Amylase Purification

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International Research Journal of Microbiology Vol. 2(3) pp. 096-103, March 2011Available online@ http://www.interesjournals.org/IRJMCopyright 2011 International Research Journals

Full Length Research Paper

Purification and characterization of

-amylase from anewly isolated AspergillusflavusF2Mbb

Nagwa M. Sidkey1; Maha A. Abo-Shadi2; Reham Balahmar3; Reham Sabry3; GhadeerBadrany3

1Botany & Microbiology Dept., Faculty of Science (Girls), Al-Azhar Univ., Egypt.

2Microbiology and Immunology Dept., Faculty of Pharmacy (Girls), Al-Azhar Univ., Egypt.

3Biology Dept., Faculty of Science (Girls), Taibah Univ., KSA.

Accepted 11 May 2011

This study reports the purification and characterization of -amylase from Aspergillus flavus,F2Mbb isolated previously from some enviro-agro-industrial wastes in Al-Madinah al-Munawarah,Saudi Arabia. The enzyme was purified to homogeneity using 60% ammonium sulfate precipitationand Sephadex G-200 gel filtration which resulted in 15.74% recovery and specific activity of 4348(units/mg protein/ml). SDS-PAGE showed a single band equal to molecular weight of about 56 kDa.The activity of the purified -amylase increased with increasing enzyme concentration andincubation time. The enzyme exhibited maximum activity at 30C and pH 6.4 with the optimumstarch concentration 15 mg/ml.

Key words: -amylases; Production; Purification; Characterization, Aspergillus flavus F2Mbb; SaudiArabia.

INTRODUCTION

-Amylases (EC3.2.1.1, 1,4-a-D-glucan-glucanohydro-lyase) are extracellular enzymes which hydrolyze starchinto a range of products such as glucose and maltose orspecific malto-oligosaccharide or mixed malto-oligosaccharides (Dey et al., 2002; Messaoud et al.,2004; Hashim et al., 2005).

Although -amylases can be derived from severalsources, such as plants, animals and microorganisms,the enzymes from microbial sources are preferred inindustrial sector and a large number of them are availablecommercially (Crueger and Crueger, 1989; Kathiresanand Manivannan, 2006). Sources of amylases in bacteria,

yeast and other fungi have been reported and theirproperties described by (Chi et al., 2007; Gupta et al.,2008; Liu and Xu, 2008). Due to their diversity, fungi havebeen recognized as a source of new enzymes with usefuland/or novel characteristics (Bakri et al., 2009).

Fungal amylases are used for hydrolyzingcarbohydrate, protein and other constitutes of soy beans

*Corresponding author E-mail:[email protected]

and wheat into peptides, amino acids, sugars and othelow molecular weight compounds in soy sauceproduction. The enzyme is also used in the preparation omiso (bean paste), tofu (bean curd), sofu (Chinesecheese) and soymilk (Negi and Banerjee, 2009). Theseenzymes also have various applications in major areas ofood processing, beverage production, animal nutritionleather, paper and pulp, textiles, detergents, etc. With theadvent of new frontiers in biotechnology, the spectrum oamylase applications has expanded into many new fieldssuch as clinical, medicinal and analytical chemistry(Pandey et al., 1999; Pandey et al., 2000 ; Gupta et al.

2003). The demand for amylase is increasing day by daybecause of its magnificent potentiality in the abovementioned industrial sectors taking into consideration thathe properties of -amylases such as thermostability andpH profile should match the application (Karakas et al.2010).

Considering the industrial importance of amylases, wewere focusing in our previous study (Sidkey et al., 2010on the possibility of using different fermented enviro-agroindustrial wastes as very cheap and available substratesfor obtaining microbial -amylases. That investigation hasled to the selection and identification of a high amylase


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producer one (A. flavus, F2Mbb) isolated from brownbread waste and capable of growing on different enviro-agro-industrial wastes. In addition, some environmentaland nutritional parameters affecting the biosynthesis of -amylases from A. flavus, F2Mbb were studied. Incompletion to that work, the purpose of this investigation

was purification and studying some parameters affectingthe activity of the pure enzyme.

MATERIALS & METHODS

Production of A. flavus, F2Mbb-amylase under solidstate fermentation (SSF) conditions

To obtain the maximum amylase production, A. flavus,F2Mbb was grown under all the optimal conditionspreviously determined by Sidkey et al. (2010) as follows:incubation temperature, 37C; pH, 6.0 using a 0.2 Mphosphate buffer; shaking condition at 200 rpm;incubation period, 6 days; inoculum size of 13.0108spore/ml on Modified Czapek-Dox wheat bran agarmedium (MCDWB) under SSF with a substrateconcentration of 20% bran (instead of sucrose); chemicaltreatment of bran with 6 N phosphoric acid; carbonsource, corn gluten; nitrogen source, NaNO3; amino acid,methionine; and a mixture of different salts (100 ppmMgSO4; 200 ppm KCl; 50 ppm K2HPO4 and 50 ppmNiSO4).

