research article solid state fermentation of a raw starch ...molecular weight of puri ed enzyme was...
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Research ArticleSolid State Fermentation of a Raw Starch DigestingAlkaline Alpha-Amylase from Bacillus licheniformisRT7PE1 and Its Characteristics
Romana Tabassum,1 Shazia Khaliq,1 Muhammad Ibrahim Rajoka,2 and Foster Agblevor3
1 National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Faisalabad, Pakistan2Department of Bioinformatics, G C University, Faisalabad, Pakistan3 Biological Engineering Department, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105, USA
Correspondence should be addressed to Romana Tabassum; [email protected]
Received 30 August 2013; Revised 25 November 2013; Accepted 26 November 2013; Published 21 January 2014
Academic Editor: Goetz Laible
Copyright ยฉ 2014 Romana Tabassum et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
The thermodynamic and kinetic properties of solids state raw starch digesting alpha amylase from newly isolated Bacilluslicheniformis RT7PE1 strain were studied.The kinetic values๐๐, ๐๐/๐ , ๐๐/๐, and ๐๐ were proved to be best with 15% wheat bran.Themolecular weight of purified enzyme was 112 kDa. The apparent ๐พ๐ and ๐max values for starch were 3.4mg mL
โ1 and 19.5 IUmgโ1protein, respectively. The optimum temperature and pH for ๐ผ-amylase were 55โC, 9.8. The half-life of enzyme at 95โC was 17h. Theactivation and denaturation activation energies were 45.2 and 41.2 kJ molโ1, respectively. Both enthalpies (ฮ๐ปโ) and entropies ofactivation (ฮ๐โ) for denaturation of ๐ผ-amylase were lower than those reported for other thermostable ๐ผ-amylases.
1. Introduction
Starch is an excellent carbon source in nature and majorstorage product of many economically important crops. Alarge-scale starch processing industry has emerged in the lastcentury [1]. Five groups of enzymes play a key role in thehydrolysis of starch. These enzymes comprise about 30% ofthe worldโs enzyme production. Alpha amylases (endo-1,4-a-D-glucan glucanohydrolase, E.C.3.2.1.1) are extracellularendo enzymes that randomly cleave the 1,4-a linkage betweenadjacent glucose units in the linear amylose chain andultimately generate glucose, maltose, and maltotriose units[2]. Alpha amylases derived from microbial sources havegreat success and replaced the chemical hydrolysis of starch instarch processing industries. Alpha amylases have a numberof applications, including liquefaction of starch in the alcohol,brewing, and sugar industries and in the textile industry fordesizing of fabrics [3]. They also have applications in laundryand detergents or as antisalting agents and baking such asbread making [3], since thermostability and alkaline charac-teristics are important features for industrial applications ofamylase isolated from alkalophilic organisms [4].
The microbial ๐ผ-amylases for industrial processes arederived mainly from Bacillus subtilis, Bacillus amylolique-faciens, and Bacillus licheniformis asthey are capable ofsecreting amylases into the culture supernatant in submerged[5]. The microbial production of alpha-amylase is greatlyinfluenced by the composition of the medium culture andenvironmental and growth kinetic parameters [5]. However,solid state fermentation provides an interesting alternative forconcentrated enzyme production and less purification costs[6]. This work reports the solid state production, kinetic andthermodynamic properties of raw starch-digesting alkalinealpha-amylase from an indigenous culture of B. licheniformisRTPE1 which possessed potentially interesting propertiesfrom ๐ผ-amylases already described.
2. Materials and Methods
2.1. Microorganism and Production Medium. IndigenousBacillus licheniformis RTPE-1strain was maintained inNIBGE stock culture collection and 16S rRNA accessionnumber EF644418 submitted at NCBI. The Bacillus
Hindawi Publishing CorporationBiotechnology Research InternationalVolume 2014, Article ID 495384, 8 pageshttp://dx.doi.org/10.1155/2014/495384
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licheniformis RTPE1 was maintained on maize starch(1%W/V) plates containing basal salts medium and 2.5%agar. The basal salt medium comprised of (g/Lโ1) K2HPO41.0, (NH4)2 PO4 1.0, MgCl2 0.5, yeast extract 10.0, andmaize starch 10. For liquid media preparation, all the mediacomponents were added and mixed one by one in one literconical flask containing 200mL distilled water. The pH ofthe medium was adjusted to 7.0 with 1N HCl/NaOH. Themedium was autoclaved at 20 p.s.i, 120โC for 20min.
