Protein & Peptide Letters, 2009, 16, 1098-1105 1098 Invertase from Hyper Producer Strain of Aspergillus niger: Physiochemical Properties, Thermodynamics and Active Site Residues Heat of Ionization Habibullah Nadeem, Muhammad Hamid Rashid*, Muhammad Riaz, Bibi Asma, Muhammad Rizwan Javed and Raheela Perveen Enzyme Engineering Lab., National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan Abstract: Here we report for the first time heat of ionization of invertase (E.C.3.2.1.26) active site residues from hyperproducer strain of Aspergillus niger (34.1 U ml-1), along with its physiochemical properties, kinetics and thermodynamics of stability-function. The Invertase showed great potential for industry as being highly efficient (kcat = 24167 s-1 at 65 °C, pH 5.0) and stable (half life= 12 h at 56°C). Keywords: Thermostability, Enthalpy, Entropy, Free energy, Ionization energy. INTRODUCTION Invertase (-fructofuranosidase: E.C.3.2.1.26) catalyse the hydrolysis of sucrose into glucose and fructose. It is one of the most widely used enzymes in the food industry, especially in the preparation of jams and candies [1], where fructose is preferred over sucrose because it is sweeter and does not crystallize easily. Using waste biomass to produce biofuel (ethanol) can reduce the use of fossil fuels, reduce greenhouse gas emissions and reduce pollution and waste management problems [2]. Biologically alcohols are produced by the action of microbes and enzymes through the fermentation of sugars. Ethanol can be used in petrol engines as a replacement for gasoline; it can be mixed with gasoline to any percentage. Gasoline with ethanol added has higher octane, hence the engine can typically burn hotter and more efficiently. Molasses, a sub-product in sugar production is rich in sucrose and has been used as a carbon source in the production of fructofuranosidase by Saccharomyces cerevisiae [3]. Invertases have high potential to be used in biofuel production because they have a tendency to enhance the glucose content in molasses, which is considered to be an excellent ethanol feedstock. Invertase has been mainly produced from Saccharomyces cerevisiae and commercial fungal strains of the Aspergillus genus, such as A. niger, A. oryzae and A. ficcum [4]. Aspergillus niger is a filamentous, non-pathogenic fungus that belongs to the “Generally Recognized As Safe (GRAS)” microbial organisms, which are used for the production of enzymes [5]. Although there are many reports on isolation and characterization of invertases from various microbes but on an average less than 1% of the potential microbes have *Address correspondence to this author at the Principal Scientist, National Institute for Biotechnology and Genetic Engineering, P.O. Box 577, Jhang Road, Faisalabad, Pakistan; Fax: +92-041-2651472; E-mails: hamidcomboh@gmail.com; mhrashid@nibge.org 0929-8665/09 $55.00+.00 been identified [6]. So the need to isolate and identify organisms, which are either hyper-producers and/or sufficiently robust to withstand conditions of the intended application and/or are producers of novel enzymes is highly significant. This current report deals with the purification, kinetic and thermodynamic properties of a highly efficient and stable fructofuranosidase from a local, hyper-producing strain of A. niger. We considered that this biocatalyst has great potential to be used in the food industry and in biofuel production. MATERIALS AND METHODS Microorganism A pure local strain of Aspergillus niger was obtained from NIBGE, Faisalabad. The culture was maintained on potato dextrose agar (PDA) plates and slants as described earlier [7]. All chemicals were purchased from MP Biomedicals, USA. Inoculum Preparation & Biomass Estimation For inoculum preparation a loop full of spores from a 5 to 6 days old A. niger slant was transferred into a 250 ml Erlenmeyer flask containing 50 ml of sterile Vogel’s medium and 