Journal of Proteomics & EnzymologyISSN: 2470-1289

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Research Article, J Biocatal Biotransformation Vol: 2 Issue: 2

Enhancing Thermal Stability of Immobilized Raw Starch Digesting Amylase (RSDA)Using Different Additives

Onyetugo C Amadi1,2, Bartho N Okolo2, Cesar Mateo1, Jose M Guisan1 and Benevides C Pessela3*
1Departamento de Biocatálisis Enzimática, Instituto de Catálisis y Petroleoquímica, ICP-CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, España
2Department of Microbiology, Faculty of Biological Sciences, University of Nigeria Nsukka, Nigeria
3Departamento de Microbiología y Biotecnología, Instituto de Investigaciones en Ciencias de los alimentos, CIAL-CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, España
Corresponding author : Dr. Benevides C Pessela
Departamento de Microbiología y Biotecnología, Instituto de Investigaciones en Ciencias de los alimentos, CIAL-CSIC, Campus Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, España
Tel: +34910017962; Fax: +34910017905
E-mail: [email protected]
Received: October 30, 2013 Accepted: December 11, 2013 Published: December 18, 2013
Citation: Amadi OC, Okolo BN, Mateo C, Guisan JM, Pessela BC (2013) Enhancing Thermal Stability of Immobilized Raw Starch Digesting Amylase (RSDA) Using Different Additives. J Biocatal Biotransformation 2:2. doi:10.4172/2324-9099.1000109

Abstract

Enhancing Thermal Stability of Immobilized Raw Starch Digesting Amylase (RSDA) Using Different Additives

Industrial and large scale applications of biocatalysts are often limited by lack of thermostability. Raw starch digesting amylase(RSDA) of Aspergillus carbonarius was immobilized on cyanogen bromide to facilitate mild covalent attachment. To improve thermal stability of immobilized and soluble RSDA different concentrations of sugar (trehalose) and polyols (polyethylene glycol, glycerol and mannitol) under varying conditions; temperature, pH and buffer ionic strength was studied.

Keywords: Aspergillus carbonarius; Thermostability; Immobilized enzyme; Polyols; Sugar

Keywords
Aspergillus carbonarius; Thermostability; Immobilized enzyme; Polyols; Sugar
Introduction
Enzymes which hydrolyze starch have been categorized into four groups which include the endo-acting (α-amylase), exoacting (β-amylase, α-glucosidase), debranching (glucoamylase) and transferase (glycosyltransferase) [1,2]. A minor percentage of these enzymes are termed- Raw starch digesting amylases (RSDA) due to their ability to catalyse the degradation of raw starch granules. They are unlike other starch hydrolases which act on only gelatinized starch. RSDAs vary from other amylases in their special affinity and interaction with the microcrystalline structure of the raw starch molecule, through the starch binding domain [3]. RSDA have gained attention in academia and industry owing to their cost effectiveness, conservation of energy and time. Raw starch hydrolysing amylases find potential applications in paper, food, textile, pharmaceutical, bioanalytical and biomedical industries [4,5]. In view of the great potentials of RSDAs, their application cannot be over exhausted. RSDAs are often produced along with other non-raw starch digesting type and proteolytic enzymes whose activity can result in the digestion of the starch binding domain and consequently loss of raw starch hydrolysing activity [6]. This is somewhat responsible for the poor stability reported for raw starch digesting amylases. Also Robertson et al. [1] associated instability of RSDAs to their catabolite repression by the products of hydrolysis, glucose and maltose. In general, application of RSDAs is limited due to low enzyme activity, poor stability, and incomplete conversion, enzyme inhibition by glucose and maltose and problems of contamination due to application at low temperature.
To be applied industrially an enzyme should function adequately, maintain their stability, selectivity as well as specificity under conditions which are entirely different from their physiological environment. However most natural enzymes are not able to meet with all the demands of an industrial biocatalyst and hence would need improvement of their functional characteristics before application [7,8]. These improvements may be achieved by employing different mechanisms, such as immobilization, covalent modification [9,10] and use of soluble additives.
