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Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis
Journal of Biotechnology 70 (1999) 53 – 60
Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis Marianna Turkiewicz *, Ewa Gromek, Halina Kalinowska, Maria Zielin´ska Institute of Technical Biochemistry, Technical Uni6ersity of Lodz, 4 /10 Stefanowskiego Street, Lodz 90 -924, Poland Received 14 October 1998; received in revised form 26 November 1998; accepted 22 December 1998
Abstract An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at − 5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Psychrophilic enzymes; Metalloprotease; Antarctic marine bacteria
1. Introduction
Abbre6iations: BSA, bovine serum albumin; BzTyrOEt, Nbenzoyl-tyrosine ethyl ester; CFU, colony forming unit; EDTA, ethylenediaminetetraacetic acid; Hb, haemoglobin; IAA, iodoacetic acid; PAGE, polyacrylamide gel electrophoresis; pCMB, p-chloromercuribezoate; PMSF, phenylmethylsulfonyl fluoride; Rm, relative mobility; TCA, trichloroacetic acid; Tris, tris(hydroxymethyl)aminomethane. * Corresponding author. Tel.: + 48-42-6366618; fax: + 4842-6313402. E-mail address: [email protected] (M. Turkiewicz)
The overwhelming majority of commercially important enzymes are produced by mesophilic microorganisms and applied in conditions resembling their environment in vivo. New biocatalysts active in unusual conditions are looked for among extremophilic microorganisms including psychrophiles and psychrotrophs (Adams et al., 1995). They constitute the most abundant group of organisms, since temperatures below 5°C occur in about 80% of biosphere and even more than 90% of marine environment (Margesin and Schinner, 1994; Brenchley, 1996).
0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 0 5 7 - 7
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M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
Cold-adapted enzymes secreted by these microorganisms, also called psychrophilic enzymes or psychrozymes, show high catalytic efficiency at low temperatures, especially at 0 – 20°C, i.e. a temperature range at which mesozymes are usually inactive, as well as reduced thermostability resulting in denaturation at moderate temperatures (Margesin and Schinner, 1994; Feller et al., 1996). Cold enzymes are potentially useful for numerous biotechnological processes, especially those which require a supply of exogenous energy, are exposed to higher risk of microbial contamination or temperature instability of reactants or products (Margesin and Schinner, 1994; Marshall, 1997). Another profit resulting from studies on microbial psychrozymes is an enrichment of pure culture collection by strains existing in permanently cold environments, e.g. marine polar regions (DeLong, 1997). Information acquired in this way are important for development of our still very limited knowledge of biodiversity.
rpm. The strain growth was observed by determination of CFUs on solid growth medium (0.3% of beef extract, 0.5% of bactotryptone and 5.51% of bacto-marine agar 2216, Difco) or by absorbance reading at 660 nm. All other chemicals employed in the studies were purchased from standard sources and were at least reagent grade.
2.2. Protease assay Proteolytic activity was determined according to Anson (1938) using urea denatured Hb (pH 7.0, 30°C, reaction time 15 min) or spectrophotometrically according to Hummel (1959) using Nbenzoyl-tyrosine ethyl ester (BzTyrOEt) in 50 mM tris(hydroxymethyl)aminomethane (Tris)–HCl buffer pH 8.0 at 20°C (increase of absorbance at 254 nm). In both methods, the activity was expressed in mmol of product (L-tyrosine present in trichloroacetic acid (TCA)-soluble hydrolysis products or N-benzoyl-L-tyrosine, respectively) liberated from respective substrate for 1 min in standard conditions.
2. Materials and methods
2.3. Protein determination
2.1. Organism and culti6ation
Protein was assayed according to Lowry et al. (1951) using bovine serum albumin (BSA) as a standard, or spectrophotometrically at 280 nm in fractions eluted from chromatography columns.
Gram-negative rods Sphingomonas paucimobilis, strain no. 116, from the collection of the Department of Antarctic Biology of Polish Academy of Sciences in Warsaw, were used for the studies. This strain was isolated from a stomach of Antarctic krill, Euphausia superba Dana by Stuart Donachie at the Arctowski Polish Antarctic Station. The strain classification was done using API ZONE strips (Api System, Biomerieux, France) at 15°C as well as microscopic observations and Gram staining (Donachie, 1995). Shaken cultures of the strain were performed at pH 8.0 in a liquid medium containing synthetic marine water (Instant Ocean, Aquarium System Inc., France), 0.2% of bactopeptone and 1% of casein digest (Difco). One hundred and twenty ml of the medium in a 500 ml flask was inoculated with a standard inoculum (4% v/v, 1.5 ×105 CFU (colony forming units) ml − 1) and cultivated at 10°C in a shaker (INFORS-Switzerland) at 160
2.4. Electrophoresis Polyacyrlamide gel electrophoresis (PAGE) was run in nondenatured conditions, according to Laemmli (1970), using 7.5% gels (0.6×8 cm) in 0.1 M borate buffer pH 9.0 and current intensity 2.5 mA per gel. Gels were fixed and stained with Coomassie brillant blue R-250. Proteolytic activity was assayed in 2 mm slices of nonstained gels against denatured Hb (2% v/w, 30°C, pH 7.0) as a substrate.