8-10 acid washed glass beads were added. The composition of the growth medium was (gl-1): glucose (20.0), trisodium citrate (2.5), KH2PO4 (5.0), NH4NO3 (2.0), (NH4)2SO4 (4.0), MgSO4.7H2O (0.2), peptone (2.0), microelement solution (10 ml) and vitamin solution (5.0 ml). The pH of the medium was adjusted to 5.0 using 1M HCl/1M NaOH. The flask was incubated at 30 °C on an orbital shaker at 150 rpm for 48h to get a homogenous cell suspension. The biomass content of the inoculum was estimated on the basis of absorptiometry (cells turbidity) by measurement of the light transmitted at 610 nm [8]. The standard curve for biomass estimation was prepared as follows: 50 ml culture was harvested and centrifuged at 15,300
g at 4 °C for 15 © 2009 Bentham Science Publishers Ltd. Invertase from Hyper Producer Strain of Aspergillus niger min. The supernatant was separated and the pellet was resuspended in 0.89% (w/v) saline solution and centrifuged (23 times) as above. Then the pellet was freeze dried and ground to fine powder with pestle and mortar. Appropriate stock solutions were made, which were used to prepare the final stock (1 mg ml-1). Various dilutions of the final stock (1-10 folds) were made and the optical density (OD) of each dilution was noted at 610 nm. Distilled water was used as a blank and calibration curve was plotted between OD and mg of cells. Invertase Production Invertase or sucrase was produced under submerged growth conditions in 250 ml Erlenmeyer flasks containing Vogel’s medium with sucrose (2% w/v) as sole carbon source. Flasks were autoclaved for 15 min at 121 °C, 1.1 kgfcm-2 and inoculated (92 mg cells per 50 ml growth medium). The flasks were incubated at 30±1 °C for 60 hours. Isolation of Crude Invertase After 60 hours of cultivation, the crude enzyme was extracted from the growth medium by filtration through Whatman filter paper. The extract was centrifuged at 25,900  g for 15 min at 4 °C to remove the suspended particles. Invertase Assay Activity of the enzyme was determined as described [9]. Briefly, an appropriate amount of invertase was reacted with 1% (w/v) sucrose solution in 50 mM Na-acetate buffer (pH 5.0) at 50 °C for 15 min. The reaction was quenched by placing the tubes in boiling water for 5 min and then cooled immediately in ice. The released glucose was measured using glucose measuring kit (Fluitest® GLU, Made in Germany). One unit was defined as the amount of invertase that releases 1
mol of glucose min-1 from sucrose at 50 °C, pH 5.0. Protein Assay Protein was estimated as described by Bradford [10] using bovine serum albumin as a standard. Purification of Invertase Crude invertase was subjected to a four-step purification procedure. After optimization of invertase precipitation solid ammonium sulfate was added bit by bit to the crude enzyme to make 30% saturation at 0 °C. The invertase solution was left overnight at 4 °C and then centrifuged at 25,900g for 20 min. The pellet of precipitated proteins was discarded and more ammonium sulfate was added to the supernatant to make 70% saturation at 0 °C. The solution was kept overnight at 4 °C and centrifuged as described above. This time, the supernatant was discarded, whereas the pellet containing invertase was dissolved in 50 mM Na-acetate buffer (pH 5.0) and dialyzed over night against distilled water to remove salts. The invertase after ammonium sulfate precipitation was loaded on a Hiload anion exchange (Q-Sepharose) column of Fast Protein Liquid Chromatography (FPLC) system, using a Protein & Peptide Letters, 2009, Vol. 16, No. 9 1099 superloop of 50 ml at a flow rate of 2 ml min-1. A linear gradient of NaCl (0-1M) in 20 mM Tris-HCl, pH 7.5 was used as elution buffer. The fractions containing invertase were pooled. Solid ammonium sulfate was added to the dialyzed sample from the Hiload column to a final concentration of 2 M. The enzyme solution was filtered through 0.22
