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Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted β-galactosidase
Biomolecular Engineering 20 (2003) 317 /324 www.elsevier.com/locate/geneanabioeng
Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted b-galactosidase Marianna Turkiewicz a,*, Jo´zef Kur b, Aneta Bialkowska a, Hubert Cies´lin´ski b, Halina Kalinowska a, Stanislaw Bielecki a a
Institute of Technical Biochemistry, Technical University of Lo´dz´, Stefanowskiego Street 4/10, 90-924 Lo´dz´, Poland b Department of Microbiology, Technical University of Gdan´sk, Narutowicza 11/12, Gdan´sk, Poland
Abstract The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 b-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura , synthesizes an intracellular cold-adapted b-galactosidase, which efficiently hydrolyzes lactose at 0 /20 8C, as indicated by its specific activity of 21 /67 U mg 1 of protein (11 /35% of maximum activity) in this temperature range, as well as kcat of 157 s 1, and kcat/Km of 47.5 mM 1 s 1 at 20 8C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 8C, thus below the optimum growth temperature of the bacterium (15 8C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 8C (190 U mg 1 of protein) and pH 6.0 /8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b b-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K , Na , Mn2 */40% increase, Mg2 */15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu2. Noteworthy, Ca2 ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 8C for at least 30 days without any stabilizers. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Cold-adapted b-galactosidase; Pseudoalteromonas sp.; Lactose hydrolysis
1. Introduction Marine and terrestrial Antarctic ecosystems are a rich source of cold-evolved microorganisms, whose specific molecular mechanisms confer thriving at low temperatures via an adjustment of their metabolism to harsh environmental conditions [1]. One of the reasons of a burgeoning interest in these microbes is a wealth of potentially useful biomolecules synthesized by them, including enzymes with unique kinetic and molecular properties, which are attractive catalysts for enthalpydeficient conditions [2,3]. Principal captivating facets of these biocatalysts include maximal activity at temperatures 20/30 8C below that optimal for mesophilic
* Corresponding author. Tel.: /48-42-631-3440; fax: /48-42-6366618. E-mail address: [email protected] (M. Turkiewicz).
enzymes, and a high catalytic efficiency in the range 0 /20 8C, so under conditions resulting in a drop in activity of their mesophilic counterparts. The poor thermal stability of psychrophilic enzymes, which facilitates their rapid inactivation by a moderate rise in temperature is also advantageous in some technologies. Investigations into the ‘‘cold’’ enzymes have been mainly focused on deciphering of the relevant molecular alterations that provide the high catalytic activity in the polar climate, and on their commercial applications. One of particularly interesting enzymes is a psychrophilic b-galactosidase, potentially useful for fast lactose digestion below 20 8C, to produce lactose-free milkderived foods for individuals with lactose intolerance (approximately 30% of the world population), to avoid lactose crystallization in dairy foodstuffs, and to degrade this bulk pollutant in dairy sewage. Currently applied for lactose hydrolysis, mesophilic Kluyveromyces lactis b-galactosidase shows poor activity at 20 8C, and
1389-0344/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1389-0344(03)00039-X
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therefore, its replacement with a psychrophilic counterpart will shorten the process of lactose cleavage run in cold, in order to eliminate any contamination with mesophilic microflora, and to avoid non-enzymatic browning products, formed at higher temperature. Studies on isolation, characterization, and applications of psychrophilic b-galactosidases [4 /7], and also their immobilization [8], have not given any enzyme with properties meeting requirements of industry, such as high activity and stability at 4/8 8C and at pH 6.7 /6.8, facilitating complete degradation of lactose during transport and storage of milk, or sewage treatment. That prompted our studies on b-galactosidase-producing strains from the Antarctic microorganisms collection, belonging to the Institute of Technical Biochemistry (ITB), and comprising 107 marine and soil microbial species. It is one of the two Polish assemblies of Antarctic isolates. Our experiments were aimed at selection of the best psychrophilic b-galactosidase producer, purification of the enzyme to homogeneity, and its kinetic and molecular characterization.
2. Materials and methods
2.1. Organism selection and culture conditions The strain 22b from the ITB collection of Antarctic microorganisms (107 strains, including 45 marine bacteria), was isolated from the intestinal tract of Antarctic krill Thysanoessa macrura , caught in the Admiralty Bay waters (King George Island, Southern Shetlands, 62810?S, 58828?W), and selected from other isolates because: a) It hydrolyzed 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) at 6 8C in plate tests on agar medium containing 1% lactose, 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), 2.3% bacto nutrient agar (Difco) and 3.5% marine salt (Instant Ocean, Aquarius System Inc., France). b) It produced an intracellular b-galactosidase in agitated cultures at 6 8C in IPTG-free liquid medium with an initial pH of 7.6, referred to as a standard one (S), containing 1% lactose as a main carbon source and b-galactosidase inducer, 0.2% bactopeptone, 0.1% yeast extract, and 3.5% marine salt. The cultures were carried out for 8 days in an incubator Infors AG CH-4103 (Switzerland) at 130 rpm. b-Galactosidase activity was assayed in culture medium supernatants and cell-free extracts. c) Cell-free extracts from its biomass displayed the highest specific b-galactosidase activity at 0 /20 8C (10 min reaction, ONPG as a substrate).
To compare the enzyme biosynthesis yields, the bacterium 22b was also cultured in the medium S enriched with IPTG, and in the medium H, described by Hoyoux et al. [4], containing 2% lactose, 1% tryptone, 0.5% yeast extract and 3% marine salt. In all the cases, the agitated cultures were carried out at 6 8C for 8 days (130 rpm, incubator Infors) in 500 ml flasks containing 200 ml of culture medium inoculated with 0.25% (v/v) inoculum (bacterial cells suspension with A660 of 2.2), prepared by washing the cells from an agar slant (2.3% Bacto Nutrient Agar enriched with 3.5% marine salt) incubated for 2 weeks. After cultivation, the cells were harvested by centrifugation at 10 000/g for 45 min at 4 8C. The cell pellet was washed with 0.05 M potassium phosphate buffer, pH 7.6, containing 15 mM EDTA and 200 mM Mg2, suspended in the same buffer and stored, if necessary, at /20 8C until use. 2.2. Characterization and identification of the strain 22b Characterization of a fresh culture of marine isolate 22b involved Gram staining, API 20NE and APIZYM tests (BioMerieux, France) as well as an estimation of alanine aminopeptidase and cytochrome oxidase activities. Proteolytic, lipolytic, and amylolytic properties of the isolate were examined at 0 and 10 8C, on plates with nutrient agar, enriched with skimmed milk, tributirine, or starch, respectively. The temperature profile of growth was determined in the range 0/37 8C, by means of stationary cultures in the standard liquid culture medium. The genus of the strain was assessed based on the sequence of 1600 bp of 16S rRNA gene, amplified by using PCR technique, and employing the primers fD1 and rP2 [9] designed to regions ghe of this gene, conserved among eubacteria. The alignment of the 16S rDNA sequence with that found in databases, such as Ribosomal Data Project (RDP) and GenBank database at the National Center for Biotechnology Information (NCBI), was facilitated by the Basic Alignment Search Tool (BLAST) program [10]. 2.3. Assays of b-galactosidase activity b-Galactosidase activity in hydrolysis of o-nitrophenyl-b-D-galactopyranoside (ONPG, 10 mM) was estimated in 0.05 M potassium phosphate buffer, pH 7.6. After the temperature of 0.6 ml aliquots of the substrate solution achieved 30 8C, 0.15 ml samples of enzymatic extract were added, and the reaction was carried out for 10 min at this temperature. The hydrolysis was terminated with 1.5 ml of 0.6 M Na2CO3, and the absorbency of the mixture was measured at 420 nm. One unit (U) of the activity denoted 1 mmol of o -nitrophenol liberated from the substrate for 1 min under the standard reaction conditions. To determine the values of Michaelis constant (Km) in the ONPG hydrolysis at 10 and 20 8C, the
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substrate concentrations from 0.1 to 50 mM were employed, and the reaction was monitored at 420 nm during initial 10 min by using the spectrophotometer (Beckman DH 7500, GB). The temperature of the cuvette holder was kept constant with an accuracy of 0.01 8C by using the thermostate Julabo F25 (Germany). The activity against lactose (110 mM) was assayed in the same buffer, and the hydrolysis reaction (10 min, 30 8C) was stopped by an incubation of the reaction mixture at 100 8C for 3 min. An amount of glucose liberated by the enzyme was estimated with an enzymatic test (Glucose EP, POCH, Gliwice, Poland) employing glucose oxidase and peroxidase. Glucose concentration was determined based on A530 measurements of the color intensity of a dye produced by oxidation of a chromogen (4-aminopyrine). Lactose concentrations in the range 1/250 mM were used to determine Km value at 20 8C. The activity was expressed in mmoles of glucose released from lactose for 1 min under standard conditions.