Additives are often referred to as soluble species added to enzyme solution which has effect on the thermal stability of the protein structure and interact non covalently with groups of the protein. This can be a valuable method for thermostabilization where immobilization or covalent modification is not feasible [11]. When native proteins undergo conformational changes to less active or inactive form and becomes unfolded (denatured) as a result of operational activity or transportation, activity of such may be recovered or retained in the presence of soluble additives, thus prolonging the effective use of the enzyme. Although the degree of stabilization may not be as effective as covalent methods, yet stabilization by soluble additives can be advantageous, as it may not only increase the notional half-life of the enzyme by several folds but also impact significantly economically. Additionally, an already immobilized enzyme may be further stabilized by use of an appropriate additive.
Thermal stability of an enzyme is greatly influenced by the solvent structure. Polyhydric alcohols and sugars modify the solvent structure there by acting as stabilizing agents. Their stabilizing effect depend on the nature of the enzyme studied, its hydrophilic/hydrophobic character and on the degree of its interaction with the additive [12]. Modification of the microenvironment of enzyme using sugars and polyhydric alcohols has been studied with considerable success in understanding the mechanism of thermal inactivation [13-15].
Aspergillus carbonarius (Bainier Thom IMI 1366159) which is able to produce copious amounts of RSDA was previously isolated [16]; the amylase is capable of one step hydrolysis of starch to simple sugars (glucose and maltose) over a wide range of pH and temperature conditions [17]. Regardless of these qualities the enzyme is faced with problems of instability which in turn affects its application in industrial processes. To further stabilize this enzyme immobilization and covalent modification of this enzyme has been studied [18].
Most studies have focused on effect of additives on soluble enzyme. Immobilized enzymes offers numerous advantages including improved stability and activity; however under operational and storage condition immobilized enzyme may experience loss of activity. Although chemical modification is used in addition to immobilization in stabilizing proteins, chemical modification may require change in the overall properties of the enzyme or modification of the key residues this is often more rigorous and may take a longer time. The use of appropriate additives provides a simple and cheap means of stabilizing an already immobilized enzyme thereby increasing the effective use of the biocatalyst. In the present investigation we report for the first time the stabilizing effect of various additives (polyethylene glycol, glycerol, mannitol and trehalose) on covalently immobilized and soluble RSDA from Aspergillus carbonarius.
Materials and Methods
Materials
Cyanogen bromide (BrCN) activated Sepharose 4B was purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). All other reagents were of analytical grade.
Methods
All experiments were performed at least in triplicate and the results were presented as its mean value. Experimental error was never over 10%.
Organism and growth conditions: RSDA was obtained from culture filtrate of A. carbonarius (Bainier Thom IMI 1366159) grown using the submerged fermentation (SmF) method. Approximately 107 spores/mL from a 3-day-old culture was inoculated into 500 mL Erlenmeyer flask containing 100 mL fermentation medium which comprised, (gl-1): Raw cassava starch (Manihotutilissima Crantz), 20; yeast extract, 5; KH2PO4, 7; CaCl2.6H2O, 0.3; and MgSO4.7H2O, 0.3 in de-ionized water. Cultures were incubated at 30oC with rotary shaking at 100 rpm for 96 h, after which mycelial pellets were separated by filtration through Whatman No. 1 filter paper. The filtrate was used as a source of crude extracellular raw starch digesting amylase.
Isolation of enzyme: The crude enzyme filtrate was partially purified by adsorption to MANAE-Agarose through interaction by ion exchange at a ratio of 1 g of support for each 10 mL of enzyme filtrate. The enzyme was desorbed at a 1 M concentration of NaCl solution, dialysed using 0.5 mM phosphate buffer and subsequently stored at 4oC for further use.