2.5. Protease purification Purification of the protease was carried out at 4°C and in presence of Ca2 + ions (0.5 mM) as a stabilizing factor. The protein was precipitated
M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
from the centrifuged (5000 × g, 30 min) culture medium with ammonium sulphate (80% of saturation, 1 h, 0°C), dissolved in 10 mM sodium phosphate buffer pH 7.0 and desalted on Sephadex G-25 (0.9×20 cm) equilibrated with the same buffer. Five ml protein fractions (A280 above 0.8) were pooled and applied to a bacitracin-Sepharose 4B column (0.9×10 cm, the buffer as above), prepared according to Stepanov and Rudenskaya (1983). The column was prewashed with the starting buffer (flow rate 20 ml h − 1) and the bound proteins were eluted using this buffer enriched with 25% (v/v) of isopropanol and 1 M of NaCl. Active fractions were pooled, dialysed against the starting buffer and stored in small aliquots at −40°C.
3. Results
3.1. Effect of temperature on growth and protease secretion S. paucimobilis 116 was grown at temperatures from 0 to 30°C. The highest proteolytic activity in the culture medium was observed in a range from 5 to 10°C (about 0.25 – 0.26 units ml − 1, Fig. 1). At 15°C which is optimal for the strain growth, as well as at 0°C, the activity was twice lower. At 30°C the strain did not excrete extracellular proteases. Above 30°C its growth was not observed. At 10°C which was chosen as a standard
Fig. 1. Effect of S. paucimobilis 116 growth temperature on synthesis of extracellular proteases. Proteolytic activity was assayed in culture medium after 72 h of cultivation, in standard conditions, using urea denatured Hb.
55
Fig. 2. Relationship between the strain growth at 10°C and the proteolytic activity in the culture medium during 7 days of cultivation. Proteolytic activity was assayed in standard conditions using urea denatured Hb.
strain cultivation temperature, the protease synthesis occurred to run simultaneously to the growth and reached the maximum after 72 h, i.e. at a late logarithmic phase of bacterial growth (Fig. 2). Protein pattern of the third day supernatant of the culture medium shows Fig. 3. Two protein bands were active against denatured Hb. The proteolytic activity present in this supernatant was inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF) (34% of inhibition) and 1 mM EDTA (ethylenediaminetetraacetic acid) (38% of inhibition) which proves that S. paucimobilis 116
Fig. 3. Electophoretic pattern of proteins and its relationship with proteolytic activity in the culture medium after 72 h of the strain growth.
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M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
Fig. 4. Elution profile of proteins (A280) and proteolytic activity in affinity chromatography of the strain proteases on bacitracin-Sepharose 4B. The arrow shows the start of elution with 25% (v/v) isopropanol.
S. paucimobilis 116 protease was most active at 25–30°C for 15 min, and at 15–20°C for 60 min, against urea denatured Hb (Fig. 6). At − 10 to 0°C the enzyme retained 30–47% of maximal activity. The apparent energy of activation for the hydrolysis of urea denatured Hb calculated from an Arrhenius plot was 24.3 kJ mol − 1. The S. paucimobilis 116 metalloprotease is very thermolabile, since the complete stability was observed for 15 min up to 30°C and for 60 min up to 20°C (Fig. 7). At 45°C only 38% of the activity remained after 15 min, and after 60 min it was inactive. The metalloprotease digested Hb at pH from 5.0 to 10.5 with maximum at pH 6.5–7.0
produces extracellular serine proteases and metalloproteases. p-Chloromercuribezoate (p-CMB) and iodacetic acid (IAA) did not influence the activity.
3.2. Metalloprotease properties The highly purified metalloprotease was obtained using the procedure described in Section 2. The enzyme was isolated from 760 ml of the supernatant from 72 h S. paucimobilis 116 culture medium (Table 1). Specific activity of the pooled active fractions after affinity chromatography (Fig. 4) was about 20-fold higher in comparison to the starting supernatant, both against urea denatured Hb and BzTyrOEt. Although bacitracin-Sepharose 4B coupled all the proteolytic activity (Fig. 4), the fractions eluted from this column contained only 10% of it. The eluted proteolytic activity was in 92% inhibited by EDTA (final concentration 1 mM, enzyme-inhibitor incubation time 1 h) and not influenced by PMSF. It proves that only the metalloprotease was eluted from the column and the serine protease present in the starting material remained bound to bacitracin-Sepharose 4B. The yield of purification was low (3 – 4%, Table 1). PAGE of this highly purified enzymic preparation run in non-denatured conditions showed one strong protein band with relative mobility (Rm) 0.58 contaminated with trace amount of another protein with Rm 0.83 (Fig. 5).
