World
Journal
and Biofechnology
of Microbiology
9, 153-155
Selection of a mutant strain of Lipomyces kononenkoae with dextranase synthesis resistant to catabolite repression O.N. Zinchenko,*
O.V. Krivosheeva
and A.G. Lobanok
A mutant strain of Lipomyces kononenkoae 2896-3 synthesizing dextranase but resistant to catabolite was obtained using N-nitroso-Nmethylurea treatment. Enzyme biosynthesis in media with dextran carbon sources was then characterized. The capacity of the mutant to produce dextranase when hydrolysed corn starch is demonstrated. Key words:
Catabolite
repression,
dextranase,
mutant, yeast.
Catabolite repression is a common control mechanism of enzyme biosynthesis. Dextranase production by micromycetes and yeasts is subject to catabolite repression (Lobanok & Zinchenko 1983) but may be induced both by readily utilizable carbon sources and their non-metabolizable analogues, namely 2-deoxyglucose and glucosamine (Hackney & Freeman 1980). These anti-metabolites may be used for selection of mutants with de-repressed enzyme synthesis. Such mutants, as a rule, synthesize and secrete extracellular enzymes with higher activities and at earlier stages of the growth cycle than wild-type strains. Cheap fermentation media containing glucose in high concentrations can therefore be used for commercial production of such enzymes. The aim of this paper was to select a mutant of Lipomyces kononenkoae which had good dextranase synthesis resistant to catabolite repression.
Materials Organisms
and Methods and growth
Lipomyces konorwkoae 15 (from the culture collection of the Institute of Microbiology, Byelorussian Academy of Sciences) and Lipomyces kononenkoae 2896 (from the culture collection of the Soil Biology Department, Moscow State University) were grown on nutrient medium 1, containing (g/l): glucose, IO; (NH&SO,, 3.0; MgS0,7H,O, 0.7; NaCl, 0.5; KH,PO,, 1.0; K,HPO,, 0.1; biotin, 0.001 (medium 1). Selective medium 2, for screening mutants not O.N. Zinchenko. of Microbiology, 220067, Minsk, @ 1993 Rapid
O.V. Krivosheeva and A.G. Lobanok are with the Institute Byelorussian Academy of Sciences, Zhodinskaya 2, Byelorussia; fax: (0172) 64 4766. ‘Corresponding author. Communications
of Oxford
Ltd
repression and other grown on
sensitive to catabolite repression, also contained 10 g dextran/l as a carbon source and 0.1 g 2-deoxyglucose/l. Dextranase activity in the mutants was determined by growing yeast on medium 3, comprising (g/l): dextran, 20; (NH&SO,, 3.0; MgS0,.7H,O, 0.7; NaCI, 0.5; KH,PO,, 1.0; K,HPO,, 0.1; corn steep liquor, 20. Solid media contained 2% (w/v) agar. Yeast cultivation was carried out in 250-ml shake-flasks at 28°C. Starch Hydrolysis for Nutrient Media Corn starch was hydrolysed at 70-75°C with Bacillus amylase added to suspension at 600 U/100 g substrate.
subtilis
Mutagenesis For the UV mutagenic procedure, yeasts were grown on liquid medium 1 up to mid-exponential phase, transferred to solid medium I and treated with UV light. The resulting colonies were seeded on solid selective medium 2. For mutagenesis with N-nitroso-N-methylurea (NMU), cells were harvested by centrifuging, washed twice with distilled water, resuspended in water to obtain suspension containing 4 x 10' cells/ml and 1 mg mutagen/ml was added. Cells were held for 60 min in the dark af 28”C, then washed three times with distilled water, resuspended in the same volume of water and subsequently a I ml aliquot was transferred into 50 ml of selective medium 2. The cells were grown at 28°C for 112 h with shaking and 0.1 ml aliquots were then dispensed into Petri dishes with agar medium 2. After 5 days’ incubation, colonies growing on dextran in the presence of 2-deoxyglucose were obtained. Analytical
Methods
Dextranase activity was assayed as described by Lobanok et al. (1981). Reducing substances were estimated by the ferricyanide method (Kerr 1950). Yeast biomass was determined by measuring optical density of the suspension at 670 nm. Biomass concentration was expressed in mg/ml, using an experimental conversion coefficient of 0.38.
