Extracellular protease production by psychrotrophic bacteria from glaciers

International Biodeterioration & Biodegradation 29 (1992) 177-189

Extracellular Protease Production by Psychrotrophic Bacteria from Glaciers

R. Margesin* & F. Schinner Institute of Microbiology. University of Innsbruck, Technikerstral3e 25. A-6020 lnnsbruck. Austria (Received 21 August 1991; accepted 9 October 1991)

A BS T R A C T The effects ~?f temperature, time and medium composition on protease .formation and on the growth of 25 psychrotrophic bacteria from glaciers were investigated. In addition to their abili~ to grow at low temperatures, psychrotrophic strains demonstrated a cold dependent enzyme production. More than growth, the excretion of proteases into the medium was shown to be dependent on the cultivation temperature. Protease production by all 25 strains was inlTuenced by medium composition, which demonstrates the temperature dependen~ T of this process.

INTRODUCTION Organisms have adapted to their environments in such a way that metabolic processes, reproduction and survival strategies are optimal in their natural biotopes. The adaptation to extreme conditions led to the development of extremophilic organisms preferring these habitats. One factor that requires particularly high standards of adaptation, is temperature. Psychrotrophic organisms are differentiated from mesophiles by their ability to grow and multiply at temperatures around 0°C. Psychrotrophic micro-organisms from glacial areas are confronted with permanently cold temperatures, with a constant change from freezing to *To whom correspondence should be addressed. 177

International Biodeterioration & Biodegradation 0964-8305/92/$05.00© 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain.

178

R. Margesin. F. Schinner

thawing, as well as with permanently fluctuating substrate conditions. Organisms proliferating in this environment have developed survival strategies of their own by adaptation to low temperatures with regard to growth and enzyme activity. Many psychrotrophic bacteria are known to produce extracellular proteases (Kato etal., 1972~ Jensen etal., 1980: Margesin etal., 1991). Beside their ecological importance, such bacteria may contribute to the deterioration of food (Juffs, 1976; McKellar, 1982: Fairbairn & Law, 1987). Extracellular proteases of psychrotrophic micro-organisms have mainly been investigated with regard to their isolation and characterization, whereas little information is available on the synthesis of exoproteases and on factors influencing the enzyme production (Juffs, 1976~ McKellar, 1982: Fairbairn & Law, 1987). Regulation of protease production has been studied mainly on single organisms. There are only a few comparative investigations (Juffs, 1976). The effect of environmental and of nutritional conditions have been investigated on the protease formation and on the growth of 25 psychrotrophic bacteria isolated from glaciers.

MATERIALS A N D M E T H O D S

Organisms Among 308 protease-producing bacteria isolated from cryoconite of glaciers (black organic accumulations) and from ice water in the European Alps, 25 strains were selected on the basis of their high proteolytic activity (Margesim 1990). Strains were identified as Pseudomonas fluorescens (seven strains), Ps. paucimobilis (seven strains), Ps. cepacia (four strains), Flavobacterium spp. (three strains), Xanthomonas maltophilia (two strains), Aeromonas hydrophila (one strain) and Bacillus sp. (one strain) (Margesin et al., 1991).

Growth conditions In each experiment, cells were first grown at 20°C in nutrient broth for 20 h on a rotary shaker (180 rev min-~). These precultures were inoculated at a final concentration of 1-2.5% into 100 ml Erlenmeyer flasks containing 20 ml of medium. The flasks were incubated on a shaker at 180 rev minfor 72 h. The results presented are the m e a n of three experiments (maximum variation 12% from mean).

