Ectoine-mediated protection of enzyme from the effect of pH and temperature stress: a study using Bacillus halodurans xylanase as a model

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In this work, the protective effect of ectoines against pH stress and high temperature has been investigated using an alkaline active xylanase from Bacillus halodurans as a model. The disaccharide, trehalose—a well-known stabiliser for biomolecules (Kaushik and Bhat 2003), has also been included in the study. The enzyme was selected as a model because it is efficiently active at high pH and is highly stable in the pH range of 5.5–10.5 at 50 °C.

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Nội dung Text: Ectoine-mediated protection of enzyme from the effect of pH and temperature stress: a study using Bacillus halodurans xylanase as a model

Appl Microbiol Biotechnol DOI 10.1007/s00253-012-4528-8 BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS Ectoine-mediated protection of enzyme from the effect of pH and temperature stress: a study using Bacillus halodurans xylanase as a model Doan Van-Thuoc & Suhaila O. Hashim & Rajni Hatti-Kaul & Gashaw Mamo Received: 19 July 2012 / Revised: 5 October 2012 / Accepted: 18 October 2012 # Springer-Verlag Berlin Heidelberg 2012 Abstract Compatible solutes are small, soluble organic Keywords Compatible solutes . Ectoine . Hydroxyectoine . compounds that have the ability to stabilise proteins against Xylanase . Protein stabilisation various stress conditions. In this study, the protective effect of ectoines against pH stress is examined using a recombi- nant xylanase from Bacillus halodurans as a model. Introduction Ectoines improved the enzyme stability at low (4.5 and 5.0) and high pH (11 and 12); stabilisation effect of hydrox- Organisms adapted to thrive in extreme environments have yectoine was superior to that of ectoine and trehalose. In the evolved strategies that allow them to survive and flourish in presence of hydroxyectoine, residual activity (after 10 h the harsh ecology they inhabit. Halophiles, a group of extrem- heating at 50 °C) increased from about 45 to 86 % at pH 5 ophiles growing in high salt environment have two adaptive and from 33 to 89 % at pH 12. When the xylanase was strategies to cope with high external salt concentration—one incubated at 65 °C for 5 h with 50 mM hydroxyectoine at is the accumulation of inorganic ions, used by members of the pH 10, about 40 % of the original activity was retained extremely halophilic Archaea of family Halobacteriaceae and while no residual activity was detected in the absence of the Bacteria order Haloanaerobiales (Empadinhas and da additives or in the presence of ectoine or trehalose. The Costa 2008; Oren 2002), and the other involving the accumu- xylanase activity was slightly stimulated in the presence of lation of small, highly soluble osmolytes, also known as 25 mM ectoines and then gradually decreased with increase compatible solutes, is found in the vast majority of this group in ectoines concentration. The thermal unfolding of the of microorganisms (Oren 1999, 2006, 2008; Roberts 2005). enzyme in the presence of the compatible solutes showed Today, several organisms belonging to different taxonomic a modest increase in denaturation temperature but a larger groups of Archaea, Bacteria and Eukarya are known to pro- increase in calorimetric enthalpy. duce and/or accumulate one or more types of compatible solutes. These compounds are zwitterionic, noncharged or anionic and are represented by various classes of organic D. Van-Thuoc : S. O. Hashim : R. Hatti-Kaul : G. Mamo (*) compounds including polyols, sugars, amino acids, betaines, Department of Biotechnology, ectoines and their derivatives (Lentzen and Schwarz 2006; Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, 221 00 Lund, Sweden Roberts 2005). The diversity of these compounds has been e-mail: gashaw.mamo@biotek.lu.se increasing as more organisms, especially thermophilic and hyperthermophilic Bacteria and Archaea, have been examined D. Van-Thuoc (Empadinhas and da Costa 