Improved thermodynamic stability of subtilisin Carlsberg by covalent modification

Enzyme and Microbial Technology 39 (2006) 301–307

Improved thermodynamic stability of subtilisin Carlsberg by covalent modification S. Srimathi a,b , G. Jayaraman a,∗ , P.R. Narayanan b,∗∗ a

Centre for Protein Engineering and Biomedical Research, The Voluntary Health Services, Adyar, Chennai 600113, India b Tuberculosis Research Centre, Mayor V.R. Ramanathan Road, Chetput, Chennai 600031, India Received 6 June 2005; accepted 28 October 2005 This article is dedicated to Prof. P.V. Sundaram.

Abstract The present work is aimed at improving the kinetic, thermal and thermodynamic stability of subtilisin Carlsberg (SCB) obtained from Bacillus licheniformis by means of simple, inexpensive but effective covalent coupling to oxidized sucrose polymers (OSP) of varying sizes (OSP400 and OSP70) as well as polyglutaraldehyde (PGA). In the presence of 10 mM calcium the half-life of the enzyme at 60 ◦ C increased by 6.06-fold, 5.20-fold and 2.92-fold when coupled with OSP400, OSP70 and PGA, respectively. Even in the absence of added calcium the stability against thermal inactivation was found to be greater for the modified enzymes as evident from the increase in the energy of activation for the inactivation process (Eai ). Guanidium thiocyanate-induced unfolding indicated Cm values of 1.3 M, 1.8 M, 1.5 M and 1.4 M for the native and enzymes modified with OSP400, OSP70 and PGA, respectively. Thermally induced unfolding was delayed for the modified enzymes as evident from the shift in Tm of 8.45 ◦ C, 5.91 ◦ C and 4.66 ◦ C for OSP400, OSP70 and PGA modified enzymes. The results indicate that among the modifiers used OSP400 was most effective in stabilizing the enzyme and interestingly the increase in stability reported here is comparable to the most stabilized subtilisin variants obtained by site-directed mutagenesis. © 2005 Elsevier Inc. All rights reserved. Keywords: Carbohydrates; Chemical modification; Conformational stability; Heat inactivation; Subtilisin

1. Introduction Commercial exploitation of enzymes, with a potential for use in a wide variety of applications began to be recognized as a genuine area of research and development around the early 1980s. One of the enzymes that could be used in a consumer product with a vast market was the alkaline protease of the subtilisin family. What was seen as having a market worth millions of dollars has clearly outstripped all calculations and is today valued as billions of dollars. However, only enzymes with optimal levels of activity and stability will be commercially attractive. Glycoproteins perform a wide variety of biological functions and are often superior in stability compared to the carbohydrate-free ∗ Corresponding author. Present address: Biotechnology Unit, School of Biotechnology and Chemical Engineering, Vellore Institute of Technology, Vellore 632014, India. Tel.: +91 44 2254 2252; fax: +91 44 2254 2018. ∗∗ Corresponding author. Tel.: +91 416 2202 2608; fax: +91 416 224 3092. E-mail addresses: [email protected] (G. Jayaraman), [email protected] (P.R. Narayanan).

0141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.10.027

proteins [1]. In fact, it has been suggested that the carbohydrate part stabilize an otherwise less stable polypeptide chain [2]. It is reasonable to expect that deliberate attachment of carbohydrates in vitro to a carbohydrate-free protein might help in the manipulation of catalytic and stability properties of enzymes. There are reports of enhanced chemical and biochemical properties of enzymes when simple sugars and polysaccharides are attached covalently [3–11]. Bacterial subtilisins are in general extracellular serine proteases with broad substrate specificity with preferences for large aromatic or aliphatic amino acids in the P1 site of the substrate [12]. Subtilisin Carlsberg was the first serine protease from bacterial source to be discovered and it consists of a single polypeptide chain comprising 274 amino acids with a Mr of 27,292 [13]. They are produced as preprosubtilisins. A unique propeptide consisting of 77 amino acids acts as a chaperone to facilitate the folding of the active protease [12]. The mature subtilisin after auto processing of the precursor has 274 residues with the catalytic triad consisting of histidine, aspartic acid and serine characteristic of serine proteases. It also possesses three