2.2. Preparation of Inoculums. The basal salt medium con-taining maize starch (1%W/V) was dispensed in 50mLquantity into 250mL Erlenmeyer flasks. A single colonyfrom fresh agar culture plate was transferred into the abovemedium and incubated at 37โC for 24 h on an orbital shaker(Gallenkamp, UK, 150 rpm).
2.3. Growth of Organism in Solid State Fermentation (SSF).The SSF studies were carried out in one-litre Erlenmeyerflasks containing different concentrations of wheat bran,maize bran, and maize starch. Each carbon source wasmoistened with 25mL basal salt solution. The flasks wereautoclaved for 20min and cooled. For time course studythe flasks were inoculated with 5mL of fresh inoculums(5mg cell mass mLโ1) and incubated at 37โC for 7 days. Theincubator was humidified by sterile water. For the extractionof extracellular ๐ผ-amylase from solid state fermentationexperiments, after desired intervals (24, 48, 72, 96, 120, 144,and 168 h), the flasks were taken out in triplicate and theenzymewas extracted (three washings) with 100mL of 10molphosphate buffer (pH 7.0) after vigorous shaking on an orbitalshaker (150 rpm, 4โC).The extracted enzyme was centrifuged(at 15,000รg for 10min at 4โC) to get clear supernatantfor enzyme assay. All the experiments were run in threereplicates.
2.4. Assay for Alpha-Amylase Activity. The activity of ๐ผ-amylase was measured on the basis of liberated reducingsugars [7] using 3,5-dinitrosalicylic acid (DNS) using starchas the substrate after 10min incubation. One unit of ๐ผ-amylase activity was defined as the amount of enzyme, whichreleased 1 ๐mol of maltose or glucose equivalent per minunder the assay conditions. Glucose was measured usingglucose oxidase kit.
2.5. ProteinDetermination. Theprotein in the enzyme prepa-ration was quantified by the Bradford method [8] usingbovine serum albumin as the standard.
2.6. Purification of ๐ผ-Amylase. All purification steps wereconducted at room temperature. Enzyme extract from above(1000mL) was purified by a combination of ammonium sul-phate precipitation, ion-exchange, and gel filtration columnchromatography as described previously [9] with 20-foldincrease in specific activity (138 to 2769U (mgโ1 protein)with30% recovery).
2.7. pH Optima Studies. The effect of pH on the stability of ๐ผ-amylase was determined by assay of ๐ผ-amylase activity afterincubating the enzyme in the reaction system of different pHvalues at 50โC for 1 h [10].
2.8. Effect of Substrate Concentration. ๐ผ-Amylase was assayedin 100mM phosphate buffer of pH 7, with variable amounts(0.02%โ1%) of soluble starch solution. The data was plottedaccording to Line-Weaver Burk plot as described [11].
2.9. Effect of Activators and Inhibitors on ๐ผ-Amylase Activity.The enzyme solutions were incubated for 10min along withactivators and inhibitors with the final concentration of 1mMat 55โC and then assayed in the presence of metal ions for๐ผ-amylase activity. The various activators or inhibitors usedwere HgCl2, CaCl2, CuSO4, MgCl2, ZnCl2, FeSO4, CaCl2,MnCl2, SDS, and EDTA.
2.10. SDS-Denaturing-Renaturing Polyacrylamide Gel Elec-trophoresis (SDS-DR-PAGE). Subunit molecular weight of ๐ผ-amylase was determined by subjecting crude and purified ๐ผ-amylase to 10% SDS-PAGE using Hoeffer small apparatus.After electrophoresis, the gel was incubated for 30min in100mmol potassium phosphate buffer (pH 7.5) containing20% isopropanol for the removal of SDS. After removingSDS, the part of the gel containing different molecular weightmarkers was stained over night with Comassie ๐ -250. Thegel was destained with a solution of methanol, acetic acidand water with a ratio of 9 : 2 : 9, respectively. The part of gelcontaining ๐ผ-amylase was transferred to potassium iodidesolution (Gramโs iodine) for activity staining [11].