Determination of RSDA activity: The enzyme activity of the derivative was determined by indirect test at 25oC, detecting the glucose released from the hydrolysis of 1% starch in 50 mM sodium citrate buffer, pH 6.5. The released glucose was determined spectrophotometrically by the increase in absorbance at 405nm, oxidation of ABTS by a system of glucose oxidase and peroxidase bond (in the presence of oxygen and glucose, glucose oxidase is converted to gluconic acid, releasing oxygen peroxide, this is transferred to the ABTS, by the action of peroxidise producing colour). The reaction mixture consist of 0.5 ml of glucose oxidase 2.5 g /L, 0.5 ml of peroxidise 2.5 g/L, both in sodium phosphate buffer 50 mM, pH 7, 0.4 ml of 1 mM ABTS in 50 mM sodium phosphate buffer, pH 6 and 1.0 ml of 1% starch in 50 mM sodium citrate buffer, pH6, 5. The reaction was initiated by adding an appropriate volume of enzyme derivative (25-200 μL) and the activity determined by taking the slope of the linear region of the curve (absorbance versus time).
Immobilization on CNBr-activated agarose: About 1 g of washed CNBr-activated agarose 4 BCL was prepared according to manufacturer’s instruction (Pharmacia, Sweden), and it was incubated in 5 mL of 0.1 M sodium phosphate buffer at pH 7.0 and 10 mL of RSDA solutions, for 1 h at room temperature under gentle stirring. After that, the immobilized preparation was filtered and remaining reactive groups were blocked with 1 M ethanolamine at room temperature for 2 h by gentle stirring. Finally, the derivative was washed with sodium phosphate buffer 0.1 M, pH 7.0.
Effect of additive on the thermal stability of the immobilized enzyme: The effect of different additives, polyethylene glycol (600, 1500KDa), glycerol mannitol and trehalose were studied over a concentration range of 10-50% at different temperature, pH and buffer ionic strength. One gram of CNBr derivative was incubated in 10 ml of the indicated additive at indicated pH, buffer condition and temperature. Samples of suspension were withdrawn at different time intervals and their residual activity determined as described earlier. Stability was given as half-life.
Effect of additives on soluble enzyme: The effect of different additives, polyethylene glycol (600, 1500 KDa), glycerol, mannitol, trehalose were studied over a concentration range of 10-50% at different temperature, pH and buffer ionic strength. Ten millilitre of soluble enzyme was incubated in the indicated additive at indicated pH, buffer condition and temperature. Samples of solution were withdrawn periodically and their residual activity determined as earlier described. Stability was given as half-life.
Results
Effect of additives on the thermal stability of native and immobilized RSDA
In order to study the effect of additives on RSDA, as a first approach the enzyme was covalently immobilized on CNBr-agarose. A hundred percent covalent attachment was achieved with the support.
Effect of Polyethylene glycol (PEG) on thermal stability of RSDA
In an attempt to improve the thermal stability of RSDA immobilized on CNBr agarose, 600 and 1500 KDa PEG was used at a concentration of 10 and 30%. The derivative together with the additive was suspended in different buffers, acetate and citrate in a 10 mM concentration pH 5 at 53°C. Irrespective of the different buffers used the PEG did not have much stabilizing effect on the immobilized enzyme as shown in Figure 1. On the average half-life of the derivative in the presence of PEG was approximately 60 min. However in the presence of citrate buffer 10% 1500 KDa PEG was able to retain 49% of its initial activity after 360 min incubation.
Figure 1: Effect of the additive PEG on the thermal stability of CNBr derivative of RSDA at 53°C in the presence of (A) acetate buffer 10 mM pH 5; closed diamond symbol represents the cyanogen bromide (CNBr) derivative of RSDA suspended in acetate buffer only, closed square represents the derivative in 30% 600 KDa PEG in acetate buffer, closed triangle represents derivative in 10% 1500 KDa PEG in acetate buffer, open circle represents derivative in 30% 1500 KDa PEG in acetate buffer and (B) citrate buffer 10 mM pH 5; closed diamond is the derivative CNBr in citrate buffer only, closed square is derivative in 30% 600 KDa PEG in citrate buffer, closed triangle is derivative in 10% 1500 PEG in citrate buffer and open circle is derivative in 30% 1500 PEG in citrate buffer.
