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Enzyme and Microbial Technology 41 (2007) 558–564

Kinetic and thermodynamic properties of novelglucoamylase from Humicola sp.

Muhammad Riaz, Raheela Perveen, Muhammad Rizwan Javed,Habibullah Nadeem, Muhammad Hamid Rashid ∗

Enzyme Engineering Group, National Institute for Biotechnology and Genetic Engineering (NIBGE),P.O. Box 577, Jhang Road, Faisalabad, Pakistan

Received 29 December 2006; received in revised form 26 April 2007; accepted 5 May 2007

bstract

Extra-cellular glucoamylase of Humicola sp. produced under submerged growth condition (10.44 U mg−1 protein) was purified to homogeneityevel by using three-step purification procedure. Crude enzyme was subjected to ammonium sulphate precipitation, Hiload anion exchange chro-atography and hydrophobic interaction column chromatography on FPLC purification system. The purified glucoamylase was monomeric in nature

ecause native molecular mass on gel column chromatography and sub-unit mass on SDS-PAGE were the same, i.e. 72.8 kDa. Activation energyor soluble starch hydrolysis was 21.09 kJ mol−1, while temperature quotient (Q10) was 1.01. The enzyme was stable over a pH range of 3.5–5.9 andave pH optimum of 4.7. Temperature optimum of the glucoamylase was 55 ◦C. Vmax for soluble starch hydrolysis was 56.63 U mg−1 protein andm was 0.26 mg (soluble starch) ml−1. The turn over (kcat) was 69 s−1. The pKa1 and pKa2 of ionizable groups of active site controlling Vmax were.65 and 6.3, respectively. Thermodynamic parameters for soluble starch hydrolysis were as follows: �H* = 18.36 kJ mol−1, �G* = 69.06 kJ mol−1,

S* = −154.56 J mol−1 K−1, �G∗

E−S = −3.71 kJ mol−1 and �G∗E−T = −26.41 kJ mol−1. Thermodynamic parameters (�H*, �G*, �S*) for irre-

ersible inactivation of glucoamylase at different temperatures (45–55 ◦C) were also determined. Current report has novelty as it explained for therst time kinetics and thermodynamics of soluble starch hydrolysis and irreversible inactivation of glucoamylase from Humicola sp.2007 Elsevier Inc. All rights reserved.

at[aAcctcv

eywords: Purification; Activation energy; Enthalpy; Entropy; Free energy

. Introduction

Glucoamylases [�-(1,4)-d-glucan glucohydrolase (EC.2.1.3)], also known as amyloglucosidases, glucamylases,altases, saccharogenic amylases and �-amylases are an

mportant group of starch-degrading enzymes [1]. Theseatalyze the hydrolysis of �-1,4, �-1,6 glucosidic linkages fromhe non-reducing ends of starch and related oligo and polysac-harides into short chain saccharides [2,3]. Glucoamylases aresed to produce crystalline glucose and high fructose cornyrup. Moreover, these are extensively used in the production

f different antibiotics and amino acids; in brewing, textile,ood, paper and pharmaceutical industries [4–6].

∗ Corresponding author. Tel.: +92 41 2651475–79x229; fax: +92 41 2651472.E-mail addresses: mhrashid@nibge.org, hamidcomboh@gmail.com

M.H. Rashid).

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141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2007.05.010

Glucoamylases are widely distributed among many species ofnimals, plants and microorganisms [7]. Among microbes bac-eria, yeast and fungi are capable of producing these enzymes8,9]. Filamentous fungi, however, constitute the major sourcemong all microorganisms. Microbial strains of genus, viz.spergillus and Rhizopus are mainly used for the commer-ial production of glucoamylases [10]. Many fungal species areapable of producing glucoamylases under different fermenta-ion conditions. Most attempts involved seeking fungal speciesapable of hydrolyzing raw starch at elevated temperature. Thearious fungi synthesizing glucoamylases that are active atigher temperatures include Aspergillus awamori, Aspergillusoetidus, Aspergillus niger, Aspergillus oryzae, Aspergillus ter-eus, Mucor rouxians, Mucor javanicus, Neurospora crassa,hizopus delmar and Rhizopus oryzae [11].