2.11. Half-Life. Purified ๐ผ-amylase was redissolved in100mmol phosphate buffer (pH 7) and assayed forthermostability. Optimum temperature of ๐ผ-amylasewas determined in 100mM potassium phosphate buffer (pH7.0) at selected temperatures (50, 55 60, 65, 70, 75, 80, 85,90, 95 and 100โC) of reaction system [11, 12]. ๐ผ-Amylase wasexamined by incubating the enzyme in the above buffer, atcertain temperatures (50โ90โC) and the enzyme sampleswere taken after 15, 30, 60, 120, and 180min of incubation forthe assay of enzyme activity.
2.12. Thermodynamics of Enzyme. Arrhenius relationshipwas used to calculate the activation energy (๐ธ๐) requiredby the enzyme to hydrolyze starch as described earlier[11]. Thermal inactivation of the enzyme was determinedby incubating the enzyme solutions in above buffer at aparticular temperature. Aliquots were withdrawn at differenttimes, cooled on ice, and assayed for enzyme activity at55โC as described above. This procedure was repeated atdifferent temperatures ranging from 45 to 100โC. The datawere fitted to first-order plots and analyzed. The first-orderrate constants (๐๐) were determined by linear regression ofln (V) versus time of incubation (t). The thermodynamicdata were calculated by rearranging the Eyring absolute rate
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equation to study the overall thermodynamic parameters inthe range 45โ95โC:
๐พ๐=๐โ ๐๐ต/โ๐ฮ๐โ
๐
eฮ๐ปโ
๐ โ ๐,
ln(๐๐
๐) = ln (๐๐ต
โ) +
ฮ๐โ
๐ โ
ฮ๐ปโ
๐ โ 1
๐,
(1)
where ๐๐, ๐, ๐๐ต, โ, ฮ๐โ, ฮ๐ปโ, and ๐ are specific reaction
velocity, absolute temperature, Boltzmann constant, enthalpyof activation, and gas constant, respectively.
3. Results and Discussion
Various factors including particle size, inoculum density,moisture content, and enzyme extraction parameters wereemployed when wheat bran, maize starch, and maize branwere used. For comparison of data to make extrapolations,IUg-1 cells (๐๐/๐ฅ), IUg-1 substrate consumed (๐๐/๐ ), volu-metric productivityโs (๐๐), and specific productivity (๐๐) ofthe enzyme have been presented in Tables 1(a) and 1(b).Wheat bran (15%) proved to be the best carbon source tosupport maximum values of all kinetic parameters relatedto product formation. Maximum values of ๐ผ-amylase ๐๐(1302 IU Lโ1 h), ๐๐/๐ฅ (1333 IU
gโ1 cells), ๐๐/๐ (22588 IUgโ1
substrate consumed), and ๐๐ (357 IUgโ1 cells hโ1 specific)
were also improved (Table 1(a)). Growth yield coefficient(๐๐ฅ/๐ ), volumetric rate of substrate utilization (๐๐ ), specificrate of substrate utilization (๐๐ ), specific growth rate (๐), andvolumetric productivity of extracellular protein (๐๐๐) werealso increased with 15% wheat bran (Table 1(b)).
The suitability of a particular substrate in a SSF process forthe production of bacterial amylases appeared to be governedby the physicochemical requirement of the microorganismsused.Theuniversal suitability ofwheat branmay be due to thefact that it contains sufficient nutrients and is able to remainloose even in moist conditions, thus providing a large surfacearea. When compared to earlier studies in SSF [13], enzymeproduction was enhanced up to several in SSF and a 2.4-foldimprovement in specific productivity was noted comparedwith submerged fermentation.