Extracellular protease production hy psychrotrophic bacteria

179

Effect of time and temperature Strains were grown at 10, 20 and 30°C in medium B described below. Growth and proteolytic activity were measured at 24 h intervals. The pH of the cultures was measured with a glass electrode. Effect of medium composition Strains were grown at their respective optimum temperature for protease production in the following three media: Medium A: casein (Hammarsten) 0.5%, yeast extract 0-05%, MgSO4.7H20 0.005%, NaC1 0.005%, FeSO4.7H20 0.001% (pH 7.2). M e d i u m B: lactose 2.5%, glycerol 2.5%, corn steep liquor 2%, sodium caseinate 1%, soybean meal 0.5%, CaC12.2H20 0-1%, MgSO4.7H20 0.1% (pH 6-9). M e d i u m C: lactose 0.5%, glycerol 0.5%, soybean meal 1%, yeast extract 0.1%, MgSO4.7H20 0.01%, NaC1 0.05% (pH 6.9). Additionally, the media contained a buffer system composed of K H 2 P O 4 0"025% and Na2HPO4.12H20 0.25%. Determination of growth Optical density at 660 nm was used as an index of growth. Protease assay Cell-free culture supernates prepared by centrifugation at 10 000g and 4°C for 20 min were assayed for proteolytic activity using azocasein as substratc (Margesin and Schinncr, 1991). Reactions were carried out at 30°C and pH 7 for30 min. One azocasein digestion unit (ACU) of activity was defined as the increase of 0"001 per min in the absorbance at 340 nm. Assays were performed in triplicate and averaged.

RESULTS Effects of time and temperature on growth and protease production Rates of protease production and of growth at different temperatures were obtained by the temperature coefficient value Q t0. Since proteolytic

180

R Margesin, F. Schinner

activities of the investigated strains were often very high at low temperatures, the reciprocal values (1/Qi0) were calculated. While all 25 strai~as grew at 10 a n d at 20°C, only 14 strains were able to grow at 30°C after 48 h of cultivation. The latter strains grew better at 20 than at 30°C. After 96 h, 16 strains showed c o m p a r a b l e cell densities at 10 and at 20°C (I/Q., < 1.20) while 9 strains showed a distinctly better growth at 10 than at 20°C (Table 1). More than growth, the excretion of proteases into the m e d i u m was shown to be d e p e n d e n t on the cultivation temperature. Contrary to all 25 strains growing at 10 and 20°C and forming protease at 10°C, four of them did not produce protease at 20°C. Of these strains, two were unable to grow at 30°C, and two produced no enzyme at 30°C although they grew at this temperature. Of the 14 strains growing at 30°C, only four formed protease. With the exception of the Bacillus sp. strain, protease formation was highest at 10°C. A cultivation temperature of 30°C was not the best temperature for protease formation for any of the strains. Only six and three strains respectively showed a higher protease production at 20 than at 10°C (1/Qi. < i) after 72 h and 96 h respectively. Ten strains were characterized by a markedly higher protease formation at 10 than at 20°C at comparable cell densities (Table 2). The relationship between proteolytic activity a n d growth rates was used as an approximate assessment of the variation in protease formation relative to the growth. These ratios changed with the temperature as well as with time of incubation. The highest proteolytic activities were achieved in the stationary growth phase corresponding to an incubation time of 72-96 h. W h e n cultivating strains at 10°C, growth a n d protease formation were delayed by 24 h in c o m p a r i s o n to those at 20 and 30°C. The pH values in the culture fluids increased in parallel with growth a n d the excretion ofprotease into the m e d i u m up to p H 9. Similar results were obtained by Whooley & M c L o u g h l i n (1983) who found a positive correlation between p H value a n d protease formation by Ps. aeruginosa.

Effect of medium on protease production

Protease production by all 25 strains was shown to be d e p e n d e n t on the m e d i u m composition. Of the thl:ee media tested, the majority of strains (18) produced the highest a m o u n t of protease when cultivated in m e d i u m C. A m o n g them. eight strains produced more protease in the nutrient-poor m e d i u m A than in the nutrient-rich m e d i u m B: the other

Extracellular protease production by psychrotrophic bacteria

181

ten strains p r o d u c e d no protease in m e d i u m A. All 25 strains formed protease w h e n cultivated in m e d i u m C. Six strains formed the highest a m o u n t s of protease in the most complex of the media (B); protease excretion of these strains was depressed in m e d i u m A. Only one strain preferred the nutrient-poorest of the media tested (A) for protease production, whereas 19 strains formed none or only small a m o u n t s of protease in this m e d i u m (Fig. 1).

