Improvement of thermostability and activity of pectate lyase in the presence of hydroxyapatite nanoparticles

Bioresource Technology 116 (2012) 348–354

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Improvement of thermostability and activity of pectate lyase in the presence of hydroxyapatite nanoparticles Arka Mukhopadhyay, Anjan Kumar Dasgupta, Dhrubajyoti Chattopadhyay, Krishanu Chakrabarti ⇑ Department of Biochemistry, University College of Science, Calcutta University, 35 Ballygunge Circular Road, West Bengal, Kolkata 700 019, India

a r t i c l e

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Article history: Received 29 November 2011 Received in revised form 29 March 2012 Accepted 29 March 2012 Available online 9 April 2012 Keywords: Pectate lyase Hydroxyapatite nanoparticle Thermostability Retention of activity

a b s t r a c t The activity and half-life of pectate lyase (PL) from Bacillus megaterium were nine- and 60-fold, respectively, higher at 90 °C in the presence of hydroxyapatite nanoparticles (NP-PLs) than in the presence of 1 mM CaCl2. Thermodynamic analysis of the nanoparticle-induced stability revealed an enhanced entropy–enthalpy compensation by the NP-PLs since a reciprocal linearity of the enthalpy–entropy change to 90 °C was observed. Without nanoparticles, the linearity range was 70 °C. Such compensation reflected the maintenance of the native structure of proteins. The remarkable enhancement of activity and stability of the NP-PL system at high temperatures may be utilized commercially e.g. in the food industry or the processing of natural fibers that may require a thermotolerant enzyme. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Pectin, an important constituent of the plant cell wall, is a heteropolysaccharide, which contains a-1-4 linked galacturonate chains, that are highly methyl esterified. Pectinolytic enzymes, such as pectin lyase (PL) and polygalacturonase (PG) catalyze the degradation of pectin. PG generally hydrolyzes pectic acid and PL, specific for methyl esterified substrates, catalyzes the cleavage of a-D-(1, 4) glycosidic bonds by b-elimination of the pectin substances (Pereira et al., 2002; Basu et al., 2011). These pectinolytic enzymes are produced by a variety of microbes and play an important role in biotechnological applications such as in the food and beverages industries for improving the yield and clarification of fruit juice and in the textile industries to facilitate degumming of natural fibers like ramie, jute as an alternative to conventional retting (Soriano et al., 2005; Basu et al., 2011). Thermostable enzymes are stable and active at temperatures considerably higher than their optimum temperatures (Saboto et al., 1999; Haki et al., 2003). An enzyme or protein may be termed as thermostable if a high definite unfolding (transition) temperature (Tm) or a long half-life at a preferred high temperature is observed (Turner et al., 2007). Thermostable enzymes can be used at temperatures that enhance substrate solubility and reaction rates while allowing

Abbrevations: NP, nanoparticle; HAp, hydroxyapatite; NP-PL, HAp nanoparticle treated pectate lyase; PL, untreated pectate lyase; TBA, thio-barbituric acid; PGA, poly-galactouronic acid. ⇑ Corresponding author. Tel.: +91 33 9831535059; fax: +91 33 24614849. E-mail address: [email protected] (K. Chakrabarti). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.03.094

for pretreatment of raw materials. Contamination by mesophiles can also be curtailed under these conditions (Demirijan et al., 2001; Haki et al., 2003). Scouring of natural fibers improves water absorbency and whiteness of textiles by removing non-cellulosic substances from many natural fibers (Basu et al., 2008). Chemicals like soda-ash, oxalic acid and caustic soda, used for chemical scouring give rise to polluting effluents while weakening the strength of the finished fiber. The use of pectin-removing enzymes as an alternative has thus gained importance. However, the thermal instability of the enzyme impairs it is scouring efficiency. Nano-materials exhibit properties such as large surface-to-volume ratios, high surface reaction activity, high catalytic efficiency, and strong adsorption ability (Pendry, 1999; Hudson et al., 1997; Feldstein et al., 1997; Fukumi et al., 1994; Hagland et al., 1993). Protein adsorption on nanoparticles can lead to improved activity (Lynch and Dawson, 2008) and improvements in thermal stabilities of the enzymes were observed after adsorption (Chronopoulou et al., 2011). Enzyme stability is maximized with nano-scaled supports with possible modulation of the catalytic specificity (Konwarh et al., 2009). Hydroxyapatite (HAp), Ca10(PO4)6(OH)2, is a compound which can be prepared in a nano-form that is apparently non-toxic (Sheik and Kim, 2010). Although chemical and molecular biology methods have been applied to pectate lyase, thermostability concomitant with high activity were not achieved (Basu et al., 2008). In the present study, pectate lyase was treated with hydroxyapatite nanoparticles. The objective of this work was to develop a highly active thermostable pectate lyase preparation for potential application in industry.