Despite there enormous utility, industrial use of glucoamy-ases confronts two major problems. First, at highly dissolvedolids concentrations, they do not yield all glucose, but insteadondense some of the glucose to various di-, tri-, and tetrasac-

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M. Riaz et al. / Enzyme and Micr

harides [12]. These polysaccharides are undesirable becausehey are not as sweet as glucose and cannot be further processedo fructose [13]. Second, being used at higher temperaturesuring starch saccharification process these are not as stables �-amylase and glucose isomerase [12]. Hence, the ultimatebjectives are to produce glucoamylases having higher ther-ostability. This would allow a higher process temperature,hich might reduce the production of by products, lower theroduction time and reduce the risk of contamination [13].

Industrial enzymatic hydrolysis of starch is influenced byariables related to the chemical and physical starch naturerelative proportion of amylose and amylopectin or gran-le crystallinity) and its suspensions (viscosity, resistance toetrogradation or gelatinization degree) but also by those vari-bles associated with the catalytic process (pH, temperature,nzyme/substrate ratio or enzymatic deactivation and inhibitionhenomena) [14].

Enzyme production and characterization is a growing fieldf biotechnology. Interest in thermostable enzymes has beenncreased tremendously, since resistance to thermal inactivationas become a desirable property in many industrial applications15]. It is necessary to have enzyme kinetic information for anynzymatic process. Poor enzyme stability under standard con-itions of pH, temperature and pH inhibition affects the endroduct yield [16].

This work presents the purification and characterization oflucoamylase from Humicola sp. with respect to its ability toatalyze soluble starch for its conversion into glucose. Ther-odynamic parameters are also presented for soluble starch

ydrolysis and the irreversible inactivation of this enzyme. Theurrent study will help to determine the operating range of glu-oamylase in terms of pH, temperature and compatibility withther enzymes to exploit its efficiency in a variety of food pro-essing applications.

. Materials and methods

.1. Microorganism

Pure culture of Humicola sp. was obtained from First Fungal Culture Bank,epartment of Mycology, University of The Punjab, Lahore. The culture wasaintained on potato dextrose agar (PDA) plates and slants as described earlier

17]. All chemicals were purchased from Sigma Chemical Co., Missouri, USA.

.2. Inoculum preparation

Inoculum was prepared by transferring loop full of spores from 5 to 6 days oldlant cultures into 250 ml Erlenmeyer flasks containing 50 ml of sterile Vogel’sedium and 8–10 acid washed glass beads [17]. The composition of inocu-

um medium was (g l−1): glucose (20.0), trisodium citrate (2.5), KH2PO4 (5.0),H4NO3 (2.0), (NH4)2SO4 (4.0), MgSO4·7H2O (0.2), peptone (2.0), microele-ent solution (10 ml) and vitamin solution (5.0 ml). The pH of the medium was

djusted to 5.0 using 1 M HCl/1 M NaOH. The flasks were incubated at 35 ◦Cn a rotary shaker at 150 rpm for 36 h to get homogenous cell suspension. Theell suspension having 106 to 107 cells ml−1 was used as inoculum in the growthedia for production of glucoamylase.

.3. Glucoamylase production

Glucoamylase was produced under submerged fermentation conditions in50 ml Erlenmeyer flasks containing Vogel’s media but glucose was replaced

pwwa

Technology 41 (2007) 558–564 559

ith wheat bran. Flasks were plugged with cotton and sterilized by autoclavingor 15 min at 121 ◦C and 1.1 kgf cm−2. After sterilization, the flasks were coolednd inoculated with inoculum having 106 to 107 cells ml−1 at a concentration of0% (v/v) and incubated at 37 ± 1 ◦C for 72 h at pH 5.