m filter paper and was applied to a hydrophobic interaction (Phenyl Superose) column using a superloop of 50 ml at a flow rate of 1.0 ml min-1. The elution was carried out with a linear gradient of ammonium sulphate (2-0 M) in 50 mM sodium phosphate buffer pH 7. Active fractions were collected. The dialyzed sample after HIC was loaded on a Mono Q column, using a superloop of 50 ml at a flow rate of 1.0 ml min-1. A linear gradient of NaCl (0-1 M) in 20 mM Tris-HCl, pH 7.5 was used as an elution buffer. The fractions containing invertase were pooled. The pooled fractions after each purification step were dialyzed extensively against distilled water at 4 °C and total enzyme activity and proteins were estimated in the pools. Sub-Unit Molecular Mass The purity of the purified invertase and its sub-unit molecular mass was determined by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as described [11]. Protein markers of Fermentas (10-200 kDa) were used as standard. The gel containing different molecular weight markers was stained with 0.1% Coomassie brilliant blue R-250 solution. The subunit molecular mass of invertase was also confirmed by using 10% SDS denaturing-renaturing PAGE (SDS-DR-PAGE). The sample was prepared by incubating purified invertase at 50 °C for 45 min with SDS sample buffer in the presence of -mercaptoethanol (2% v/v). PAGE was run at constant voltage (70 V) and the temperature was controlled (10 °C) by using a refrigerated circulating water bath. The gel was re-natured to remove the SDS by treating with 20% (v/v) isopropanol in 50 mM sodium acetate buffer, pH 5.0 (two washes of 45 min each). Then the gel was immersed in 50 mM sodium acetate buffer pH 5.0 to remove isopropanol (two changes of buffer each of 45 min). Invertase activity staining was done as follows: The re-natured gel was over-laid on agarose gel (0.8% w/v) prepared in Naacetate buffer containing sucrose (1.0% w/v) as a substrate. The gels were incubated at 50 °C for 90 min with continuous sprinkling of buffer to avoid drying of the gels and after incubation the agarose gel was cut as a replica copy of the renatured SDS-gel, and stained by immersing in the glucose detection kit solution. A pink-colored activity band appeared after 15 min of staining. Optimum Temperature, Activation Energy & Temperature Quotient (Q10) The optimum temperature and activation energy (Ea) of invertase were determined by incubating an appropriate amount of the enzyme in 1% sucrose solution at various temperatures ranging from 25-70 °C in 50 mM Na-acetate buffer for 15 min at pH 5.0. The activation energy was calculated by using an Arrhenius plot as described earlier [7]. The effect of the temperature on the rate of the reaction was expressed in terms of Q10, which is the factor by which the rate 1100 Protein & Peptide Letters, 2009, Vol. 16, No. 9 Nadeem et al. increases or decreases, due to an increase or decrease in temperature by 10 °C. The Q10 was calculated by rearranging the equation given by Dixon and Webb [12]. Q10 = Antilog (E
10/RT2) (1) Where, (Kd) were determined as described [15, 16]. The energy of activation for irreversible thermal denaturation Ea(d) was determined from an Arrhenius plot. Thermodynamics of irreversible thermal stability of invertase was determined by rearranging the Eyring’s absolute rate equation derived from the transition state theory [14]. E = Ea = Activation Energy of sucrose hydrolysis Kd = (kbT/h).e (-H*/RT).e (S*/R) * Optimum pH The effect of pH on sucrose hydrolysis was determined by assaying the invertase at various pHs ranging from 2-11, and pH optima were determined at 50, 55, 60 and 65 °C. The buffers used were as follows: Glutamic acid/HCl (pH 2-2.9), Na-acetate/acetic acid (pH 3.2-5.3), MES/KOH (pH 5.6-6.5), MOPS/KOH (pH 6.8-7.4), HEPES/KOH (pH 7.7-8.3), Glycine/NaOH (pH 8.6-9.8) and CAPSO/NaOH (pH 10.1-11.0). Heat of Ionization (