302

S. Srimathi et al. / Enzyme and Microbial Technology 39 (2006) 301–307

Ca2+ binding sites for stabilizing the three-dimensional structure [14]. Subtilisin has been the subject of several protein engineering studies [12,15–18]. As the substrate specificity is very broad and they display a high stability at neutral or at higher pH values, they are useful as protein degrading additives to the detergent formulations in the washing powder industry [12,19]. In the present study we have chosen a bacterial serine protease, subtilisin from Bacillus licheniformis (subtilisin Carlsberg, SCB) for covalent modification and analyzed the effect of carbohydrate coupling on the catalytic and thermodynamic stability properties. In this paper, we explain the results of covalent modification of subtilisin Carlsberg with polyglutaraldehyde (PGA) and oxidized sucrose polymers of two different sizes (400 kDa and 70 kDa) by reductive alkylation of the ␧-NH2 groups of the lysines. We have dealt with both catalytic and structural stability in terms of the half-life period of thermal inactivation, melting temperature and resistance to chemical denaturants during unfolding. 2. Materials and methods 2.1. Enzyme purification Subtilisin, an alkaline protease from B. licheniformis was supplied by Spic Science Foundation, Chennai, India, as a concentrated broth. The enzyme was purified after ammonium sulfate precipitation followed by gel filtration chromatography using Sephadex G-200. The purity of the protein was checked by SDS-PAGE, which showed a single band around 27 kDa. The absolute protein concentration was determined by the method of Lowry et al. [20] using bovine serum albumin as the standard. FicollTM (400 kDa and 70 kDa) was purchased from Pharmacia, Sweden. Glutaraldehyde, acrylamide, urea, guanidium chloride, guanidium thiocyante, sodium cyanoborohydride and casein were obtained from Sigma, St. Louis. All other chemicals were of high analytical grade and were purchased from E. Merck/SRL, India.

2.2. Enzyme assay Catalytic activity of subtilisin was determined using casein as the substrate. Five micrograms of enzyme was incubated with 0.5 ml of 2% casein in 50 mM borate buffer containing 10 mM CaCl2 at pH 8.0 (at 37 ◦ C) for 20 min maintaining a total assay volume of 1.4 ml. At the end of the incubation time the hydrolytic reaction was terminated by precipitation with 0.6 ml of 25% TCA. After centrifugation the absorbance of the supernatant was read at 280 nm. The activity was expressed as micromoles of tyrosine released per liter per second with help of a tyrosine standard.

2.3. Covalent modification of subtilisin by reductive alkylation The modifiers chosen for coupling to native subtilisin are oxidized forms of a synthetic sucrose polymer FicollTM of two different sizes (400 kDa and 70 kDa referred to as OSP400 and OSP70, respectively) and polyglutaraldehyde (prepared according to the method of Tor et al. [21]). Preparation and coupling of the oxidized sucrose polymers were as given in Venkatesh and Sundaram [5]. A molar ratio of 1:0.077 (enzyme:modifier) for OSP coupling and 1:25 for PGA were used. The modifications were performed in 50 mM borate buffer (pH 8.0) containing 10 mM CaCl2 . The OSP modified enzymes were purified by gel filtration chromatography. The fractions showing maximum proteolytic activity were pooled and referred to as OSP400 SCB and OSP70 SCB. After modification with PGA the solution was extensively dialyzed against the working buffer (10 mM borate buffer, pH 8.0, containing 10 mM CaCl2 ). The degree of modification was estimated by reaction with fluorescamine (λex = 390 nm) and by subsequent analysis of the emission intensity at 475 nm [22].

2.4. Effect of substrate, temperature and pH on activity The effect of substrate concentration on the activity was checked using 8.5–42.4 ␮M casein and analyzed by the Lineweaver–Burk plot. The influence of temperature on the activity was checked in the temperature range 4–80 ◦ C and the activation constants obtained were used for constructing the Arrhenius plot. Ea , the activation energy was calculated as given in Sundaram and Srimathi [10]. pH-induced changes in the activity was studied using 50 mM borate buffer containing 10 mM CaCl2 adjusted to the required pH.

2.5. Inactivation kinetics The native and modified enzymes (100 ␮g/ml) were inactivated in the presence and absence of excess calcium (10 mM) at specific temperatures (40–70 ◦ C) in a water bath. Aliquots of 50 ␮l were withdrawn at regular time intervals and cooled immediately to 4 ◦ C. The residual activity was assayed with caesin at 37 ◦ C. The activation parameters for thermal inactivation were calculated using the appropriate equations [10,23].