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2. Methods 2.1. Reagents The HAp nanoparticle dispersion, 10 wt% in H2O, <200 nm (Acc No: 702153), PCR primers (forward and reverse), poly-galactouronic acid (PGA) for pectate lyase assay, apple pectin and silver nitrate for silver staining were obtained form Sigma–Aldrich (Germany). Beef extract, agar powder and glutaraldehyde were purchased from Himedia, India. All other reagents for bacterial culture media, agar, beef extract and salts were purchased from local companies and were of analytical grade. 10–200 kDa protein markers were from Fermentas (Germany) (#SM0661). 2.2. Isolation of pectinolytic strain Soil samples were collected from a termite field of North 24Parganas, West Bengal, India, and added to sterile 0.9% saline water and plated on YP agar (NaCl 0.5%, yeast extract 1.0%, pectin 0.75% and cycloheximide 0.005%; pH 8.5). The plates were incubated at 37 °C for 2 days. After the formation of colonies, 0.05% ruthenium red (Bruhlmann et al., 1994) was poured over the agar surface and pectinolytic strains were identified by their halo-formation. Isolate AK2 produced the largest halo and was cultured in liquid (YP) medium for carrying out pectate lyase activity assays.

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were added to stop the reaction. The sample was centrifuged at 3000 g for 10 min at 4 °C. The clear supernatant was mixed with 0.04 M TBA and 0.1 M HCl followed by 30 min of heating in boiling water. Color formation was detected at 550 nm. One unit of activity was defined as the amount of enzyme that caused a change in absorbance of 0.01 under the conditions of the assay. 2.4.2. Measurement of Ca concentration by Atomic Absorption Spectra (AAS) Ca contents were determined utilizing an A Analyst 200 (Perkin Elmer) and comparison with data obtained with standard solutions provided by Perkin Elmer. 2.4.3. Effect of nano-particles on pectate lyase activity HAp at concentrations of 2.2, 4.4, 6.6, 8.8 and 11 lg/ml were incubated with purified PL (200 ll of 0.145 mg/ml) at 50–90 °C and the enzyme activity of each system was measured as described in Section 2.4.1. 2.4.4. Effect of temperature on NP-PL The optimum temperature of NP-PL was determined by carrying out the standard assay in Tris–HCl buffer (25 mM, pH 8.5), at temperatures ranging from 40 to 90 °C. At each temperature, the assay mixture was incubated for 2 h. The effect of temperature on PL without NPs was observed as the control.

2.3. Ribotyping To identify strain AK2, 16S rDNA gene sequencing was carried out. Genomic DNA was prepared from all different isolates by the sodium dodecyl sulfate proteinase K-cetyltrimethylammonium bromide (CTAB) method (Basu et al., 2008). Partial amplification of the 16S rRNA gene was performed with the thermal cycler ABI 9700 (ABI, Foster City, USA). The amplified and gel-eluted PCR fragments were sequenced with an ABI 3100 Genetic Analyzer. Sequencing reaction was performed by using the Big Dye terminator cycle sequencing Kit V3.1 (Applied Biosystems, Foster City, USA). The nucleotide sequence was deposited in the GenBank under the accession number JN798190. 2.4. Purification of pectate lyase from Bacillus megaterium AK2

2.4.5. Thermal stability of NP-PL To measure the retention of enzyme activity (with PGA as substrate) at high temperature, NP-PL was incubated for 1–5 h at 90 °C. After the treatment, the enzyme activities were determined as described in Section 2.4.1. The results were expressed relative to the values of PL without NPs. The same set of experiments was performed using 0.015% apple pectin (Sigma–Aldrich) as substrate. 2.5. Kinetics and activation–inactivation parameters of NP-PL The Km–Vmax, activation energy Ea and the activation/deactivation kinetics of both NP-PL and PL were studied using the standard reaction mix as described in Section 2.4.1. For the study of enzyme kinetics in NP-PL, the buffer (25 mM Tris–HCl, pH 8.5) contained 8.8 lg/ml HAp NP. The enzyme concentration was 0.145 mg/ml for both systems.