Fig. 5. PAGE of the highly purified metalloprotease from S. paucimobilis 116 (pooled active fractions after bacitracin-sepharose 4B column).
Purification step
Culture supernatant Salted out protein Sephadex G-25 Bacitracin-Sepharose 4B
Volume (ml)
760 10 43 5.4
Protein (mg)
313.20 29.84 11.70 0.46
Activity Specific (units mg−1 of protein)
Total (units)
Hb
BzTyrOEt
Hb
BzTyrOEt
0.23 1.40 2.33 5.60*
0.04 0.15 0.28 0.72*
71.82 41.84 27.30 2.60
11.70 4.39 3.29 0.33
* The activity was inhibited by EDTA (in 92%) and not by PMSF.
Yield (%)
Purification (fold)
Hb
BzTyrOEt
Hb
BzTyrOEt
100 59 38 3.6
100 38 28 3
– 6 10 24
– 4 8 19
M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
Table 1 Purification of a metalloprotease from S. paucimobilis 116
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M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
Fig. 6. Effect of temperature on the activity of the S. paucimobilis 116 metalloprotease. The activity was assayed for two reaction times: (a) 15; and (b) 60 min, using urea denatured Hb pH 7.0.
(Fig. 8). Optimal pH in BzTyrOEt hydrolysis was 8.0 and at pH 10.5 the enzyme showed 30% of the maximal activity (Fig. 8). At 4°C the protease was stable at pH 6.0–8.0 for 60 min, and at pH 9.5 exhibited 91% of maximal activity (data not presented). The preferred substrate of the highly purified metalloprotease from S. paucimobilis 116 was denatured Hb (Table 2). Its activity against casein, fibrinogen and native Hb was 20 – 30% lower. BSA was digested to a small extent.
4. Discussion A metalloprotease active against various proteins at neutral and slightly alkaline pH was isolated from extracellular proteases secreted by
Fig. 8. Influence of pH on the activity of the S. paucimobilis 116 metalloprotease. The enzyme activity was assayed against urea denatured Hb (a) and BzTyrOEt (b).
S. paucimobilis strain 116, isolated from stomachs of Antarctic krill E. superba Dana. S. paucimobilis 116 synthesizes extracellular proteases with highest intensity at a temperature markedly lower (5–10°C) than corresponding to its maximal growth rate (15°C), similarly to many other coldadapted microorganisms, including Antarctic ones (Margesin and Schinner, 1994; Feller et al., 1996). It is presumed that intensification of enzyme biosynthesis at low temperatures is one of the main mechanisms of adaptation of these organisms to surrounding environment (Margesin and Schinner, 1994; Brenchley, 1996). Secretion of proteases by S. paucimobilis 116 starts already in the phase of adaptation and its maximum coincides with the end of logarithmic phase, falling on 72 h. This period of time is shorter than observed e.g. by Margesin and Schinner (1992a) for a metalloprotease from Alpine Table 2 Activity of the S. paucimobilis 116 metalloprotease against selected proteins
Fig. 7. Thermostability of the S. paucimobilis 116 metalloprotease. The enzyme was incubated at different temperatures (0–50°C) for: (a) 15; and (b) 60 min and the residual activity towards urea denatured Hb was determined in standard conditions (30°C, pH 7.0).
Substrate
Activitya (units ml−1)
Hb denatured Hb native Casein Fibrinogen BSA
0.249 0.160 0.200 0.177 0.017
a Assay conditions: 30°C, pH 7.0, 2% solutions of protein substrates, 15 min reaction.