.4. Isolation of crude enzyme

At the end of fermentation, the crude enzyme was extracted from growthedia by filtration through Whatman filter paper. The extract was centrifuged at

3,000 rpm (25,900 × g) for 15 min at 4 ◦C to remove the suspended particles.

.5. Glucoamylase assay

Glucoamylase activity was determined by using the method described pre-iously [18]. Appropriate amount of the enzyme was reacted with 1% (w/v)oluble starch solution in 50 mM Na-acetate buffer (pH 5.0) at 40 ◦C for 40 min.he reaction was quenched by placing the tubes in boiling water for 5 min, and

hen immediately cooled in ice. The released glucose was measured using alucose measuring kit (Fluitest® GLU, made in Germany).

One unit is defined as the enzyme amount that releases 1 �mol of glucoseer minute from soluble starch at 40 ◦C, pH 5.0.

.6. Protein assay

Protein was estimated as described by Bradford [19] using bovine serumlbumin as a standard.

.7. Purification of glucoamylase

The harvested crude enzyme was subjected to three-step purification proce-ure comprised of fractional precipitation by ammonium sulphate, Hiload anionxchange and hydrophobic interaction chromatography on fast protein liquidhromatography (FPLC) system.

.7.1. Ammonium sulphate precipitationPrecipitation of the glucoamylase was optimized. Ammonium sulphate was

dded in the crude enzyme to 70% saturation at 0 ◦C and left over night at◦C. Precipitated enzyme was collected by centrifugation (25,900 × g, 20 min),issolved in 50 mM Na-acetate buffer (pH 5.0) and dialyzed over night againsthe same buffer.

.7.2. Hiload anion-exchange chromatographyThe dialyzed sample after ammonium sulphate precipitation was loaded

n Hiload anion-exchange (Q-Sepharose) column, using superloop of 50 ml at aow rate of 2 ml min−1. The linear gradient of NaCl (0–1 M) in 20 mM Tris–HCl,H 7.5 was used as elution buffer. The fractions containing glucoamylase wereooled and dialyzed against Na-acetate buffer. Total enzyme units and totalroteins were determined.

.7.3. Hydrophobic interaction chromatographySolid ammonium sulphate was added to the dialyzed sample from Hiload

olumn to a final concentration of 2 M. The enzyme solution was filtered through.45 �m filter paper and was charged on hydrophobic interaction (Phenyl Super-se) column of FPLC at a flow rate of 0.5 ml min−1. The elution was carriedut with a linear gradient of ammonium sulphate (2–0 M) in 50 mM sodiumhosphate buffer pH 7. Active fractions were collected and dialyzed againsta-acetate buffer.

.8. Native molecular mass determination

Pooled fractions from HIC column were loaded on gel chromatogra-hy (Superose) column for native molecular mass determination and elutedith 100 mM Tris–HCl buffer pH 7 having 0.15 M NaCl. The flow rateas 0.5 ml min−1. Active fractions were collected and dialyzed against Na-

cetate buffer. Different molecular weight markers (carbonic anhydrase, 29 kDa;

5 obial

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60 M. Riaz et al. / Enzyme and Micr

hicken egg albumin, 45 kDa; bovine serum albumin, 66 kDa; alkaline phos-hatase, 100 kDa and alcohol dehydrogenase, 150 kDa) were used for standardurve formation.

.9. Sub-unit molecular mass determination

Purity of the purified enzyme and its sub-unit molecular mass was deter-ined by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis

SDS-PAGE) as described by previously [20]. Protein markers of Fermentasith molecular mass ranging from 10 to 200 kDa were run as standard. Theel containing different molecular weight markers was stained with Coomassierilliant blue R-250 solution.

.10. Characterization of glucoamylase

Physiochemical, kinetic and thermodynamic properties of glucoamylaserom Humicola sp. were determined as mentioned below.