Pectate lyase was purified from a 100-ml YP culture (Basu et al., 2008). Cell-free supernatant was mixed with 10 ml CM-Sepharose pre-equilibrated with Tris–HCl buffer (25 mM, pH 8.5) and kept at 4 °C overnight. Then 10 ml of the mixture was loaded onto a column (10 ml bed volume). Bound compounds were eluted with Tris–HCl buffer (25 mM, pH 8.5) containing 0–1 M NaCl. Fractions of 1 ml were collected and those showing pectate lyase activity (Basu et al., 2008) were concentrated using a Macrosep 10 K unit and loaded onto a glass column packed with Sephadex G-75 (bed volume 30 ml) and equilibrated with the same Tris–HCl buffer. Elution of the proteins was done using Tris–HCl buffer (25 mM, pH 8.5). SDS polyacrylamide gel electrophoresis (PAGE) in a 12% gel was performed by the method of Laemmli (1970). Protein markers and protein bands were stained by silver staining.

2.5.1. Km, Vmax and activation energy (Ea) The kinetic parameters, Km, Vmax and the activation energy (Ea) were measured according to Liao et al. (1997). The substrate (PGA) concentration was used from 0.015% to 1.25% to determine Km and Vmax. Since the optimum temperature of purified PL from AK2 was 75 °C, one set of experiments for Km and Vmax was carried out at this temperature. As high activity for NP-PL was observed at 90 °C, a separate set of experiments was performed at this temperature. The activation energy (Ea) was evaluated for the temperature range of 50–90 °C. The PGA concentration used for this calculation was 0.75% (Basu et al., 2008).

2.4.1. Activity assay of pectate lyase The activity of purified pectate lyase was measured by the TBA (thio-barbituric acid) method. Briefly, purified pectate lyase was incubated with 0.015% poly-galactouronic acid (PGA) in the presence of hydroxyapatite nanoparticles (NP-PL) or in the presence of 1 mM CaCl2 (PL) in 25 mM Tris–Cl buffer (pH-8.5) for 2 h at 55 °C. The assay volume was 1 ml including buffer, substrate and enzyme. After incubation, 9% (w/v) zinc sulfate and 0.5 M NaOH

2.5.2. Activation/inactivation kinetics of NP-PL PL and NP-PL were subjected to temperatures between 40 and 90 °C (313–363 K) for up to 10 min prior to running the assay. Inactivation parameters comprising half-life (t1/2), decay rate constant (k), energy of deactivation (Ed), enthalpy (DH), entropy (DS) and free energy change (DG) were obtained according to Ortega et al. (2004). The PGA concentration used for this purpose was 0.75% (Ortega et al., 2004).

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3. Results and discussions 3.1. Identification of strain AK2 The sequence of the amplified partial 16S rDNA fragment was found to be similar to B. megaterium (more than 96%) when compared to rDNA sequence database in GenBank. 3.2. Purification of pectate lyase from AK2 Pectate lyase activity was found after ion-exchange chromatography with 50.2% recovery. Two fractions showed pectate lyase activity after gel filtration chromatography. The recovery of enzyme activity was about 6.8%. SDS–PAGE analysis showed a single band, indicating complete purification of the enzyme. Compared to protein markers, the size of the purified enzyme was around 40 kDa (Fig. 1). A more than 18-fold purification was achieved (Table 1). 3.3. Ca content in HAp NP From AAS absorbance data it was observed 1 mM CaCl2 contain 234.3% higher Ca2+ than Hydroxyapatite NP. 3.4. Effect of NP on PL activity Purified pectate lyase activity increased with the inclusion of HAp NP as compared to the control set with 1 mM Ca2+, without NP (Basu et al., 2008). Within the temperature range of 50–90 °C, the optimum concentration of NP was 8.8 lg/ml (NP_20) (Fig. 2a). With respect to NP_0, PL activity increased 268% ± 1.22% for 2.2 lg/ml (NP_5), 283% ± 1.2% for 4.4 lg/ml (NP_10), 291% ± 1.31% for 6.6 lg/ml (NP_15), 304% ± 1.205% for 8.8 lg/ml (NP_20) and 270% ± 1.28% for 11 lg/ml (NP_25). In case the of NP_25, PL activity decreased by 8.3% ± 1.02% as compared to NP_20, 8.8 lg/ml. It has