2.6. Intrinsic fluorescence and chaotrope-induced unfolding Protein fluorescence measurements of native and modified SCB were made using a protein concentration of 7.79 ␮M in 10 mM borate buffer at pH 8.0 containing 10 mM CaCl2 at 25 ◦ C in a Shimadzu spectrofluorophotometer (RF1501). The protein samples were excited at 280 nm and the emission spectra was recorded in a wavelength range of 300–450 nm. The tertiary structure unfolding of proteins was followed by observing the changes in the fluorescence intensity and λmax in the presence of various concentrations of GdmSCN/GdmCl incubated for 3 h at 25 ◦ C. The unfolding curves were created using the equations given by Santoro and Bolen, by assuming a simple two-state mechanism [24]. The best fit of the denaturation profile was used to assess the conformational stability.

2.7. Circular dichroism and thermal unfolding The effect of modification on the secondary and tertiary structure was compared with that of native SCB by recording the CD spectra in the far UV (190–250 nm) and near UV (250–300 nm), respectively. A protein concentration of 9.16 ␮M in 50 mM borate buffer (pH 8.0) containing 10 mM CaCl2 and a path length of 1 mm in the far UV region and 10 mm in the near UV region were used. Thermally induced unfolding of native and modified SCB were carried out by monitoring the ellipticity (θ) changes at 222 nm over a temperature range of 20–100 ◦ C using Jasco J-715 model spectropolarimeter equipped with a Peltier type temperature controller, Jasco-PTC 348WI. A protein concentration of 6.1 ␮M in 10 mM borate buffer containing 10 mM CaCl2 at pH 8.0 was used for all the unfolding measurements. The temperature was increased at a rate of 50 ◦ C/h with a step resolution of 1 ◦ C.

3. Results and discussion 3.1. Properties of native and modified enzyme conjugates The degree of modification as determined by reaction with fluorescamine indicated that approximately three, two and four lysines per molecule out of a total of nine lysines in native SCB were modified, respectively, in OSP400 SCB, OSP70 SCB and PGA SCB. The decrease in Vmax observed for the modified subtilisin could be a consequence of the changes in substrate binding affinity and protein conformation upon modification (Table 1). The order of Vmax is native SCB > OSP400 SCB > OSP70 SCB > PGA SCB. Estimation of the other Michaelis parameters (kcat and Km ) was not done since it could lead to erroneous results as casein is a large substrate and therefore after ini-

S. Srimathi et al. / Enzyme and Microbial Technology 39 (2006) 301–307 Table 1 Changes in the catalytic and activation parameters for SCB Enzyme

Vmax (␮M s−1 )

Ea a (kJ/mol)

Eai b (kJ/mol)

Native SCB OSP400 SCB OSP70 SCB PGA SCB

1.85 1.52 1.32 1.27

73.54 68.04 66.79 69.06

224.99 (95.84) 328.85 (130.47) 293.94 (121.39) 266.35 (99.50)

The duplicate assays were concordant and showed <5% error. a Arrhenius activation energy for thermal activation in the presence of 10 mM CaCl2 . b Activation energy for inactivation in the presence of 10 mM CaCl and values 2 given in parentheses are in the absence of added calcium.

tial hydrolysis a mixed population of substrate the molecules might be present. The enzyme subtilisin and its modified counterparts showed activity over a wide range of pH (pH 3.5–11.0) and all of them had maximum proteolytic activity at pH 10.5. Modification with these neutral polymers did not change the pH dependence of activity though they showed 20–30% higher activity in the pH range examined (data not shown). Temperature dependence of activity indicated that the maximum activity was observed at 67 ◦ C for native as well as the modified SCB and the T50 value (temperature at which 50% activity is lost) remained at 76.6 ◦ C. The Arrhenius activation energy, Ea was lowered by ∼4–6 kJ/mol for the modified enzymes compared to 73.54 kJ/mol for the native enzyme (Table 1). The activation enthalpy (H= ) decreased slightly for the modified enzymes (data not shown). This indicates that the activation energy barrier is slightly decreased for the modified enzymes. 3.2. Thermal inactivation and the kinetics of inactivation Thermal inactivation of the proteolytic activity of SCB was analyzed according to the kinetic model ki

N−→I where N is the native protein, I the thermally inactivated protein and ki is the inactivation rate constant. The inactivation appears to be pseudo-first-order with respect to [E] over the temperature range studied both in the absence and presence of added calcium. In the presence of 10 mM CaCl2 , decreased inactivation