.10.1. Optimum temperature, activation energy and temperature quotientQ10)

Optimum temperature and activation energy (Ea) of glucoamylase wereetermined by incubating appropriate amount of the enzyme with 1% solubletarch at various temperatures ranging from 25 to 65 ◦C in 50 mM Na-acetateuffer for 40 min at pH 5. Ea was calculated by using Arrhenius plot as describedarlier [21]. The effect of temperature on the rate of reaction was expressedn terms of temperature quotient (Q10), which is the factor by which the ratencreases due to a raise in the temperature by 10 ◦C. Q10 was calculated byearranging the equation given by Dixon and Webb [22]:

10 = antilogε

(E × 10

RT 2

)(1)

here E = Ea = activation energy.

.10.2. Optimum pHThe optimum pH was determined by measuring activity at 55 ◦C using

arious buffers: glutamic acid/HCl (pH 2–2.9), Na-acetate/acetic acid (pH.2–5.3), MES/KOH (pH 5.6–6.5), MOPS/KOH (pH 6.8–7.4), HEPES/KOHpH 7.7–8.3), glycine/NaOH (pH 8.6–9.8) and CAPSO/NaOH (pH 10.1–11.0).he ionizable groups of essential active site residues (pKa1 and pKa2) involved

n the catalysis were determined as described by Dixon and Webb [22].

.10.3. Kinetics of starch hydrolysisThe kinetic constants (Vmax, Km, kcat and kcat/Km) were determined by incu-

ating fixed amount of glucoamylase with varied concentrations of soluble starchs a substrate ranging from 0.005 to 0.075% (w/v) at 55 ◦C, pH 5.0 as describedreviously [23].

.10.4. Thermodynamics of starch hydrolysisThe thermodynamic parameters for substrate hydrolysis were calculated by

earranging the Eyring’s absolute rate equation derived from the transition stateheory [24]:

cat =(

kbT

h

)e(−�H∗/RT ) e(�S∗/R) (2)

here kb is the Boltzmann’s constant (R/N) = 1.38 × 10−23 J K−1, T the absoluteemperature (K), h the Planck’s constant = 6.626 × 10−34 J s, N the Avogadro’sumber = 6.02 × 1023 mol−1, R the gas constant = 8.314 J K−1 mol−1, �H* thenthalpy of activation and �S* is the entropy of activation:

H∗ = Ea − RT (3)

G∗ (free energy of activation) = −RT ln(

kcath

kbT

)(4)

S∗ = �H∗ − �G∗

T(5)

ve

Technology 41 (2007) 558–564

The free energy of substrate binding and transition state formation wasalculated using the following derivations:

G∗E–S (free energy of substrate binding) = −RT ln Ka (6)

here Ka = 1/Km:

G∗E–T (free energy for transition state formation) = −RT ln

(kcat

Km

)(7)

.10.5. Thermodynamics of enzyme stabilityThermal inactivation of enzyme was determined by incubating the enzyme

olution in 10 mM Tris–HCl buffer (pH 7) at various temperatures (45, 48, 50,3 and 55 ◦C) in the absence of substrate. Aliquots were withdrawn at differentime intervals, cooled on ice for 30 min and assayed for glucoamylase activityt 40 ◦C. The data was fitted to first-order plot and inactivation rate constantsKd) were determined as described previously [25,26]. The energy of activationor irreversible inactivation Ea(d) was determined by Arrhenius plot (Fig. 6).

Thermodynamics of irreversible inactivation of the glucoamylase was deter-ined by rearranging the Eyring’s absolute rate equation derived from the

ransition state theory [24]:

d =(

kbT

h

)e(−�H∗/RT ) e(�S∗/R) (8)

H*, �G*, and �S* of irreversible inactivation were calculated by applyingqs. (3)–(5) with the modifications that in Eq. (3). Ea(d) was used instead of Ea

nd in Eq. (4) Kd was used in place of kcat.

. Results and discussion

.1. Purification of glucoamylase

The extra-cellular glucoamylase from locally isolated Humi-ola sp. grown under submerged growth conditions was purifiedp to homogeneity level. The purification procedure is describedn Section 2. The purification of glucoamylase is summarizedn Table 1 and its purification to apparent homogeneity on SDS-AGE stained by Comassie Blue R-250 was completed in threeteps (Fig. 1). The overall purification was 39-fold with 33%ecovery. The specific activity was enhanced to 406.9 U mg−1

rotein. Glucoamylase having similar recovery (31.8%) but6.2-fold purification was obtained from Fusarium solani inve-step purification procedure [27].