O.N. Zinchenko, 0.X
Krivosheeua and A.G. Lobanok
Table
1. Dextranase
activity
Strain
Dextran
of mutant
strains
on liquid
(2% w/v)
Biomass OWmU
nutrient
Dextran
Activity
media (2%
w/v)
with
dextran.
+ glucose
Biomass OwW
(2%
w/v)
Activity
(U/ml)
Wmg)
(Ufml)
36.9
5.7
9.9
2.8
Ww)
Original 15
6.5
Mutant 15-1
0.3
6.1
19.6
3.2
11.8
39.9
3.4
6.5 7.9
43.9 34.1
6.8 4.3
11.0 12.5
42.8 32.5
3.9 2.6
7.9 6.8
36.9 26.6
4.7 3.9
12.9 11.4
32.6 20.4
2.5 1.8
15-12 15-14
6.1 6.8
21.9 26.6
3.6 3.9
10.6
23.2 23.2
2.2 2.2
15-15
11.4 7.2
34.1
3.0
10.6 11.0
5.1 2.4 3.4
10.6 10.3 11.0
25.5 23.2
2.3 2.2
6.1 6.5
36.9 14.9 21.9
25.6 20.4
2.5 1.9
15-20 15-22
7.6 6.8
36.9 34.1
4.9 5.0
10.6
15-23 16-24
6.5 6.8
39.2 19.6
6.0 2.9
32.6 35.4 32.6
3.1
9.9 10.6 12.2
1525
7.2
17.3
2.4
11.4
20.4 20.4
6.8
41.1
6.0
10.3
4.6
0.4
Mutant 2896-l
6.8
6.5 6.1
45.2 52.6
3.6
6.8
43.9 41.5
12.5
2896-2 2896-3 2896-4
6.8 6.5
43.9 43.9
6.5 6.8
6.5
41.5
6.4
15-Z l&l 15-5 15-8
15-16 16-18 15-19
3.6 3.1 1.7 1.8
Original 2896
2896-l
Results
1
9.1 8.4
3.6 4.6
45.2 42.9
4.7 1.6
13.5
and Discussion
l.. kononenkoae 15 and 1. kononenkoae 2896 are characterized by highly efficient biosynthesis of dextranase when grown on fermentation medium 3. Enzyme formation was repressed by up to 90% when 2% (w/v) glucose was added and totally repressed by the 2-deoxyglucose at 0.01% (w/v) in solid medium 2. This effect did not correlate with growth inhibition since both strains grow well on glucose media with 0.01% (w/v) 2-deoxyglucose. 2-Deoxyglucose has been previously used to screen mutants of Lipomyces starkeyi with dextranase synthesis resistant to catabolite repression, UV-irradiation being used as the mutagenic factor (Laires 1983). UV- and NMUtreatments were applied to obtain L. kononenkoae mutants resistant to catabolite repression. With UV, exposure for 1 min did not affect cell viability, but after 5 min all cells were killed. Exposure for 2 min gave survival rates of 0.4% and 0.5% for strains 15 and 2896, respectively. However, colonies resistant to catabolite repression were
154
14.8 9.9
World Jorrmal of Micmbrology and Biotechnology. Vol 9, 1993
i = $, lE. 2 $ 8 3 1
Time Figure 1. Growth (U, L. kononenkoae growing symbols; strain 2896-3,
n)
(h)
and glucose consumption on 1% (w/v) glucose. Strain closed symbols.