Distribution of proteolytie activity Protease formation was shown to be strongly influenced by the environmental a n d nutritional conditions. Nevertheless, the single strains exhibited differing proteolytic activities. The majority of strains (18), a m o n g t h e m all P. paucimobilis strains, formed proteases in the lower range (up to 2500 A C U ml-t). Only a few species were characterized by a very high protease excretion (up to 10000 A C U ml-~). The highest proteolytic activity a m o n g the 25 strains was shown by A. hydrophila, p r o d u c i n g 15 000 A C U ml -~ (Table 3).

DISCUSSION A limited n u m b e r of studies have been concerned with the nutritional requirements for protease production by psychrotrophic bacteria. Our studies on the effect of nutritional conditions on protease formation indicate that this process is temperature dependent. Similar results were obtained with psychrotrophic Ps. fluorescens (McKellar, 1982; Fairbairn & Law, 1987). A l t h o u g h the strains investigated were isolated from habitats with low organic substrate concentrations, nutrient-rich media stimulated the excretion of protease to a greater degree t h a n nutrientp o o r media. Protease p r o d u c t i o n by m a n y psychrotrophic p s e u d o m o n a d s was shown to be an essential function of the organic nitrogen content of the m e d i u m (Juffs, 1976; Fairbairn & Law, 1987). However, the incubation temperature can modify the influence of m e d i u m c o m p o n e n t s on protease production (Juffs, 1976). Most of the strains were characterized by a low o p t i m u m temperature for growth as well as for protease production, thus indicating a high resistance to low-temperature stress. Heat sensitivity of cold-adapted bacteria was found to be caused by inhibition of protein-synthesizing

045/12 047/08 049/03 053/08 054/01 150/11 159/08 164/03 164/13 165/14 167/28

Strain

TABLE !

Flavobacterium sp. Xanthomonas maltophilia Pwudomonas paucimobilis Flavobacterium sp. Xanthomonas maltophilia Ps'eudomonas.fluorescens Aeromonas hydrophila Pwudomonas fiuorescens P~'eudomonas cepacia Pseudomonasfluorewens Ps'eudomonas cepacia

Identification

0-34 0.26 0.21 0-31 / 0.34 0.17 O. 33 0.22 0.22 0-28

24 h I).86 0.88 0.94 0-87 0.83 0.74 0.68 0- 88 0.88 0.95 1-06

48 h 0.97 1-03 1.34 1-01 1.05 1.05 1.23 1-00 1.04 1.20 1.12

72 h

1.02 1.07 1.64 1-22 1.29 0.95 1.20 O. 95 1.01 1.41 1.14

96 h

Growth at IO°C/growth at 20°C

-----0.87 1.77 O. 86 1-21 I. I0 1.17

24 h

-----1.12 1.35 I. 04 1.32 1.32 1.12

48 h

Growth at 20°C/g

T e m p e r a t u r e C o e f f i c i e n t s o f G r o w t h o f 25 P s y c h r o t r o p h i c B a c t e r i a . S t r a i n s were G r o w n in M e d i u m C at I£ (/: N o G r o w t h at 10°C; - - N o G r o w t h at 3 0 ° C )

n Mean value SD

172/15 175/09 177/21 177/27 177/30 178/02 178/13 180/01 182/51 189/06 189/07 189/20 190/10 190/22