The novel aspect of the study was that spore formingindigenous Bacillus licheniformis RTPE1 strain producedmaximum amount of extracellular ๐ผ-amylase when growthrate declined principally in the stationary phase (see repre-sentative Figure 1(a)), though maximum enzyme productionwas observed in the fermentation medium producing max-imum amounts of biomass. In the earlier findings, mostly๐ผ-amylase production was growth associated and highestenzyme activities were obtained in the exponential phase andeven in the early stage of growth phase [5]. Very few findingsrevealed that the production of ๐ผ-amylase is nongrowthassociated. Further enhancement in the catalytically activeenzyme can be obtained by exploiting nitrogen sources andmaintenance of proper aeration in the fermentation vessel.
3.1. SDS-Polyacrylamide Gel Electrophoresis of the Purified๐ผ-Amylase of Indigenous Bacillus licheniformis RTPE1 Strain.Purified ๐ผ-amylase was subjected to SDS-PAGE as describedin materials and methods. A single band of purified ๐ผ-amylase was observed in lane 1 (Figure 1(b)A). It was foundthat the molecular weight of ๐ผ-amylase was 112 kDa andenzyme was active in situ and showed zone of clearanceeven in crude samples (Figure 1(b)B). Purified ๐ผ-amylase hasbeen shown to be homogenous by SDS-PAGE. It has highermolecular weight than most of the ๐ผ-amylases from Bacillussp (40โ140 kDa) [15].
3.2. Alkaline Nature of the Indigenous Bacillus lichenformisRT 7PE1 ๐ผ-Amylase. Studies of alkalophilic microorganismshad resulted in the discovery of extra-cellular enzymes thatare characterized by maximum pH for activity and stabilityoccurring on the alkaline side. Although various extra-cellular enzymes of alkalophilic microorganism have beendescribed and only a few are alkaline amylases [16]. Maxi-mum activity was observed at pH 9.0 and 60โC (Figures 2(a)and 2(b)), which is very suitable for industrial use. In previousstudy, the effect of temperature and pH on the activity of ๐ผ-amylase, produced by Bacillus sp. KR-8104 in a solid statefermentation system, was optimized [17]. Comparing theresults obtained from the optimization of crude ๐ผ-amylaseactivity in solid state and submerged fermentation systemsrevealed some differences in pH and temperature optima formaximizing the crude enzyme activity.
3.3. Effect of Metal Ions and Effectors on ๐ผ-Amylase ActivityandThermostability. The thermostability of purified enzymewas enhanced in the presence of 5mM Ca2+ at 90โC. Theenzyme activity was strongly inhibited by Hg2+, Pb2+, Zn2+,Cu2+, EDTA, and SDS (Table 2) [12].
But in the presence of 5mMCa2+ enzyme retained 97%of its activity even after incubation of 180min at 90โC. Ingeneral high concentrations of bothmonovalent and divalentsalts were inhibitory to the reaction.The inhibition dependedupon the nature of the salt, either due to anionic strengtheffect or specific cation effect [10].
3.4. Liquefaction of Starch. Liquefaction of the 10% maizestarch was done with purified ๐ผ-amylase (200 IU gโ1 starch)from indigenous Bacillus licheniformis RT7PE1 strain. Theenzyme gave 100% dextrose equivalent (DE) values from 10%maize starch hydrolyzed completely after 360min of incu-bation. Starch at 30% (w/v) was not completely hydrolyzedto glucose (Figure 2(c)). High performance liquid chro-matographic studies proved that the starch hydrolysis majorproduct was maltose and oligosaccharides. The productionof sugars from starch sources is an industry that exists in itspresent form due to the application of industrial enzymologyto solve process related problems. As the industry matures,the demand for more efficient enzymes leading to higherquality products and lower production costs for the starchprocessing will increase [18].
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Table 1: (a) Comparative fermentation kinetic parametersโ of B. licheniformis RTPE1 for ๐ผ-amylase formation following growth ondifferent substrates in solid state fermentation under optimized fermentation conditions. (b) Comparative fermentation kinetic parametersof B. licheniformis RTPE1 for ๐ผ-amylase formation following growth on different substrates in solid state fermentation under optimizedfermentation conditions.