303

rate constants and a dramatic improvement in t1/2 (Table 2) were observed at 55 ◦ C and 60 ◦ C for all the modified enzymes. Subtilisins contain three metal binding sites and binding to divalent calcium increases the thermodynamic stability of the enzyme [14,25]. It has been shown that thermal inactivation of subtilisin BPN decreased ∼1000-fold in the presence of 100 mM CaCl2 [17]. In the presence of calcium the half-life at 55 ◦ C and 60 ◦ C were 3.6-fold and 3.48-fold higher for the native SCB compared to that in the absence of added calcium (Table 2). The delayed inactivation in the presence of calcium could be due to increased global stability of the protein upon calcium binding. OSP400 SCB showed maximum stability compared to all the other enzymes. The order of stabilization towards thermal inactivation was found to be OSP400 > OSP70 > PGA > native. The half-life periods and the SFs with respect to the native enzyme are given in Table 2. The energy of activation for inactivation process (Eai ) obtained from the Arrhenius plot (data not shown) was found to increase for the modified enzymes indicating the higher energy barrier for inactivation to take place (Table 1). Increase in Eai was greater for OSP400 SCB than OSP70 SCB and PGA SCB. The free energy and enthalpy of activation for the inactivation increases for the modified enzymes indicating increased energy barrier and higher kinetic thermal stability. Reduced inactivation rates of the modified enzymes may be due to the extra rigidity conferred by the covalent bonds between the protein and the modifier molecule. Though there are numerous reports of improved stability in the presence of additives and polyols (3–11), it could be argued that the covalent coupling procedure can produce stabilizing effects due to: (1) direct interaction with protein by forming covalent bonds, (2) polyhydroxy group mediated stabilization by changing the solvent properties [26] and (3) increasing the enthalpy of inactivation. This procedure also, like in the co-solvent-induced stabilization, seems to shift the equilibrium between native enzyme (N) and the unfolded enzyme (U) more towards the native state. 3.3. Two-state unfolding detected by steady-state fluorescence The fluorescence emission spectra of native and sucrose polymer modified subtilisins show an emission maximum at 328 nm.

Table 2 Modification-induced changes in the stability of subtilisin Enzyme

Half-life periods in hours No added Ca2+

Native SCB OSP400 SCB OSP70 SCB PGA SCB

Cm (M)

Tm (◦ C)

1.30 0.39 (1.66) 1.48 1.41

67.78 76.23 73.69 72.44

10 mM Ca2+

55 ◦ C

60 ◦ C

55 ◦ C

60 ◦ C

2.69 5.17 (1.91) 3.60 (1.33) 1.92 (0.71)

1.00 2.15 (2.13) 1.87 (1.87) 1.29 (1.28)

9.73 87.90 (9.02) 55.23 (5.67) 22.22 (2.28)

3.48 21.09 (6.06) 18.09 (5.20) 10.17 (2.92)

The thermal inactivation was carried out in the absence of added calcium and in the presence of 10 mM calcium. Half-life period at each temperature was calculated from inactivation constants obtained from the plots of time vs. log% activity using the formula t1/2 = ln 2/ki . The stabilization factor, SF (given in parentheses), is equal to t1/2 (modified)/t1/2 (native). Values reported are the average of the duplicates. The unfolding was carried out in 10 mM borate buffer at pH 8.0 in the presence of 10 mM CaCl2 . G (H2 O), Cm and m were obtained from the GdmSCN-induced unfolding monitored by fluorescence emission changes. Tm was estimated from the temperature-induced changes in θ 222 . All data represent the average of duplicates with <5% error.

304

S. Srimathi et al. / Enzyme and Microbial Technology 39 (2006) 301–307

Fig. 1. Fluorescence emission spectra of subtilisin (1) in the native state and in the presence of: (2) 6 M GdmHCl, (3) 1.98 M NaSCN and (4) 1.98 M GdmSCN. Excitation was carried out at 280 nm using a protein concentration of 7.79 ␮M in 10 mM borate buffer at pH 8.0 containing 10 mM CaCl2 . The corresponding wavelengths of maximum emission are 328 nm, 338 nm, 328 nm and 350 nm.