2006). Department of Biotechnology and Microbiology, Compatible solutes protect not only cells but also proteins Faculty of Biology, Hanoi National University of Education, 136 Xuan Thuy, Cau Giay, and other labile molecules from the deleterious effects of Hanoi, Vietnam environmental stress. Protection of cells, enzymes and DNA by compatible solutes against the effect of heat, freezing and S. O. Hashim desiccation has been proven (Lippert and Galinski 1992; Louis Department of Chemistry and Biochemistry, School of Pure and Applied Sciences, Pwani University College, et al. 1994; Welsh 2000), and this has promoted a growing P.O. Box 195-80108, Kilifi, Kenya interest in using these solutes in various biotechnological
Appl Microbiol Biotechnol applications. Particularly, ectoines (ectoine and its hydroxy the B. halodurans xylanase (2.2 μg/ml) and 50 mM of the derivative, hydroxyectoine) have been studied extensively. compatible solutes, respectively at 65 °C in glycine–NaOH Ectoines have been shown to stabilise enzymes against the buffer, pH 10. Samples were taken at different time intervals denaturing effects of heating, freezing, freeze–thawing, drying, for determination of the residual xylanase activity. oxidative damage and proteolysis (Andersson et al. 2000; The effect of the solutes (50 mM) at pH values lower and Göller and Galinski 1999; Kolp et al. 2006; Lippert and higher than the optimal pH range for stability of the enzyme Galinski 1992). was studied by incubation with the enzyme (2.2 μg/ml) in The pH of biological systems affects the biochemical 50 mM buffer (sodium acetate at pH 4.5 and 5 and glycine– reaction rates and stability of biological machineries like NaOH at pH 11 and 12) at 50 °C for 10 h followed by proteins and other macromolecular structures having pro- determination of residual activity. found importance to the very existence of life. Particularly, All the analyses were performed in triplicates. enzymes, the biological catalysts in myriad biochemical reactions of life are known to be active and stable only Xylanase activity assay within a range of pH, often around neutrality. The great majority of enzymes are neither active nor stable at ‘ex- The xylanase activity was determined based on the release treme’ pH conditions. On the other hand, a variety of bio- of reducing sugar from xylan using the dinitrosalicylic acid technological applications require the use of enzymes that (DNS) method (Miller 1959). A mixture of appropriately are operationally stable at extreme pH. For example, kraft diluted xylanase and 1 % (w/v) birchwood xylan dissolved pulping requires xylanases that are active and stable under in 50 mM glycine–NaOH buffer, pH 10 was incubated at the conditions of high pH and high temperature (Kulkarni et 70 °C for 10 min, and the reaction was stopped by adding al. 1999). Similarly, enzymes for detergent formulation and DNS reagent and placing the tubes in a boiling water bath leather tanning should be active and stable at high pH for 5 min. Subsequently, the tubes were cooled by placing in (Horikoshi 1999). cold water for 10 min and absorbance of the samples read at In this work, the protective effect of ectoines against pH 540 nm (Mamo et al. 2006b). A calibration curve was made stress and high temperature has been investigated using an using xylose as the standard. One unit of xylanase activity alkaline active xylanase from Bacillus halodurans as a was defined as the amount of enzyme releasing 1 μmol model. The disaccharide, trehalose—a well-known stabiliser reducing sugar/min under the standard assay conditions. for biomolecules (Kaushik and Bhat 2003), has also been included in the study. The enzyme was selected as a model Effect of pH and high temperature on stability of the ectoines because it is efficiently active at high pH and is highly stable in the pH range of 5.5–10.5 at 50 °C. Furthermore, it shows The stability of the ectoines was determined at 70 °C in a clear pH-dependent stability profile at higher temperature; 50 mM glycine–NaOH buffer, pH 10 or 12. Samples were at 65 °C the enzyme retained over 60 % of its initial activity withdrawn at different time intervals and analysed by HPLC after 3 h at pH 9, but there was no detectable activity at (Perkin-Elmer HPLC system) on an Aminex HPX-87C col- pH 10 (Mamo et al. 2006b). umn (Bio-Rad) maintained at 65 °C and using 5 mM calci- um chloride as the mobile phase at a flow rate of 0.3 ml/min. The solutes were detected at 210 nm on a UV detector. The Materials and methods decrease in the level of ectoines was measured based on the percent decrease in peak area of the samples. Ectoine treated Materials with 0.1 M KOH was used as control. The pH of the samples was measured using Mettler Ectoine and hydroxyectoine were purchased from Fluka Toledo SevenEasy pH meter equipped with InLab® (Buchs, Switzerland). Birchwood xylan and trehalose were Routine Pro pH probe. obtained from Sigma (Sigma, Germany). A recombinant xylanase from an alkaliphilic B. halodurans (DSM-18197) Differential scanning calorimetry was purified following the procedure described in a previ- ous report (Mamo et al. 2006a). The effect of ectoine and hydroxyectione, respectively, on the thermal stability of the B. halodurans xylanase (13.5 μM) was Effect of compatible solutes on the pH and thermal stability investigated at pH 9 and 10 using a Microcal VP-DSC instru- of B. halodurans xylanase ment (Northampton, MA). All solutions were degassed prior to analyses and DSC scans were performed from 20 to 100 °C The enzyme stabilising effect of ectoine, hydroxyectoine at a rate of 1 °C/min. The data generated were analysed using and trehalose, at high temperature was studied by incubating Origin™ software. Appropriate buffer scans were subtracted
Appl Microbiol Biotechnol from sample scans prior to determination of molar excess heat capacities (Cp) by normalising the experimental thermograms with enzyme concentration and volume of the calorimeter cell. The apparent denaturation temperature (Tm) was determined as the temperature corresponding to maximum Cp (Cpmax) and the calorimetric enthalpy (ΔHcal) was calculated by integrating the area under the peak. A cubic baseline was subtracted prior to the determination of thermodynamic parameters. Results Stabilisation of the xylanase by compatible solutes against the denaturing effect of pH and temperature Fig. 2 Protective effect of compatible solutes on B. halodurans xyla- nase at low and high pH values. The enzyme (2.2 μg/ml) was incubat- In agreement with our previous results (Mamo et al. 2006b), ed in the absence and presence of 50 mM compatible solutes at 50 °C B. halodurans xylanase was found to completely lose its for 10 h after which the residual activity of the enzyme was deter- mined. The buffers used were 50 mM sodium acetate (pH 4.5 and 5), activity within 5 h when incubated at 65 °C and pH 10. and glycine–NaOH (pH 11 and 12) Incubation in the presence of 50 mM ectoine, hydroxyec- toine and trehalose, respectively, showed only hydoxyec- toine to have some protective effect against deactivation of stability—the residual activity increased from about 45 to the enzyme, retaining 40 % of the original activity after 5 h 86 % when incubated at pH 5 and 33 to 89 % at pH 12. At incubation (Fig. 1). pH 4.5, the enzyme retained 8–18 % of its original activity The stabilising effect of the compatible solutes was then in the presence of the ectoines. Trehalose provided little tested at both low and high pH conditions, which from our stabilisation effect at pH 5 and 11 but not at pH 4.5 and 12. earlier studies have shown a clear denaturing effect on B. In order to ensure that the stabilisation achieved above halodurans xylanase (Mamo et al. 2006b). After 10 h incu- was conferred by the ectoines, the stability of the ectoines bation at 50 °C, the enzyme in the absence of additives had was tested at high pH and temperature. While treatment with completely lost its activity at pH 4.5 while about 30 % of the 0.1 M KOH resulted in a near-complete (>95 %) depletion original activity was maintained at pH 12, but the enzyme of the ectoines within 5 h incubation at 70 °C, about 83 % of retained higher