ISSN 0003-6838, Applied Biochemistry and Microbiology, 2006, Vol. 42, No. 3, pp. 246–251. © MAIK “Nauka /Interperiodica” (Russia), 2006. Original Russian Text © L.I. Sapunova, A.G. Lobanok, I.O. Kazakevich, E.A. Shlyakhotko, A.N. Evtushenkov, 2006, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2006, Vol. 42, No. 3, pp. 279–284.
Biosynthetic Features and Properties of Xylose Isomerases from Arthrobacter nicotianae, Escherichia coli, and Erwinia carotovora subsp. atroseptica L. I. Sapunova*, A. G. Lobanok*, I. O. Kazakevich*, E. A. Shlyakhotko*, and A. N. Evtushenkov** * Institute of Microbiology, National Academy of Sciences of Belarus, Minsk, 220141 Belarus e-mail:
[email protected] ** Belarussian State University, Minsk, 220080 Belarus e-mail:
[email protected] Received September 9, 2005
Abstract—The characteristics of xylose isomerase biosynthesis in the bacteria Arthrobacter nicotianae BIM B-5, Erwinia carotovora subsp atroseptica jn42xylA, and Escherichia coli HB101xylA have been studied. The bacteria produced the enzyme constitutively. Out of the carbon sources studied, D-glucose and D-xylose were most favorable for the biosynthesis of xylose isomerase in E. carotovora subsp. atroseptica, but the least appropriate in terms of the enzyme production efficiency in E. coli. Minimum and maximum levels of xylose isomerase formation in A. nicotianae were noted, respectively, during D-xylose and sucrose utilization. An addition to the D-xylose-containing nutrient medium of 0.1–1.5% D-glucose did not affect the enzyme synthesis in A. nicotianae, but suppressed it in Erwinia carotovora subsp. atroseptica (by 7% at the highest concentration) and Escherichia coli (by 63 and 75% at concentrations of 0.1 and 1.0%, respectively). The enzyme proteins produced by the bacteria exhibited the same substrate specificity and electrophoretic mobility (PAGE) as xylose isomerase A. nicotianae, although insignificant differences in the major physicochemical properties were noted. DOI: 10.1134/S0003683806030045
Xylose isomerase (D-xylose: ketole isomerase, EC 5.3.1.5) is widely used in the production of xylose– fructose syrup (a natural sugar replacement in dietetic food products, prophylactic food additives, and clinical nutrition products). The increasing demand for sweeteners calls for refining the technologies used for their production; the search for more productive and technologically flexible producer strains is one of approaches to meeting this goal [1]. The bacterium A. nicotianae BIM B-5, characterized by the high level of constitutive xylose isomerase synthesis, was selected by us previously [2]. We obtained recombinant strains E. coli çÇ101xylA and E. carotovora subsp. atroseptica jn42xylA, inheriting the gene xylA of the bacterium A. nicotianae B-5. The cultivation times of these recombinants were, respectively, 48 and 24 h less than that of the parent strain [3, 4]. In this work we sought to study the biosynthetic features and major physicochemical properties of the enzymes produced in A. nicotianae BIM B-5, E. coli
çÇ101xylA, and E. carotovora subsp. atroseptica jn42xylA. MATERIALS AND METHODS The major objects of this study were the gram-positive bacterium Arthrobacter nicotianae Giovanozzi– Sermanni BIM B-5 (a xylose isomerase producer), and gram-negative bacteria Escherichia coli (Migula) Costellani et Chalmers çÇ101xylA and Erwinia carotovora subsp. atroseptica (van Hall) Dye jn42xylA. The cultures were stored in collections of nonpathogenic microorganisms of the Institute of Microbiology (National Academy of Sciences of Belarus) and the Chair of Molecular Biology of the Faculty of Biology of Belarussian State University. Submerged cultivation of the bacteria was performed in 250-ml Erlenmeyer flasks at 180–200 rpm for 24–72 h; A. nicotianae and E. carotovora subsp. atroseptica were grown at 28–30ºC and E. coli at 36−37°ë.