PGA SCB exhibited emission maximum at 324 nm. The tertiary structure of subtilisin does not show any change in the tryptophan environment after modification. We have tried unfolding the protein to see the extent of stabilization of the folded conformation over the unfolded. Efforts to unfold subtilisin with GdmCl or urea were futile. Even 6.0 M GdmCl induced only partial unfolding (Fig. 1) of the enzyme as evident from the λmax values (shift in λmax value from 328 nm to 338 nm) and from the absence of a stable post-transition region (data not shown). In order to induce complete unfolding of subtilisin, we used GdmSCN. Thiocyanate, being a larger anion, preferentially bind to the peptide backbone of the protein and may thus induce unfolding of proteins [27] and has been proved to be a stronger chaotropic agent than GdmCl. In addition to the inherent protein unfolding property of GdmSCN, quenching of protein fluorescence by thiocyanate ions is also observed. In order to distinguish these two phenomena (denaturation and quenching), we followed the changes in the fluorescence intensity at 328 nm (λmax of native subtilisin) and the shift in the in λmax upon titration with NaSCN and GdmSCN. As the concentration of NaSCN was increased there was a steady decrease in the fluorescence intensity at 328 nm without appreciable shift in λmax values. Inclusion of about 2 M NaSCN decreased the fluorescence intensity by 26% (Fig. 1). Fluorescence quenching is generally associated with decrease in the fluorescence intensity and does not alter the wavelength of maximum emission [28]. However, presence of equivalent concentration of GdmSCN induced appreciable shift in the λmax (350 nm) in addition to a 63% decrease in the fluorescence intensity (Fig. 1). Such red shifts in the in λmax are attributed to the unfolding of protein molecules in which the tryptophan residues become more solvent exposed. Thus, it is very clear that the parameters observed are associated with the unfolding of subtilisin and not with mere quenching. The tertiary structure unfolding of native and modified SCB in the presence of GdmSCN appears to follow a two-state model (Fig. 2). The unfolding of the native and chemically modified

Fig. 2. Two-state unfolding of native and modified SCB in the presence of GdmSCN monitored from the changes in the fluorescence intensity at 328 nm (λex = 280 nm). The changes in the emission wavelength upon unfolding are also given (symbols without curve fitting).

subtilisin were monitored both by the changes in the fluorescence intensity value at 328 nm and the wavelength of maximum emission (Fig. 2). The mid-point of transition calculated from the fluorescence intensity changes and from the λmax values were identical (with standard deviations less than 2%). The midpoint of unfolding, Cm , shifted from 1.30 M for native SCB to 1.66 M for OSP400 SCB, 1.48 M for OSP70 SCB and 1.41 M for PGA SCB (Table 2). This clearly showed that all the modified enzymes are stabilized against tertiary structure unfolding of which OSP400 SCB is the most stable with a shift in Cm of 0.36 M. The slope of the transition region is similar for the native and modified enzymes indicating that the co-operativity of unfolding has not changed upon modification with OSP70 and PGA. From these results, it can be concluded that the additional covalent links present in the modified enzymes delay the unfolding process by offering extra resistance to denaturation. The relatively rigid modified enzymes are a result of the multipoint attachments through the surface ␧-NH2 groups. Among the modified enzymes the difference in stability may be due to the extra rigid conformation achieved in the case of OSP400 compared to OSP70 followed by PGA. The increased surface area of the modifier reduces the solvent exposed protein surface and preferentially excludes the protein from the solvent [27]. 3.4. Effect of modification on thermal stability The far UV CD spectra depicting the peptide backbone conformations of the native and modified SCB did not show visible changes in the basic pattern (inset, Fig. 3). The near UV CD spectra of the native and modified subtilisins were almost similar (data not shown). Covalent coupling of sugars to subtilisin did not induce substantial changes in the protein secondary structure being consistent with the unchanged tryptophan emission maximum. The measurements of Tm for the native and modified counterparts of SCB show that the latter have delayed melting of the secondary structure compared to native SCB. The decrease in the negative ellipticity at 222 nm with respect to temperature is shown in Fig. 3. But the overall unfolding pattern of the

S. Srimathi et al. / Enzyme and Microbial Technology 39 (2006) 301–307

Fig. 3. Thermal unfolding curves for the native and modified subtilisin. Fraction of native enzyme was calculated using the molar ellipticity at 222 nm. Inset: far UV CD spectra at 25 ◦ C recorded in 10 mM borate buffer containing 10 mM CaCl2 at pH 8.0.