Effect of the additive glycerol on the thermal stability of RSDA
Figure 2 shows the effect of glycerol on the thermal stability of immobilized RSDA suspended in acetate and citrate buffer 10 mM pH 5 and incubated at 53°C. The addition of Glycerol to the derivative under the study conditions resulted in an increase in the half-life from 180 to 360 min with optimal condition at 50% glycerol citrate buffer.
Figure 2: Effect of glycerol on the thermal stability of CNBr derivative of RSDA at 53°C in (A) acetate buffer; closed diamond represents CNBr derivative of RSDA in acetate buffer only, closed square represents derivative in 10% glycerol in acetate buffer, closed triangle is derivative in 30% glycerol in acetate buffer, open circle is derivative in 50% glycerol in acetate buffer and (B) citrate buffer 10 mM pH 5; closed diamondis the CNBr derivative in citrate buffer only, closed square is derivative in 10% glycerol in citrate buffer, closed triangle is derivative in 30% glycerol in citrate buffer and open circle is derivative in 50% glycerol in citrate buffer.
Figure 3 shows the protective effect of the glycerol on the thermal stability of CNBr derivative of RSDA at 50°C in the presence of tris buffer 10 and 100 mM pH 7. All concentrations of glycerol in the presence of tris 10 mM prolonged the half-life of the derivative from 180-360 min with optimal activity of 89% observed using 50% glycerol. On the contrary increase in the buffer ionic strength (tris 100 mM) resulted in rapid inactivation of the derivative leaving only 48% of its initial activity within 60 min of incubation. However in the presence of glycerol at a concentration of 50 and 30% stability was improved retaining 86 and 56%, respectively of their initial activity after 360 min incubation.
Figure 3: Effect of glycerol on the thermal stability of CNBr derivative of RSDA at 50°C in (A) tris buffer 10 mM pH 7; closed diamond represents CNBr derivative of RSDA in tris buffer only, closed square represents derivative in 10% glycerol in tris buffer, closed triangle is derivative in 30% glycerol in tris buffer, open circle is derivative in 50% glycerol in tris buffer and (B) tris buffer100 mM pH 7; closed diamondis the CNBr derivative in tris buffer only, closed square is derivative in 10% glycerol in tris buffer, closed triangle is derivative in 30% glycerol in tris buffer and open circle is derivative in 50% glycerol in tris buffer.
Figure 4 shows the effect of mannitol on the thermal stability of CNBr derivative treated with acetate and citrate buffer 10 mM pH 5 incubated in 53°C. Under these conditions the derivative was able to maintain an average of 36 and 49% of the initial activity respectively after 60 min incubation. However stability was improved upto 360 min and maximum activity of 83% was observed using 30% mannitol in the presence of citrate buffer.
Figure 4: Effect of mannitol on the thermal stability of CNBr derivative of RSDA at 53°C in (A) acetate buffer 10 mM pH 5; closed diamond represents CNBr derivative of RSDA in acetate buffer only, closed square represents derivative in 10% mannitol in acetate buffer, closed triangle is derivative in 20% mannitol in acetate buffer, open circle is derivative in 30% mannitol in acetate buffer and (B) citrate buffer 10 mM pH 5; closed diamondis the CNBr derivative in citrate buffer only, closed square is derivative in 10% mannitol in citrate buffer, closed triangle is derivative in 20% mannitol in citrate buffer and open circle is derivative in 30% mannitol in citrate buffer.