Bacillus sp. P~eudomonas cepacia Ps'eudomonasfiuorescens bTavobacterium sp. P~eudomonasfluorescens P~eudomonasfiuorescens P~eudomonas paucimobilis Pseudomonas paucimobilis Pseudomonas)quorescens Ps'eudomonas paucimobilis P~eudomonas cepacia P~'eudomonas paucimobilis Pseudomonas paucimobilis P~eudomonas paucimobilis 19 0-29 0.18

0.10 0. 15 0-94 / 0-26 / 0.11 / 0.46 0-23 0.33 0.19 / / 25 0.85 0.17

0.30 0.88 1.25 0.68 0.91 0.66 0.88 0.70 0.93 0.90 1.04 0.91 0.83 0.70 25 1. I 1 0.17

0-88 1.05 1.25 I-31 1.00 1.07 1-70 0.92 1.05 I. 16 1.06 1.14 1.15 1-09 25 I. 18 0-18

1.15 1.03 1-16 1.51 1.06 1-09 1-30 1.08 I. 09 1.03 1-12 1-36 1-46 I. 14 14 1-24 0.52

0.93 2-84 0.96 -I. 11 ---0.90 -I. 17 -1.46 1.06 14 1.32 0.40

1-30 2.67 1.20 -1.22 ---I. 1 I -1.24 -1.38 I. 13

TABLE

2

* *#

*# *#

/#

*

0.01 * *#

* *#

049/03 0 53 /0 8

054/01

150/11

159/08 164/03 164/13

165/14 167/28

24 h

0.37 #

0-26 0.39 0.78

O.84

3.75

* #

2.53 #

48 h

0.41 #

0-90 1.13 3-95

0- 64

6.61

1-54 #

6.57 #

72 h

0-63 #

1.14 1.58 3.55

1.29

5.41

1-20 #

8-21 #

96 h

Protease production at lO°C/protease production at 20°C

0 45 /1 2 0 47 /0 8

Strain

x #x

2-54 x #x

×

--

---

---

24 h

× #×

3.64 x ×

X

--

---

---

48 h

#x

×

4.23 × ×

X

--

---

---

72 k

Protease production at 20°C/protease p+

T e m p e r a t u r e C o e f f i c i e n t s o f P r o t e a s e P r o d u c t i o n b y 25 P s y c h r o t r o p h i c B a c t e r i a . S t r a i n s w e r e G r o w n in M e d i u m ( (/: N o G r o w t h at 10°C: - - : N o G r o w t h a t 3 0 ° C : *: N o A c t i v i t y at 10° C : # : N o A c t i v i t y at 2 0 ° C : × : N o A c t

* *#

*#

#*

/

/#

182/51 189/06

189/07

189/20

190/10

190/22

Mean value SD

3

0.07 0.05

/#

180/01

n

*

/# *#

/#

177/27

178/02 178/13

0.10

177/21

177/30

* 0.09

172/15 175/09

0.87 0.98

17

#

0.27

0.50

1.74

I-26 *#

*

O- 14 0.83

0.60

*

0.07

O. 15 0"36

2.42 2.01

21

#

1.69

1.62

4.57

3.58 1-00

4.85

4.65 2-16

0-77

1.77

0.77

0.39 4.29

3-03 2-47

21

#

1-33

2.05

7.06

4-59 1.00

5-56

6.34 1.71

0.76

1-91

1.13

0.86 6" 51

2 1.96 0.83

#x

×

--

#×

x --

--

---

x

--

X

1"37 X

2.26 0.98

4

#X

2.26

--

x

1.69 --

--

---

x

--

x

1.44 X

4 2.29 1.33

#X

1.38

--

×

2-06 --

--

---

x

--

X

1.47 X

186

R. Margesin, F. Schinner

°4g/°3H.~..Jmm Strain

164/lz

167/28

~/////////~

178/02 -q 178/13

--q 189/06 189/20 ~ 190/10

/~/////////////A I

0

100

i

I

I

I

200

300

400

500

600

Protease activity (ACU m1-1)

Strain

047/08-~ 053/08

W

+/////////A

054/01 ~__q//

/////////////////////////j

150/11

I

172/15 175/09 ~ 180/01 189/07

~

190/22 0

500

1 000

1 500

2 000

2 500

Protease activity (ACU m1-1)

Fig. 1. Protease production by 25 psychrotrophic bacteria in medium A (E3), medium B ([]) and medium C (11). Strains were cultivated for 72 h at their respective temperature optimum.