been reported, that bacterial pectate lyase requires calcium (Ca2+) for activity (Sakai et al., 1993; Stutzenberger, 1987). Similar results had already been observed with different strains of B. pumilus (Dave and Vaughn, 1971. Basu et al., 2008) and B. polymyxa (Nagel and Vaughn, 1961). Previously it had been observed that 1 mM Ca2+ ion concentration was optimum for pectate lyase from Bacillus pumilus (Basu et al., 2008). In the present study, it was found that in the presence of Ca2+, HAp NP could not induce pectate lyase activity, whereas without Ca2+, NP enhanced the activity 304% ± 1.205% at 90 °C (Fig. 2b). The optimum concentration of HAp NP, i.e. 8.8 lg/ml (NP_20), enhanced the enzyme activity at 90 °C by 304% ± 1.205% compared to the control with 1 mM Ca2+ without NP. When comparing the values of Ca concentration (HAp versus CaCl2), it is clear that NP-PL needed only 1/3 of the Ca (as Hap) to induce pectate lyase activity, even at high temperatures as compared to the PL system supplemented with Ca2+. 3.5. Effect of temperature on NP-PL For NP-PL, activity increased 45.5% ± 0.5% at 50 °C, 68.3% ± 0.45% at 60 °C, 84.6% ± 0.45% at 70 °C, 98.5% ± 0.48% at 80 °C and 117.3% ± 0.55% at 90 °C, with respect to enzyme activity at 40 °C. In contrast, the activity of PL decreased steadily above 50 °C. The values for percent decrease of enzyme activity (with respect to enzyme activity at 40 °C), were 7.8% ± 0.305% at 60 °C, 23% ± 0.84% at 70 °C, 32.8% ± 0.75% at 80 °C and 59.3% ± 0.602% at 90 °C. Thus the presence of nano-calcium appeared to confer enhanced activity to pectate lyase under conditions that normally favor loss of activity (Fig. 3). 3.6. Retention of activity at 90°C by NP-PL (substrate PGA) At a holding temperature of 90 °C, purified PL retained its activity for 2 h. In contrast, NP-PL retained its activity for 4 h at 90 °C and the enzyme activity increased gradually with time. However, after 4 h the activity decreased slightly. After 4 h, the NP-treated enzyme had five times greater activity than the untreated enzyme (Fig. 4). 3.6.1. Retention of activity at 90 °C by NP-PL (substrate pectin) When pectin was used as a substrate, the activity of NP-PL showed fivefold higher activity than PL after 2 h, whereas after 4 h incubation, NP-PL activity was 11-fold higher than that of PL.(Table 2). After 5 h, NP-PL activity decreased 3-fold as compared to the activity at 4 h. This indicated that NP-PL showed enhanced activity at high temperature with both synthetic and natural substrates. 3.7. Kinetics and activation energy of NP-PL

Fig. 1. SDS–PAGE (12%) showing purification of pectate lyase from AK2.

3.7.1. Km, Vmax and activation energy (Ea) The Km value decreased while the Vmax value increased in NP-PL as compared to PL at both 75 °C and 90 °C (Table 3). Thus nanoparticle pre-treatment enhanced enzyme-substrate affinity and lowered the activation energy compared to that of untreated enzyme. Within the NP-PL system, with increased temperature from 75 °C to 90 °C, the Km value decreased 27.2% and Vmax value increased 69.7%. At 75 °C, the Km value of NP-PL decreased 63% while Vmax increased 255.1% over that of the PL system. At 90 °C, the NP-PL system showed an 80% decrease in Km and a 716.4% increase in Vmax over the PL system. With increased temperatures from 75 °C to 90 °C, the Km of PL increased 39.3% whereas Vmax of PL decreased 26.1%. The activation energy (Ea) of NP-PL was almost threefold less than that of PL. According to the Arrhenius plot, PL showed an Ea

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A. Mukhopadhyay et al. / Bioresource Technology 116 (2012) 348–354 Table 1 Purification of pectate lyase from Bacillus megaterium AK2. Purification procedure

Total volume (ml)

Total protein (mg)

Total activity (unit)

Specific activity (unit/mg)

Purification (fold)

Yield (%)

Crude CM-sepharose Gel filtration (Sephadex G-75)