activity in the presence of 50 mM compat- the ectoine and 64 % of the hydroxyectoine were detected ible solutes (Fig. 2). Hydroxyectoine provided the highest after 10 h incubation at pH 12. The stability of ectoines at pH 10 was significantly higher and more than 90 % of the ectoines remained unaffected by the treatment. Even the potential pH drift of the buffer that might happen due to ectoines degradation was investigated. As shown in Fig. 3, the pH drift was insignificant after 10 h of incubation at 70 °C in the buffer with initial pH of 10, however at the initial pH of 12, the pH falls by over 1 unit within the first 2 h. Effect of varying concentrations of ectoines on xylanase stability and activity The xylanase was incubated with varying concentration of the ectoines (25–300 mM) at pH 10 and 65 °C for 5 h after which the residual enzyme activity was determined. As shown in Fig. 4, increased stability of the xylanase was observed right from the lowest concentration of hydroxyectoine tested, and Fig. 1 Effect of compatible solutes on thermal stability of B. halodur- with increasing hydroxyectoine concentration to 300 mM the ans xylanase at pH 10 and at 65 °C. The enzyme (2.2 μg/ml) was residual activity increased to 60 % of the original activity. incubated in 50 mM glycine–NaOH buffer, pH 10 in the absence or presence of 50 mM ectoine, hydroxyectoine and trehalose, respective- Ectoine, on the other hand, did not provide any stabilising ly. Samples were withdrawn every hour, and the residual activity of the effect up to a concentration of 150 mM, and higher concen- enzyme was determined trations resulted in a maximum of about 10 % residual activity.
Appl Microbiol Biotechnol Fig. 3 Change in the pH of the ectoine or hydroxyectoine contaning buffer during incubation at 70 °C. Samples were taken at different time intervals, and the pH was measured using pH meter. The symbols, Fig. 5 Relative activity of B. halodurans xylanase in the presence of filled triangles and filled circles, stand for ectoine and hydroxyectoine, varying concentrations of ectoine and hydroxyectoine, respectively. respectively, when the initial pH of the buffer was about 12; whereas The activity of the enzyme was measured at 70 °C with 1 % (w/v) filled squares and filled diamonds stand for ectoine and hydroxyec- birchwood xylan dissolved in 50 mM glycine–NaOH buffer, pH 10 toine, respectively, when the initial pH was 10 Effect of ectoines on the thermodynamic properties On the other hand, when the xylanase assay at 70 °C and of the xylanase pH 10 was performed in a reaction mixture containing different concentrations of the ectoines, highest xylanase The presence of ectoines at 0.2 M concentration during the activity was observed at 25 mM ectoines and was followed thermal unfolding of B. halodurans xylanase at pH 9 and 10 by a gradual decrease in activity with increase in concentra- resulted in a modest increase in Tm (0.5–0.9 °C), but led to a tion (Fig. 5). The enzyme assay was subsequently run at significant increase in ΔHcal-more than 2-fold increase being different pH values in buffers with 25 mM ectoine, hydrox- seen in the presence of hydroxyectoine (Fig. 7a, b). The shape yectoine and trehalose, respectively. While there was no of the thermogram generated by thermal unfolding of the en- apparent effect of the ectoines on the activity at pH 4.5, zyme in presence of 0.2 M hydroxyectoine at pH 9 is markedly the enzyme activity increased to a certain extent in a pH more asymmetric at temperatures below the Tm (Fig. 7a, inset). range of 5–12 (Fig. 6). The maximum increase in activity (44 %) was observed at pH 12 in the presence of hydrox- yectoine and trehalose. Fig. 6 Effect of compatible solutes on the activity of B. halodurans xylanase at different pH. The enzyme was incubated at 70 °C with 1 % Fig. 4 Effect of the concentration of compatible solutes on thermosta- (w/v) birchwood xylan dissolved in 50 mM buffers in the absence and bility of B. halodurans xylanase. The enzyme (2.2 μg/ml) was incu- presence of 25 mM ectoine, hydroxyectoine or trehalose, respectively. bated in 50 mM glycine–NaOH buffer, pH 10 in the presence of The buffers used were sodium acetate (pH 4.5 and 5) and glycine– varying concentrations of ectoine or hydroxyectoine at 65 °C for 5 h NaOH (pH 9, 11 and 12). For each pH, the enzyme activity without after which the residual activity of the enzyme was measured compatible solute is considered as 100 %