M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
strain of Pseudomonas fluorescens (96 h) or for subtilisin from Antarctic Bacillus TA 39 (150 h, Feller et al., 1996). Electrophoretic analysis and inhibitory tests proved that S. paucimobilis 116 produces at least two extracellular proteases, i.e. a metallo- and a serine enzyme. However we have managed to purify only the metalloenzyme. The serine protease coupled with bacitracin-Sepharose 4B but was immobile in a milieu of the eluent employed in our studies. According to Stepanov and Rudenskaya (1983) ligand from bacitracin-Sepharose possesses various amino acid residues and therefore binds not only serine proteases, but some other too which was revealed in model experiments on papain, subtilisin and B. subtilis metalloprotease. Kinetic properties such as very weak thermostability and low optimal temperature (15 – 30°C depending on reaction time) of the S. paucimobilis 116 metalloprotease point to its increased adaptation to low temperatures in comparison to many other enzymes, also originating from permanently cold, e.g. Antarctic habitats. Reported metalloproteases from other strains inhabiting cold environments usually exhibit higher optimal temperatures. Good examples are proteases from Alpine strain of P. fluorescens 114, most active at 40 – 45°C for 30 min (Hamamoto et al., 1994) and from Xanthomonas maltophilia with maximal activity at 50°C for 15 min (Margesin and Schinner, 1991). However, a protease isolated by Ka¨rst et al. (1994) from an unidentified Antarctic bacterial strain was most active against proteins at 28°C. Another important feature pointing to the psychrophilic character of the metalloprotease from S. paucimobilis 116 is its high activity at temperatures from 0 to 5°C (47 – 60% of maximal activity), resembling the strain physiological temperature range, as well as the ability to protein hydrolysis below 0°C (e.g. at−10°C the enzyme shows 30% of maximal activity against urea denatured Hb) which was not reported for other coldadapted proteases. For comparison, a metalloprotease from P. fluorescens 114 exhibited 20 – 30% of maximal activity at temperatures from 0 to 10°C (Hamamoto et al., 1994), enzymes from Alpine strains showed 15 – 18% of maximal activ-
59
ity at 10°C (Margesin and Schinner, 1992b), the protease from X. maltophilia did not hydrolyse azocasein at 0°C and at 10°C retained only about 5% of maximal activity (Margesin and Schinner, 1991). The value of activation energy calculated for the S. paucimobilis 116 (24.3 kJ mol − 1) is lower than obtained for enzymes from X. maltophilia (61.9 kJ mol − 1, Margesin and Schinner, 1991), psychrophilic P. fluorescens strains (36.9– 38.0 kJ mol − 1, Margesin and Schinner, 1992a) or Antarctic psychrophile Bacillus TA41 (38.5 kJ mol − 1, Davail et al., 1994). In all the cases, values of activation energies of mesozymes tested under the same conditions were even higher (Margesin and Schinner, 1991, 1992a; Davail et al., 1994). The reduction of energy barrier displayed by the S. paucimobilis 116 metalloprotease and correlated with its low temperature optimum and weak thermostability may result from more flexible conformation of the molecule in comparison to enzymes mentioned above. A potentially useful property of the S. paucimobilis 116 metalloprotease might be its relatively high activity towards native Hb, equal to about 70% of the activity against urea denatured Hb. Cold-adapted proteases may be applied in many fields, e.g. in production of detergents for washing at low temperatures, in tannery, in the food industry (haze removal from beer, bakery, cheese-making, production of fermented foods, meat tenderisation) or high protein waste degradation (Margesin and Schinner, 1994; Brenchley, 1996). Our studies demonstrate that proteases synthesized by marine Antarctic bacteria might become an interesting alternative to currently applied mesophilic enzymes.
References Adams, M.W.W., Perler, F.B., Kelly, R.M., 1995. Extremozymes: expanding the limits of biocatalysis. Bio/ Technology 13, 662 – 668. Anson, M.L., 1938. Estimation of pepsin, trypsin papain and cathepsin with haemoglobin. J. Gen. Physiol. 22, 79 – 82. Brenchley, J.E., 1996. Psychrophilic micro-organisms and their cold-active enzymes. J. Ind. Microbiol. 17, 432 – 437. Davail, S., Feller, G., Narinx, E., Gerday, C., 1994. Cold adaptation of proteins. J. Biol. Chem. 269, 17448 – 17453.
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M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60 Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 165 – 275. Margesin, R., Schinner, F., 1991. Characterization of a metalloprotease from psychrophilic Xanthomonas maltophilia. FEMS Microbiol. Lett. 79, 257 – 262. Margesin, R., Schinner, F., 1992a. Production and properties of an extracellular metalloprotease from a psychrophilic Pseudomonas fluorescens. J. Biotechnol. 24, 207 – 210. Margesin, R., Schinner, F., 1992b. A comparison of extracellular proteases from three psychrotrophic strains of Pseudomonas fluorescens. J. Gen. Appl. Microbiol. 38, 209 – 225. Margesin, R., Schinner, F., 1994. Properties of cold-adapted micro-organisms and their potential role in biotechnology. J. Biotechnol. 33, 1 – 14. Marshall, C.J., 1997. Cold-adapted enzymes. Trends Biotechnol. 15, 359 – 363. Stepanov, V.M., Rudenskaya, G.N., 1983. Proteinase affinity chromatography on bacitracin-Sepharose. J. Appl. Biochem. 5, 420 – 428.