100 10 1.5

148 13.8 0.5

6780 3405 426

45.8 246.74 852

1 5.38 18.6

100 50.22 6.28

Fig. 2. Pectate lyase activity (a) as a function of nanoparticle concentration (b) at elevated temperature in the presence of Hydroxyapatite nanoparticle and Ca2+ (values are averages of results from triplicate trials; error bars indicate the SD values).

value of 6.1 kJ/mole whereas NP-PL showed a value of 19.825 kJ/mole for Ea (Table 3). From the thermodynamic point of view, NP supplementation made the pectate lyase more stable at high temperatures. At 90 °C, the Km of NP-PL became lower than that at 75 °C and Vmax became higher. This result indicates that substrate affinity became steady and the rate of reaction was fast at the high temperature, whereas the untreated pectate lyase was not as substrate-specific and fast-reacting at high temperature. The activation energy (Ea) of NP-PL became around 200% lower than that of PL. This observation signifies that NP-PL would be more easily activated than untreated PL. 3.7.2. Activation/inactivation kinetics The kinetics of PL and NP-PL systems were examined between 50 and 90 °C. The semi-logarithmic plots of residual activity versus

time (between 50 and 90 °C) for both cases were linear. The plots suggested that PL was heat inactivated with first order kinetics, whereas the NP-PL was heat activated with first order kinetics. The half-life (t1/2) according to the plots increased with increasing temperature for the NP-PL (Fig. 5a). The activity of PL decreased with increasing temperatures as reflected in the halflife (t1/2) values. Beyond 70 °C, enzyme activity was severely inhibited. In contrast, the thermal stability of NP-PL increased with temperature. The deactivation energy (Ed) of NP-treated pectate lyase was calculated from a linear portion of the Arrhenius plot and was around 36.106 kJ/mol and Ed of untreated enzyme was 57.556 kJ/mol (Fig. 5b(i) and (ii)) The half-life of inactivation was 20.26 min at 50 °C as compared to 288.75 min at 90 °C for the NP-PL. The gradual decrement of the dissociation constant (k) with increasing temperatures, the high deactivation energy (Ed) and the increase of t1/2 value with temperature of NP-PL imply that nanoparticles made

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Fig. 3. Pectate lyase activity as a function of temperature. (values are averages of results from triplicate trials; error bars indicate the SD values).

Fig. 4. Retention of activity of nanoparticle treated pectate lyase (NP-PL) versus untreated pectate lyase (PL) systems at 90 °C. (values are averages of results from triplicate trials; error bars indicate the SD values).

Table 2 Retention of activity at 90 °C by nanoparticle treated pectate lyase (NP-PL) (substrate pectin). Enzyme system

Pectate lyase activity after 2 h

Pectate lyase activity after 4 h

Pectate lyase activity after 5 h

NP-PL PL

69.54 ± 0.48 14.8 ± 0.397

76.4 ± 0.65 7.8 ± 0.54

26.8 ± 0.802 4.64 ± 0.73

Table 3 Km–Vmax and activation energy values of nanoparticle treated pectate lyase (NP-PL) and un-treated pectate lyase (PL) systems. Enzyme (pectate lyase)

Km @ 75 °C (mg/ml)

Vmax @75 °C (unit/ml)

Km @ 90 °C (mg/ml)

Vmax @ 90 °C (unit)

NP-PL PL

0.11 0.3

76 21.4

0.08 0.418

129 15.8

Ea (KJ mol

1

)

19.825 6.1

this enzyme highly stable at higher temperatures. Therefore it needed high energy to denature and the low t1/2 value at higher temperature stands for its instability.

Fig. 5. (a) Comparison of half life (t1/2) of nanoparticle treated pectate lyase (NP-PL) and un-treated pectate lyase (PL) systems over 50–90 °C (b)Arrhenius plot for deactivation energy (Ed); (i) nanoparticle treated pectate lyase (NP-PL), (ii) untreated pectate lyase (PL). (values are averages of results from triplicate trials; error bars indicate the SD values).