Figure 5 shows the effect of the additive mannitol on the thermal stability of the derivative in tris buffer 10 and 100 mM pH 7 incubated at 50°C. All the concentrations of mannitol in the presence of tris 10 mM had a positive effect on the thermal stability of the derivative, optimum stability was observed using 30% mannitol with 91% of initial activity retained for period of 360 min. On the other hand in the presence of tris 100 mM, the thermal stability of the derivative was prolonged by a 56-fold increase in the presence of 30% mannitol.
Figure 5: Effect of Mannitol on the thermal stability of CNBr derivative of RSDA at 50°C in (A) tris buffer 10 mM pH 7; closed diamond represents CNBr derivative of RSDA in tris buffer only, closed square represents derivative in 10% mannitol in tris buffer, closed triangle is derivative in 20% mannitol in tris buffer, open circle is derivative in 30% mannitol in tris buffer and (B)tris buffer 100 mM pH 7; closed diamondis the CNBr derivative in tris buffer only, closed square is derivative in 10% mannitol in tris buffer, closed triangle is derivative in 20% mannitol in tris buffer and open circle is derivative in 30% mannitol in tris buffer.
Figure 6 shows the effect of the additive trehalose on the thermal stability of RSDA from A. carbonarius immobilized on cyanogen bromide agarose (CNBr derivative) and treated in acetate and citrate buffer 10 mM pH 5 incubated at 53°C. In the presence of acetate buffer half-life of the derivative was 110 min. Addition of trehalose at varying concentration presented a protective effect that was increased upto 360 min. Optimum stability was obtained using 30% trehalose and 68% of initial activity was retained. With citrate buffer there was a rapid loss of activity the derivative was only able to retain 32% of its initial activity by 60 min incubation. Optimal stability was obtained using 20% trehalose with an increased protective effect of 360 min with 68% of activity retained.
Figure 6: Effect of trehalose on the thermal stability of CNBr derivative of RSDA at 53°C in (A) acetate 10 mMpH 5; closed diamond represents CNBr derivative of RSDA in acetate buffer only, closed square represents derivative in 10% trehalose in acetate buffer, closed triangle is derivative in 20% trehalose in acetate buffer, open circle is derivative in 30% trehalose in acetate buffer and (B) citrate buffer 10 mM pH 5; closed diamondis the CNBr derivative in citrate buffer only, closed square is derivative in 10% trehalose in citrate buffer, closed triangle is derivative in 20% trehalose in citrate buffer and open circle is derivative in 30% trehalose in citrate buffer.
The effect of the additive trehalose on the thermal stability of CNBr derivative treated in tris buffer 10 and 100 mM pH 7 incubated in 50oC is depicted in Figure 7, the concentration of trehalose to improve the stability of the derivative was 30% in both buffer concentration, but tris 10 mM had a higher stabilizing effect of 69%.
Figure 7: Effect of the additive trehalose on the thermal stability of CNBr derivative of RSDA at 50°C, (A) tris buffer 10 mM pH 7; closed diamond represents CNBr derivative of RSDA in tris buffer only, closed square represents derivative in 10% trehalose in tris buffer, closed triangle is derivative in 20% trhalose in tris buffer, open circle is derivative in 30% trehalose in tris buffer and (B) tris buffer 100 mM pH 7; closed diamond is the CNBr derivative in tris buffer only, closed square is derivative in 10% trehalose in tris buffer, closed triangle is derivative in 20% trehalose in tris buffer and open circle is derivative in 30% trehalose in tris buffer.
Effect of additives on thermal stability of native RSDA
The best concentration of additives was further used to study their effect on soluble/native RSDA in the presence of different buffer and their respective pH and temperature conditions. Figure 8 shows the effect of different additives on native RSDA in acetate and citrate buffer 10 mM pH 5 and 53°C. Under these conditions, all additives improved the thermal stability of the native RSDA. Optimum stability of the native amylase was obtained using 30% glycerol, up to 70% initial activity was retained after 300 min incubation while the use of 30% mannitol had the least stabilizing effect; only 54% of the initial activity was retained. In citrate buffer 10 mM and 53°C the native amylase was able to maintain 42% of the initial activity by 60 min of incubation, in the presence of this buffer maximum stability was observed using 30% trehalose and an initial activity of 77% was retained after 300 min incubation. While the use of 30% glycerol offered the least protective effect retaining only 56% of the initial activity.