DeLong, E.F., 1997. Marine microbial diversity. Trends Biotechnol. 15, 203 – 207. Donachie, S.P., 1995. Ecophysiological description of marine bacteria from Admiralty Bay (Antarctica) and the digestive tracts of selected Euphausiidae, Philosophy Doctor Thesis, Polish Academy of Sciences, Warsaw. Feller, G., Narinx, E., Arpigny, J.L., Aittaleb, M., Baise, E., Genicot, S., Gerday, Ch., 1996. Enzymes from psychrophilic organisms. FEMS Microbiol. Rev. 18, 189–202. Hamamoto, T., Kaneda, M., Horikoshi, K., Kudo, T., 1994. Characterization of a protease from a psychrotroph Pseudomonas fluorescens 114. Appl. Environ. Microbiol. 60, 3878 – 3880. Hummel, B.C.W., 1959. A modified spectrophotometric method for determination of chymotrypsin and thrombin. Can. J. Biochem. Physiol. 37, 1393–1400. Ka¨rst, U., Woehl, M., Czempinski, K., Schmid R.D., 1994. Characterization of extracellular hydrolases from marine psychrophilic bacteria. In: Seventh International Congress of Bacteriology and Applied Microbiology, Praha, Communicate MO122.
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Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis Marianna Turkiewicz *, Ewa Gromek, Halina Kalinowska, Maria Zielin´ska Institute of Technical Biochemistry, Technical Uni6ersity of Lodz, 4 /10 Stefanowskiego Street, Lodz 90 -924, Poland Received 14 October 1998; received in revised form 26 November 1998; accepted 22 December 1998
Abstract An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at − 5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Psychrophilic enzymes; Metalloprotease; Antarctic marine bacteria
1. Introduction
Abbre6iations: BSA, bovine serum albumin; BzTyrOEt, Nbenzoyl-tyrosine ethyl ester; CFU, colony forming unit; EDTA, ethylenediaminetetraacetic acid; Hb, haemoglobin; IAA, iodoacetic acid; PAGE, polyacrylamide gel electrophoresis; pCMB, p-chloromercuribezoate; PMSF, phenylmethylsulfonyl fluoride; Rm, relative mobility; TCA, trichloroacetic acid; Tris, tris(hydroxymethyl)aminomethane. * Corresponding author. Tel.: + 48-42-6366618; fax: + 4842-6313402. E-mail address: [email protected] (M. Turkiewicz)
The overwhelming majority of commercially important enzymes are produced by mesophilic microorganisms and applied in conditions resembling their environment in vivo. New biocatalysts active in unusual conditions are looked for among extremophilic microorganisms including psychrophiles and psychrotrophs (Adams et al., 1995). They constitute the most abundant group of organisms, since temperatures below 5°C occur in about 80% of biosphere and even more than 90% of marine environment (Margesin and Schinner, 1994; Brenchley, 1996).
0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 0 5 7 - 7
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M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
Cold-adapted enzymes secreted by these microorganisms, also called psychrophilic enzymes or psychrozymes, show high catalytic efficiency at low temperatures, especially at 0 – 20°C, i.e. a temperature range at which mesozymes are usually inactive, as well as reduced thermostability resulting in denaturation at moderate temperatures (Margesin and Schinner, 1994; Feller et al., 1996). Cold enzymes are potentially useful for numerous biotechnological processes, especially those which require a supply of exogenous energy, are exposed to higher risk of microbial contamination or temperature instability of reactants or products (Margesin and Schinner, 1994; Marshall, 1997). Another profit resulting from studies on microbial psychrozymes is an enrichment of pure culture collection by strains existing in permanently cold environments, e.g. marine polar regions (DeLong, 1997). Information acquired in this way are important for development of our still very limited knowledge of biodiversity.
rpm. The strain growth was observed by determination of CFUs on solid growth medium (0.3% of beef extract, 0.5% of bactotryptone and 5.51% of bacto-marine agar 2216, Difco) or by absorbance reading at 660 nm. All other chemicals employed in the studies were purchased from standard sources and were at least reagent grade.
2.2. Protease assay Proteolytic activity was determined according to Anson (1938) using urea denatured Hb (pH 7.0, 30°C, reaction time 15 min) or spectrophotometrically according to Hummel (1959) using Nbenzoyl-tyrosine ethyl ester (BzTyrOEt) in 50 mM tris(hydroxymethyl)aminomethane (Tris)–HCl buffer pH 8.0 at 20°C (increase of absorbance at 254 nm). In both methods, the activity was expressed in mmol of product (L-tyrosine present in trichloroacetic acid (TCA)-soluble hydrolysis products or N-benzoyl-L-tyrosine, respectively) liberated from respective substrate for 1 min in standard conditions.
2. Materials and methods
2.3. Protein determination
2.1. Organism and culti6ation
Protein was assayed according to Lowry et al. (1951) using bovine serum albumin (BSA) as a standard, or spectrophotometrically at 280 nm in fractions eluted from chromatography columns.