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dissolved in the same buffer, but without PMSF and EDTA, and applied on agarose coupled with p-aminobenzyl-1-thio-b-D-galactopyranoside (PABTG-agarose, Sigma) column (0.9 /10 cm). After washing with 200 ml of the starting buffer, the column was eluted with 0.1 M sodium borate, pH 10.0. The enzyme-containing fractions were pooled, concentrated on a 30 kDa filter, and purified to homogeneity by molecular sieving on Sepharose Cl-6B column (0.6 /80 cm, flow rate of 0.16 ml min 1), previously equilibrated with 0.05 M potassium phosphate buffer, pH 7.6, and calibrated with molecular mass standard proteins (67 /450 kDa). 2.6. Other analytical methods Protein concentration was determined according to Bradford [11], using BSA as a standard. SDS-PAGE was carried out following Laemmli [12] method, on slabs (10 /5.5 cm) of 10% polyacrylamide gel. The samples were denatured for 5 min at 100 8C in the presence of 10% SDS and 0.5% 2-mercaptoethanol.
2.4. b-Galactosidase extraction from the biomass Cell-free extracts from the bacterium biomass, harvested after cultures carried out at 6 8C in the standard liquid medium, were prepared by using two different methods: 1) the wet biomass extraction (4 8C, 24 h) with 0.5% sodium cholate in 0.05 M potassium phosphate buffer, pH 7.6, enriched with 15 mM EDTA, 0.2 M Mg2, and 1 mM PMSF. 2 ml aliquots of the buffered cholate solution were used per 1 g samples of wet biomass. The residual insoluble cell debris was discarded after centrifugation (10 000/g, 4 8C, 30 min). 2) The wet biomass sonification (twice for 2.5 min, 0 8C, Vibrocell 72480, Bioblock Scientific, USA) in the same buffer as above, enriched with 1 mM PMSF and 2 mM EDTA. The centrifugation conditions remained unchanged, and the insoluble cell debris was discarded. 2.5. b-Galactosidase purification All purification steps were carried out at 4 8C. The cholate extract obtained from 26 g of fresh wet Pseudoalteromonas sp. 22b biomass was applied on DEAE-Sepharose column (0.8 /30 cm) previously equilibrated with 0.05 mM potassium phosphate buffer, pH 7.6, containing 1 mM PMSF and 2 mM EDTA. An elution was carried out with a linear NaCl gradient (0 / 0.2 M, 100 ml) in the starting buffer, and with a flow rate of 0.22 ml min 1. b-Galactosidase-containing fractions, eluted within the range 0.08 /0.095 M NaCl, were concentrated on a filter with a cut-off of 30 kDa,
3. Results 3.1. Selection of an efficient b-galactosidase producer The bacterium 22b was one of 41 strains, which cleaved X-Gal at 6 8C in plate tests, and one of 19 of them, which synthesized b-galactosidase in the lactosecontaining and IPTG-free liquid culture medium. None of the 41 isolates secreted an extracellular b-galactosidase to the culture medium. The specific b-galactosidase activity (determined under optimum conditions) in crude cell-free extracts of the isolate 22b (0.9 U mg 1 of protein, 40 8C) was higher than that of other strains. At temperatures 0 /20 8C, the b-galactosidase present in these extracts showed 11 /40% of maximum activity (0.10/0.36 U mg 1 of protein). The b-galactosidase from the strain 22b was easily extractable from cells, with a satisfactory yield, by using the buffered sodium cholate solution, enriched with EDTA and Mg2, as described in Section 2. The protein patterns visualized by SDS-PAGE proved that the cholate extracts contained much less different proteins as compared with that obtained by sonification (in the latter case the specific activity of b-galactosidase was approximately twice lower; results not presented), thus facilitating the enzyme purification. 3.2. Characterization of the isolate 22b The cells of the isolate 22b are Gram-negative (this was also confirmed by the positive result of BioMerieux test, which revealed the activity of alanine aminopeptidase), motile, and very short rods. The bacterium forms
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smooth, glittering, creamy colonies with radial edges on agar medium. API 20NE and APIZYM tests showed that its properties were typical of Pseudoalteromonas species. Plate tests presented both proteolytic and esterolytic/lipolytic (at pH 7.0 and 10.0) activities of the bacterium 22b, apart from the b-galactosidase activity. The bacterium 22b was categorized as a psychrotolerant strain with Topt of 15 8C, Tmax of 30 8C, and capability of thriving at 0 8C. An alignment of the 16S rRNA gene sequence of the isolate 22b (GenBank, accession number AF 443784) with the sequences available in the RDP and GenBank databases at NCBI, demonstrated that the isolate 22b should be classified as a Pseudoalteromonas sp., and its closest relative was P. elyakovii . 3.3. b-Galactosidase biosynthesis The enzyme biosynthesis yields achieved at 6 8C, in agitated cultures in 1% lactose-containing standard liquid medium, either enriched with 1 mM IPTG or not, were similar, and amounted to 79.8 U l 1 of culture medium (specific activity of 0.52 U mg 1 of protein), and 76.4 U l 1 (specific activity of 0.49 U mg 1), respectively. IPTG added to the culture medium after initial 48 h of the culture did not cause any increase in the b-galactosidase biosynthesis yield. Table 1 presents results of the bacterium 22b cultures, carried out for 8 days at 6 8C in two different liquid culture media, S and H (the latter was used for bgalactosidase biosynthesis by Pseudoalteromonas haloTable 1 The comparison of Pseudalteromonas sp. 22b b-galactosidase biosynthesis yields in two different culture media H and Sa Parameter
Medium S
Medium H
Medium composition (%) Bactopeptone Tryptone Yeast extract Marine salt Lactose PH
0.2 / 0.1 3.5 1.0 7.0
/ 1.0 0.5 3.0 2.0 7.0
Culture results Biomass (wet mass) (g l 1)
16.3
70.9
b-Galactosidase activity U mg 1 of protein U mg 1 of wet mass U l 1 of medium U l 1 h 1
0.47 5.07 82.64 0.43
0.26 0.70 49.60 0.26
Relative costs 1 l of culture medium 100 U of activity
1 1
3.5 5.9
a
The cultures were carried out at 6 8C and 130 rpm (incubator Infors) for 8 days, in 500 ml Erlenmayer flasks, each containing 200 ml of culture medium.