(0, 0) by 2896, open
Mutant
with
dexfranase
synthesis
“5
Table 3. Growth and accumulation (2896-3) and parent (2896) strains Strain
Starch (% w/v) PH
30
2896
Biomass (WmU
2
E 2
c>
5
.? 2
,
II w
72
dextranase
accumulation
29
Time
Figure
2. Growth
n)
(0,
by L. kononenkoaeon symbols;
strain
and
(h) (0,
0)
1% (w/v) dextran media. Strain 2896, open 2896-3,
closed
symbols.
not detected. NMU as a mutagen yielded 334 mutants resistant to 2-deoxyglucose. Mutants were tested for potential ability to hydrolyse dextran in the presence of glucose by plating on solid media with 1% (w/v) Blue dextran and 1% (w/v) glucose. From the parent strain 15, 87 mutant colonies were obtained that were variants forming clear zones in Blue dextran. Similarly, 14 mutants with derepressed dextranase synthesis were obtained from parent strain 2896. Repression of dextranase synthesis in the mutants was not observed, even with 10% (w/v) glucose. For quantitative activity measurement in mutants capable of efficient dextran hydrolysis in the presence of 10% (w/v) glucose (diameter of clear zone: 15 to 20 mm), growth in liquid medium 3 was carried out (Table 1). Although none of the tested mutants exceeded the parent strains in activity, dextranase synthesis was only slightly repressed by glucose. Stable mutant strain 2896-3 was chosen for further
Strain Activity (U/ml)
pH
5.2 4.2
4.0 2.0
1.9
2.8
4.9
3.4 2.6 2.5
5 10
=‘
of dextranase on starch media.
a.5 8.7
by
mutant
2896-3
Biomass (Wml)
Activity (U/ml)
4.8
13.0 40.7
11.6 12.5
61.6
experiments. De-repression was not affected by disturbances of glucose transport since substrate consumption by the mutant and parent strain proceeded similarly (Figure 1); the only difference was that the mutant strain produced slightly more biomass than the parent strain. Comparative analysis of the dextranase biosynthesis dynamics in the mutant and parent strains on 1% (w/v) dextran medium revealed that the mutant was characterized by earlier initiation of enzyme synthesis (Figure 2). The most significant differences between the strains were observed in the production of dextranase on media with various carbon sources (Table 2); the data presented in Table 2 shows 1% to 75% higher activities in the mutant strain than in parent strain for all the carbon sources examined. Corn starch, predigested with amylase, was also tested as a carbon source (Table 3). Mutant 2896-3 produced high activities of dextranase on media without dextran, the specific inducer. Enzyme synthesis was more efficient on 10% (w/v) saccharified starch compared with dextran media. The mutant may be considered a promising strain for industrial dextranase production, since the economics of the process could be significantly improved by using hydrolyzed starch instead of dextran, the most expensive media constituent.
References R.C. & Freeman, R.F. 1980 Gratuitous catabolite repression by glucosamine of maltose utilization in Saccharomyces cereuisiae. Journal of General Microbiology 121, 479-482.
Hackney,
Kerr,
R.W.
1950
7he Chemistry
and Industry
of Starch.
New
York:
Academic Press. Laires,
A. 1983
Use of D-glucosamine
and 2-deoxyglucose in the Lipomyces starkeyi derepressed for the production of extracellular endodextranase. Zeitschrift fiir Allgemeine Microbiologic 29, 601-603.
selective isolation of mutants of the yeast Table 2. Dextranase sources.
biosynthesis
on media
with various
carbon
Lobanok, A.G. & Zinchenko, Carbon source (2% w/v)
Activity Strain
Dextran Glucose
Maltose Sucrose Soluble
starch
2896
(U/ml) Strain
2896-3
41.1 6.8 5.9
42.0 a.5 10.3
6.3 7.8
7.6 11.6
O.N. 1983 Certain aspects of
dextranase biosynthesis control in micromycetes. In Proceedings of the International SymposiLtm on Regulation of Microbial Mefabolism by Environmental Factors, p, 81. Pushtchino. Lobanok, A.G., Zinchenko, O.N. & Shishlo, V.I. 1981 Some properties of dextranases from the fungus Penicillium piscarittm BIM G-102. Prikladnaya Biokhimiya and Microbiologiya (Moscow) 17, 210-215. (Received
in revisedform
27July
1992;
accepted
8 August
World journal of’ Microbiology and Biofechnolqy, Vol 9, 1993
1992)
155
Journal
and Biofechnology
of Microbiology
9, 153-155
Selection of a mutant strain of Lipomyces kononenkoae with dextranase synthesis resistant to catabolite repression O.N. Zinchenko,*
O.V. Krivosheeva
and A.G. Lobanok
A mutant strain of Lipomyces kononenkoae 2896-3 synthesizing dextranase but resistant to catabolite was obtained using N-nitroso-Nmethylurea treatment. Enzyme biosynthesis in media with dextran carbon sources was then characterized. The capacity of the mutant to produce dextranase when hydrolysed corn starch is demonstrated. Key words:
Catabolite
repression,
dextranase,
mutant, yeast.