Gram-negative rods Sphingomonas paucimobilis, strain no. 116, from the collection of the Department of Antarctic Biology of Polish Academy of Sciences in Warsaw, were used for the studies. This strain was isolated from a stomach of Antarctic krill, Euphausia superba Dana by Stuart Donachie at the Arctowski Polish Antarctic Station. The strain classification was done using API ZONE strips (Api System, Biomerieux, France) at 15°C as well as microscopic observations and Gram staining (Donachie, 1995). Shaken cultures of the strain were performed at pH 8.0 in a liquid medium containing synthetic marine water (Instant Ocean, Aquarium System Inc., France), 0.2% of bactopeptone and 1% of casein digest (Difco). One hundred and twenty ml of the medium in a 500 ml flask was inoculated with a standard inoculum (4% v/v, 1.5 ×105 CFU (colony forming units) ml − 1) and cultivated at 10°C in a shaker (INFORS-Switzerland) at 160
2.4. Electrophoresis Polyacyrlamide gel electrophoresis (PAGE) was run in nondenatured conditions, according to Laemmli (1970), using 7.5% gels (0.6×8 cm) in 0.1 M borate buffer pH 9.0 and current intensity 2.5 mA per gel. Gels were fixed and stained with Coomassie brillant blue R-250. Proteolytic activity was assayed in 2 mm slices of nonstained gels against denatured Hb (2% v/w, 30°C, pH 7.0) as a substrate.
2.5. Protease purification Purification of the protease was carried out at 4°C and in presence of Ca2 + ions (0.5 mM) as a stabilizing factor. The protein was precipitated
M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
from the centrifuged (5000 × g, 30 min) culture medium with ammonium sulphate (80% of saturation, 1 h, 0°C), dissolved in 10 mM sodium phosphate buffer pH 7.0 and desalted on Sephadex G-25 (0.9×20 cm) equilibrated with the same buffer. Five ml protein fractions (A280 above 0.8) were pooled and applied to a bacitracin-Sepharose 4B column (0.9×10 cm, the buffer as above), prepared according to Stepanov and Rudenskaya (1983). The column was prewashed with the starting buffer (flow rate 20 ml h − 1) and the bound proteins were eluted using this buffer enriched with 25% (v/v) of isopropanol and 1 M of NaCl. Active fractions were pooled, dialysed against the starting buffer and stored in small aliquots at −40°C.
3. Results
3.1. Effect of temperature on growth and protease secretion S. paucimobilis 116 was grown at temperatures from 0 to 30°C. The highest proteolytic activity in the culture medium was observed in a range from 5 to 10°C (about 0.25 – 0.26 units ml − 1, Fig. 1). At 15°C which is optimal for the strain growth, as well as at 0°C, the activity was twice lower. At 30°C the strain did not excrete extracellular proteases. Above 30°C its growth was not observed. At 10°C which was chosen as a standard
Fig. 1. Effect of S. paucimobilis 116 growth temperature on synthesis of extracellular proteases. Proteolytic activity was assayed in culture medium after 72 h of cultivation, in standard conditions, using urea denatured Hb.
55
Fig. 2. Relationship between the strain growth at 10°C and the proteolytic activity in the culture medium during 7 days of cultivation. Proteolytic activity was assayed in standard conditions using urea denatured Hb.
strain cultivation temperature, the protease synthesis occurred to run simultaneously to the growth and reached the maximum after 72 h, i.e. at a late logarithmic phase of bacterial growth (Fig. 2). Protein pattern of the third day supernatant of the culture medium shows Fig. 3. Two protein bands were active against denatured Hb. The proteolytic activity present in this supernatant was inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF) (34% of inhibition) and 1 mM EDTA (ethylenediaminetetraacetic acid) (38% of inhibition) which proves that S. paucimobilis 116
Fig. 3. Electophoretic pattern of proteins and its relationship with proteolytic activity in the culture medium after 72 h of the strain growth.
56
M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
Fig. 4. Elution profile of proteins (A280) and proteolytic activity in affinity chromatography of the strain proteases on bacitracin-Sepharose 4B. The arrow shows the start of elution with 25% (v/v) isopropanol.
S. paucimobilis 116 protease was most active at 25–30°C for 15 min, and at 15–20°C for 60 min, against urea denatured Hb (Fig. 6). At − 10 to 0°C the enzyme retained 30–47% of maximal activity. The apparent energy of activation for the hydrolysis of urea denatured Hb calculated from an Arrhenius plot was 24.3 kJ mol − 1. The S. paucimobilis 116 metalloprotease is very thermolabile, since the complete stability was observed for 15 min up to 30°C and for 60 min up to 20°C (Fig. 7). At 45°C only 38% of the activity remained after 15 min, and after 60 min it was inactive. The metalloprotease digested Hb at pH from 5.0 to 10.5 with maximum at pH 6.5–7.0
produces extracellular serine proteases and metalloproteases. p-Chloromercuribezoate (p-CMB) and iodacetic acid (IAA) did not influence the activity.