planktis TAE 79 [4]). Assays of b-galactosidase activity in cholate extracts prepared from two biomass pellets, harvested from these media, revealed that the medium H, which contained more nutrients, favored proliferation of the cells, since four times more of biomass was obtained from this medium as compared with the medium S. However, the latter medium provided seven times higher b-galactosidase activity (5.07 and 0.70 U per 1 g of cells wet mass, respectively). Also the enzyme productivity was higher in medium S (0.43 vs. 0.26 U l 1 h1), and the costs of production of 100 units of bgalactosidase activity were approximately six times lower. The yield of Pseudoalteromonas sp. 22b b-galactosidase synthesis depended on temperature, and was approximately 80% higher at 6 8C than at 15 8C, the latter being optimum for the strain growth. At 6 8C, the maximum enzyme synthesis declined on the eighth day of the agitated culture, corresponding to the end of the logarithmic phase of growth (results not presented). 3.4. Purification of b-galactosidase The enzyme was purified to homogeneity by using the three-step procedure, presented in Table 2. The most efficient step was an affinity chromatography on Sepharose coupled with a specific competitive b-galactosidase inhibitor */PABTG [4,13]. The best results (larger elution yield, more narrow protein peak) were achieved by eluting the enzyme with 0.1 M sodium borate, pH 10.0, used for this purpose also by other authors [13,14]. In contrast, an application of 100 mM lactose solution, which was used to elute the Pseudomonas haloplanctis TAE 79 b-galactosidase by Hoyoux et al. [4], for our experiments, brought about an approximately twice lower enzyme elution recovery. Due to the presence of a minor amount of another protein with a similar molecular mass (Fig. 1) in the Pseudoalteromonas 22b b-galactosidase preparation, derived by the affinity chromatography, it was further subjected to molecular sieving on Sepharose Cl-6B column, previously calibrated with protein molecular mass standards. The latter step provided both the enzyme homogeneity, and the determination of molecular mass of the native protein, which appeared to be 4909/5 kDa. Since SDSPAGE of the denatured enzyme demonstrated the mass close to 115 kDa (Fig. 1), so the Pseudoalteromonas sp. 22b b-galactosidase is believed to be a tetrameric protein. The homogeneous enzyme is stable at 4 8C for at least 30 days, without any stabilizers. 3.5. Properties of the homogeneous b-galactosidase The enzyme hydrolyzes only three substrates, i.e. PNPG (the best activity), ONPG, and p-nitrophenyl b-D-galacturonide (only weak activity), out of 12 tested
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Table 2 Purification of Pseudoalteromonas sp. 22b b-galactosidase Purification step
Sodium cholate extract DEAE-Sepharose Affinity chromatography Sepharose Cl-6B
Volume (ml)
32 20 3 9
Protein (mg)
166.4 7.8 0.65 0.22
b-Galactosidase activity Specific (U mg 1)
Total (U)
0.45 13.0 75.0 115.0
101.5 101.4 48.8 23.0
Yield (%)
Purification fold
100 100 48 25
/ 29 167 255
The enzyme was obtained from 16 g of fresh biomass, harvested from 1 l of culture medium after 8 days of agitated culture.
chromogens. The chromogens tested are adducts of nitrophenol and various mono- and disaccharides (Table 3). The activity of the bacterium 22b b-galactosidase is markedly enhanced by thiol compounds (10 mM), particularly dithiotreitol (70% rise in activity; Table 4), some metal ions (5 mM), such as Na , K , and Mn2 (approximately 40% increase in activity, Table 5), and to a lesser extent by Mg2 ions (only 15% rise in activity). 5 mM Ca2 have no impact on the enzyme activity, whereas Pb2, Ni2 and Zn2 ions reduce it significantly (to 17/34% of an initial activity), and Cu2 (even 1 mM) ions completely inactivate the enzyme in 1 min. The b-galactosidase is strongly inhibited by 4-chloromercuribenzoic acid (pCMB) (Table 4) that confirms an importance of free thiol groups for the enzyme activity. The optimum temperature for ONPG hydrolysis by the Pseudoalteromonas sp. 22b b-galactosidase is 40 8C (190 U mg 1 of protein), i.e. 20 8C below that of mesophilic E. coli enzyme (Fig. 2). The enzyme from the
isolate 22b displays 11/35% of maximum activity (21 / 67 U mg 1 of protein) at 0/20 8C, and remains active even at /5 8C (8% of maximum activity). It is stable for 60 min at temperatures up to 40 8C, and undergoes rapid denaturation above this temperature point. The carbohydrase is completely inactivated in 2 min at 50 8C. The optimum pH range of the enzyme activity is relatively wide (6.0 /8.0, Fig. 3), similarly to its pHstability range. The Antarctic b-galactosidase is stable at 4 8C for 60 min at pH 5.9 /9.5 (Fig. 3), and for 24 h at pH 6.5 /9.5. Although at 20 8C, an affinity of the Pseudoalteromonas sp. 22b b-galactosidase towards ONPG is one order of magnitude larger as compared with that towards lactose (Table 6), the rate of the latter substrate hydrolysis is only twice smaller (157 and 312 s 1, respectively, Table 6). This enzyme’s facet is particularly captivating due to its potential harnessing for lactose degradation in milk or whey. Initial results of our
Fig. 1. SDS-PAGE protein patterns of fractions obtained after successive purification steps of Pseudoalteromonas sp. 22b b-galactosidase. (1) Cholate extract; (2) pooled fractions after ion-exchange chromatography; (3) pooled fractions after affinity chromatography; (4) the enzyme preparation derived by molecular sieving.
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Table 3 The relative activity of Pseudoalteromonas sp. 22b b-galactosidase against different chromogens (nitrophenol derivatives of mono- and disaccharides) Substrate
Relative activitya (%)
o -Nitrophenyl-b-D-galactopyranoside p -Nitrophenyl-b-D-galactopyranoside p -Nitrophenyl-b-D-galacturonide p -Nitrophenyl-b-L-arabinopyranoside p -Nitrophenyl-b-D-cellobioside p -Nitrophenyl-b-D-lactopyranoside p -Nitrophenyl-b-D-mannopyranoside p -Nitrophenyl-b-D-glucopyranoside p -Nitrophenyl-a-D-galactopyranoside p -Nitrophenyl-b-L-fucopyranoside p -Nitrophenyl-b-D-xylopyranoside p -Nitrophenyl-b-D-glucuronide
100 150 1.5 0 0 0 0 0 0 0 0 0
Fig. 2. An effect of temperature on the Pseudoalteromonas sp. 22b bgalactosidase activity and stability.
a The activity was assayed under standard conditions (30 8C, 10 min, pH 7.6, substrate concentration of 10 mM). The b-galactosidase activity against ONPG was 115 U mg1 of protein.
Table 4 An effect of thiol compounds and pCMB on Pseudoalteromonas sp. 22b b-galactosidase activity Reagent
Residual activitya (%)
None Dithiotreitol 2-Mercaptoethanol Glutathion Cysteine PCMB
100 170 125 110 104 24
a The enzyme was incubated for 60 min at 30 8C with the reagent (10 mM), and the residual activity was assayed under standard conditions.
Table 5 An effect of metal ions on Pseudoalteromonas sp. 22b b-galactosidase activity Metal ion
Residual activitya (%)
None Na K Mn2 Mg2 Ca2 Zn2 Pb2 Ni2 Cu2
100 142 134 139 115 100 34 30 17 0
a The enzyme was incubated for 60 min at 30 8C with 5 mM metal ion, and the residual activity was assayed under standard conditions.
experiments on hydrolysis of model 4% lactose solutions (the concentration addressing lactose content in milk and whey), confirm this assumption, since at 20 8C and pH 7.6, the homogeneous b-galactosidase hydrolyzed
Fig. 3. An effect of pH on the Pseudoalteromonas sp. 22b bgalactosidase activity and stability (60 min at 4 8C). Table 6 Kinetic constants in ONPG and lactose hydrolysis, catalyzed by Pseudoalteromonas sp. 22b b-galactosidase at 20 8C Substrate
Km (mM)
kcat (s 1)
kcat/Km (mM 1 s 1)
ONPG Lactose
0.28 3.3
312 157
1114 47.5
during 6 h approximately 35% of lactose present in reaction mixture, when applied in a ratio of 1 U ml 1 of 4% lactose solution. Further optimization of hydrolysis conditions may enhance this result.