.2. Native and sub-unit molecular mass

The native molecular mass of glucoamylase determined byel filtration chromatography on Pharmacia’s FPLC unit andub-unit molecular mass from SDS-PAGE was 72.75 kDa, whichrovided an evidence for monomeric nature of the glucoamylase.ingle band on SDS-PAGE confirmed its purity to homogeneity

evel (Fig. 1). In contrast, several polymorphic or isozymic formsf glucoamylases ranging from 2 to 5 have been reported in lit-rature [28,29]. The molecular mass (72.75 kDa) of the enzymes close to the values reported 71 kDa from Hormoconis resinae30] and 75 kDa from Scytalidium thermophilum [31].

.3. Effect of pH

The hydrolysis of soluble starch by purified glucoamylase atarious pHs ranging from 2 to 11 at 40 ◦C was determined. Thenzyme exhibited optimum activity in a pH range of 3.5–5.9

M. Riaz et al. / Enzyme and Microbial Technology 41 (2007) 558–564 561

Table 1Purification of glucoamylase from Humicola sp.

Step Total protein (mg) Total units (U) Specific activity (U mg−1) Purification factor % recovery

Crude enzyme 117.80 1230 10.44 1.00 100(NH4)2SO4 precipitation 49.10 905 18.43 1.77 74H 695 304.80 29.20 57H 411 406.90 39 33

A

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FL

F

iload column chromatography 2.28ydrophobic interaction column chromatography 1.01

ll quoted values were taken after dialysis against distilled water.

ith the maximum activity at pH 4.7 (Fig. 2). Activity of thenzyme was inhibited rapidly below and above optimum pHange. The pKa1 and pKa2 values of acidic and basic limbs of thective site residues determined by Dixon plot were 3.65 and 6.3,espectively. During saccharification, the pH of the liquid slurrys adjusted to 4.2–4.5, to move it closer to pH optimum for A.iger GA. Using enzymes that function over a wider range wouldliminate this adjustment step. Most of the fungal glucoamylasesave optimum pH values ranging from 4 to 6 [6,32,33].

.4. Temperature optima, activation energy andemperature quotient (Q10)

Optimum temperature of the purified glucoamylase from

umicola sp. for hydrolysis of soluble starch was found to be5 ◦C. Glucoamylase of exactly similar optimum temperatureas been characterized [34]. An Arrhenius plot (Fig. 3) clearlyuggested that beyond transition point at 55 ◦C Vmax declined,

ig. 1. Ten percent SDS-PAGE analysis of purified Humicola sp. glucoamylase.ane 1: molecular markers and lane 2: purified glucoamylase.

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3

s

Ffse

ig. 2. Dixon plot of glucoamylase at 40 ◦C for the determination of pKas ofctive site residues that control Vmax. Data presented are average values ± S.D.f n = 3 experiments.

ndicating the inactivation of enzyme at higher temperatures. Eaf the enzyme for soluble starch hydrolysis was 21.09 kJ mol−1,hich is significantly lower than required by glucoamylases

rom Thermomyces lanuginosus (61 kJ mol−1) [35] and fromrachniotus sp. (57 kJ mol−1) [36]. The temperature quotient for

he enzyme was 1.01, which was near to that of Thermomucornicae-sedudaticae; 1.35 [37].

.5. Kinetic constants of starch hydrolysis

The Km and Vmax values of glucoamylase from Humicolap. determined through Lineweaver–Burk plot for the hydroly-

ig. 3. Arrhenius plot for the determination of activation energy (Ea)or the hydrolysis of soluble starch, Ea = −slope × R, where R (gas con-tant) = 8.314 J K−1 mol−1. Data presented are average values ± S.D. of n = 3xperiments.