3.2. Metalloprotease properties The highly purified metalloprotease was obtained using the procedure described in Section 2. The enzyme was isolated from 760 ml of the supernatant from 72 h S. paucimobilis 116 culture medium (Table 1). Specific activity of the pooled active fractions after affinity chromatography (Fig. 4) was about 20-fold higher in comparison to the starting supernatant, both against urea denatured Hb and BzTyrOEt. Although bacitracin-Sepharose 4B coupled all the proteolytic activity (Fig. 4), the fractions eluted from this column contained only 10% of it. The eluted proteolytic activity was in 92% inhibited by EDTA (final concentration 1 mM, enzyme-inhibitor incubation time 1 h) and not influenced by PMSF. It proves that only the metalloprotease was eluted from the column and the serine protease present in the starting material remained bound to bacitracin-Sepharose 4B. The yield of purification was low (3 – 4%, Table 1). PAGE of this highly purified enzymic preparation run in non-denatured conditions showed one strong protein band with relative mobility (Rm) 0.58 contaminated with trace amount of another protein with Rm 0.83 (Fig. 5).
Fig. 5. PAGE of the highly purified metalloprotease from S. paucimobilis 116 (pooled active fractions after bacitracin-sepharose 4B column).
Purification step
Culture supernatant Salted out protein Sephadex G-25 Bacitracin-Sepharose 4B
Volume (ml)
760 10 43 5.4
Protein (mg)
313.20 29.84 11.70 0.46
Activity Specific (units mg−1 of protein)
Total (units)
Hb
BzTyrOEt
Hb
BzTyrOEt
0.23 1.40 2.33 5.60*
0.04 0.15 0.28 0.72*
71.82 41.84 27.30 2.60
11.70 4.39 3.29 0.33
* The activity was inhibited by EDTA (in 92%) and not by PMSF.
Yield (%)
Purification (fold)
Hb
BzTyrOEt
Hb
BzTyrOEt
100 59 38 3.6
100 38 28 3
– 6 10 24
– 4 8 19
M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
Table 1 Purification of a metalloprotease from S. paucimobilis 116
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M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
Fig. 6. Effect of temperature on the activity of the S. paucimobilis 116 metalloprotease. The activity was assayed for two reaction times: (a) 15; and (b) 60 min, using urea denatured Hb pH 7.0.
(Fig. 8). Optimal pH in BzTyrOEt hydrolysis was 8.0 and at pH 10.5 the enzyme showed 30% of the maximal activity (Fig. 8). At 4°C the protease was stable at pH 6.0–8.0 for 60 min, and at pH 9.5 exhibited 91% of maximal activity (data not presented). The preferred substrate of the highly purified metalloprotease from S. paucimobilis 116 was denatured Hb (Table 2). Its activity against casein, fibrinogen and native Hb was 20 – 30% lower. BSA was digested to a small extent.
4. Discussion A metalloprotease active against various proteins at neutral and slightly alkaline pH was isolated from extracellular proteases secreted by
Fig. 8. Influence of pH on the activity of the S. paucimobilis 116 metalloprotease. The enzyme activity was assayed against urea denatured Hb (a) and BzTyrOEt (b).
S. paucimobilis strain 116, isolated from stomachs of Antarctic krill E. superba Dana. S. paucimobilis 116 synthesizes extracellular proteases with highest intensity at a temperature markedly lower (5–10°C) than corresponding to its maximal growth rate (15°C), similarly to many other coldadapted microorganisms, including Antarctic ones (Margesin and Schinner, 1994; Feller et al., 1996). It is presumed that intensification of enzyme biosynthesis at low temperatures is one of the main mechanisms of adaptation of these organisms to surrounding environment (Margesin and Schinner, 1994; Brenchley, 1996). Secretion of proteases by S. paucimobilis 116 starts already in the phase of adaptation and its maximum coincides with the end of logarithmic phase, falling on 72 h. This period of time is shorter than observed e.g. by Margesin and Schinner (1992a) for a metalloprotease from Alpine Table 2 Activity of the S. paucimobilis 116 metalloprotease against selected proteins
Fig. 7. Thermostability of the S. paucimobilis 116 metalloprotease. The enzyme was incubated at different temperatures (0–50°C) for: (a) 15; and (b) 60 min and the residual activity towards urea denatured Hb was determined in standard conditions (30°C, pH 7.0).
Substrate
Activitya (units ml−1)
Hb denatured Hb native Casein Fibrinogen BSA
0.249 0.160 0.200 0.177 0.017
a Assay conditions: 30°C, pH 7.0, 2% solutions of protein substrates, 15 min reaction.
