environm. Microbial. 42, 682-687. Witter, L. D., Anderson, C. B. (1987): Osmoregulation by microorganisms at reduced water activity. In "Food Microbiology" Ed: Montville, T. 1. 1, 1-34, Boca Raton: CRC Press Inc.
Microbiol. Res. 153 (1998) 1
69