modified enzymes was comparable to that of native, which is sharp following an all – or – none model. This showed that the unfolding mechanism is not altered but only shifted to higher temperature due to induced stability through the lysine links, which offer additional protection. Native SCB showed a Tm of 67.78 ◦ C and the improved Tm for the modified enzymes are as given in Table 2. A Tm of 8.45 ◦ C was estimated for OSP400 SCB, which went down to 5.9 ◦ C for OSP70 SCB, followed by 4.6 ◦ C for PGA SCB. Though there was a defined post-transition component, significant residual structure was inferred for the temperature denatured states of native and covalently modified subtilisins, as evident from a molecular ellipticity value of about −4.5 × 10−5 mdeg cm2 dmol−1 (for all these enzymes). Residual structures in the denatured states have been reported for many other proteins [29]. The unchanged T50 values but increased Tm (relative to the native enzyme) for the modified analogs of SCB is acceptable since the kinetic stability need not reflect the thermodynamic or conformational stability of the enzyme molecule. Moreover, even minor change(s) in the enzyme conformation can result in loss of activity without affecting the global stability. The higher stability towards thermal inactivation gives only the ability of the modifier in delaying the events leading to loss of activity which is always estimated after initial inactivation of the enzyme in the absence of substrate. The observed values also suggest that the dynamics of the catalytic domain might not have been affected by covalent chemical modification and therefore the increase in the conformational stability, as indicated by the increase in the Tm value, might be a consequence of decreased dynamics at other part of the molecule not involved in either substrate binding/catalysis. 3.5. General mechanism of stabilization There are numerous hypotheses to explain the stabilizing effects imparted on the protein conformation by polyhydric alcohols. Any generalization of the reasons for the extra stability look ambiguous because of the variation in the surface patterns of

305

many proteins studied so far [30]. Preferential interactions generally destabilize the conformation whereas preferential hydration or preferential exclusion tends to have a stabilizing effect. The covalent links decrease the solvent exposed protein surface and thus make the interior more compact and lowers the accessibility to any harmful agents leading to protein collapse. It seems to be a general rule that stabilizing substances are preferentially excluded from the protein vicinity or, in other words that the protein is preferentially hydrated due to their ability to increase the surface tension of water [31]. The stabilization acquired by subtilisin in the present study might be due to the preferential hydration of the modified enzymes and reduced solvent accessibility due to increased degree of water organization induced by the sugars [30]. The increased Tm and Cm could also be due to loss of hydrogen bond rupturing capacity of water in the presence of sugar [1] as well as due to the formation of hydrogen bonds between the hydroxyl groups of the sugar part and the hydrophilic amino acid residues on the enzyme surface [32]. Formation of additional hydrogen bonds between the modifier and the enzyme and preferential hydration of the protein could be the reasons for the positive shift in Eai (higher t1/2 ) and Cm . However, it could not be ruled out that the increased stability could also be due to decreased autolysis of the modified subtilisin molecule. 3.6. Comparison with other covalently modified proteases Coupling of subtilisin Carlsberg with PEG activated by cyanuric chloride (CC-PEG) nitrophenol carbonate (NPC-PEG) has been reported to increase the half-life of the native enzyme, depending on the inactivation temperature, by 3–28-fold [33]. The onset of thermal inactivation and melting of protein conformation were reported to be delayed by approximately 10 ◦ C though the Tm values remained unaltered [33]. Effect of covalent modification of papain [4], trypsin [5,11] and chymotrypsin [6] with OSP400 has been reported recently. In all these proteases studied, OSP400 had been effective in stabilizing the enzymes against inactivation by increasing their thermodynamic stability. However, the stoichiometric ratio of the enzyme to oxidized sucrose polymer in the studies involving papain, trypsin and chymotrypsin is 1:0.5. But about seven times lower concentration of OSP400 was used in the present study. Further increase in the concentration of OSP400 resulted in precipitation of the enzyme samples due to “salting out” effect. Therefore, the concentration of sucrose polymer that is required to enhance the stability of enzymes could vary from protein to protein where the size and surface properties play an important role. The increase in the enthalpy of inactivation of OSP400 modified enzymes over the native enzymes show the following trend: ␣-chymptrypsin (80.50%) > papain (45.80%) > trypsin (41.75%) > subtilisin (36.13%). In addition, modification of savinase, a variant of subtilisin did not increase the thermodynamic stability of the enzyme (unpublished results). The observed trend in stabilization suggests that the increase in thermodynamic stability need not be merely due to the increased number of covalent and non-covalent interactions between the modifier and the enzyme but also the native enzyme characteristics that are deter-

306

S. Srimathi et al. / Enzyme and Microbial Technology 39 (2006) 301–307

mined by the primary sequence and the folded conformation. However, more detailed investigation is necessary to explain the basis of the observed differential ability of these sugar polymers to confer stability.

Acknowledgement This work was supported by a grant from the Department of Biotechnology, Ministry of Science and Technology, Government of India (D.O. No. BT/R&D/15/19/94).