Appl Microbiol Biotechnol a 700 599.4 600 ΔHcal (kcal/mol) / Tm (°C) 500 400 287.2 300 258.4 200 100 76.5 77 77.2 0 xylanase xylanase + 0.2 M xylanase + 0.2 M ectoine hydroxyectoine b 500 434.2 400 ΔHcal (kcal/mol) / Tm (°C) 320.1 300 215.5 200 100 71.6 72.1 72.5 0 xylanase xylanase + 0.2 M xylanase + 0.2 M ectoine hydroxyectoine Fig. 7 Thermal unfolding of B. halodurans xylanase in presence and calculated calorimetric enthalpies, ΔHcal (in kilocalories per mole). absence of ectoines by differential scanning calorimetry at (a) pH 9 and Inset, thermograms generated by thermal unfolding of the xylanase in (b) 10 in a Microcal VP-DSC instrument. The enzyme was used at a absence (solid line) and presence of 200 mM ectoine (dashed line) or concentration of 13.5 μM in 20 mM glycine–NaOH buffer. White bars hydroxyectoine (dotted line) represent Tm (in degree Celsius) values while black bars represent the It is worth noting that the lower temperature region of the scans of the enzyme from 25 to 90 °C resulted in the recovery endotherm for this enzyme exhibits a great degree of revers- of about 83.3 % of the endotherm (data not shown). A DSC ibility at pH 9. The extent of reversibility of thermal unfolding transition is considered calorimetrically reversible if 85–90 % was determined in a separate experiment, by reheating and of the endotherm is recovered in the heating run (Ibarra- subsequent cooling of the same enzyme sample in the DSC Molero and Sanchez-Ruiz 2006). Thus, this enzyme exhibits sample cell, at defined temperatures. The maximum heat a high degree of reversibility within the pre-transition region. capacity (Cpmax) is fully recovered when the enzyme is The effect of ectoine and hydroxyectoine concentration on reheated up to 85 °C. In addition, performing two consecutive the Tm value of the xylanase at pH 10 was also considered.
Appl Microbiol Biotechnol The highest Tm values of 73.3 and 75.1 °C were obtained in ectoine. If the addition of hydroxyl group on the ectoine the presence of 1 M ectoine and hydroxyectoine, respectively, structure enhances the preferential hydration effect, then the in comparison to 71.6 °C in the absence of the solutes. stability remains to be elucidated. Trehalose is known to be Presence of 2 M ectoine during thermal unfolding of the an excellent protein stabiliser as compared with other dis- enzyme resulted in protein aggregation after unfolding, while accharides, polyols, proline and betaine (Borges et al. 2002); 2 M hydroxyectoine resulted in a flattened thermogram (data however, in this study, the sugar was not as effective as the not shown). ectoines in providing stabilisation against high pH. Ectoines are known to degrade at high pH into N-acetyl- diaminobutyric acid by consuming a hydroxyl group (Kunte Discussion et al. 1993), which may lower the pH of the buffer. We noted that the ectoines were degraded in 0.1 M KOH but were Stability of proteins at high pH is of interest both from significantly stable at pH 12 (83 and 64 % of ectoine and fundamental and applied perspectives. Protein engineering hydroxyectoine remaining after 10 h) in glycine–NaOH and immobilisation have been used to improve enzyme buffer. Nevertheless, the pH was decreased by over 1 pH stabilities at high pH (Bhandari et al. 2008; Gülich et al. unit and may partly contribute to the stabilisation observed 2002; Mateo et al. 2007). However, these techniques of in the presence of ectoines. When the initial pH was set at protein stabilisation are relatively complex, expensive, time 10, the degradation of the ectoines and the concomitant consuming and sometimes unpredictable. On the other hand, reduction of the buffer pH were very low (Fig. 3); hence, the use of simple additives