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The nutritive medium had the following composition (%): peptone, 1.0; yeast extract, 0.5; K2HPO4, 0.3; and MgSO4 · 7H2O, 0.1 (the initial pH value was equal to 6.8). D-xylose, xylitol, D-glucose, and sucrose were used as carbon sources (1% of each, unless otherwise indicated; the amount is expressed as the relative content of carbon in the medium). A water suspension of bacteria grown for three days on peptone–yeast agar (A. nicotianae and E. carotovora subsp. atroseptica) or beef–peptone agar (E. coli) served as an inoculum, which was introduced in an amount equivalent to 2 vol %. When the cultivation was completed, the optical density of the culture liquid was measured; the cells were separated by centrifugation (6000 g, 20 min), washed, used for determining xylose-isomerase activity, and then processed further (preparation of cell-free extracts and purification of the enzyme). The amount of biomass was determined using a calibration plot relating the optical density of bacterial cell suspensions to the number of bacteria (D540 for A. nicotianae; D600 for E. carotovora subsp. atroseptica and E. coli) and expressed in mg per 1 ml culture liquid (mg/ml). The preparations of purified xylose isomerase were obtained by the method developed previously [5, 6] and stored at –20°ë. Electrophoretic procedures (PAGE and SDS-PAGE) followed conventional methods [7, 8]. Tris–glycine (pH 8.5) was used as an electrode buffer. The duration of the separation ranged from 1.5–2 h at 20 mA (gel size, 130 × 130 mm). Following SDS-PAGE the gel was washed with distilled water, fixed in 15% trichloroacetic acid for 2 h, stained with Coomassie Brilliant Blue G-250 (Sigma, United States), and washed again with a 1 : 3 : 12 mixture of acetic acid, ethanol, and distilled water. Proteins used as molecular-weight markers (Amersham Pharmacia Biotech, Sweden) included myosin (212 kDa), α2-macroglobulin (17 kDa), β-galactosidase (116 kDa), transferrin (76 kDa), and glutamine dehydrogenase (53 kDa). Following PAGE the gel was washed with distilled water and incubated at 70°ë in a 12 : 5 : 2 : 1 mixture of distilled water, 0.2 M potassium–sodium-phosphate buffer (pH 7.8), 0.1 M MgSO4 · 7H2O, and 0.1 M D-glucose for 10–20 min. Thereafter, the gel was washed in distilled water and treated in the dark with a 0.1% solution of 2,3,5-triphenyltetrazolium chloride in 1 M NaOH (30°ë, 1 min). Xylose isomerase spots were vizualized in the gels by the pinkish-red color of formazan, produced as a result of the oxidation of the colorless 2,3,5-triphenyltetrazolium by D-xylulose (product of D-xylose isomerization) [9, 10]. APPLIED BIOCHEMISTRY AND MICROBIOLOGY
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In studying the specificity of xylose isomerase, the following substrates were used: D-glucose, D-xylose, D-mannose, L-arabinose, L-galactose, maltose, L-rhamnose, D-ribose, or 2-deoxy-D-glucose. In order to compare the major physicochemical properties of xylose isomerase specimens, effects of the temperature and pH of the reaction medium on the enzyme activity were studied (20–90°ë, pH 2.0–11.0). Temperature stability was assessed by the residual activity determined after 15–120 min of the heating of xylose isomerase solutions in 0.2 M potassium– sodium-phosphate buffer (pH 7.8) at 50, 60, 70, and 80°ë. In determining pH stability, the solution of the enzyme was incubated in the buffer (pH 5.0–11.0) at 70 and 80°ë for 1 h, after which the residual activity was determined. The buffer systems used included 0.2 M citrate–phosphate buffer (Na2HPO4–citric acid), potassium–sodium-phosphate buffer, and a universal buffer. The content of protein in the solutions of the enzyme was determined spectrophotometrically by absorption at 280 nm or by using the Bradford