HI) of Active Site Residues The pKa values of acidic and basic limbs of invertase active site residues were determined at different temperatures (50, 55, 60 and 65 °C) as described by Dixon and Webb. The effect of temperature on the pH optima was determined as mentioned above. HI was determined by plotting pKa values of active site residues determined at different temperatures against 1/T [12]. HI = -slope
2.303R (2) Kinetics & Thermodynamics of Sucrose Hydrolysis The kinetic constants (Vmax, Km, kcat and kcat/Km) were determined by incubating fixed amounts of invertase with varied concentrations of sucrose as a substrate ranging from 0.025 to 1.5% (w/v) at 65 °C, pH 5.0 as described previously [13]. The thermodynamic parameters for substrate hydrolysis were calculated by rearranging the Eyring’s absolute rate equation derived from the transition state theory [14]. kcat = (kbT/h)e(-H*/RT).e(S*/R) * H = Ea – RT (4) * G (Free energy of activation) = -RT ln (kcat h/kb.T) * * (3) * S = (H - G )/T (5) (6) The free energy of substrate binding and transition state formation was calculated using the following derivations: G*E-S (free energy of substrate binding) = -RT ln Ka (7) Where, Ka = 1/Km G*E-T (free energy for transition state formation) = -RT ln(kcat /Km) (8) Thermodynamics of Irreversible Thermal Stability of Invertase The enzyme solution in 10 mM Tris-HCl buffer (pH 7) was incubated at various temperatures (56, 59, 62, 65 °C) in the absence of substrate. Aliquots were withdrawn at different time intervals, cooled on ice for 30 min and assayed for -fructofuranosidase (invertase) activity at 50 °C. The data were fitted to a first order plot and inactivation rate constants * (9) * H , G , and S of irreversible thermal stability were calculated by applying Eq. # 4, 5 and 6 with the modifications that in Eq # 4 Ea(d) was used instead of Ea and in Eq # 5 Kd was used in place of kcat. RESULTS AND DISCUSSION Production & Purification of Invertase The local isolate of Aspergillus niger proved to be a hyper producer strain and maximum extra-cellular invertase production (34.1 U ml-1) under submerged growth conditions occurred after 60 h of incubation on sucrose (2 % w/v) as sole carbon source. Bhatti et al. [17] reported 9.9 U ml-1 invertase from Fusarium solani after 96 h of growth on 2 % molasses. On the other hand, Aspergillus ochraceus produced 12.5 U ml-1 invertase [18], while 2.4 U ml-1 were produced by Aspergillus niger IMI303386 on 2 % sucrose [19]. The invertase of A. niger was purified up to homogeneity by a four step purification procedure Table (1). The onset and complete invertase precipitation occurred at 30% and 70% ammonium sulfate saturation at 0 °C, respectively and invertase was 2.27 fold purified. The invertase was eluted at 440 mM NaCl on FPLC Hiload anion exchange chromatography Fig. (1Ai). Afterwards, the enzyme was purified by hydrophobic interaction FPLC chromatography using a linear ammonium sulfate gradient (2-0 M) was used. A single broad peak of invertase was obtained at 850 mM ammonium sulfate Fig. (1Aii). Further purification was done by FPLC Mono Q anion exchange chromatography using a linear gradient of 0-1 M NaCl and invertase was eluted at 783.5 mM NaCl Fig. (1Aiii). Crude invertase was 101 fold purified with a recovery of 22% and the specific activity of purified invertase was 28,465 U mg-1 protein (Table 1). The purity of the enzyme was checked on 10% SDS-PAGE, which was stained with Coomassie blue and a single band was obtained Fig. (1B). Quang et al. [19] purified -fructofuranosidase from Aspergillus niger IMI 303386 up to 50 fold by using a threestep purification procedure and recovery of the enzyme was 42%. A single protein band of invertase was found on 10% SDS-PAGE and the subunit molecular mass of the enzyme was 116 kDa, which was also confirmed by doing SDS-DRPAGE and the same result was found Fig. (1B). Sub-unit molecular mass of the invertase was close to other reported