Catabolite repression is a common control mechanism of enzyme biosynthesis. Dextranase production by micromycetes and yeasts is subject to catabolite repression (Lobanok & Zinchenko 1983) but may be induced both by readily utilizable carbon sources and their non-metabolizable analogues, namely 2-deoxyglucose and glucosamine (Hackney & Freeman 1980). These anti-metabolites may be used for selection of mutants with de-repressed enzyme synthesis. Such mutants, as a rule, synthesize and secrete extracellular enzymes with higher activities and at earlier stages of the growth cycle than wild-type strains. Cheap fermentation media containing glucose in high concentrations can therefore be used for commercial production of such enzymes. The aim of this paper was to select a mutant of Lipomyces kononenkoae which had good dextranase synthesis resistant to catabolite repression.
Materials Organisms
and Methods and growth
Lipomyces konorwkoae 15 (from the culture collection of the Institute of Microbiology, Byelorussian Academy of Sciences) and Lipomyces kononenkoae 2896 (from the culture collection of the Soil Biology Department, Moscow State University) were grown on nutrient medium 1, containing (g/l): glucose, IO; (NH&SO,, 3.0; MgS0,7H,O, 0.7; NaCl, 0.5; KH,PO,, 1.0; K,HPO,, 0.1; biotin, 0.001 (medium 1). Selective medium 2, for screening mutants not O.N. Zinchenko. of Microbiology, 220067, Minsk, @ 1993 Rapid
O.V. Krivosheeva and A.G. Lobanok are with the Institute Byelorussian Academy of Sciences, Zhodinskaya 2, Byelorussia; fax: (0172) 64 4766. ‘Corresponding author. Communications
of Oxford
Ltd
repression and other grown on
sensitive to catabolite repression, also contained 10 g dextran/l as a carbon source and 0.1 g 2-deoxyglucose/l. Dextranase activity in the mutants was determined by growing yeast on medium 3, comprising (g/l): dextran, 20; (NH&SO,, 3.0; MgS0,.7H,O, 0.7; NaCI, 0.5; KH,PO,, 1.0; K,HPO,, 0.1; corn steep liquor, 20. Solid media contained 2% (w/v) agar. Yeast cultivation was carried out in 250-ml shake-flasks at 28°C. Starch Hydrolysis for Nutrient Media Corn starch was hydrolysed at 70-75°C with Bacillus amylase added to suspension at 600 U/100 g substrate.
subtilis
Mutagenesis For the UV mutagenic procedure, yeasts were grown on liquid medium 1 up to mid-exponential phase, transferred to solid medium I and treated with UV light. The resulting colonies were seeded on solid selective medium 2. For mutagenesis with N-nitroso-N-methylurea (NMU), cells were harvested by centrifuging, washed twice with distilled water, resuspended in water to obtain suspension containing 4 x 10' cells/ml and 1 mg mutagen/ml was added. Cells were held for 60 min in the dark af 28”C, then washed three times with distilled water, resuspended in the same volume of water and subsequently a I ml aliquot was transferred into 50 ml of selective medium 2. The cells were grown at 28°C for 112 h with shaking and 0.1 ml aliquots were then dispensed into Petri dishes with agar medium 2. After 5 days’ incubation, colonies growing on dextran in the presence of 2-deoxyglucose were obtained. Analytical
Methods
Dextranase activity was assayed as described by Lobanok et al. (1981). Reducing substances were estimated by the ferricyanide method (Kerr 1950). Yeast biomass was determined by measuring optical density of the suspension at 670 nm. Biomass concentration was expressed in mg/ml, using an experimental conversion coefficient of 0.38.