4. Discussion Our collection of pure Antarctic cultures is a relatively rich source of b-galactosidase producers, since as much as 41 of 107 strains, subjected to screening, cleaved XGal in the plate test. The isolate 22b was superior to other microorganisms with respect to an amount of enzyme synthesized by the biomass unit, the enzyme specific activity, and its absolute activity at temperatures 0 /20 8C. The bacterium 22b was identified as Pseudoalteromonas sp., a genus abundant in the Antarctic coastal
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waters [15]. T. macrura , which is one of three genera of Antarctic krills, also lives in these waters, and the specimen bearing the bacterium 22b either could be accidentally infected with it, or its intestinal tract was its natural niche, similarly to that detected in E . superba stomach, inhabited by numerous microorganisms, whose presumable role is synthesis of enzymes facilitating degradation of food particles [16]. The highest yield of b-galactosidase synthesis by the psychrotolerant strain 22b (good growth at 0 8C, Topt of 15 8C, Tmax of 30 8C), is observed at 6 8C, so much below its optimum growth temperature, similarly to an Antarctic, b-galactosidase-producing, halotolerant, psychrophilic bacterium P. haloplanktis TAE 79 (maximum b-galactosidase synthesis at 4 8C) [4]. In contrast to the latter strain, the synthesis of b-galactosidase by Pseudoalteromonas sp. 22b is not affected by the initial presence or later supplement of IPTG in the culture medium, whereas Hoyoux et al. [4] report on 2 /3 fold increase in the enzyme activity in response to the enrichment of the culture medium with this compound after 44 h of culture. Noteworthy, Pseudoalteromonas sp. 22b grew very well in the medium applied for P. haloplanktis TAE 79 cultures by Hoyoux et al. [4], but the medium optimized by us was superior for bgalactosidase synthesis (seven times higher yield). The most efficient b-galactosidase synthesis by Pseudoalteromonas sp. 22b was observed in the presence of 1% lactose (both principal carbon source and the enzyme inducer) in the culture medium, that provides relatively low costs of the enzyme production. The Pseudoalteromonas sp. 22b b-galactosidase is very specific since it hydrolyses almost exclusively bgalactose derivatives, such as lactose, ONPG, and PNPG (the preferred substrate). Apart from them, a minor activity was detected only against PNP-galacturonide. The optimum temperature for the enzyme activity of 40 8C, is 20 8C below that of known mesophilic bgalactosidases. Also thermal stability of the Antarctic enzyme is weaker, since it undergoes fast denaturation above 40 8C. To avoid enzyme inactivation caused by its inherent lability, common among cold-evolved proteins, its purification was done in three steps, the most efficient of that was the second step */an affinity chromatography on a carrier bearing a competitive b-galactosidase inhibitor (PABTG), providing enzyme coupling, and recommended by other authors, who purified b-galactosidases of various origin [4,8]. However, to obtain the narrow enzyme peak, containing almost a half of the activity applied on the column, the Pseudoalteromonas sp. 22b b-galactosidase had to be eluted from the PABTG-agarose with 0.1 M sodium borate, pH 10.0, though the latter pH exceeded (0.5 pH unit) that correlated with total enzyme stability at 4 8C for 60 min. The buffered 100 mM lactose solution (pH 7.5, 1 M
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KCl), which was applied for b-galactosidase recovery by Hoyoux et al. [4], was the worse eluent in our experiments, presumably due to a higher affinity of the P. haloplanktis TAE 79 enzyme towards lactose (2.4 mM at 25 8C vs. 3.3 mM, at 20 8C, of the isolate 22b). SDSPAGE of Pseudoalteromonas sp. 22b b-galactosidase eluted from the PABTG-carrier, revealed it was contaminated with a trace amount of another protein (without this activity), bound due to the reported earlier, non-specific adsorption of some other proteins on this matrix [8]. The contamination was readily removed by molecular sieving, ultimately giving the homogeneous enzyme with 25% recovery of an initial activity exhibited by the sodium cholate extract prepared from fresh cells, and 255 times larger specific activity. The enzyme stability was excellent since its activity remained unchanged without any stabilizers for at least 30 days at 4 8C, whereas a decline in Pseudoalteromonas sp. TAE 79b b-galactosidase activity amounted to 60% after 2 weeks [8]. Comparison of the results of SDS-PAGE with that of molecular sieving demonstrated the tetrameric structure of Pseudoalteromonas sp. 22b b-galactosidase, detected also in E. coli and P. haloplanktis TAE 79 enzymes [4]. Also with respect to the stimulating impact of some metal ions, such as K , Na , Mn2 and Mg2, the protein behavior is similar to these two Mg2-containing b-galactosidases. The Pseudoalteromonas sp. 22b enzyme activity remains unaltered in the presence of 5 mM Ca2, so this stable at 4 8C glycoside hydrolase can be harnessed for lactose hydrolysis in refrigerated milk. However, any contact with heavy metal ions, especially with Cu2, should be avoided due to their detrimental effect to this enzyme. The beneficial influence of thiol compounds (particularly dithiotreitol) indicate that free thiol groups are essential for the carbohydrase activity. Despite the aforementioned, lower affinity of Pseudomonas sp. 22b b-galactosidase towards lactose (3.3 mM at 20 8C) as compared with that of P. haloplanktis TAE 79 (2.4 mM at 25 8C) [4], the turnover number of our enzyme is 4.5 times larger than that of the latter bgalactosidase (kcat of 157 s 1 at 20 8C vs. 33 s 1 at 25 8C). The ratio of both enzymatic efficiency (kcat/Km) values is similar (47.5 vs. 13.7 s 1 mM 1, respectively). Noteworthy, at 25 8C the mesophilic E. coli b-galactosidase displays very poor affinity, turnover number and efficiency in lactose degradation (13 mM, 2 s 1, and 0.15 s 1 mM 1, respectively) as compared with both the psychrozymes. It should be emphasized that the Pseudomonas sp. 22b enzyme displays even better performance in ONPG hydrolysis as is indicated by kcat/Km value as high as 1114 s 1 mM 1. This high catalytic efficiency is believed to reflect an enhanced flexibility of cold-adapted proteins, which in turn facilitates these conformational changes within the catalytic site, that provide catalysis at relatively low
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energy input. The flexibility of Pseudoalteromonas sp. 22b b-galactosidase seems to be promising with respect to its potential application for galacto-oligosaccharides synthesis in organic solvents, known to stiffen the molecular edifice of proteins. Another advantage of Pseudoalteromonas sp. 22b bgalactosidase is its relatively wide pH optimum of activity (6.0 /8.0) and stability (6.5 /9.5 for 24 h at 4 8C), meeting the requirements of milk treatment. Although, the native Pseudoalteromonas sp. 22b enzyme efficiently hydrolyzes lactose in its model 4% solutions, the Antarctic bacterium is not a suitable source of commercial cold-adapted b-galactosidase preparation, since its cultures should be run at 6 8C to achieve a good enzyme biosynthesis yield. Therefore, the studies on isolation of the gene encoding the b-galactosidase, and its expression in a mesophilic host are in progress.
5. Conclusions 1) The capability of b-galactosidase synthesizing was found to be relatively widespread among the Antarctic microorganisms from the ITB culture collection, since as much as approximately 40% of these microbes cleaved X-Gal in the plate test. 2) Kinetic properties of the homogeneous b-galactosidase produced by the marine bacterium Pseudoalteromonas sp. 22b, and particularly its relatively large activity in the range 0/20 8C, and high catalytic efficiency in lactose hydrolysis at 20 8C, indicate that the enzyme (native or more probablyrecombined) can be potentially applied for degradation of this sugar in large scale.
Acknowledgements This work was supported by a grant no 021/P06/99/24 from the Polish Committee of Scientific Researches.
