ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2014, том 50, № 1, с. 34-38
UDC 576.80:577.15
PURIFICATION AND CHARACTERIZATION OF AN EXTREMELY STABLE GLUCOSE ISOMERASE FROM Geobacillus thermodenitrificans TH2
© 2014 L. Konak, Y. Kolcuo glu, E. Ozbek, A. Colak, B. Ergenoglu
Department of Chemistry, Karadeniz Technical University, 61080 Trabzon, Turkey
e-mail: acolak@ktu.edu.tr Received March 28, 2013
The D-glucose/D-xylose isomerase was purified from a thermophilic bacterium, Geobacillus thermodenitrificans TH2, by precipitating with heat shock and using Q-Sepharose ion exchange column chromatography, and then characterized. The purified enzyme had a single band having molecular weight of 49 kDa on SDS-PAGE. In the presence of D-glucose as a substrate, the optimum temperature and pH of the enzyme were found to be 80°C and 7.5, respectively. The purified xylose isomerase of G. thermodenitrificans TH2 was extremely stable at pH 7.5 after 96 h incubation at 4°C and 50°C. When the thermal stability profile was analyzed, it was determined that the purified enzyme was extremely stable during incubation periods of 4 months and 4 days at 4°C and 50°C, respectively. The Km and Vmax values of the purified xylose isomerase from G. thermodenitrificans TH2 were calculated as 32 mM and 4.68 ^mol/min per mg of protein, respectively. Additionally, it was detected that some metal ions affected the enzyme activity at different ratios. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.
DOI: 10.7868/S0555109914010061
Xylose isomerase (EC 5.3.1.5), also known as glucose isomerase, is an enzyme that reversibly catalyzes the isomerization of D-xylose to D-xylulose in vivo. It also converts D-glucose into D- fructose in vitro [1]. This enzyme has been used commercially because of its glucose isomerization activity to produce a high-fructose corn syrup (HFCS) [2] as a sweetener in soft drinks and other food products [3]. Xylose isomerase also has an industrial interest to produce xylulose which can be fermented to ethanol by yeast. Due to the industrial significance of the enzyme, xylose isomerases from various sources have been purified, biochemically characterized, clonned and their catalytic and physicochemical properties have also been reviewed [4—6].
Most commercially used xylose isomerases have been isolated from mesophilic microorganisms, including Steptomyces, Flavobacterium and Actinoplanes species [1, 7]. It is easily seen that the activity and stability of these enzymes require metal ions and the pH optima of the enzymes are in the range of 7.5—9.0. Higher temperatures and alkaline pHs lead to the formation of by-product in the resulting fructose solution. Therefore, industrial process of the fructose is limited to 60°C and requires neutral or slightly acidic pHs [8].
Thermophilic microorganisms which intrinsically produce thermostable enzymes evolve and adapt themselves to the extreme environment of their natural habitats. It is known that enzymes derived from
thermophilic organisms are more resistant than meso-philic enzymes. In industry, developing of one step processes for the conversion of starch to HFCS and ethanol production via conversion of xylose to xylulose is useful [9—11]. Hence, investigation of xylose isomerase from new source acting at high temperature is very important.
The aim of the study was to purify and biochemically characterize pH- and thermo-stable xylose isomerase from Geobacillus thermodenitrificans TH2, (obtained from Qamur thermal springs in Aydin, Turkey) growing at 55 °C and utilizing xylose. The enzyme was active and stable at high temperatures and nearly neutral pHs which are desirable for the usage in the food and ethanol industry.
MATERIALS AND METHODS
Materials. Substrates were purchased from Sigma (USA) and other reagents used were of analytical grade.
Bacterial strain, culture conditions and enzyme extraction. Geobacillus thermodenitrificans TH2 was isolated from Qamur thermal springs in Aydin, Turkey. Bacteria were grown aerobically at 55° C in enriched medium [12] until the optical density reaching of 0.6— 0.9 at 600 nm [13]. Then the culture was induced by adding 0.5% (v/v) D-xylose into growing medium and incubating for ~4 h. The cells were harvested in late exponential growth phase by centrifugation at 17000 x g for
3 min at 4°C and resuspended in cold resuspension buffer containing 50 mM MOPS (pH 7.0) with 100 mM glucose, 10 mM MgSO4 and 1 mM CoCl2. Sonication was applied to the resuspended cells in an ice bath (80% amplitude, 1.0 cycle for 5 min) and sonicate was centrifuged at 10000 x g for 15 min. The supernatant was used as a crude enzyme extract.
Enzyme assay. The formation of D-fructose from D-glucose was measured by using the colorimetric assay ofDische and Borenfreund [14]. 100 ^L of reaction mixture including 5 ^L enzyme solution was incubated for 30 min at 80°C. After incubation, the reaction was stopped with 100 ^L perchloric acid. The amount of fructose formed was determined by the cystein-carba-zole-sulfuric acid method [14]. One unit (U) of enzyme activity was defined as 1 ^mol fructose formed per minute.