(a)
Carbon source ๐๐IU Lโ1 hโ1
๐๐/๐
IU gโ1substrate utilized
๐๐/๐
IU gโ1cells
๐๐
IU gโ1 cells hโ1specific productivity
5% wheat bran 535 ยฑ 25 1500 ยฑ 10 11904 ยฑ 25 357 ยฑ 2510% wheat bran 1262 ยฑ 35 1550 ยฑ 12 19375 ยฑ 30 775 ยฑ 2815% wheat bran 1302 ยฑ 29 1333 ยฑ 15 22588 ยฑ 35 1242 ยฑ 3010% maize starch 427 ยฑ 41 1260 ยฑ 14 10500 ยฑ 26 315 ยฑ 215% maize bran 867 ยฑ 30 1420 ยฑ 12 18933 ยฑ 29 719 ยฑ 1210% maize bran 990 ยฑ 45 1100 ยฑ 13 16500 ยฑ 31 858 ยฑ 2315% maize bran 851 ยฑ 48 1450 ยฑ 14 17682 ยฑ 32 725 ยฑ 25Each value is a mean of three replicates. (ยฑ) stands for standard deviation among replicates. โ๐๐ = IU l
โ1 hโ1, ๐๐/๐ = IU gโ1 substrate utilized, ๐๐/๐ = IU g
โ1
cell, and ๐๐ = specific productivity = IU gโ1 cells hโ1 and were determined as described previously [14].
(b)
Carbon source Biomass g ๐๐ฅ/๐ ๐๐ ๐๐ ๐ QP๐g DW/Lh g/g g/L/h g/gh (hโ1) g/L/h
5% wheat bran 0.079 0.126 0.281 0.238 0.0300 0.15310% wheat bran 0.084 0.080 0.351 0.560 0.040 0.27115% wheat bran 0.095 0.062 0.554 0.887 0.055 0.30010% maize starch 0.053 0.082 0.600 0.500 0.041 0.3155% maize bran 0.052 0.120 0.285 0.250 0.030 0.14510% maize bran 0.063 0.075 0.487 0.506 0.038 0.26015% maize bran 0.0635 0.056 0.650 0.092 0.050 0.295๐๐ฅ/๐ : growth yield coefficient,๐๐ : volumetric rate of substrate utilization, ๐๐ : specific rate of substrate utilization, ๐: specific growth rate, and QP๐: volumetricproductivity of extracellular protein. Each value is a mean of three replicates. (ยฑ) stands for standard deviation among replicates.
25
20
15
10
5
00 24 48 72 96 120 144 168
Time (h)
BLA
U/g
ram
of w
heat
bra
n
(a)
205 kDa
116 kDa97kDa
84kDa66kDa55kDa
45kDa36kDa
29kDa
24kDa
(A) (B)
Purified marker Activity staining
(b)
Figure 1: (a) Kinetics of alpha-amylase production in solid state fermentation using wheat bran. The initial pH of the medium was 7.0 andtemperature 37โC. I: alpha-amylase, ฮ : ๐ (cell mass), and โป : ๐ (substrate) present in the fermentation medium. Error bars show standarddeviation among three replicates. (b) Purification profile of enzyme on SDS-PAGE: A, lane 1: purified enzyme; lane 2: molecular weightmarkers; B, lane 1: activity staining of purified enzyme.
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110
100
90
80
70
60
50
40
30
20
105 6 7 8 9 10 11 12
pH
Rela
tive a
ctiv
ity (%
)
(a)
110
100
90
80
70
60
50
40
30
20
1009080706050403020
10
0
Rela
tive a
ctiv
ity (%
)
Temperature (โC)
(b)
Time (min)0
060 1 420 480
44
22
DE
valu
es
(c)
Figure 2: (a) Effect of pHon enzyme activity, (b) effect of temperature on enzyme activity, and (c) liquefaction ofmaize starch used at differentconcentrations: 10% maize starch, โป 20% maize starch, and I 30% maize starch. Enzyme was used 200 IU per g substrate at 55โC.
Table 2: Effect of metal ions (used at 5mM) on enzyme activity.