562 M. Riaz et al. / Enzyme and Microbial Technology 41 (2007) 558–564

Fig. 4. Lineweaver–Burk plot of glucoamylase from Humicola sp. All sampleswere assayed in 50 mM sodium acetate buffer, pH 5, at 40 ◦C containing variableato

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3

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mounts of soluble starch. The intercept on the y-axis corresponds to 1/Vmax andhe intercept on the x-axis to −1/Km. Data presented are average values ± S.D.f n = 3 experiments.

is of soluble starch at 55 ◦C were 0.26 mg soluble starch ml−1

nd 56.63 U mg−1 protein, respectively (Fig. 4). The kcat was9 s−1 and specificity constant (kcat/Km) was 265. The Km, kcatnd kcat/Km recently reported for F. solani glucoamylase at 55 ◦Cere 1.6 mg ml−1, 217 s−1 and 136, respectively [38]. The speci-city constant of glucoamylase from Humicola sp. was aboutouble than that of F. solani, which showed higher affinity ofhe enzyme towards starch hydrolysis.

.6. Thermodynamics of starch hydrolysis

The enthalpy of activation (�H*), Gibbs free energy�G*) and entropy of activation (�S*) for soluble starchydrolysis by glucoamylase of Humicola sp. were calculateds 18.36 kJ mol−1, 69.06 kJ mol−1 and −154.56 J mol−1 K−1,espectively. The �H*, �G* and �S* for starch hydrolysis bylucoamylase from Arachniotus citrinus previously quoted were5.64 kJ mol−1, 75 kJ mol−1 and −124 J mol−1 K−1, respec-ively [34]. The lower enthalpy value of glucoamylase fromumicola sp. showed that the formation of transition state or

ctivated complex between enzyme–substrate was very efficient.oreover, lower �G* value suggested that the conversion of its

ransition complex into products was more spontaneous thanhat of A. citrinus. The feasibility and extent of a chemicaleaction is best determined by measuring change in Gibbs freenergy (�G*) for substrate hydrolysis, i.e. the conversion of ES-omplex into products. The lower is the free energy change theore feasible is the reaction, i.e. the conversion of the reactant

o product will be spontaneous. The entropy was slightly lower,hich explained that the transition complex had lesser disorder.The free energy for activation of substrate binding (�G∗

E–S)nd the free energy for the formation of activation complex�G∗

E–T) were −3.71 and −26.40 kJ mol−1, respectively, whilehe values previously determined for glucoamylase from A. cit-

inus were −2.95 and −17.7 kJ mol−1, respectively [39]. Thealues of �G∗

E–S and �G∗E–T again confirmed the greater affin-

ty of enzyme towards soluble starch for hydrolysis and itspontaneous conversion into glucose.

ow5s

oamylase from Humicola sp. The enzyme solution was incubated at variousemperatures (45–55 ◦C) in 10 mM Tris–HCl buffer (pH 7). Data presented areverage values ± S.D. of n = 3 experiments.

.7. Thermodynamics of enzyme stability

Thermostability represents the capability of an enzymeolecule to resist against thermal unfolding in the absence of

ubstrate, while thermophilicity is the ability of an enzyme toork at elevated temperatures in the presence of substrate [40].onomeric glucoamylase from Humicola sp. was incubated at

emperatures ranging from 45 to 55 ◦C. Pseudo-first-order plotsere applied to determine the extent of thermal inactivation

Fig. 5). The enzyme was very stable at 45 ◦C and exhibitedalf life of 30.2 h. The half life (t1/2) of an enzyme, at a givenemperature, is the time it takes for the activity to reduce to aalf of its original/initial activity. At higher temperatures halfife decreased sharply, i.e. 213.5 min at 48 ◦C and just 10.5 mint 55 ◦C.