Purification procedures of A. flavus, F2Mbb -amylase

All experiments were carried out in triplicates and allpurification procedures were carried out at 4 C asfollows:

Preparation of the crude enzyme: At the end ofincubation period, the culture was centrifuged at 5000rpm for 20 minutes at 4 C. The resulting culturesupernatant was filtered through Whatmann No 1 filterpaper and the filtrate used as crude enzyme solution. Theobtained filtrate was then estimated for both proteincontent and amylolytic activity.

Ammonium sulfate precipitation: Ammoniumsulfate was added to the crude culture supernatant to

different 20-100% saturations according to the method ofGomori (1955).

The precipitate of crude enzyme was dissolved in aminimum volume of 0.2 M phosphate buffer (pH 6.0); anddialyzed overnight in a dialysis bag against the samephosphate buffer at pH 6.2 at 4 C. The obtained -amylase enzyme preparation was concentrated againstcrystals of sucrose and was kept in the refrigerator at 4C for further purification steps.

Sephadex G-200 chromatography: Theconcentrated enzyme preparation was loaded onto

Sidkey et al. 097

Sephadex G-200 column (Pharmacia, Sweden, 2.5 x 45cm) pre-equilibrated with 0.2 M phosphate buffer (pH6.0), and eluted with the same buffer containing 0.2 MNaCl according to the method of El-Safey and Amma(2002). Active fractions exhibiting amylolytic activity werecollected, combined and assayed for both -amylase

activity and protein content.

Enzyme activity and protein estimation

Protein content of the enzyme extracts was estimated bythe method of Lowry et al. (1951) after preliminaryprecipitation with 50% trichloroacetic acid using bovineserum albumin (BSA) as the standard.

While the -amylase activity was performed by thestarch clearing zone technique adopted by El-Safey andAmmar (2002), the specific activity of the enzyme proteinwas determined as El-Safey and Ammar (2004) andexpressed in terms of units/mg protein/ml according tothe following equation: Specific activity = enzyme activity(U) / protein content (mg/ml).

Determination of the molecular weightof the purifiedenzyme

The molecular weight of the purified enzyme wasestimated by sodium dodecyl sulfate-polyacrylamide geelectrophoresis (SDS-PAGE) using a 10% (w/vacrylamide gel that was performed as described byLaemmli (1970) and modified later by Studier (1973).

Factors affecting -amylaseactivity

The following parameters were investigated and amylase activity was assayed at the end of eachincubation period:

Enzyme concentrations: Serial dilutions wereperformed in terms of mg protein viz. 0.0075, 0.015, 0.030.060 and 0.12 with the same substrate concentration

(1% starch solution) with incubation at 37C, pH 6 for 1

hour.Substrate concentrations: The purified enzyme was

incubated with different soluble starch concentrations

(mg/ml) viz. 1, 5, 10, 15, 20, 25, 30. The reaction mixturewas incubated for 1 hour at 37 C.

Incubation temperature: The optimal incubationtemperatures of the -amylase was determined byassaying activity using 1% (w/v) starch solution over the

range of 10-60 C at pH 6.pH values: The optimal pH of the enzyme was also

determined by assaying activity using 1% (w/v) starch

solution using phosphate buffer (0.2M) at 37 C at pH6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, and 7.6.


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098 Int. Res. J. Microbiol.

Table 1. Ammonium sulfate fractionation pattern of -amylase produced by A. flavusF2Mbb.

Purificationfold

Specific activity(U/mg protein/ml)

Protein content(mg/ml)

Enzyme activity(U)

Amm. sulfateconcentration

(%)126.460.4812.70.0

0.7720.480.6212.720

0.8923.710.6214.7407.64202.050.88177.8606.24165.00.96158.480

4.01106.10.8287.0100

Figure 1. Fractionation pattern of A. flavus, F2Mbb -amylase on G-200 column chromatography.

Incubation periods: The reaction mixture wasincubated at different incubation time 2, 4, 6, 12, 18, 24,

and 30 hours at pH 6 and 37 C.

Kinetic determinations

The initial reaction rate of amylase was determined atdifferent starch concentration ranging from 1 to 30 mg/ml.The kinetic constants Km and Vmax were estimated fromthe Lineweaver-Burk plot by plotting reciprocal ofsubstrate concentration versus velocity.

RESULTS AND DISCUSSION

Recently, interest and demand for enzymes with novelproperties are very high in various industries and it leadsto the discovery of various types of the amylases withunique properties. Each application of amylases requiresproperties with respect to specificity (Ashwini et al.,2011). Amylases from microbial sources, especially fungi(Aspergillus spp.), have gained much attention becauseof the availability and high productivity of fungi, which arealso amenable to genetic manipulation (Moreira et al.,1999; Moreira et al., 2001; Kathiresan and Manivannan,2006).