O.N. Zinchenko, 0.X
Krivosheeua and A.G. Lobanok
Table
1. Dextranase
activity
Strain
Dextran
of mutant
strains
on liquid
(2% w/v)
Biomass OWmU
nutrient
Dextran
Activity
media (2%
w/v)
with
dextran.
+ glucose
Biomass OwW
(2%
w/v)
Activity
(U/ml)
Wmg)
(Ufml)
36.9
5.7
9.9
2.8
Ww)
Original 15
6.5
Mutant 15-1
0.3
6.1
19.6
3.2
11.8
39.9
3.4
6.5 7.9
43.9 34.1
6.8 4.3
11.0 12.5
42.8 32.5
3.9 2.6
7.9 6.8
36.9 26.6
4.7 3.9
12.9 11.4
32.6 20.4
2.5 1.8
15-12 15-14
6.1 6.8
21.9 26.6
3.6 3.9
10.6
23.2 23.2
2.2 2.2
15-15
11.4 7.2
34.1
3.0
10.6 11.0
5.1 2.4 3.4
10.6 10.3 11.0
25.5 23.2
2.3 2.2
6.1 6.5
36.9 14.9 21.9
25.6 20.4
2.5 1.9
15-20 15-22
7.6 6.8
36.9 34.1
4.9 5.0
10.6
15-23 16-24
6.5 6.8
39.2 19.6
6.0 2.9
32.6 35.4 32.6
3.1
9.9 10.6 12.2
1525
7.2
17.3
2.4
11.4
20.4 20.4
6.8
41.1
6.0
10.3
4.6
0.4
Mutant 2896-l
6.8
6.5 6.1
45.2 52.6
3.6
6.8
43.9 41.5
12.5
2896-2 2896-3 2896-4
6.8 6.5
43.9 43.9
6.5 6.8
6.5
41.5
6.4
15-Z l&l 15-5 15-8
15-16 16-18 15-19
3.6 3.1 1.7 1.8
Original 2896
2896-l
Results
1
9.1 8.4
3.6 4.6
45.2 42.9
4.7 1.6
13.5
and Discussion
l.. kononenkoae 15 and 1. kononenkoae 2896 are characterized by highly efficient biosynthesis of dextranase when grown on fermentation medium 3. Enzyme formation was repressed by up to 90% when 2% (w/v) glucose was added and totally repressed by the 2-deoxyglucose at 0.01% (w/v) in solid medium 2. This effect did not correlate with growth inhibition since both strains grow well on glucose media with 0.01% (w/v) 2-deoxyglucose. 2-Deoxyglucose has been previously used to screen mutants of Lipomyces starkeyi with dextranase synthesis resistant to catabolite repression, UV-irradiation being used as the mutagenic factor (Laires 1983). UV- and NMUtreatments were applied to obtain L. kononenkoae mutants resistant to catabolite repression. With UV, exposure for 1 min did not affect cell viability, but after 5 min all cells were killed. Exposure for 2 min gave survival rates of 0.4% and 0.5% for strains 15 and 2896, respectively. However, colonies resistant to catabolite repression were
154
14.8 9.9
World Jorrmal of Micmbrology and Biotechnology. Vol 9, 1993
i = $, lE. 2 $ 8 3 1
Time Figure 1. Growth (U, L. kononenkoae growing symbols; strain 2896-3,
n)
(h)
and glucose consumption on 1% (w/v) glucose. Strain closed symbols.