Purification of xylose isomerase. The crude extract of G. thermodenitrificans TH2 was heated for 15 min at 75°C and denaturated proteins were collected by cen-trifugation (20000 x g, 20 min). Afterwards, the supernatant was precipitated by using an equal volume of cold acetone (chilled at —30°C) and incubated for 2 h at 4°C. The acetone precipitate was collected by cen-trifugation (10000 x g, 15 min, 4 °C). The supernatant was decanted and the pellet was left overnight to remove acetone at 4°C. Dried pellet resuspended in resuspension buffer and loaded on a Q-Sepharose FF anion exchange column (1.5 x 30 cm, Sigma, USA) equilibrated with 20 mM MOPS (pH 7.0) buffer containing 100 mM glucose, 10 mM MgSO4 and 1 mM CoCl2 at a flow rate of 1 mL/min. After loading the column, it was washed with the same buffer to elute unbound proteins. Bound proteins were eluted by linear NaCl salt gradient (0—0.6 M) in the same buffer. The presence of proteins in eluates was monitored by measuring absorbance at 280 nm. The fractions were examined for xylose isomerase activity as explained above. The active fractions were pooled and concentrated by ultrafiltration (ultracell membrane 10000 MWCO Millipore, Amicon, USA).
Lowry's method was used to determine the protein concentration at 650 nm. Calibration curve was generated with BSA standard solutions.
SDS-PAGE. Denaturing SDS-PAGE was performed as described by Laemmli [15] using a 12% polyacrylamide gel. Coomassie brilliant blue R-250 was used for protein staining, and the estimation of molecular weight of the polypeptide was performed by using calibration curve of the protein standards.
Effects of pH and temperature on enzyme activity. Xylose isomerase activity of G. thermodenitrificans TH2 was measured at various pH values ranging from 4.0 to 9.0. 50 mM of the following buffers were used at indicated pH; sodium acetate (pH 4.0-5.5), MOPS (pH 6.0-7.5) and Tris-HCl (pH 8.0-9.0).
Influence of temperature on enzyme activity was determined at the optimum pH in a temperature range
of40-90°C with 10°C increments. The results for optimum pH and temperature studies were presented as percentage of relative xylose isomerase activity and the optimum values were used for further characterization studies.
Effect of substrate concentration on xylose isomerase activity. The reaction was performed at constant protein and various glucose concentrations (2-250 mM) at optimum pH and temperature. For determination of the kinetic data, 1/specific activity (1/V) values were plotted versus 1/[S]. Michaelis-Menten constant (Km) and the maximum velocity (Vmax) were calculated from Lin-eweaver-Burk plot [16].
pH and thermal stability. pH stabilities were examined by incubating the purified enzyme of G. thermodenitrificans TH2 at 4, 50 and 80°C up to 15 days in 50 mM MOPS buffer (pH 7.5). At the end of the each storage periods, the activities were assayed at incubation pH. The percentage residual enzyme activity was calculated by comparison with unincubated enzyme activity.
To determine the thermal stability of the bacterial enzyme, the enzyme eluate was incubated at different temperatures (4, 50 and 80°C) in 50 mM MOPS buffer (pH 7.0) for 1, 4, 24, 48, 72 and 96 h incubation periods. The activity after incubation was assayed under standard reaction conditions and the percentage residual activity was compared with control activity without incubation.
Effects of metal ions on enzyme activity and stability. Metal ion effects on the G. thermodenitrificans TH2 xylose isomerase activity and stability were tested in the presence of Mg2+, Mn2+, Co2+, Hg2+, Zn2+, Ca2+, Ni2+, Fe2+ and Cu2+ of 5 mM final concentration in the reaction mixture. Enzyme activity determined in the absence of metal ions was defined as 100% and the residual activity was calculated by comparison with standard assay mixture containing no metal ion.
RESULTS AND DISCUSSION
Purification. In this study, the purified xylose isomerase of G. thermodenitrificans TH2 was obtained by using heat treatment, acetone precipitation and ion exchange chromatography. Fractions having high enzyme activity were pooled as purified xylose isomerase. Purification steps are summarized in Table. All procedure resulted in 24.3-fold purification, with a yield of 90% and a specific activity of 66.3 U/mg of protein. In literature, xylose isomerase was purified 39.2-fold with yield of 20% from Opuntia vulgaris [17] and 25.6-fold with yield of 17.9% from Cereuspterogonous [18].
Electrophoresis analysis. The purity of the enzyme was confirmed by the presence of sharp single band on SDS-PAGE (Fig. 1). The single band on SDS-PAGE suggests that the xylose isomerase of G. thermodenitrificans TH2 is monomer of approximatel
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