values i.e.120-130 kDa from Aspergillus niger IMI303386 [19] and 71-111 kDa from Aspergillus niger AS0023 [20]. Three bands of 95, 65 and 37 kDa molecular mass were reported for -fructofuranosidase of A. japonicus MU-2 [21]. The invertase of Aspergillus niger expressed in E. coli displayed a molecular mass of 75 kDa estimated by SDS-PAGE [22]. A single wide band of -fructofuranosidase from Candida utilis with molecular mass of 150 kDa is also reported [23]. Invertase from Hyper Producer Strain of Aspergillus niger Table 1. Protein & Peptide Letters, 2009, Vol. 16, No. 9 1101 Purification of Invertase from Aspergillus niger Grown on Sucrose Under Submerged Conditions Total Protein (mg) Total Units (U) Specific Activity (U mg-1) Purification Factor % Recovery Crude enzyme 291.6 82188 282 1.00 100 (NH4)2 SO4 precipitation 98.10 62900 641 2.27 77 Hiload anion exchange column chromatography 12.60 46013 3652 12.95 56 Hydrophobic interaction column chromatography 4.53 31038 6852 24.30 38 Mono Q anion exchange column chromatography 0.641 18246 28465 101 22 Step All quoted values were after dialysis against distilled water. Figure 1. A) Fast Protein Liquid Chromatography of invertase; i, Hiload anion exchange chromatography on a Q-Sepharose column using a 0-1 M NaCl gradient ii, Hydrophobic interaction chromatography on a Phenyl Superose column using a 2-0 M (NH4)2SO4 gradient iii, MonoQ anion exchange chromatography using a 0-1 M NaCl gradient. B) a, 10 % SDS-PAGE of purified invertase of Aspergillus niger. Lane-1: Fermentas protein markers #SM0661 (10-200 kDa); Lane-2: purified invertase. b, activity staining of A. niger invertase applied on 10% SDS-DR-PAGE. 1102 Protein & Peptide Letters, 2009, Vol. 16, No. 9 Nadeem et al. Effect of pH Hydrolysis of sucrose by purified invertase at various pHs ranging from 2–11 was determined at different temperatures (50
65 °C), and at 65 °C (temp optimum) the enzyme exhibited optimal activity in the pH range 2.9-5.6, while maximum activity was at pH 4.1. Our results agreed with that reported for -fructofuranosidase from A. niger AS0023. We found a drift in pH optimum towards the acidic side when the temperature was lowered below the optimum temperature. The pH optimum at 55 & 60 °C was 3.5, while it was 2.9 at 50 °C. -fructofuranosidase from A. niger showed optimal activity at pH 5.5 [24]. Activity of the enzyme was inhibited rapidly below and above the optimum pH range. Heat of Ionization (
HI) of Active Site Residues Here we report for the first time about the heat of ionization of the invertase active site residues. The pKa1 and pKa2 of the active site residues controlling Vmax were determined as described by Dixon and Webb and at 65 °C their values were 2.6 and 7.0, respectively Fig. (2). The pKa values of both acidic and basic limbs showed an increasing trend with the increase in temp. At 50, 55, 60 and 65 °C, pKa1 values were 1.55, 1.95, 2.35 and 2.6, while pKa2 values were 6.3, 6.4, 6.9 and 7.0, respectively. The heat of ionization for the acidic limb (HI(AL)) and the basic limb (HI(BL)) of A. niger invertase was determined as described by Dixon and Webb and Figure 3. Dixon plot for the determination of Heat of ionization of A. niger invertase active site residues. Data presented are average values ± SD of n = 3 experiments. was equal to 148.58 and 108.68 kJ mol-1, respectively Fig. (3). Figure 4. Arrhenius plot to determine the effect of temperature on activity and activation energy for sucrose hydrolysis by invertase of A. niger. Data presented are average values ± SD of n = 3 experiments. Figure 2. Dixon plot for the determination of pKa of ionizable groups of A. niger invertase active site residues. Data presented are average values ± SD of n = 3 experiments. Effect of Temperature The optimum temperature of invertase from A. niger for hydrolysis of sucrose was found to be 65 °C and its activity was lost rapidly at 70 °C. -fructofuranosidase with exactly the same optimum temperature (60–65 °C) was reported in Aspergillus japonicus [21]. However, -fructofuranosidase purified from Candida utilis [23] showed optimum activity at 70 °C and that of Aspergillus niger AS0023 at 55 °C [20], whereas maximum activity at 50 °C was also reported from Aspergillus niger IMI303386 [19]. The activation energy (Ea) of invertase for sucrose hydrolysis determined from an Arrhenius plot was 33.76 kJ mol-1 Fig. (4). The Ea for sucrose hydrolysis by invertase from Aspergillus niger was 8.32 fold higher than required by -fructofuranosidase from Fusarium solani, 5.45 fold higher than -fructofuranosidase isolated from baker’s yeast, while 1.24 fold higher than that from Rhodotorula glutinis [17,25,26]. This indicates that larger amount of energy was required to form the activated complex by -fructofuranosidase obtained from A. niger as compared to other reported -fructofuranosidases. The temperature quotient for the enzyme was 1.01. Kinetics & Thermodynamics of Substrate Hydrolysis The Michaelis Menten constant (Km) and Vmax of invertase from Aspergillus niger for sucrose hydrolysis at 65 °C were determined from a Lineweaver Burk plot and were equal to 0.117 M sucrose and 12500 U mg-1 protein, respectively Fig. (5). The Km of A. niger invertase for sucrose was smaller than that of A. japonicus (0.227 M) [21], while was higher than 0.020 M for Schwanniomyces occidentalis - Invertase from Hyper Producer Strain of Aspergillus niger fructofuranosidase [1] and 0.060 M for A. niger fructofuranosidase [27]. This indicated that the invertase produced by A. niger has higher affinity for sucrose than that of A. japonicus, while has lower affinity than that of S. occidentalis. The Kcat of A. niger invertase was 24,167 s-1 and specificity constant (Kcat / Km) was 206553. Extremely high turn over confirmed that the A. niger invertase was very efficient in sucrose hydrolysis and has great potential for industrial application. Protein & Peptide Letters, 2009, Vol. 16, No. 9 1103 elevated temperatures in the presence of substrates [29]. Pseudo first order plots Fig. (6) were applied to determine the extent of thermal inactivation and results regarding thermal inactivation of the invertase are presented in Table (2). The thermal inactivation of enzyme is accompanied by the disruption of non covalent linkages, including hydrophobic interactions, with a concomitant increase in the enthalpy of activation i.e., H* [7]. The opening up of enzyme structure is accompanied by an increase in the disorder (randomness) or entropy of activation (S*) [30]. The same trend is observed by us in case of A. niger -fructofuranosidase between the temperature range of 56-65 °C Table (2). The enzyme was very stable at 56 °C and exhibited half life of 11.9 hours. At higher temp half life decreased sharply i.e.,105 min at 59 °C, 13 min at 62 °C and just 10 min at 65 °C. Figure 5. Lineweaver-Burk plot for the determination of kinetic constants (Vmax, Km) for sucrose hydrolysis at 65°C, pH 5 by invertase of A. niger. Data presented are average values ± SD of n = 3 experiments. The turn over (Kcat) was calculated using the relation: Vmax/[e]. Invertase concentration [e] in the reaction mixture was 8.621
10-3 H mole. We could not find any report on the thermodynamic characterization of microbial invertases. Recently we have reported thermodynamic properties of sugarcane soluble acid invertase (SAI) for sucrose hydrolysis [28]. The Gibbs free energy (G*) of A. niger invertase at 65 °C was very low (54.77 kJ mol-1) as compared to the sugarcane SAI at 55 °C (71.2 kJ mol-1). As enzymes speedup the reactions by lowering down the free energy, therefore the A. niger invertase was very efficient due to its lower G*, which explained that its transition state (ES*) was spontaneously converted into the products. Similarly, the change in enthalpy (H*) for sucrose hydrolysis