Metal ions % Relative activityControl 100 ยฑ 3CaCI2โ 2H2O 115 ยฑ 4CoCl2โ 6H2O 23 ยฑ 1FeSO4โ 5H2O 20 ยฑ 3HgCI2 0 ยฑ 0KCI 67 ยฑ 3CuSO4 0 ยฑ 0MgSO4 97 ยฑ 4EDTA 30 ยฑ 2SDS 20 ยฑ 2Each value is a mean of three readings; ยฑ: stands for standard deviationamong replicates.
3.5. Effect of Starch Concentration on Catalytic Activity. Thereaction was dependent on the amount of starch present inthe reaction mixture. A Line-weaver Burk plot (Figure 3(a))of the data reveals a ๐พ๐ of 3.4mg starch mL
โ1. The ๐maxis 19.5 ๐molminโ1. So the amylase exhibited Michaelis-Menten-type kinetics. The kinetic parameters of ๐ผ-amylase
activation ๐max and ๐พ๐ that were 263 ๐molemgโ1 enzyme
minโ1 and 0.97mg/mL, respectively, were reported fromthermopile Bacillus sphaericus [14]. The ๐max and ๐พ๐ valuesfor soluble starch was found to be 4.11mg/min and 3.076mg,respectively, for ๐ผ-amylase isolated from B. amyloliquefacienspreviously reported by Gangadharan et al. [19].
3.6. Effect of Temperature on ๐ผ-Amylase Activity and Stability.Stability of the enzyme at higher temperature up to 100โCwasexamined, and the enzyme was optimally active at 55โ60โC(130 IUmLโ1) (Figure 3(b)) and showed 14% relative enzymeactivity at 100โC. The temperature-dependent properties ofthis enzyme show some similarity to Bacillus licheniformis ๐ผ-amylase (BLA) produced by recombinant E. coli which wasactive over a temperature range of 50โ80โC and had optimalactivity at 60โC [20].
3.7. Half-Life and Activation Energy. The protein midpointinactivation temperature (๐๐), activation energy, conforma-tional stability at elevated temperatures, activation parame-ters for catalytic activity, transition state formation energy,stability of the native state ensemble are potential indices
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0.50
0.40
0.30
0.20
0.10
0.00โ1.00 โ0.30 0.40 1.10 1.80 2.50
1/(S) (mg/mL)
1/V
(U/m
i/min
ร10โ1)
(a)
140
120
100
80
60
40
20
050 60 70 80 90 100
Temperature (โC)
Rela
tive a
ctiv
ity (%
)
(b)
5.00
4.50
4.00
3.50
3.00
2.50
2.000.25 0.27 0.29 0.31 0.33 0.35
1/T (1/K) (E โ 2)
ln(V
max
)
(c)
โ9.00
โ9.42
โ9.83
0.26 0.27 0.28 0.29 0.30 0.31
1/T (1/K) (E โ 2)
ln(kd/T
)
โ10.25
โ10.67
โ11.08
โ11.50
(d)
Figure 3: (a) Line weaver-Burk plot for calculation of๐พ๐ and๐max of enzyme. (b) Determination of protein midpoint (๐๐) for denaturation.Relative activity at each temperature was calculated as described in Materials and Methods and plotted against temperatures. ๐๐ is thattemperature at which held of enzyme is defolded. (c) Arrhenius plots for calculation of activation energy. (d) Arrhenius plots for calculationof activation enthalpy and entropy of alpha-amylase inactivation.
for thermostable biocatalysts. The ๐๐ value was 89โC
(Table 3(a)). For Bacillus licheniformis amylase (BLA) at pH7.0, the ๐๐ of 103
โC was obtained [20].Activation energy for catalysis of soluble starch was
45.2 kJmolโ1 (Table 3(a)). The activation energy for B.licheniformis CUMC305 enzyme was calculated as 5.1 ร105 J/mol. The investigated most thermostable ๐ผ-amylasesfrom Bacillus licheniformis enzymes exhibit activation ener-gies (๐ธ๐) between 208 and 364 kJmol
โ1 and pronounceddifferences in the corresponding unfolding rates. After theinflexion in temperature (Figure 3(c)), the enzyme becomesdenatured and shows less activity towards conversion ofsubstrate into product with decrease in activation energy(41.2 kJmolโ1) which infers thermostability of the testenzyme [21].