3.7. Comparison with SDM-induced stabilization Owing to the industrial importance of subtilisin, there have been several attempts to improve its thermodynamic stability. Changes in catalytic efficiency have been reported for the cysteine mutants (S156C, S166C and M222C) of Bacillus lentus subtilisin when chemically modified with methanethiosulfonate reagents [34]. Several single mutants have been constructed within the full-length sequence of subtilisin. Increase in the thermal stability (t1/2 ) was observed for many of the variants constructed at many independent sites of the enzyme as a result of enhanced packing of the hydrophobic residues, creation of additional salt bridge(s) or the introduction of hydrogen bond(s) [18]. Stabilization achieved by these mutational studies was highly localized and therefore the contribution to the global stability was only minimal as indicated by the stabilization factors of less than two. The stabilising mutations reported by Alexander et al. [35] are in the range of 2–5.0 in terms of their half-life. Studies involving the truncated sequence, 75–83 subtilisin by the same authors indicate stabilization in the range of 1–3.5 for single-point mutations. A logical combination of many single-point mutations in 75–83 subtilisin lead to a variant with 10-fold increase in stability. It is interesting to note that the values of stabilization factors obtained through a one step chemical modification in the present study lie in the range of 2.0–9.0 at 55 ◦ C and 2.5–6.0 at 60 ◦ C that are superior to these mutants. Moreover, increase in the Tm values for the OSP400 SCB by 8.45 ◦ C is greater than those reported by Almog et al. [36]. It should be emphasized that the stabilization gained through modification with either OSP70 or PGA is also comparable to many of the single-point variants of subtilisin. 4. Conclusion It has been postulated that the hydrophilic glycans confer favourable physicochemical properties on biologically active proteins by glycosylation. The changes introduced in the properties of subtilisin Carlsberg by attaching polymeric sucrose and polymeric glutaraldehyde in vitro, had in fact produced appreciable stabilizing effects by influencing the heat stability of the enzymatic action and the protein conformation. Though the changes in the thermodynamics of catalysis and conformation of the catalytic protein signify two different phenomena, it has been shown in the present study that chemical modification of the enzyme has a favourable effect on the biologically active native enzyme. The possibility of enhancing the enzyme stability without much loss in its specific activity is of special interest for the use of these enzymes in industrial applications. So the minimum kinetic stability or longterm stability can be made possible through covalent coupling procedures.

References [1] Pedrosa C, Fernanda G, Felice D, Trisciuzzi C, Ferreira ST. Selective neoglycosylation increases the structural stability of vicillin, the 7S-storage globulin from pea seeds. Arch Biochem Biophys 2000;382:203–10. [2] Nalivaeva NN, Turner AJ. Post translational modifications of proteins: acetylcholine esterase as a model system. Proteomics 2001;1:735–47. [3] Marshall JJ, Rabinowitz ML. Enzyme stabilization by covalent attachment of carbohydrate. Arch Biochem Biophys 1975;167:777–9. [4] Rajalakshmi R, Sundaram PV. Stability of native and covalently modified papain. Protein Eng 1995;10:1039–47. [5] Venkatesh R, Sundaram PV. Modulation of stability properties of bovine trypsin after in vitro structural changes with a variety of chemical modifiers. Protein Eng 1998;11:691–8. [6] Sundaram PV, Venkatesh R. Retardation of thermal and urea induced inactivation of ␣-chymotrypsin by modification with carbohydrate polymers. Protein Eng 1998;11:699–705. [7] Venkatesh R, Sundaram PV. Improved stability of soybean and horse radish peroxidases by covalent chemical modification. Ann N Y Acad Sci 1998;864:517–20. [8] Villalonga R, Gomez L, Ram´ırez HL, Villalonga ML. Stabilization of ␣amylase by chemical modification with carboxymethylcellulose. J Chem Technol Biotechnol 1999;74:635–8. [9] Yamazaki T, Tsugawa W, Sode K. Increased thermal stability of glucose dehydrogenase by crosslinking chemical modification. Biotechnol Lett 1999;21:199–202. [10] Sundaram PV, Srimathi S. Analysis of catalytic and structural stability of covalently modified enzymes. In: Svendsen A, editor. Enzyme functionality: design, engineering and screening. New York: Marcel Dekker; 2004. p. 632–61. [11] Venkatesh R, Srimathi S, Yamuna A, Jayaraman G. Enhanced catalytic and conformational stability of Atlantic cod trypsin upon neoglycosylation. Biochim Biophys Acta 2005;1722:113–5. [12] Wells JA, Estell DA. Subtilisin—an enzyme designed to be engineered. Trends Biochem Sci 1988;13:291–7. [13] Smith EL, Delange RJ, Evans H, Landon M, Markland FF. Subtilisin Carlsberg. V. The complete sequence; comparison with subtilisin BPN ; evolutionary relationships. J Biol Chem 1968;243:2184–91. [14] McPhalen CA, James MNG. Structural comparison of two serine proteinase–protein inhibitor complexes: eglin-C-sunbtilisin Carlsberg and CI-2-subtilisin novo. Biochemistry 1988;27:6582–98. [15] Seizen RL, de Vas WM, Leunissen JAM, Dijkstra BW. Homology modeling and protein engineering strategy of subtilases, the family of subtilisin like proteinases. Protein Eng 1991;4:719–37. [16] Pantoliano MW, Ladrer Rl, Bryan PN, Rollence ML, Wood JF, Poulos TL. Protein engineering in subtilisn BPN : enhanced stabilization through the introduction of two cysteines to form disulfide bond. Biochemistry 1987;26:2077–82. [17] Pantoliano MW, Whitlow M, Wood JF, Rollence ML, Finzel BL, Gilliland GL, et al. The engineering of binding affinity at metal ion binding sites for the stabilization of proteins: subtilisin as a test case. Biochemistry 1988;27:8311–7. [18] Bryan PN. Protein engineering of subtilisin. Biochim Biophys Acta 2000;1543:203–22. [19] Bryan PN, Rollence ML, Pantoliano MW, Wood J, Finzel BC, Gilliland GL, et al. Protease of enhanced stability. Characterization of a thermostable variant of subtilisin. Proteins 1986;1:326–34. [20] Lowry OH, Rosenberg NJ, Farr AL, Smith F. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265–75.