References [1] Margesin R, Schinner F. J Biotechnol 1994;33:1 /14. [2] Feller G, Narriux E, Arpigny JL, Aittleb M, Baise E, Genicot S, Gerday C. FEMS Microbiol Rev 1996;18:189 /202. [3] Feller G, Gerday C. Cell Mol Life Sci 1997;53:830 /41. [4] Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F, Francois JM, Baise E, Feller G, Gerday C. Appl Environ Microbiol 2001;67:1529 /35. [5] Coombs JM, Brenchley JE. Appl Environ Microbiol 1999;65:5443 /50. [6] Sheridan PP, Brenchley JE. Appl Environ Microbiol 2000;66:2438 /44. [7] Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR. Curr Opin Biotechnol 2002;13:253 /61. [8] Fernandes R, Geucke B, Delgado O, Coleman J, Hatti-Kaul R. Appl Microbiol Biotechnol 2002;58:313 /21. [9] Weisburg WG, Barns SM, Pelletier DA, Lane DJ. J Bacteriol 1991;173:697 /703. [10] Altschul S, Gish W, Miller W, Myers E, Lipman D. J Mol Biol 1990;215:403 /10. [11] Bradford MM. Anal Biochem 1976;72:248 /54. [12] Laemmli UK. Nature (London) 1970;237:680 /5. [13] Steers E, Jr, Cuatrecasas P, Pollard HB. J Biol Chem 1971;246:196 /200. [14] Cazorla D, Feliu J, Villaverde A. Biotechnol Bioeng 2001;72:255 / 60. [15] Bowman J, Macammon S, Brown M, Nichols D, Mcmeekin T. Appl Environ Microbiol 1997;63:3068 /78. [16] Rakusa-Suszczewski S. Pol Polar Res 1988;9:409 /12.
Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted b-galactosidase Marianna Turkiewicz a,*, Jo´zef Kur b, Aneta Bialkowska a, Hubert Cies´lin´ski b, Halina Kalinowska a, Stanislaw Bielecki a a
Institute of Technical Biochemistry, Technical University of Lo´dz´, Stefanowskiego Street 4/10, 90-924 Lo´dz´, Poland b Department of Microbiology, Technical University of Gdan´sk, Narutowicza 11/12, Gdan´sk, Poland
Abstract The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 b-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura , synthesizes an intracellular cold-adapted b-galactosidase, which efficiently hydrolyzes lactose at 0 /20 8C, as indicated by its specific activity of 21 /67 U mg 1 of protein (11 /35% of maximum activity) in this temperature range, as well as kcat of 157 s 1, and kcat/Km of 47.5 mM 1 s 1 at 20 8C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 8C, thus below the optimum growth temperature of the bacterium (15 8C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 8C (190 U mg 1 of protein) and pH 6.0 /8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b b-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K , Na , Mn2 */40% increase, Mg2 */15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu2. Noteworthy, Ca2 ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 8C for at least 30 days without any stabilizers. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Cold-adapted b-galactosidase; Pseudoalteromonas sp.; Lactose hydrolysis
1. Introduction Marine and terrestrial Antarctic ecosystems are a rich source of cold-evolved microorganisms, whose specific molecular mechanisms confer thriving at low temperatures via an adjustment of their metabolism to harsh environmental conditions [1]. One of the reasons of a burgeoning interest in these microbes is a wealth of potentially useful biomolecules synthesized by them, including enzymes with unique kinetic and molecular properties, which are attractive catalysts for enthalpydeficient conditions [2,3]. Principal captivating facets of these biocatalysts include maximal activity at temperatures 20/30 8C below that optimal for mesophilic
* Corresponding author. Tel.: /48-42-631-3440; fax: /48-42-6366618. E-mail address: [email protected] (M. Turkiewicz).
enzymes, and a high catalytic efficiency in the range 0 /20 8C, so under conditions resulting in a drop in activity of their mesophilic counterparts. The poor thermal stability of psychrophilic enzymes, which facilitates their rapid inactivation by a moderate rise in temperature is also advantageous in some technologies. Investigations into the ‘‘cold’’ enzymes have been mainly focused on deciphering of the relevant molecular alterations that provide the high catalytic activity in the polar climate, and on their commercial applications. One of particularly interesting enzymes is a psychrophilic b-galactosidase, potentially useful for fast lactose digestion below 20 8C, to produce lactose-free milkderived foods for individuals with lactose intolerance (approximately 30% of the world population), to avoid lactose crystallization in dairy foodstuffs, and to degrade this bulk pollutant in dairy sewage. Currently applied for lactose hydrolysis, mesophilic Kluyveromyces lactis b-galactosidase shows poor activity at 20 8C, and
1389-0344/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S1389-0344(03)00039-X
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therefore, its replacement with a psychrophilic counterpart will shorten the process of lactose cleavage run in cold, in order to eliminate any contamination with mesophilic microflora, and to avoid non-enzymatic browning products, formed at higher temperature. Studies on isolation, characterization, and applications of psychrophilic b-galactosidases [4 /7], and also their immobilization [8], have not given any enzyme with properties meeting requirements of industry, such as high activity and stability at 4/8 8C and at pH 6.7 /6.8, facilitating complete degradation of lactose during transport and storage of milk, or sewage treatment. That prompted our studies on b-galactosidase-producing strains from the Antarctic microorganisms collection, belonging to the Institute of Technical Biochemistry (ITB), and comprising 107 marine and soil microbial species. It is one of the two Polish assemblies of Antarctic isolates. Our experiments were aimed at selection of the best psychrophilic b-galactosidase producer, purification of the enzyme to homogeneity, and its kinetic and molecular characterization.
2. Materials and methods
2.1. Organism selection and culture conditions The strain 22b from the ITB collection of Antarctic microorganisms (107 strains, including 45 marine bacteria), was isolated from the intestinal tract of Antarctic krill Thysanoessa macrura , caught in the Admiralty Bay waters (King George Island, Southern Shetlands, 62810?S, 58828?W), and selected from other isolates because: a) It hydrolyzed 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) at 6 8C in plate tests on agar medium containing 1% lactose, 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), 2.3% bacto nutrient agar (Difco) and 3.5% marine salt (Instant Ocean, Aquarius System Inc., France). b) It produced an intracellular b-galactosidase in agitated cultures at 6 8C in IPTG-free liquid medium with an initial pH of 7.6, referred to as a standard one (S), containing 1% lactose as a main carbon source and b-galactosidase inducer, 0.2% bactopeptone, 0.1% yeast extract, and 3.5% marine salt. The cultures were carried out for 8 days in an incubator Infors AG CH-4103 (Switzerland) at 130 rpm. b-Galactosidase activity was assayed in culture medium supernatants and cell-free extracts. c) Cell-free extracts from its biomass displayed the highest specific b-galactosidase activity at 0 /20 8C (10 min reaction, ONPG as a substrate).
To compare the enzyme biosynthesis yields, the bacterium 22b was also cultured in the medium S enriched with IPTG, and in the medium H, described by Hoyoux et al. [4], containing 2% lactose, 1% tryptone, 0.5% yeast extract and 3% marine salt. In all the cases, the agitated cultures were carried out at 6 8C for 8 days (130 rpm, incubator Infors) in 500 ml flasks containing 200 ml of culture medium inoculated with 0.25% (v/v) inoculum (bacterial cells suspension with A660 of 2.2), prepared by washing the cells from an agar slant (2.3% Bacto Nutrient Agar enriched with 3.5% marine salt) incubated for 2 weeks. After cultivation, the cells were harvested by centrifugation at 10 000/g for 45 min at 4 8C. The cell pellet was washed with 0.05 M potassium phosphate buffer, pH 7.6, containing 15 mM EDTA and 200 mM Mg2, suspended in the same buffer and stored, if necessary, at /20 8C until use. 2.2. Characterization and identification of the strain 22b Characterization of a fresh culture of marine isolate 22b involved Gram staining, API 20NE and APIZYM tests (BioMerieux, France) as well as an estimation of alanine aminopeptidase and cytochrome oxidase activities. Proteolytic, lipolytic, and amylolytic properties of the isolate were examined at 0 and 10 8C, on plates with nutrient agar, enriched with skimmed milk, tributirine, or starch, respectively. The temperature profile of growth was determined in the range 0/37 8C, by means of stationary cultures in the standard liquid culture medium. The genus of the strain was assessed based on the sequence of 1600 bp of 16S rRNA gene, amplified by using PCR technique, and employing the primers fD1 and rP2 [9] designed to regions ghe of this gene, conserved among eubacteria. The alignment of the 16S rDNA sequence with that found in databases, such as Ribosomal Data Project (RDP) and GenBank database at the National Center for Biotechnology Information (NCBI), was facilitated by the Basic Alignment Search Tool (BLAST) program [10]. 2.3. Assays of b-galactosidase activity b-Galactosidase activity in hydrolysis of o-nitrophenyl-b-D-galactopyranoside (ONPG, 10 mM) was estimated in 0.05 M potassium phosphate buffer, pH 7.6. After the temperature of 0.6 ml aliquots of the substrate solution achieved 30 8C, 0.15 ml samples of enzymatic extract were added, and the reaction was carried out for 10 min at this temperature. The hydrolysis was terminated with 1.5 ml of 0.6 M Na2CO3, and the absorbency of the mixture was measured at 420 nm. One unit (U) of the activity denoted 1 mmol of o -nitrophenol liberated from the substrate for 1 min under the standard reaction conditions. To determine the values of Michaelis constant (Km) in the ONPG hydrolysis at 10 and 20 8C, the
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substrate concentrations from 0.1 to 50 mM were employed, and the reaction was monitored at 420 nm during initial 10 min by using the spectrophotometer (Beckman DH 7500, GB). The temperature of the cuvette holder was kept constant with an accuracy of 0.01 8C by using the thermostate Julabo F25 (Germany). The activity against lactose (110 mM) was assayed in the same buffer, and the hydrolysis reaction (10 min, 30 8C) was stopped by an incubation of the reaction mixture at 100 8C for 3 min. An amount of glucose liberated by the enzyme was estimated with an enzymatic test (Glucose EP, POCH, Gliwice, Poland) employing glucose oxidase and peroxidase. Glucose concentration was determined based on A530 measurements of the color intensity of a dye produced by oxidation of a chromogen (4-aminopyrine). Lactose concentrations in the range 1/250 mM were used to determine Km value at 20 8C. The activity was expressed in mmoles of glucose released from lactose for 1 min under standard conditions.