technique [11] and expressed in OD units or µg per 1 ml. The reaction mixture for measuring xylose isomerase activity contained 0.2 ml 1.0 M D-xylose, 0.5 ml 0.2 M potassium–sodium-phosphate buffer (pH 7.8), 0.1 ml 0.1 M MgSO4 · 7H2O, 20 mg cells, and distilled water, which gave a final volume of 2.0 ml. The isomerization reaction was performed for 1 h at 70°ë. One unit (U) of xylose isomerase activity was taken to be equal to the amount of the enzyme, which converted 1 µmol D-xylose into D-xylulose in 1 min under the conditions described above. The activity of the enzyme was expressed in U/mg protein or OD (D540) units; the relative activity was calculated as a percentage of the maximum activity. D-xylulose and other ketosugars were determined by the cysteine–carbazole method [12]. Each value is a mean of three to five triplicate measurements. RESULTS AND DISCUSSION We demonstrated previously that the synthesis levels of the cell-bound xylose isomerase by A. nicotianae, E. carotovora subsp. atroseptica, and E. coli are similar, provided that the cultivation times of the latter strains are reduced [3, 4]. Our study of enzyme biosynthesis by these bacteria, involving diverse sources of carbon, demonstrated that xylose isomerase was produced not only in media containing the specific substrate (D-xylose) or its structural analogue xylitol, but also in their absence (Fig. 1). The
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Fig. 2. Effect of D-glucose concentration (%) on the synthesis of xylose isomerases (1, U/mg; 2, U/ml) by Arthrobacter sp. (a), E. coli (b), and E. carotovora subsp. atroseptica (c).
level of enzyme synthesis and the productivity of the cultures varied with the carbon sources used. The production of xylose isomerase by the bacterium A. nicotianae was maximum in the medium with sucrose (0.73 U/mg) and minimum in the presence of D-xylose (0.47 U/mg). The productivity of this culture likewise depended on the source of carbon, with minimum and maximum values differing twofold (Fig. 1a). Figure 1b demonstrates that xylitol was the most favorable source of carbon for xylose isomerase synthesis by E. coli (1.39 U/mg), whereas D-glucose and D-xylose were the least appropriate (the levels of the enzyme production amounted to 0.22 and 0.46 U/mg, respectively). Conversely, maximum levels of enzyme synthesis by the bacterium E. carotovora subsp. atroseptica were obtained precisely with D-glucose or D-xylose as the sole source of carbon (0.81 and 0.71 U/mg, respectively). The activities of the enzyme in E. coli and E. carotovora subsp. atroseptica differed when the producer strains were grown in the presence of diverse carbon sources.
As a rule microorganisms exhibit poor growth on media containing D-xylose as both the sole source of carbon and the inducer of xylose isomerase synthesis. In order to increase the production of an enzyme, nutritive media are supplemented with small amounts of readily metabolized sources of carbon [13, 14]. As Fig. 2 demonstrates, increasing the amount of a specific substrate in a medium produced divergent effects in the cultures: biomass accumulation was increased in A. nicotianae and decreased in E. coli, whereas E. carotovora subsp. atroseptica remained unaffected. The synthesis of xylose isomerase in all the bacteria under study was suppressed on increasing the content of D-glucose from 0.1 to 1.5%. Productivity maxima were observed at 1.0% (A. nicotianae) and 0.1% (E. coli and E. carotovora subsp. atroseptica). The effects of D-glucose supplementation of nutritive media containing 0.2% D-xylose were again producer strain-specific. In A. nicotianae the presence of the readily metabolized carbon source stimulated, rather than suppressed, the formation of xylose isomerase at all of the concentrations tested (Fig. 3a).