indicated that the energy required by A. niger invertase was lower (30.95 kJ mol-1) than that of sugarcane SAI (52.6 kJ mol-1), which explained that the transition state (ES*) of A. niger invertase was more actively formed. The entropy (S*) of activation of sucrose hydrolysis by A. niger invertase was -70.46 J mol-1 K-1, while S* of the SAI was -57 J mol-1 K-1, which explained that the higher activity of A. niger invertase was due to the lowering down of free energy but was not entropically driven. Figure 6. Pseudo first order plots for irreversible thermal inactivation of A. niger invertase. The enzyme solution was incubated at various temperatures (56 to 65 °C) in 10 mM Tris-HCl buffer (pH 7). Data presented are average values ± SD of n = 3 experiments. Thermal denaturation of enzymes can be considered to occur in two steps [31] as shown below: N
U*I Where, N is the native enzyme, U* is the unfolded inactive enzyme, which could be reversibly refolded upon cooling and I is the inactivated enzyme formed after prolonged exposure to heat and therefore cannot be recovered upon cooling. Free energy for activation of substrate binding (G*E-S) and for the formation of transition complex (G*E-T) were 1.16 and -6.61 kJ mol-1, respectively for A. niger invertase, whereas, 10.8 kJ mol-1 and 2.6 kJ mol-1, respectively were for the SAI. The lower free energy values for A. niger invertase again provided the evidence for its higher activity. The activation energy for irreversible thermal denaturation ‘Ea(d)’ of the invertase was determined by applying Arrhenius plot Fig. (7) and Gibbs free energy (G*) for activation of thermal unfolding of enzyme was 99.84 kJ mol-1 . With an increase in temperature, a slight decrease in free energy was observed. The enthalpy of activation of thermal unfolding (H*) of the enzyme at 56 °C was 482.9 kJ mol-1 . Its value remained almost same upto 65 °C. The entropy of activation (S*) for unfolding of transition state of the fructofuranosidase was 1.164 kJ mol-1 K-1 at 56 °C, which was slightly increased at 62 °C Table (2). But the increase or decrease in entropy was not significant. Irreversible Thermostability of Invertase CONCLUSION Thermostability is the ability of enzyme molecule to resist against thermal unfolding in the absence of substrate, while thermophilicity is the capability of enzymes to work at Due to very high catalytic activity of A. niger invertase (-fructofuranosidase), we considered it as a strong candidate for application in food industry as well as biofuel pro- 1104 Protein & Peptide Letters, 2009, Vol. 16, No. 9 Table 2. Nadeem et al. Kinetics and Thermodynamics of Irreversible Thermal Stability of Invertase from Aspergillus niger Temp (K) Kd (min-1) t
(min) H* (kJ mol-1) G* (kJ mol-1) S* (kJ mol -1K-1) 329 0.00096 719 482.98 99.84 1.165 332 0.00658 105 482.96 95.47 1.167 335 0.05390 13 482.94 90.50 1.171 338 0.06820 10 482.91 90.68 1.16 Where, Kd = first order rate constant for inactivation, t
= half life = 0.693/Kd. Thermodynamic parameters were calculated using equations 4, 5 and 6. duction from molasses. Especially, it has high potential for sucrose processing industry to increase sweetness level of sugar. [7] [8] [9] [10] [11] [12] [13] [14] Figure 7. Arrhenius plot for determination of activation energy ‘Ea(d)’ for irreversible thermal denaturation of A. niger invertase. Data presented are average values ± SD of n = 3 experiments. [15] [16] ACKNOWLEDGEMENTS The work presented is a part of Ph.D. studies of Mr. Habibullah Nadeem. The project was partly funded by Higher Education Commission (HEC), Pakistan under the Indigenous Scholarship Scheme and Pakistan Atomic Energy Commission. Technical assistance of Mr. Ghulam Ali Waseer is appreciated. [17] [18] REFERENCES [19] [1] [2] [3] [4] [5] [6] Klein, R.; Deibel, M.; Sarcich, J.; Zurcher, H.; Reardom, I.; Heinrikson, R. 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