S. Srimathi et al. / Enzyme and Microbial Technology 39 (2006) 301–307 [21] Tor R, Dror Y, Freeman A. Enzyme stabilization by bilayer “engagement”. Enzyme Microb Technol 1989;11:306–12. [22] Stocks SJ, Jones AJM, Ramey CW, Brooks DE. A fluorimetric assay of the degree of modification of protein primary amines with polyethylene glycol. Anal Biochem 1986;154:232–4. [23] Lonhienne T, Gerday C, Feller G. Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility. Biochim Biophys Acta 2000;1543:1–10. [24] Santoro MM, Bolen D. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl ␣chymotrypsin using different denaturants. Biochemistry 1988;27:8063–8. [25] Alexander PA, Ruan B, Bryan PN. Cation-dependent stability of subtilisin. Biochemistry 2001;40:10634–9. [26] Arakawa T, Timasheff SS. Preferential interactions of proteins with salts in concentrated solutions. Biochemistry 1982;21:6536–44. [27] Timasheff SN. Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated. Adv Protein Chem 1998;51:355–432. [28] Lacowicz JR. Principles of fluorescence spectroscopy. 1st ed. NY: Plenum Publishers; 1983. [29] Dill KA, Shortle D. Denatured states of proteins. Annu Rev Biochem 1991;60:795–825.

307

[30] Combes D, Yoovidya T, Girbal E, Willemot RM, Monsan P. Mechanism of enzyme stabilization. In: Laskin AI, Mosbach K, Thomas D, Wingard Jr LB, editors. Enzyme engineering, vol. 8. 1987. p. 59– 62. ´ Understanding and increasing protein stability. Biochim Bio[31] F´ag´ain CO. phys Acta 1995;1252:1–14. [32] Srivatsava RAK. Studies on stabilization of amylase by covalent coupling of soluble polysaccharides. Enzyme Microb Technol 1991;13:164–70. [33] Yang Z, Domach M, Auger R, Yang FX, Russell AJ. Polyethylene glycol-induced stabilization of subtilisin. Enzyme Microb Technol 1996;18:82–9. [34] DeSantis G, Berglund P, Stabile MR, Gold M, Jones JB. Site-directed mutagenesis combined with chemical modification as a strategy for altering the specificity of the S1 and S1 pockets of subtilisin Bacillus lentus. Biochemistry 1998;37:5968–73. [35] Alexander PA, Ruan B, Strausberg SL, Bryan PN. Stabilizing mutations and calcium-dependent stability of subtilisin. Biochemistry 2001;40:10640–4. [36] Almog O, Gallagher DT, Ladner JE, Strausberg SL, Alexander P, Bryan PN, et al. Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN . J Biol Chem 2002;277:27553–8.