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dissolved in the same buffer, but without PMSF and EDTA, and applied on agarose coupled with p-aminobenzyl-1-thio-b-D-galactopyranoside (PABTG-agarose, Sigma) column (0.9 /10 cm). After washing with 200 ml of the starting buffer, the column was eluted with 0.1 M sodium borate, pH 10.0. The enzyme-containing fractions were pooled, concentrated on a 30 kDa filter, and purified to homogeneity by molecular sieving on Sepharose Cl-6B column (0.6 /80 cm, flow rate of 0.16 ml min 1), previously equilibrated with 0.05 M potassium phosphate buffer, pH 7.6, and calibrated with molecular mass standard proteins (67 /450 kDa). 2.6. Other analytical methods Protein concentration was determined according to Bradford [11], using BSA as a standard. SDS-PAGE was carried out following Laemmli [12] method, on slabs (10 /5.5 cm) of 10% polyacrylamide gel. The samples were denatured for 5 min at 100 8C in the presence of 10% SDS and 0.5% 2-mercaptoethanol.
2.4. b-Galactosidase extraction from the biomass Cell-free extracts from the bacterium biomass, harvested after cultures carried out at 6 8C in the standard liquid medium, were prepared by using two different methods: 1) the wet biomass extraction (4 8C, 24 h) with 0.5% sodium cholate in 0.05 M potassium phosphate buffer, pH 7.6, enriched with 15 mM EDTA, 0.2 M Mg2, and 1 mM PMSF. 2 ml aliquots of the buffered cholate solution were used per 1 g samples of wet biomass. The residual insoluble cell debris was discarded after centrifugation (10 000/g, 4 8C, 30 min). 2) The wet biomass sonification (twice for 2.5 min, 0 8C, Vibrocell 72480, Bioblock Scientific, USA) in the same buffer as above, enriched with 1 mM PMSF and 2 mM EDTA. The centrifugation conditions remained unchanged, and the insoluble cell debris was discarded. 2.5. b-Galactosidase purification All purification steps were carried out at 4 8C. The cholate extract obtained from 26 g of fresh wet Pseudoalteromonas sp. 22b biomass was applied on DEAE-Sepharose column (0.8 /30 cm) previously equilibrated with 0.05 mM potassium phosphate buffer, pH 7.6, containing 1 mM PMSF and 2 mM EDTA. An elution was carried out with a linear NaCl gradient (0 / 0.2 M, 100 ml) in the starting buffer, and with a flow rate of 0.22 ml min 1. b-Galactosidase-containing fractions, eluted within the range 0.08 /0.095 M NaCl, were concentrated on a filter with a cut-off of 30 kDa,
3. Results 3.1. Selection of an efficient b-galactosidase producer The bacterium 22b was one of 41 strains, which cleaved X-Gal at 6 8C in plate tests, and one of 19 of them, which synthesized b-galactosidase in the lactosecontaining and IPTG-free liquid culture medium. None of the 41 isolates secreted an extracellular b-galactosidase to the culture medium. The specific b-galactosidase activity (determined under optimum conditions) in crude cell-free extracts of the isolate 22b (0.9 U mg 1 of protein, 40 8C) was higher than that of other strains. At temperatures 0 /20 8C, the b-galactosidase present in these extracts showed 11 /40% of maximum activity (0.10/0.36 U mg 1 of protein). The b-galactosidase from the strain 22b was easily extractable from cells, with a satisfactory yield, by using the buffered sodium cholate solution, enriched with EDTA and Mg2, as described in Section 2. The protein patterns visualized by SDS-PAGE proved that the cholate extracts contained much less different proteins as compared with that obtained by sonification (in the latter case the specific activity of b-galactosidase was approximately twice lower; results not presented), thus facilitating the enzyme purification. 3.2. Characterization of the isolate 22b The cells of the isolate 22b are Gram-negative (this was also confirmed by the positive result of BioMerieux test, which revealed the activity of alanine aminopeptidase), motile, and very short rods. The bacterium forms
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smooth, glittering, creamy colonies with radial edges on agar medium. API 20NE and APIZYM tests showed that its properties were typical of Pseudoalteromonas species. Plate tests presented both proteolytic and esterolytic/lipolytic (at pH 7.0 and 10.0) activities of the bacterium 22b, apart from the b-galactosidase activity. The bacterium 22b was categorized as a psychrotolerant strain with Topt of 15 8C, Tmax of 30 8C, and capability of thriving at 0 8C. An alignment of the 16S rRNA gene sequence of the isolate 22b (GenBank, accession number AF 443784) with the sequences available in the RDP and GenBank databases at NCBI, demonstrated that the isolate 22b should be classified as a Pseudoalteromonas sp., and its closest relative was P. elyakovii . 3.3. b-Galactosidase biosynthesis The enzyme biosynthesis yields achieved at 6 8C, in agitated cultures in 1% lactose-containing standard liquid medium, either enriched with 1 mM IPTG or not, were similar, and amounted to 79.8 U l 1 of culture medium (specific activity of 0.52 U mg 1 of protein), and 76.4 U l 1 (specific activity of 0.49 U mg 1), respectively. IPTG added to the culture medium after initial 48 h of the culture did not cause any increase in the b-galactosidase biosynthesis yield. Table 1 presents results of the bacterium 22b cultures, carried out for 8 days at 6 8C in two different liquid culture media, S and H (the latter was used for bgalactosidase biosynthesis by Pseudoalteromonas haloTable 1 The comparison of Pseudalteromonas sp. 22b b-galactosidase biosynthesis yields in two different culture media H and Sa Parameter
Medium S
Medium H
Medium composition (%) Bactopeptone Tryptone Yeast extract Marine salt Lactose PH
0.2 / 0.1 3.5 1.0 7.0
/ 1.0 0.5 3.0 2.0 7.0
Culture results Biomass (wet mass) (g l 1)
16.3
70.9
b-Galactosidase activity U mg 1 of protein U mg 1 of wet mass U l 1 of medium U l 1 h 1
0.47 5.07 82.64 0.43
0.26 0.70 49.60 0.26
Relative costs 1 l of culture medium 100 U of activity
1 1
3.5 5.9
a
The cultures were carried out at 6 8C and 130 rpm (incubator Infors) for 8 days, in 500 ml Erlenmayer flasks, each containing 200 ml of culture medium.