3.8. Kinetics of Starch Hydrolysis. The ratio ๐cat/๐พ๐ oftenreferred to as the โspecificity constantโ is a useful index forcomparing the relative rates of enzyme acting on alternative,
competing substrates. In present studies, ๐cat is 2395/minand the specificity constant ๐cat/๐พ๐ of this enzyme is 7068(Table 3(a)). The reported ๐cat for ๐ผ-amylase from B. amy-loliquefaciens is 2.26ร 103 sโ1 [22, 23].This could be due to theionized state of the carboxyl whose charges are positive onesand the substrate (full chair conformation of sugar residue)binds more strongly to the active site than the transition stateof the substrate (sofa form of the positively charged oxo-carbonium ion). It was found that the entropy of denaturation(ฮ๐โ) of alpha-amylase at 60โCwas significantly decreased ascompared with other enzymes indicating altered and morecompact conformation for this enzyme. This happens due tothe increase in the hydrophobicity around active site, as aresult of local conformational change due to substrate (starch)binding and is a common phenomenon.
3.9. Thermodynamics of Starch Hydrolysis by Alpha-Amylase.The organism-derived enzyme requires less free energy(ฮ๐บโ๐ธโ๐
) to form the transition state than that required by
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other organisms (Table 3(a)). Similarly, enzyme releases thehigher amount of transition state binding energy (ฮ๐บโ
๐ธโ๐) as
compared with those of others (Table 3(a)), signifying thatthe high catalytic efficiency of alpha-amylase is due tothe transition state stabilization. Accordingly, this enzymeshowed highest enzyme-substrate destabilization (ฮ๐บโ
๐ธโ๐),
whereas other enzymes showed least enzyme-substrate desta-bilization energy (Table 3(a)). The activation energy (๐ธ๐)profiles of the enzyme show that this alpha-amylase has lower๐ธ๐ values up to 90
โC.These studies provided insight into improvement in
enzyme production and the process of stabilization by thisstrain.The culture had altered the values of both entropy andenthalpy of activation for irreversible inactivation of enzymeas observed for thermostabilized enzymes [24]. Actuallythermal inactivation occurs in two steps as follows:
๐ โโ ๐ โ ๐ผ, (2)
where ๐ is the native, ๐ is the unfolded enzyme whichcould be reversibly refolded upon cooling, and ๐ผ is theinactivated enzyme formed after prolonged exposure to heatand therefore can not be recovered on cooling.
The thermal denaturation of enzymes is accompa-nied by the disruption of noncovalent linkages, includ-ing hydrophobic interactions, with concomitant increasein the enthalpy of activation [24]. The opening up ofthe enzyme structure is accompanied by an increasein the disorder, randomness or entropy of activation.The values of thermodynamic parameters were calcu-lated from Figure 3(d). The enzyme had 44.8 kJmolโ1 andโ155.6 Jโ molโ1 Kโ1 ฮ๐ปโ (enthalpy of deactivation) and ฮ๐โ(entropy of deactivation) values respectively. The valuesof ฮ๐โ and ฮ๐ปโ of ๐ฝ-glucosidase from a thermophilicculture of Aspergillus wentii [24] were 125 kJmolโ1 and65 Jโ molโ1 Kโ1, respectively. The values of BLA here aremarkedly lower, therefore, up to 95โC, the enzyme wasreasonably thermostable. These values are also significantlylower than those reported on thermostable mutant ofAspergillus awamori glucoamylase [24]. When enthalpy andentropy values for inactivation were calculated at eachtemperature, ฮ๐โ had again negative values (Table 3(b)).This suggested that there was negligible disorderness as wasthat of ๐ฝ-glucosidase from A. wentii or the transition state of๐ผ-amylase from Bacillus licheniformis was found to be moreordered as revealed by its negative ฮ๐โ (โ150 Jmolโ1 Kโ1) athigh temperature of 80โC [25].