Extracellular protease production by p,~chrotrophic bacteria Strain

o4s/12

187

-L..((...~

159/08 " / / / / / / / / / / / / / / / / / / / / / J / / / / / / / ~ 164/03

"1z//////////////////////1 Ill

165/14 " / / # / / / / / # / / / / / / / / / / / / ' / / / / / / / / f / / I / / / / / / / / / A 177/21

177/27

t

177/30 " / / I / / / / / / / / / / / / / / / / / / / / / / / / / / , / / / / / A 182/~ 1 i

i

i

i

2. 000

4 000

6 000

8 000

10 000

Protease activity (ACU m1-1)

mechanisms, by inactivation of respiratory and other enzymes, and by instability or by lack of synthesis of RNA (Stokes, 1967: Harder & Veldkamp, 1968: Malcolm, 1969: Bobier etal., 1972). No changes in growth requirements at different cultivation temperatures were observed with a psychrotrophic Ps. aeruginosa (Brown, 1957). Higher cell yields at low temperatures may be due to the increased solubility, and therefore availability, of oxygen (Sinclair & Stokes, 1963). Within certain limits,

TABLE 3 Proteolytic Activity of 25 Psychrotrophic Bacteria. Strains Were Cultivated in Their Respective O p t i m u m M e d i u m a n d at Their Respective O p t i m u m Temperature for 72 h

Proteolytic activity (ACU ml- l) N u m b e r of strains <1000 1 001-2 2 501-5 5 001-7 7 501-10 >10

500 000 500 000 000

9 9 2 2 2 1

188

R. Margesin. F. Schinner

cells appear to be able to compensate for the effect of temperature on growth by increasing the production of enzymes of the energy-generating system (Inniss & Ingraham, 1978). Low temperatures stimulate the formation of larger cells and of flagella as well as the transport of solutes across the cell m e m b r a n e (Jay, 1986). In addition to their ability to grow at low temperatures, psychrotrophic strains demonstrated a cold-dependent enzyme production. We observed that the temperature optimum for protease formation was generally considerably lower than that for growth. This may be due to the adaptation of metabolic processes to the conditions of the natural environment of the strains~ but it can also be explained on the basis of enzyme inactivation, as well as on the basis of inactivation of the enzyme-synthesizing reactions at 'high" temperatures (Stokes, 1967). However, the variations caused by changing the cultivation temperature indicate a substantial difference in cell organization and metabolism at each temperature (Kato et al.. 1972). Similar results were obtained with psychrotrophic strains ofPs. fluorescens (Juffs, 1976: McKellar, 1982) and Pseudomonas sp. (Kato etal., 1972), whereas proteolytic activities of psychrotrophic Arthrobacter sp. (Potier et al., 1987) andPs, aeruginosa (Juffs, 1976) were related directly to growth. Further investigation of the properties of the proteases produced by the 25 strains studied showed that these enzymes were characterized by a temperature optimum of around 40°C, a high sensitivity against heat treatment and an activation energy lower by 10-20 kJ mol -~ on average than proteases from mesophilic sources (Margesin et al., 1991). Therefore, the existence of dual strategies of cold adaptation is supposed, on the one hand, to increase enzyme production at low temperatures and, on the other, to provide a low temperature optimum at the level of enzyme activity (Reichardt, 1989). Hydrolases with the observed properties could be important for potential applications in biotechnological processes requiring low temperatures.