that stabilise proteins at high pH the observed xylanase stability in this study is because of the is technically simple, fast and cheap. Ectoines are known stabilising effect of the ectoine against the effect of high pH. stabilisers of proteins against several stress factors such as Differential calorimetric studies on the xylanase at lower salinity, heat, freezing and desiccation (Göller and Galinski concentrations of the ectoines (such as 50 mM) showed no 1999; Lippert and Galinski 1992). This stabilisation effect is detectable change compared with the ectoine free control due to the exclusion of ectoines from the protein surface that (data not shown). Thus, only high concentrations of the results in preferential hydration of the protein (Galinski 1993; ectoines were considered for the thermodynamic studies. Street et al. 2006). The compatible solutes are believed to In contrast to the earlier studies reporting very significant increase the surface tension of water that disfavours the in- increase in melting temperatures of proteins in the presence crease in surface area of the protein and consequently results of high concentrations of different compatible solutes in a compact folded state (Ratnaparkhi and Varadarajan 2001). (Santoro et al. 1992), the observed increase in the melting This mechanism is also expected to improve the enzyme temperatures of the xylanase in the presence of up to 1 M stabilisation against the effect of pH stress by decreasing the ectoines was modest, with hydroxyectione showing a slight- conformational entropy of the unfolded state. We have earlier ly higher increase in Tm than ectoine. On the other hand, suggested the stability of the alkaline active enzymes at high more than 2-fold increase in the ΔHcal was achieved in the pH to be attributed to a water shield surrounding the acidic presence of 200 mM hydroxyectoine (Fig. 7). As unfolding surfaces of the enzymes (Mamo et al. 2009). It is likely that the is made more unfavourable in presence of compatible sol- presence of ectoines re-inforces the water shield of the enzyme utes (Ratnaparkhi and Varadarajan 2001), the increased and subsequently increase its stability. energy uptake observed below the Tm, would contribute to According to our earlier studies, the endoxylanase from the increased calorimetric enthalpy. Increase in calorimetric B. halodurans has good stability in the pH range of 5.5–10.5 enthalpy has previously been attributed to the stabilising at 50 °C (Mamo et al. 2006b). Its stability decreases sharply effect of additives (D’Amico et al. 2001). The stabilising above pH 10.5 and below pH 5.5. As shown in Fig. 2, effect of hydroxyectoine is also enthalpic in nature (Knapp ectoine and hydroxyectoine provided varying degree of et al. 1999), which is clearly demonstrated even for the protection to the enzyme at low as well as high pH. xylanase at high pH. Although these compounds are closely related in structure There is a general notion that compatible solutes do not (differing by a hydroxyl group), in vitro studies have shown affect the physiology of organisms even at molar concen- that hydroxyectoine often has superior protein-stabilising trations; nevertheless, the presence of these compounds properties than ectoine and other compatible solutes above certain concentrations is known to decrease the ac- (Borges et al. 2002; Knapp et al. 1999; Lippert and tivity of enzymes in vitro (Kolp et al. 2006; Kurz 2008; Galinski 1992), and the results in this study support these Schnoor et al. 2004). Similarly, a decrease in the activity of observations. Even in vivo, organisms tend to produce more the B. halodurans xylanase was observed when the concen- hydroxyectoine than ectoine when the stress condition is tration of the ectoines was above 25 mM (Fig. 5). However, more severe (Guzmán et al. 2009; Van-Thuoc et al. 2010), there is no available report on the effect of ectoines concen- which may be due to its better protection efficiency than tration on the activities of intracellular enzymes of ectoine
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