planktis TAE 79 [4]). Assays of b-galactosidase activity in cholate extracts prepared from two biomass pellets, harvested from these media, revealed that the medium H, which contained more nutrients, favored proliferation of the cells, since four times more of biomass was obtained from this medium as compared with the medium S. However, the latter medium provided seven times higher b-galactosidase activity (5.07 and 0.70 U per 1 g of cells wet mass, respectively). Also the enzyme productivity was higher in medium S (0.43 vs. 0.26 U l 1 h1), and the costs of production of 100 units of bgalactosidase activity were approximately six times lower. The yield of Pseudoalteromonas sp. 22b b-galactosidase synthesis depended on temperature, and was approximately 80% higher at 6 8C than at 15 8C, the latter being optimum for the strain growth. At 6 8C, the maximum enzyme synthesis declined on the eighth day of the agitated culture, corresponding to the end of the logarithmic phase of growth (results not presented). 3.4. Purification of b-galactosidase The enzyme was purified to homogeneity by using the three-step procedure, presented in Table 2. The most efficient step was an affinity chromatography on Sepharose coupled with a specific competitive b-galactosidase inhibitor */PABTG [4,13]. The best results (larger elution yield, more narrow protein peak) were achieved by eluting the enzyme with 0.1 M sodium borate, pH 10.0, used for this purpose also by other authors [13,14]. In contrast, an application of 100 mM lactose solution, which was used to elute the Pseudomonas haloplanctis TAE 79 b-galactosidase by Hoyoux et al. [4], for our experiments, brought about an approximately twice lower enzyme elution recovery. Due to the presence of a minor amount of another protein with a similar molecular mass (Fig. 1) in the Pseudoalteromonas 22b b-galactosidase preparation, derived by the affinity chromatography, it was further subjected to molecular sieving on Sepharose Cl-6B column, previously calibrated with protein molecular mass standards. The latter step provided both the enzyme homogeneity, and the determination of molecular mass of the native protein, which appeared to be 4909/5 kDa. Since SDSPAGE of the denatured enzyme demonstrated the mass close to 115 kDa (Fig. 1), so the Pseudoalteromonas sp. 22b b-galactosidase is believed to be a tetrameric protein. The homogeneous enzyme is stable at 4 8C for at least 30 days, without any stabilizers. 3.5. Properties of the homogeneous b-galactosidase The enzyme hydrolyzes only three substrates, i.e. PNPG (the best activity), ONPG, and p-nitrophenyl b-D-galacturonide (only weak activity), out of 12 tested
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Table 2 Purification of Pseudoalteromonas sp. 22b b-galactosidase Purification step
Sodium cholate extract DEAE-Sepharose Affinity chromatography Sepharose Cl-6B
Volume (ml)
32 20 3 9
Protein (mg)
166.4 7.8 0.65 0.22
b-Galactosidase activity Specific (U mg 1)
Total (U)
0.45 13.0 75.0 115.0
101.5 101.4 48.8 23.0
Yield (%)
Purification fold
100 100 48 25
/ 29 167 255
The enzyme was obtained from 16 g of fresh biomass, harvested from 1 l of culture medium after 8 days of agitated culture.
chromogens. The chromogens tested are adducts of nitrophenol and various mono- and disaccharides (Table 3). The activity of the bacterium 22b b-galactosidase is markedly enhanced by thiol compounds (10 mM), particularly dithiotreitol (70% rise in activity; Table 4), some metal ions (5 mM), such as Na , K , and Mn2 (approximately 40% increase in activity, Table 5), and to a lesser extent by Mg2 ions (only 15% rise in activity). 5 mM Ca2 have no impact on the enzyme activity, whereas Pb2, Ni2 and Zn2 ions reduce it significantly (to 17/34% of an initial activity), and Cu2 (even 1 mM) ions completely inactivate the enzyme in 1 min. The b-galactosidase is strongly inhibited by 4-chloromercuribenzoic acid (pCMB) (Table 4) that confirms an importance of free thiol groups for the enzyme activity. The optimum temperature for ONPG hydrolysis by the Pseudoalteromonas sp. 22b b-galactosidase is 40 8C (190 U mg 1 of protein), i.e. 20 8C below that of mesophilic E. coli enzyme (Fig. 2). The enzyme from the
isolate 22b displays 11/35% of maximum activity (21 / 67 U mg 1 of protein) at 0/20 8C, and remains active even at /5 8C (8% of maximum activity). It is stable for 60 min at temperatures up to 40 8C, and undergoes rapid denaturation above this temperature point. The carbohydrase is completely inactivated in 2 min at 50 8C. The optimum pH range of the enzyme activity is relatively wide (6.0 /8.0, Fig. 3), similarly to its pHstability range. The Antarctic b-galactosidase is stable at 4 8C for 60 min at pH 5.9 /9.5 (Fig. 3), and for 24 h at pH 6.5 /9.5. Although at 20 8C, an affinity of the Pseudoalteromonas sp. 22b b-galactosidase towards ONPG is one order of magnitude larger as compared with that towards lactose (Table 6), the rate of the latter substrate hydrolysis is only twice smaller (157 and 312 s 1, respectively, Table 6). This enzyme’s facet is particularly captivating due to its potential harnessing for lactose degradation in milk or whey. Initial results of our
Fig. 1. SDS-PAGE protein patterns of fractions obtained after successive purification steps of Pseudoalteromonas sp. 22b b-galactosidase. (1) Cholate extract; (2) pooled fractions after ion-exchange chromatography; (3) pooled fractions after affinity chromatography; (4) the enzyme preparation derived by molecular sieving.
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Table 3 The relative activity of Pseudoalteromonas sp. 22b b-galactosidase against different chromogens (nitrophenol derivatives of mono- and disaccharides) Substrate
Relative activitya (%)
o -Nitrophenyl-b-D-galactopyranoside p -Nitrophenyl-b-D-galactopyranoside p -Nitrophenyl-b-D-galacturonide p -Nitrophenyl-b-L-arabinopyranoside p -Nitrophenyl-b-D-cellobioside p -Nitrophenyl-b-D-lactopyranoside p -Nitrophenyl-b-D-mannopyranoside p -Nitrophenyl-b-D-glucopyranoside p -Nitrophenyl-a-D-galactopyranoside p -Nitrophenyl-b-L-fucopyranoside p -Nitrophenyl-b-D-xylopyranoside p -Nitrophenyl-b-D-glucuronide
100 150 1.5 0 0 0 0 0 0 0 0 0
Fig. 2. An effect of temperature on the Pseudoalteromonas sp. 22b bgalactosidase activity and stability.
a The activity was assayed under standard conditions (30 8C, 10 min, pH 7.6, substrate concentration of 10 mM). The b-galactosidase activity against ONPG was 115 U mg1 of protein.
Table 4 An effect of thiol compounds and pCMB on Pseudoalteromonas sp. 22b b-galactosidase activity Reagent
Residual activitya (%)
None Dithiotreitol 2-Mercaptoethanol Glutathion Cysteine PCMB
100 170 125 110 104 24
a The enzyme was incubated for 60 min at 30 8C with the reagent (10 mM), and the residual activity was assayed under standard conditions.
Table 5 An effect of metal ions on Pseudoalteromonas sp. 22b b-galactosidase activity Metal ion
Residual activitya (%)
None Na K Mn2 Mg2 Ca2 Zn2 Pb2 Ni2 Cu2
100 142 134 139 115 100 34 30 17 0
a The enzyme was incubated for 60 min at 30 8C with 5 mM metal ion, and the residual activity was assayed under standard conditions.
experiments on hydrolysis of model 4% lactose solutions (the concentration addressing lactose content in milk and whey), confirm this assumption, since at 20 8C and pH 7.6, the homogeneous b-galactosidase hydrolyzed
Fig. 3. An effect of pH on the Pseudoalteromonas sp. 22b bgalactosidase activity and stability (60 min at 4 8C). Table 6 Kinetic constants in ONPG and lactose hydrolysis, catalyzed by Pseudoalteromonas sp. 22b b-galactosidase at 20 8C Substrate
Km (mM)
kcat (s 1)
kcat/Km (mM 1 s 1)
ONPG Lactose
0.28 3.3
312 157
1114 47.5
during 6 h approximately 35% of lactose present in reaction mixture, when applied in a ratio of 1 U ml 1 of 4% lactose solution. Further optimization of hydrolysis conditions may enhance this result.