4. Conclusion
From the present study it is apparent that production ofalpha amylase production by solid state fermentation from15% wheat bran was higher as compared to 15% gram branby newly isolated Bacillus licheniformis RT7PE1 strain. Thewheat branwas best and cheap source for production of alphaamylase. The production of enzyme in submerged fermen-tation is expensive as compared to solid state fermentation.The contents of synthetic media are very expensive and
Table 3: (a) Kinetic and thermodynamic properties of ๐ผ-amylasefrom Bacillus licheniformisRTPE1 strain. (b) Kinetic and thermody-namic parameters for irreversible thermal inactivation of ๐ผ-amylasefrom B. licheniformis RTPE1.
(a)
Parameters Values๐cat (min
โ1)a 2395๐พ๐ (%w/v) 0.34๐cat/๐พ๐ 7043๐ธ๐ (kJmol
โ1)b 45.2๐๐ (kDa) 124pH optimum 9.5Temperature optima 55๐๐ (Midpoint Temp) 89ฮ๐บโ (kJmolโ1)e โ164.8
ฮ๐ปโ (kJmolโ1)f 44.8
ฮ๐โ (Jmolโ1 Kโ1)g โ155.6
ฮ๐บโ
๐ธโ๐(kJmolโ1)h โ30.3
ฮ๐โ
๐ธโ๐(kJmolโ1)I โ2.86
aTurnover number (๐cat) = ๐max/[e], where e is ๐ผ-amylase concentration(0.00806๐mol). NA: not available.bActivation energies (๐ธ๐) determined as described previously [11].eฮ๐บโ (activation free energy of ๐ผ-amylase hydrolysis) = โ ๐ ๐. ln
(๐catโ h)/(๐พ๐ตโ ๐), where h is planck constant (6.63ร 10โ34 Js),๐พ๐ต is boltzmanconstant (1.38 ร 10โ23 JKโ1), and R is gas constant (8.314 JKโ1 molโ1).fฮ๐ป (activation enthalpy of starch hydrolysis) ๐ธ๐ โ RT.gฮ๐ (activation entropy of starch hydrolysis) = (ฮ๐ปโ โ ฮ๐บโ)/T.
iฮ๐บ๐ธโ๐ (free energy of transition state binding) = โ RT ln ๐cat/๐พ๐.Jฮ๐๐ธโ๐ (free energy of substrate binding) = โ RT ln๐พ๐, where๐พ๐ = 1/๐พ๐.
(b)
๐๐พ๐ ๐ก1/2 ฮ๐ป
โฮ๐บโ
ฮ๐โ
10โ3 hโ1 (h) kJmolโ1 kJmolโ1 Jmolโ1 Kโ1
323 4.4 158 42.51 115.76 โ226328 4.7 147 42.47 117.53 โ229333 6.8 102 42.43 118.29 โ230343 7.8 89 42.35 121.59 โ231353 8.3 83 42.27 125.04 โ234363 18.0 38 42.18 126.33 โ240368 41.0 17 42.18 127.12 โ231+๐พ๐ (first-order rate constant for inactivation)was calculated from relation-
ship,๐พ๐โ t = ln๐, where t is time of incubation and๐ is the reaction velocity.๐ก1/2: half-life of enzyme.ฮ๐ปโ (kJmolโ1) = ๐ธ๐ (45.2 kJmol
โ1) โ RT, where ๐ธ๐ is activation energy.ฮ๐บโ (kJmolโ1) = โ RT ln (๐๐โ h)/(๐๐ตโ T)ฮ๐โ is entropy of irreversible inactivation and was calculated from ฮ๐โ =ฮ๐ปโโ ฮ๐บโ/๐.
these contents might be replaced with more economicallyagricultural waste material for the reduction of cost of themedium. The use of agricultural wastes makes solid statefermentation an attractive alternative method. There is ongoing interest in the isolation of new bacterial strains toproduce amylases for new industrial applications, such asalkaline amylases for the detergent industry. This ๐ผ-amylasecan be used as a good model protein for investigation of the
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8 Biotechnology Research International
molecular basis of alkalophilicity of moderately thermostablealkaline enzymes.
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper.
Acknowledgments
The work described is part of the M.Phil, research of Ms.Shazia Khaliq.The authors do not have any conflict of interestfor financial, commercial, and professional to publish thispaper, supported by PAEC. The technical assistance of Mr.Khalid Yaqoob is appreciated.
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