Thermal inactivation of enzymes occurred in two steps41,42] as shown below:

�U∗ → I

here N is the native enzyme, U* the unfolded inactive enzyme,hich could be reversibly refolded upon cooling and I is the

nactivated enzyme formed after prolonged exposure to heat andherefore cannot be recovered upon cooling. The thermal inac-ivation of enzymes is accompanied by the disruption of lots ofon-covalent linkages including hydrophobic interactions withoncomitant increase in the enthalpy of activation (�H*) [43].he opening up of the enzyme structure is accompanied by a

ot of increase in the disorder or entropy of activation (�S*)44,45].

Activation energy for irreversible inactivation ‘Ea(d)’ of thelucoamylase was determined by applying Arrhenius plot andibbs free energy (�G*) for activation of thermal unfolding of

nzyme was 109.66 kJ mol−1 (Fig. 6). With an increase in tem-erature, a decrease in free energy was observed. The enthalpy

f activation of thermal unfolding (�H*) of the enzyme at 45 ◦Cas 433.34 kJ mol−1. Its value remained almost same up to5 ◦C. The entropy of activation (�S*) for unfolding of tran-ition state of the glucoamylase was 1.018 kJ mol−1 K−1, which

M. Riaz et al. / Enzyme and Microbial Technology 41 (2007) 558–564 563

Table 2Kinetic and thermodynamic parameters for irreversible inactivation of glucoamylase from Humicola sp.

Temperature (K) Kd (min−1) t1/2 (min−1) �H* (kJ mol−1) �G* (kJ mol−1) �S* (kJ mol−1 K−1)

318 0.000382 1814 433.34 109.68 1.018321 0.00324 213 433.32 105.04 1.023323 0.0118 58 433.30 102.24 1.025326 0.0297 23 433.28 100.71 1.020328 0.0658 10 433

Kd (first-order rate constant for inactivation) was taken from Fig. 5, t1/2 = half life = 0Section 2.

Fig. 6. Arrhenius plot to calculate activation energy ‘Ea(d)’ for irreversibletrs

wdtILiw4

4

sestp

A

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R

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[

[

[

[

[

[

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hermal inactivation/denaturation of glucoamylase from Humicola sp. usingelationship ln Kd = −Ea/RT, where R (gas constant) = 8.314 kJ mol−1. Data pre-ented are average values ± S.D. of n = 3 experiments.

as slightly increased at 50 ◦C (Table 2). But the increase orecrease in entropy was not significant. At higher tempera-ures decrease in �G* made the enzyme thermally unstable.t was investigated previously that glucoamylase produced byactobacillus amylovorus ATCC 33621 was thermolabile and

ts activity was decreased after 10 min exposure at 60 ◦C [46]. Itas also reported that the glucoamylase from R. oryzae mutantU2 was stable only for an hour at temperature up to 40 ◦C [47].

. Conclusions

Humicola sp. glucoamylase was very efficient for solubletarch hydrolysis. Due to lower values of Km and �G*, thenzyme proved to have greater affinity to catalyze solubletarch hydrolysis. Kinetic and thermodynamic parameters ofhe enzyme suggested that it may be used commercially for theroduction of glucose in starch processing industry.

cknowledgements

The work presented is a part of Ph.D. studies of Mr. Muham-ad Riaz. The project was partly funded by Higher Educationommission (HEC), Pakistan under the Indigenous Scholar-

hip scheme and Pakistan Atomic Energy Commission. The

irst Fungal Culture Bank at University of The Punjab isindly acknowledged for the physicochemical identification ofultures. Technical assistance of Mr. Ghulam Ali Waseer isppreciated.

[

.26 99.18 1.018

.693/Kd, all other parameters were calculated from Eqs. (3)–(5) mentioned in

eferences

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[2] Meager MM, Nikolov ZL, Reilly PJ. Subsite mapping of Aspergillus nigerglucoamylase I and II. Biotechnol Bioeng 1989;34:681–8.

[3] Dubey AK, Suresh C, Kavitha R, Karanth NG, Kumar US. Evidencethat the glucoamylases and alpha-amylase secreted by Aspergillus nigerare proteolytically processed products of a precursor enzyme. FEBS Lett2000;471:251–5.

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