Pectate lyase of AK2 had a t1/2 at 80 °C of 14.19 min which was roughly 14-fold higher than that reported for BK2 pectate lyase (1.2 min) (Klug-Santner et al., 2006) whereas NP-PL had a t1/2 of 56.8 min at 80 °C. NP-PL showed highest activity at 90 °C, whereas the B. pumilus pectate lyase (Dave and Vaughn, 1971) lost 90% of its total activity at 75 °C and B. pumilus dcsr1 showed maximum activity at 50 °C (Sharma and Satyanarayana, 2006). B. pumilus DKS1 showed an optimum temperature for pectate lyase of 75 °C (Basu et al., 2008) So, among the pectate lyases of Bacillus strains (Sharma and Satyanarayana, 2006; Dave and Vaughn, 1971; Klug-Sant-

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A. Mukhopadhyay et al. / Bioresource Technology 116 (2012) 348–354 Table 4 Variation of kinetic parameters for nanoparticle treated pectate lyase (NP-PL) within 323–363 K. Pre-incubation temperature (K)

Dissociation constant [k] (min

323 333 343 353 363

0.0342 ± 0.0004 0.03 ± 0.0004 0.0142 ± 0.0015 0.0122 ± 0.0045 0.0024 ± 0.0005

1

)

Half life [t1/2] (min)

Ed (kJ/mol)

DH (kJ/mol)

DG (kJ/mol)

20.26 ± 1.33 23.1 ± 2.252 48.8 ± 1.563 56.8 ± 0.502 288.75 ± 0.735

36.106 ± 0.325

33.426 ± 1.742 33.346 ± 1.742 33.256 ± 1.742 33.176 ± 1.9 33.096 ± 1.545

100.75 ± 0.709 104.3 ± 0.638 103.09 ± 0.538 99.88 ± 0.467 93.78 ± 0.335

DH (kJ/mol)

DG (kJ/mol)

DS (J/mol/k) 208.43 ± 3.357 213.73 ± 4.209 203.54 ± 2.425 188.95 ± 3.005 167.17 ± 2.051

Table 5 Heat inactivation parameters for un-treated pectate lyase (PL). Pre-incubation temperature (K)

Dissociation constant [k] (min

323 333 343 353 363

0.01035 ± 0.00051 0.01988 ± 0.00186 0.02607 ± 0.0136 0.047 ± 0.00451 0.1455 ± 00887

1

)

Half life [t1/2] (min) 66.95 ± 2.532 34.85 ± 2.904 26.58 ± 0.581 14.19 ± 0.92 4.78 ± 0.718

Ed (kJ/mol) 57.996 ± 0.482

60.67 ± 2.896 60.756 ± 2.72 60.846 ± 3.324 60.926 ± 3.324 61.006 ± 3.324

91.596 ± 0.665 92.713 ± 0.667 94.804 ± 0.533 102.68 ± 0.295 109.27 ± 0.197

DS (J/mol/k) 471.4 ± 5.165 460.86 ± 3.558 453.79 ± 4.935 463.42 ± 3.849 469.07 ± 4.574

ner et al., 2006; Basu et al., 2008), NP-treated pectate lyase of AK2 was the most thermoactive. From Tables 4 and 5, it can be observed that with increasing temperatures, the change of free energy (DG) decreased by 11% at 90 °C compared to 50 °C in case of NP-PL whereas for PL, DG increased by 11% in the same interval. 3.7.3. Entropy–enthalpy map An entropy enthalpy map is essentially a plot of enthalpy change (DH) against the change of entropy (DS). When the compensation occurs, the overall free energy (DG = DH TDS) remains stationary (minimum). As soon as there is a structural change, this is associated with a change in free energy and thus the compensatory nature of the plot is lost the entropy–enthalpy map thus summarizes the thermodynamic changes associated with the enzymatic process in the presence and absence of nanoparticles. In presence of NP, a compensatory nature was evident as the linearity of the DH DS plot was maintained at a higher temperature. In absence of NP, a critical change in the entropy–enthalpy map was observed. Incidentally, the critical behavior corresponded to the onset of inactivation of the enzyme. The entropy–enthalpy profile for the thermal inactivation process of NP-PL showed that till 90 °C, there was entropy–enthalpy compensation. For the NP-treated enzyme, the DH and DS had opposite sign(s), implying a significant entropy enthalpy compensation Cornish-Bowden, 2002; Sharp, 2001). The profile remained monotonic between 50 and 90 °C. In contrast, in case of PL, entropy–enthalpy compensation was operative below 70 °C (Fig. 6a and b). Beyond such a critical point, the compensatory profile was lost, the entropic and enthalpic contributions assumed the same sign (both negative). The unfavorable entropic contribution perhaps indicated a loss of structure (and enzymatic activity) as maintenance of native structure normally is an entropy-driven process. Identical signs of DH and DS generally imply a weak form of compensation (Cornish-Bowden, 2002; Sharp, 2001) where a free energy balance (negative free energy change) is obtained by loss of structure and activity. Thus, above 70 °C, thermal stability of the untreated enzyme decreased rapidly. The use of enzymes industrially may require reactions to be conducted at high temperatures for a long time such as in the case of processing of fibers in the textile industries (Rombouts and Pilnik, 1986). It has been found by Solbak et al. (2005) that under alkaline conditions and high temperatures (the optimal conditions for scouring), methyl esters in pectin are quickly hydrolyzed.