Figure 8: Effect of different additives on the thermal stability of native/soluble amylase (RSDA) at 53°C in (A) acetate buffer 10 mM pH 5; closed diamond represents RSDA in acetate buffer only, closed square represents RSDA in 30% glycerol in acetate buffer, closed triangle is RSDA in 30% trehalose in acetate buffer, open circle is RSDA in 30% mannitol in acetate buffer and (B) citrate buffer 10 mM pH 5; closed diamondis the RSDA in citrate buffer only, closed square is RSDA in 30% glycerol in citrate buffer, closed triangle is RSDA in 30% trehalose in citrate buffer and open circle is RSDA in 30% mannitol in citrate buffer.
Figure 9 shows the effect of the different additive on the thermal stability of the native amylase in tris buffer 10 mM pH 7 and 50°C. The native amylase was inactivated in less than 60 min of incubation. In the presence of this buffer, 30% glycerol and trehalose had the most stabilizing effect increasing the protective effect upto 180 min, 59 and 50% of the initial activity was retained, respectively.
Figure 9: Effect of different additives on the thermal stability of native amylase (RSDA) in tris buffer 10 mM pH 7 at 50°C. Closed diamond represents RSDA in tris buffer only, closed square represents RSDA in 30% glycerol in tris buffer, closed triangle is RSDA in 30% trehalose in tris buffer, open circle is RSDA in 30% mannitol in tris buffer.
Discussion
Immobilization of enzymes improves the enzyme stability/activity however exposure to high temperature may result in inactivation of the enzyme, reducing the operational life span of the biocatalyst. Hence chemical modification is used in addition to immobilization to further stabilize proteins, although chemical modification improves the enzyme stability it involves change in properties of enzyme surface or modification of key residues which may result to partial or complete loss of activity. One major limitation of RSDA is the problem of loss of activity. In other to achieve stability and retain activity RSDA was mildly immobilized on CNBr, a one point covalent attachment which permits taking advantage of the solid phase. To further improve the immobilized enzyme the use of suitable additives provided a simple and cheap means (as against chemical modification) of further stabilizing the already immobilized RSDA and prolonging the effective use of the immobilized biocatalyst. The significance of this method is that the stabilizing agent does not modify the enzyme residues [19]. Addition of Polyols and sugars to protein solution and changing its microenvironment may facilitate and extend the thermal stability of immobilized enzyme.
In order to improve the thermostability of immobilized RSDA at an increased temperature different additives were added to the enzyme solution. The influence of different additive (PEG, glycerol, mannitol and trehalose) on the thermal stability of immobilized and soluble RSDA of Aspergillus carbonarius at the enzyme inactivation temperature (50 and 53°C) in the presence of different pH and buffer ionic strength was studied.
In the presence of acetate and citrate buffer pH 5(acidic pH) the protective effect of the additives was in this order, Mannitol>Trehalo se>Glycerol>Polyethylene glycol. While with the neutral pH (tris 10 and 100 mM) the stabilizing effect of the additive was in this order Mannitol>Glycerol>Trehalose. The protective effect offered by the additives was dependent on the added compound concentration, pH, buffer and ionic strength.
Most literatures have reported the effect of additives to be stronger at higher concentration [13,20,21]. Graber and Combes [18] observed a protective effect (defined as ratio of alpha-amylase half-life with additives to alpha-amylase half-life without additive) of sorbitol by a factor of 200 in 2.5 M sorbitol solution and a factor of 2000 when sorbitol concentration was increased to 4 M. This is in agreement with the current work where protective effect of the additive increased with increase in concentration.