4. Discussion Our collection of pure Antarctic cultures is a relatively rich source of b-galactosidase producers, since as much as 41 of 107 strains, subjected to screening, cleaved XGal in the plate test. The isolate 22b was superior to other microorganisms with respect to an amount of enzyme synthesized by the biomass unit, the enzyme specific activity, and its absolute activity at temperatures 0 /20 8C. The bacterium 22b was identified as Pseudoalteromonas sp., a genus abundant in the Antarctic coastal
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waters [15]. T. macrura , which is one of three genera of Antarctic krills, also lives in these waters, and the specimen bearing the bacterium 22b either could be accidentally infected with it, or its intestinal tract was its natural niche, similarly to that detected in E . superba stomach, inhabited by numerous microorganisms, whose presumable role is synthesis of enzymes facilitating degradation of food particles [16]. The highest yield of b-galactosidase synthesis by the psychrotolerant strain 22b (good growth at 0 8C, Topt of 15 8C, Tmax of 30 8C), is observed at 6 8C, so much below its optimum growth temperature, similarly to an Antarctic, b-galactosidase-producing, halotolerant, psychrophilic bacterium P. haloplanktis TAE 79 (maximum b-galactosidase synthesis at 4 8C) [4]. In contrast to the latter strain, the synthesis of b-galactosidase by Pseudoalteromonas sp. 22b is not affected by the initial presence or later supplement of IPTG in the culture medium, whereas Hoyoux et al. [4] report on 2 /3 fold increase in the enzyme activity in response to the enrichment of the culture medium with this compound after 44 h of culture. Noteworthy, Pseudoalteromonas sp. 22b grew very well in the medium applied for P. haloplanktis TAE 79 cultures by Hoyoux et al. [4], but the medium optimized by us was superior for bgalactosidase synthesis (seven times higher yield). The most efficient b-galactosidase synthesis by Pseudoalteromonas sp. 22b was observed in the presence of 1% lactose (both principal carbon source and the enzyme inducer) in the culture medium, that provides relatively low costs of the enzyme production. The Pseudoalteromonas sp. 22b b-galactosidase is very specific since it hydrolyses almost exclusively bgalactose derivatives, such as lactose, ONPG, and PNPG (the preferred substrate). Apart from them, a minor activity was detected only against PNP-galacturonide. The optimum temperature for the enzyme activity of 40 8C, is 20 8C below that of known mesophilic bgalactosidases. Also thermal stability of the Antarctic enzyme is weaker, since it undergoes fast denaturation above 40 8C. To avoid enzyme inactivation caused by its inherent lability, common among cold-evolved proteins, its purification was done in three steps, the most efficient of that was the second step */an affinity chromatography on a carrier bearing a competitive b-galactosidase inhibitor (PABTG), providing enzyme coupling, and recommended by other authors, who purified b-galactosidases of various origin [4,8]. However, to obtain the narrow enzyme peak, containing almost a half of the activity applied on the column, the Pseudoalteromonas sp. 22b b-galactosidase had to be eluted from the PABTG-agarose with 0.1 M sodium borate, pH 10.0, though the latter pH exceeded (0.5 pH unit) that correlated with total enzyme stability at 4 8C for 60 min. The buffered 100 mM lactose solution (pH 7.5, 1 M
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KCl), which was applied for b-galactosidase recovery by Hoyoux et al. [4], was the worse eluent in our experiments, presumably due to a higher affinity of the P. haloplanktis TAE 79 enzyme towards lactose (2.4 mM at 25 8C vs. 3.3 mM, at 20 8C, of the isolate 22b). SDSPAGE of Pseudoalteromonas sp. 22b b-galactosidase eluted from the PABTG-carrier, revealed it was contaminated with a trace amount of another protein (without this activity), bound due to the reported earlier, non-specific adsorption of some other proteins on this matrix [8]. The contamination was readily removed by molecular sieving, ultimately giving the homogeneous enzyme with 25% recovery of an initial activity exhibited by the sodium cholate extract prepared from fresh cells, and 255 times larger specific activity. The enzyme stability was excellent since its activity remained unchanged without any stabilizers for at least 30 days at 4 8C, whereas a decline in Pseudoalteromonas sp. TAE 79b b-galactosidase activity amounted to 60% after 2 weeks [8]. Comparison of the results of SDS-PAGE with that of molecular sieving demonstrated the tetrameric structure of Pseudoalteromonas sp. 22b b-galactosidase, detected also in E. coli and P. haloplanktis TAE 79 enzymes [4]. Also with respect to the stimulating impact of some metal ions, such as K , Na , Mn2 and Mg2, the protein behavior is similar to these two Mg2-containing b-galactosidases. The Pseudoalteromonas sp. 22b enzyme activity remains unaltered in the presence of 5 mM Ca2, so this stable at 4 8C glycoside hydrolase can be harnessed for lactose hydrolysis in refrigerated milk. However, any contact with heavy metal ions, especially with Cu2, should be avoided due to their detrimental effect to this enzyme. The beneficial influence of thiol compounds (particularly dithiotreitol) indicate that free thiol groups are essential for the carbohydrase activity. Despite the aforementioned, lower affinity of Pseudomonas sp. 22b b-galactosidase towards lactose (3.3 mM at 20 8C) as compared with that of P. haloplanktis TAE 79 (2.4 mM at 25 8C) [4], the turnover number of our enzyme is 4.5 times larger than that of the latter bgalactosidase (kcat of 157 s 1 at 20 8C vs. 33 s 1 at 25 8C). The ratio of both enzymatic efficiency (kcat/Km) values is similar (47.5 vs. 13.7 s 1 mM 1, respectively). Noteworthy, at 25 8C the mesophilic E. coli b-galactosidase displays very poor affinity, turnover number and efficiency in lactose degradation (13 mM, 2 s 1, and 0.15 s 1 mM 1, respectively) as compared with both the psychrozymes. It should be emphasized that the Pseudomonas sp. 22b enzyme displays even better performance in ONPG hydrolysis as is indicated by kcat/Km value as high as 1114 s 1 mM 1. This high catalytic efficiency is believed to reflect an enhanced flexibility of cold-adapted proteins, which in turn facilitates these conformational changes within the catalytic site, that provide catalysis at relatively low
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energy input. The flexibility of Pseudoalteromonas sp. 22b b-galactosidase seems to be promising with respect to its potential application for galacto-oligosaccharides synthesis in organic solvents, known to stiffen the molecular edifice of proteins. Another advantage of Pseudoalteromonas sp. 22b bgalactosidase is its relatively wide pH optimum of activity (6.0 /8.0) and stability (6.5 /9.5 for 24 h at 4 8C), meeting the requirements of milk treatment. Although, the native Pseudoalteromonas sp. 22b enzyme efficiently hydrolyzes lactose in its model 4% solutions, the Antarctic bacterium is not a suitable source of commercial cold-adapted b-galactosidase preparation, since its cultures should be run at 6 8C to achieve a good enzyme biosynthesis yield. Therefore, the studies on isolation of the gene encoding the b-galactosidase, and its expression in a mesophilic host are in progress.
5. Conclusions 1) The capability of b-galactosidase synthesizing was found to be relatively widespread among the Antarctic microorganisms from the ITB culture collection, since as much as approximately 40% of these microbes cleaved X-Gal in the plate test. 2) Kinetic properties of the homogeneous b-galactosidase produced by the marine bacterium Pseudoalteromonas sp. 22b, and particularly its relatively large activity in the range 0/20 8C, and high catalytic efficiency in lactose hydrolysis at 20 8C, indicate that the enzyme (native or more probablyrecombined) can be potentially applied for degradation of this sugar in large scale.
Acknowledgements This work was supported by a grant no 021/P06/99/24 from the Polish Committee of Scientific Researches.
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