ACKNOWLEDGEMENT This work was supported by the Biochemie G m b H , Kundl, Austria.

REFERENCES Bobier, S. R., Ferroni, G. D. & Inniss, W. E. (1972). Protein synthesis by the psychrophiles Bacillus psychrophilus and Bacillus insolitus. Can. J. Microbiol.. 18, 1837-43.

Extracellular protease production by psychrotrophic bacteria

189

Brown, A. D. (1957). Some general properties of a psychrophilic pseudomonad: the effects of temperature on some of these properties and the utilization of glucose by this organism and Pwudomonas aeruginosa. J. Gen. Microbiol., 17, 640-8. Fairbairn, D. J. & Law, B. A. (1987). The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens. J. Appl. Bacteriol., 62, 105-13. Harder, W. & Veldkamp, H. (1968). Physiology of an obligately psychrophilic Pseudomonas species. J. Appl. Bacteriol.. 31, 12-23. Inniss, W. E. & Ingraham, J. L. (1978). Microbial life at low temperatures. Mechanisms and molecular aspects. In Microbial Life in Extreme Environmerits, ed. D. J. Kushner. Academic Press, London, pp. 73-104. Jay, J. M. (1986). Characteristics and growth ofpsychrotrophic microorganisms. In Modern Food Microbiology. 3rd edn. Van Nostrand Reinhold Company, New York, pp. 579-92. Jensen, S., Feceycz, I. T. & Campbell, J. N. (1980). Nutritional factors controlling exocellular protease production by Pseudomonas aeruginosa. J. Bacteriol.. 144, 844-7. Juffs, H. S. (1976). Effects of temperature and nutrients on proteinase production by Pseudomonasfluorescens and P~. aeruginosa in broth and milk. J. Appl. Bacteriol.. 40, 23-32. Kato, N., Nagasawa, T., Tani, Y. & Ogata, K. (1972). Protease formation by a psychrophilic bacterium. Agr. Biol. Chem.. 36, 1177-84. McKellar, R. C. (1982). Factors influencing the production of extracellular proteinase by Pseudomonas fluorescens. J. Appl. Bacteriol.. 53, 305-16. Malcolm, N. L. (1969). Molecular determinants of obligate psychrophily. Nature. 221, 1031-3. Margesin, R. (1990). Proteasen psychrophiler Bakterien. PD thesis, University of lnnsbruck, Innsbruck. Austria. Margesin, R. & Schinner, F. (1991). Characterization of a metalloprotease from psychrophilic Xanthomonas maltophilia. FEMS Microbiol. Lett., 79,257-62. Margesin, R., Palma, N., Knauseder, F. & Schinner, F. (1991). Proteases of psychrotrophic bacteria from glaciers. J. Basic Microbiol., 31(5) 377-83. Potier, P., Drevet, P., Gounot, A. M. & Hipkiss, A. R. (1987). Proteolysis in cellfree extracts of the psychrophilic bacterium Arthrobacter sp. 55: effects of growth temperature and ATP. Biochem. Soc. Trans., 15, 968-9, Reichardt, W. (1989). Cold-adaptation of biopolymer-degrading bacteria from permanently cold environments, is there a biotechnical potential? In Microbiology of Extreme Environments and lts Potential for Biotechnology, ed. M. S. Da Costa, J. C. Duarte & R. A. D. Williams. Elsevier Applied Science Publishers, p. 405. Sinclair, N. A. & Stokes, J. L. (1963). Role of oxygen in the high cell yields of psychrophiles and mesophiles at low temperatures. J. Bacteriol., 85, 164-7. Stokes, J. L. (1967). Heat-sensitive enzymes and enzyme synthesis in psychrophilic microorganisms. Science, 84, 311-23. Whooley, M. A. & McLoughlin, A. J. (1983). The protonmotive force in Pseudomonas aeruginosa and its relationship to exoprotease production. J. Gen. Microbiol., 129, 989-96.