Additional substance such as sugars polyhydric alcohols are used to stabilize enzymes, these additives strengthens the hydrophobic interactions among non-polar amino acid residues. These interactions together with hydrogen bonds and ionic van der walls interactions are essential for maintaining the native, catalytically active structure of the enzyme. Thus, the strengthened hydrophobic interaction makes protein macromolecules more rigid, and therefore more resistant to thermal unfolding [22]. Furthermore the mechanism of enzyme stabilization in the presence of sugars and Polyols in aqueous media is an indirect action meaning that the additives do not change the protein conformation but influence the physicochemical properties of the system, such as solvent structure, resulting in protein stabilization.
All additives but PEG showed protective effects on the enzyme heat stability Polyethylene glycol in the different concentration under the study conditions did not have any stabilizing effect on the immobilized RSDA of Aspergillus carbonarius. Even the half-life of the derivative was less than 60 min in the presence of the additive. The decreasing activity obtained in the presence of PEG can be interpreted as resulting from increased viscosities. Increase in the viscosity of an enzyme solution may cause reduction in chemical or biological reaction rate which could result in inactivation of the enzyme [23,24]. But, again PEG has been used in the modification of enzymes; inactivation of the derivative in the presence of PEG may also result from interaction between charges on the support/enzyme surface.
The addition of glycerol and mannitol prolonged the activity of the immobilized enzyme. Optimum activity was observed in the presence of citrate buffer 30% mannitol with a 72 fold increase. Again increase in the concentration of glycerol and mannitol brought about increase in their protective effect. Glycerol and mannitol provided maximum protection at a concentration of 50% and 30%, respectively. This stabilisation appears to be due to preferential hydration of the proteins in the presence of these additives that is, the polyols are preferentially excluded from the vicinity of the protein surface. Polyols are able to maintain solvophobic interaction and are capable of forming hydrogen bonds that play the major role in supporting the native conformation of the protein thereby aid in protein stabilization. However several authors have also related the influence of polyols on thermal stability of enzyme to be as a result of hydroxyl group provided [25,26]. Graber and Combes [18] observed that the stabilizing effect of polyols on Aspergillus oryzea alphaamylase was related to the number of hydroxyl groups per molecule for a given fixed molar concentration of polyol.
The optimal additive concentration was used to study their effect on thermostability of the soluble RSDA in the presence of buffer and their inactivating temperature, 30% Trehalose had the most stabilizing effect with 77-fold increase in the presence of citrate buffer. Sugars raise surface tension of water and do not demonstrate any affinity or possible interaction with the protein molecule either in native form or in unfolded state. Lin and Timasheff [27] opined that the increase of surface tension of water in the presence of sugar could be responsible for protein stabilization. According to them in the case of sugars there is a good correlation between the negative preferential interaction and positive surface tension increment, which also led to their suggestion that the stabilization of proteins is due to increase of the surface tension of water.
Also the buffer used in this study played a significant role in influencing the enzyme stabilization. The extent to which an enzyme may be stabilized or destabilized by a buffer depends on many factors thus making the selection of buffer for formulating enzyme very challenging. Ordinarily buffers used to formulate protein should have little or no change in pH with temperature, have maximum capacity at a pH where the protein exhibit optimal stability. In essence buffers should be able to mimic natural conditions at which proteins have shown optimal stability. Several researchers have investigated the effects of various buffer solution ionic strength and pH on the thermal stability of enzymes [28,29].
Thus, protective effects offered by the additives were strongly related to the concentration of the protective compound as well as the pH and buffer conditions. Immobilized RSDA was further stabilized and effective use prolonged in the presence of additives. Trehalose offered the most protective effect on the soluble enzyme.
Acknowledgments
The authors thank the Instituto de Catalisis y Petroleoquimica, ICP-CSIC, Madrid, Spain for the Graduate Fellowship for Amadi O. C. The authors are grateful to Prof. J.M. Guisan of the Departamento de Biocatalysis, ICP-CSIC, Spain, for the assistance rendered throughout the course of this work.

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