M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60
strain of Pseudomonas fluorescens (96 h) or for subtilisin from Antarctic Bacillus TA 39 (150 h, Feller et al., 1996). Electrophoretic analysis and inhibitory tests proved that S. paucimobilis 116 produces at least two extracellular proteases, i.e. a metallo- and a serine enzyme. However we have managed to purify only the metalloenzyme. The serine protease coupled with bacitracin-Sepharose 4B but was immobile in a milieu of the eluent employed in our studies. According to Stepanov and Rudenskaya (1983) ligand from bacitracin-Sepharose possesses various amino acid residues and therefore binds not only serine proteases, but some other too which was revealed in model experiments on papain, subtilisin and B. subtilis metalloprotease. Kinetic properties such as very weak thermostability and low optimal temperature (15 – 30°C depending on reaction time) of the S. paucimobilis 116 metalloprotease point to its increased adaptation to low temperatures in comparison to many other enzymes, also originating from permanently cold, e.g. Antarctic habitats. Reported metalloproteases from other strains inhabiting cold environments usually exhibit higher optimal temperatures. Good examples are proteases from Alpine strain of P. fluorescens 114, most active at 40 – 45°C for 30 min (Hamamoto et al., 1994) and from Xanthomonas maltophilia with maximal activity at 50°C for 15 min (Margesin and Schinner, 1991). However, a protease isolated by Ka¨rst et al. (1994) from an unidentified Antarctic bacterial strain was most active against proteins at 28°C. Another important feature pointing to the psychrophilic character of the metalloprotease from S. paucimobilis 116 is its high activity at temperatures from 0 to 5°C (47 – 60% of maximal activity), resembling the strain physiological temperature range, as well as the ability to protein hydrolysis below 0°C (e.g. at−10°C the enzyme shows 30% of maximal activity against urea denatured Hb) which was not reported for other coldadapted proteases. For comparison, a metalloprotease from P. fluorescens 114 exhibited 20 – 30% of maximal activity at temperatures from 0 to 10°C (Hamamoto et al., 1994), enzymes from Alpine strains showed 15 – 18% of maximal activ-
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ity at 10°C (Margesin and Schinner, 1992b), the protease from X. maltophilia did not hydrolyse azocasein at 0°C and at 10°C retained only about 5% of maximal activity (Margesin and Schinner, 1991). The value of activation energy calculated for the S. paucimobilis 116 (24.3 kJ mol − 1) is lower than obtained for enzymes from X. maltophilia (61.9 kJ mol − 1, Margesin and Schinner, 1991), psychrophilic P. fluorescens strains (36.9– 38.0 kJ mol − 1, Margesin and Schinner, 1992a) or Antarctic psychrophile Bacillus TA41 (38.5 kJ mol − 1, Davail et al., 1994). In all the cases, values of activation energies of mesozymes tested under the same conditions were even higher (Margesin and Schinner, 1991, 1992a; Davail et al., 1994). The reduction of energy barrier displayed by the S. paucimobilis 116 metalloprotease and correlated with its low temperature optimum and weak thermostability may result from more flexible conformation of the molecule in comparison to enzymes mentioned above. A potentially useful property of the S. paucimobilis 116 metalloprotease might be its relatively high activity towards native Hb, equal to about 70% of the activity against urea denatured Hb. Cold-adapted proteases may be applied in many fields, e.g. in production of detergents for washing at low temperatures, in tannery, in the food industry (haze removal from beer, bakery, cheese-making, production of fermented foods, meat tenderisation) or high protein waste degradation (Margesin and Schinner, 1994; Brenchley, 1996). Our studies demonstrate that proteases synthesized by marine Antarctic bacteria might become an interesting alternative to currently applied mesophilic enzymes.
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M. Turkiewicz et al. / Journal of Biotechnology 70 (1999) 53–60 Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 165 – 275. Margesin, R., Schinner, F., 1991. Characterization of a metalloprotease from psychrophilic Xanthomonas maltophilia. FEMS Microbiol. Lett. 79, 257 – 262. Margesin, R., Schinner, F., 1992a. Production and properties of an extracellular metalloprotease from a psychrophilic Pseudomonas fluorescens. J. Biotechnol. 24, 207 – 210. Margesin, R., Schinner, F., 1992b. A comparison of extracellular proteases from three psychrotrophic strains of Pseudomonas fluorescens. J. Gen. Appl. Microbiol. 38, 209 – 225. Margesin, R., Schinner, F., 1994. Properties of cold-adapted micro-organisms and their potential role in biotechnology. J. Biotechnol. 33, 1 – 14. Marshall, C.J., 1997. Cold-adapted enzymes. Trends Biotechnol. 15, 359 – 363. Stepanov, V.M., Rudenskaya, G.N., 1983. Proteinase affinity chromatography on bacitracin-Sepharose. J. Appl. Biochem. 5, 420 – 428.
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