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For example, the introduction of 1.0% of D-glucose increased the level of enzyme synthesis and the culture productivity by 26 and 63%, compared to the corresponding control values. In E. coli the effects of D-glucose supplementation were opposite (Fig. 3b). Even at minimum concentrations D-glucose suppressed the enzyme synthesis. Introduction of 0.1% D-glucose decreased synthesis by 63%; further elevation of the concentration of the supplement (up to 1.0%) did not augment the inhibitory effect considerably (a 75% decrease was noted). The modified strain E. carotovora subsp. atroseptica gave yet another type of responses to supplementation (Fig. 3c). D-glucose exerted weak suppressing effects on the synthesis of xylose isomerase, which were concentration-independent (up to 7%). Decrease in the productivity of the bacteria, related to inhibition of their growth, was observed at D-glucose concentrations in excess of 0.5% (D-glucose is a catabolic repressor).
The above data allow us to conclude that the synthesis of xylose isomerase by recombinant strains of E. coli and E. carotovora subsp. atroseptica is constitutive, as in the case of A. nicotianae, but regulatory mechanisms, whereby its control is performed differ between the cultures. The structure, physicochemical properties and mechanisms of the action of xylose isomerase were studied in diverse representatives of prokaryotes [1, 15–20]. The table lists the characteristics of purified xylose isomerase preparations from A. nicotianae, E. coli, and E. carotovora subsp. atroseptica. The enzymes produced by the bacteria had identical electrophoretic mobilities in both intact and denatured forms (Fig. 4). The preferred substrate for isomerization was D-xylose; D-glucose was isomerized with a lower efficiency, and only weak effects were observed with D-ribose and L-arabinose (Fig. 5). Determination of the optimum conditions for D-xylose conversion into D-xylulose demonstrated that
Properties of xylose isomerases from A. nicotianae, E. coli, and E. carotovora subsp. atroseptica Producer Property A. nicotianae
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At 70°ë the xylose isomerases retained their activity at a pH 7.0–8.0 for 1 h; on increasing the temperature of incubation to 80°ë, 70–75% of the activity was lost (table).
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Fig. 4. Electrophoretograms of preparations of intact (I) and denatured (II) xylose isomerase of E. carotovora subsp. atroseptica (1), E. coli (2), and A. nicotianae (3), stained with 2,3,5-triphenyltetrazolium (a) or Coomassie Brilliant Blue G-250 (b); molecular weight markers (kDa) for SDS-PAGE (4, top to bottom): myosin (212), α2-macroglobulin (170), β-galactosidase (116), transferrin (76), and glutamine dehydrogenase (53).
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ACKNOWLEDGMENTS This work was supported by the Belarussian Republican Foundation for Basic Research (project no. B00233). REFERENCES
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2. Lobanok, A.G., Sapunova, L.I., Dikhtievski, Ya.O., and Kazakevich, I.O., World J. Microbiol. Biotechnol., 1998, vol. 14, no. 2, pp. 259–262.
50 25 0
Our analysis of the results obtained led us to the conclusion that constitutive syntheses of xylose isomerase by the bacteria A. nicotianae, E. coli, and E. carotovora subsp. atroseptica are controlled by different regulatory systems. The bacteria are characterized by the same levels of synthesis of the enzyme proteins, which do not exhibit significant difference in either electrophoretic mobility, substrate specificity, or major physicochemical properties. Given the considerable differences between the producers in the duration of cultivation and the preferred substrates, the availability of the three strains makes it possible to select an economically efficient variant of the enzyme as appropriate in each exact case.
I
II
III
IV
Fig. 5. Substrate specificity of xylose isomerases of A. nicotianae (1), E. coli (2), and E. carotovora subsp. atroseptica (3): I, xylose; II, glucose; III, ribose; and IV, L-arabinose.
xylose isomerases of A. nicotianae and E. coli exhibited maximum activity at 70°ë and pH 7.5. The optimum conditions for the enzyme produced by E. carotovora subsp. atroseptica were achieved at higher temperatures and alkalinity (75°ë and pH 8.0; table). When studied at the pH optima, the enzymes produced by the bacteria under study retained their activity at 70°ë for no less than 2 h. Following 2 h of incubation at 80°ë, the residual activities of xylose isomerase from A. nicotianae, E. coli, and E. carotovora subsp. atroseptica amounted to 30.5, 30.1, and 37.6%, respectively.
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