(0, 0) by 2896, open
Mutant
with
dexfranase
synthesis
“5
Table 3. Growth and accumulation (2896-3) and parent (2896) strains Strain
Starch (% w/v) PH
30
2896
Biomass (WmU
2
E 2
c>
5
.? 2
,
II w
72
dextranase
accumulation
29
Time
Figure
2. Growth
n)
(0,
by L. kononenkoaeon symbols;
strain
and
(h) (0,
0)
1% (w/v) dextran media. Strain 2896, open 2896-3,
closed
symbols.
not detected. NMU as a mutagen yielded 334 mutants resistant to 2-deoxyglucose. Mutants were tested for potential ability to hydrolyse dextran in the presence of glucose by plating on solid media with 1% (w/v) Blue dextran and 1% (w/v) glucose. From the parent strain 15, 87 mutant colonies were obtained that were variants forming clear zones in Blue dextran. Similarly, 14 mutants with derepressed dextranase synthesis were obtained from parent strain 2896. Repression of dextranase synthesis in the mutants was not observed, even with 10% (w/v) glucose. For quantitative activity measurement in mutants capable of efficient dextran hydrolysis in the presence of 10% (w/v) glucose (diameter of clear zone: 15 to 20 mm), growth in liquid medium 3 was carried out (Table 1). Although none of the tested mutants exceeded the parent strains in activity, dextranase synthesis was only slightly repressed by glucose. Stable mutant strain 2896-3 was chosen for further
Strain Activity (U/ml)
pH
5.2 4.2
4.0 2.0
1.9
2.8
4.9
3.4 2.6 2.5
5 10
=‘
of dextranase on starch media.
a.5 8.7
by
mutant
2896-3
Biomass (Wml)
Activity (U/ml)
4.8
13.0 40.7
11.6 12.5
61.6
experiments. De-repression was not affected by disturbances of glucose transport since substrate consumption by the mutant and parent strain proceeded similarly (Figure 1); the only difference was that the mutant strain produced slightly more biomass than the parent strain. Comparative analysis of the dextranase biosynthesis dynamics in the mutant and parent strains on 1% (w/v) dextran medium revealed that the mutant was characterized by earlier initiation of enzyme synthesis (Figure 2). The most significant differences between the strains were observed in the production of dextranase on media with various carbon sources (Table 2); the data presented in Table 2 shows 1% to 75% higher activities in the mutant strain than in parent strain for all the carbon sources examined. Corn starch, predigested with amylase, was also tested as a carbon source (Table 3). Mutant 2896-3 produced high activities of dextranase on media without dextran, the specific inducer. Enzyme synthesis was more efficient on 10% (w/v) saccharified starch compared with dextran media. The mutant may be considered a promising strain for industrial dextranase production, since the economics of the process could be significantly improved by using hydrolyzed starch instead of dextran, the most expensive media constituent.
References R.C. & Freeman, R.F. 1980 Gratuitous catabolite repression by glucosamine of maltose utilization in Saccharomyces cereuisiae. Journal of General Microbiology 121, 479-482.
Hackney,
Kerr,
R.W.
1950
7he Chemistry
and Industry
of Starch.
New
York:
Academic Press. Laires,
A. 1983
Use of D-glucosamine
and 2-deoxyglucose in the Lipomyces starkeyi derepressed for the production of extracellular endodextranase. Zeitschrift fiir Allgemeine Microbiologic 29, 601-603.
selective isolation of mutants of the yeast Table 2. Dextranase sources.
biosynthesis
on media
with various
carbon
Lobanok, A.G. & Zinchenko, Carbon source (2% w/v)
Activity Strain
Dextran Glucose
Maltose Sucrose Soluble
starch
2896
(U/ml) Strain
2896-3
41.1 6.8 5.9
42.0 a.5 10.3
6.3 7.8
7.6 11.6
O.N. 1983 Certain aspects of
dextranase biosynthesis control in micromycetes. In Proceedings of the International SymposiLtm on Regulation of Microbial Mefabolism by Environmental Factors, p, 81. Pushtchino. Lobanok, A.G., Zinchenko, O.N. & Shishlo, V.I. 1981 Some properties of dextranases from the fungus Penicillium piscarittm BIM G-102. Prikladnaya Biokhimiya and Microbiologiya (Moscow) 17, 210-215. (Received
in revisedform
27July
1992;
accepted
8 August
World journal of’ Microbiology and Biofechnolqy, Vol 9, 1993
1992)
155