Fig. 6. Entropy–enthalpy compensation plots for (a) nanoparticle treated pectate lyase (NP-PL) (b) un-treated pectate lyase (PL) systems.

Another invention (Bjornvad et al., 2003) provides methods for treating cellulosic fibers to remove non-cellulosic compounds. For optimal efficiency, the fiber was treated with enzyme under the following conditions: (i) a temperature above about 70 °C, preferably above about 80 °C, (ii) preferably for long time at that high temperature and (iii) a pH above about 7.0, preferably above 8.0, and most preferably above about 9.5. The thermoactive and (persistently) thermostable NP-PL has an alkaline pH optimum (8.5– 8.7) and low activation energy which suggests that this strain

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could potentially be utilized efficiently for removal of pectin from natural sources or for other reactions that require the breakdown of pectin. Taking all these observations into consideration, it may be concluded that HapNP specifically enhanced the activity of PL in a concentration-dependent manner (with an optimum concentration) while imparting thermostability. Not only was this a unique feature of the NP-PL system, it was singularly different from the activation observed with Ca2+ ions. In fact, Ca2+ was an inhibitor of the activation process at high temperatures. This indicates a specific role of the HapNP in promoting and maintaining the activation of both the catalytic and structural features of the NP-PL. This hypothesis is further supported by the lower Ea, lower Km and higher Vmax values for the NP-PL system as compared to the untreated PL system. The nanoparticle-mediated retention of enzyme activity at high temperature implies that the nanoparticle interacts with the enzyme active site. The question why a nanoparticle formulation of calcium is more effective in stabilizing the activity as compared to ionic calcium needs further investigation. One plausible explanation may be that an electrical double layer effect posed by clusters of calcium ions may be a deterrent to efficient contact of calcium with the active site. An indirect support for this idea (active site contact with calcium) stems from the observation that other nanoparticles e.g. copper nanoparticles are ineffective in inducing higher thermostability of the enzyme (data not shown). Copper-dependent enzymes (e.g. laccase) show similar enhanced thermostability effects only in presence of copper nanoparticle and not in presence of other nanoparticles (e.g. calcium nanoparticle) (data not shown). The thermodynamic data relating to NP-PL system, presented here, highlights for the first time, the role of Hap in enhancing the entropy–enthalpy compensation for this enzyme. 4. Conclusion This study suggests that HAp nanoparticle supplementation induces thermostability of pectate lyase from B. megaterium AK2. This enzyme can also retain high activity at elevated temperatures for an extended period of time (4 h) in the presence of HAp NP. These findings have the potential to be utilized for developing an eco-friendly process for the textile, paper food and wastewater industries. Acknowledgements This study was supported by the Grant from Centre for Research on Nanoscience and Nanotechnology (CRNN) under University of Calcutta. The authors are grateful to Dr. P. Acharyya, Department of Horticulture, Institute of Agricultural Science, University of Calcutta for the use of AAS equipment. The authors are also grateful to the CAS programme of Department of Biochemistry of University of Calcutta, UGC, FIST and laboratory colleagues. References Basu, S., Ghosh, A., Bera, A., Saha, M.N., Chattopadhyay, D.J., Chakrabarti, K., 2008. Thermodynamic characterization of a highly thermoactive extracellular pectate lyase from a new isolate Bacillus pumilus DKS1. Bioresour. Technol. 99, 8088– 8094. Basu, S., Roy, A., Ghosh, A., Bera, A., Chattopadhyay, D.J., Chakrabarti, K., 2011. Arg235 is an essential catalytic residue of Bacillus pumilus DKS1 pectate lyase to degum ramie fibre. Biodegradation 22, 153–161.

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