Since A. flavus, F2Mbb proved to be the most potent amylase producer strain (Sidkey et al., 2010), it wasselected for the purpose of production, purification and

investigating properties of the enzyme.Concerning the precipitation of -amylase by using

different concentrations of ammonium sulfate, 60%concentration gave the highest enzyme activity of 202.05(U/mg protein/ml) as illustrated in table 1.

Our results obtained by fractionation on a sephadexG-200 column chromatography revealed that fractions (712) represent the most active fractions (Figure 1).

Enzyme purification using 60% ammonium sulfate foprecipitation and subsequent Sephadex G-200 gefiltration resulted in 15.74% recovery, specific activity o4348 U/mg protein/ml as illustrated in table 2.

With regards to the findings of other workers, Ibrahim

et al. (1990) succeeded to precipitate and purify amylasesecreted by Streptomyces aureofaciens77 using a 50-70% saturation ammonium sulfate and the amylasepurification on Sephadex G-200 column chromatographyresulted in an increase of purification up to 74 foldSimilarly, Sidkey et al. (1997) purified -amylase from Aflavus, S-7 by a process of ammonium sulfateprecipitation at 80% saturation and sephadex G-200column chromatography resulting in a purified enzymewith specific activity of 28.6 (units/mg protein/ml) and 6.7purification folds. El-Safey and Ammar (2004) also


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Table 2. A summary of the purification steps of A. flavus, F2Mbb -amylase.

Figure 2. SDS-PAGE of pure A. flavus, F2Mbb -amylase.(1) Molecular size marker. (2) Enzymeafter Sephadex G-200 chromatography.

purified -amylase using ammonium sulfate precipitationresulted in specific activity of 3670.51 (units/mg

protein/ml) and purification folds 5.66 times, dialysisagainst sucrose resulted in specific activity 4023.63(units/mg protein/ml) and purification folds 6.20 times,sephadex G200 filtration resulted in a specific activity of6471.6 (units/mg protein/ml) with purification folds 9.97times. In a recent study, by Nouadri et al. (2010) -amylase purified by ammonium sulfate precipitation,dialysis, sephadex G-100 and DEAESepharose CL-6Bcolumn chromatography resulted in an enzyme withspecific activity of (154.2 units/ml/mg protein) with (38.5folds) purification .

The purified enzyme in our work (the most activefractions 7-12 after collection and concentration) was

then subjected to SDS-PAGE for molecular weightdetermination. From the gel (Figure 2), it could bepredicted that the relative molecular weight of fungalamylase was ~ 56 kDa. This result is supported byVihinen and Mantsala (1989) who found that molecularweights of microbial -amylases are usually 50-60 kDa asshown directly by analysis of cloned -amylase genesand deduced amino acid sequences.

This observation also corroborates that reported byKhoo et al. (1994) who purified -amylase enzymeproduced by A. flavus using ammonium sulfateprecipitation and ion-exchange chromatography and

found that the enzyme is homogeneous on SDS-PAGEwith a molecular weight of 52.5 +/- 2.5 kDa.

With regards to the findings of other workers, theenzyme obtained by Liu and Xu (2008) showed amolecular weight of 56 kDa by SDS-PAGE aftepurification using ammonium sulfate precipitation, ionexchange and gel filtration chromatography from a newlyisolated Bacillussp.YX-1. Chakraborty et al. (2009) foundthat SDS-PAGE and zymogram activity staining of thei-amylase enzyme from strain marine Streptomyces spD1 showed a single band equal to molecular weight of 66kDa. Abou-Zeid (1997) found that the molecular weight ofthe A. flavus-amylase was approximately 75 +/- 3 kDaby SDS-PAGE. More recently, Varalakshmi et al. (2009found that partial purification of the alpha-amylase

obtained from A. niger JGI 24 using ammonium sulfatefractionation results in enzyme with a molecular weight o43 kDa by SDS-PAGE. Prakash et al. (2009) purified twokinds of amylase activity, designated amylase I andamylase II, from culture filtrates to homogeneity withmolecular masses of 72 and 62 kDa, respectively fromhalophilic bacterial strain Chromohalobacter sp. TVSP101. Analyses of enzyme from Penicillium camembertPL21 in study of Nouadri et al. (2010) by SDS-PAGEelectrophoresis revealed one band of molecular mass o60.5 kDa. Recently, Kikani and Singh (2011) had

Precipitationsteps

VolumeTotal

activity(unit/ml)

Totalprotein(mg/ml)

Specific activity(Unit/mg-1 protein)

Purificationfolds

Recovery(%)

Cell free filtrate 500 6350 240 27 1 100

(NH4)2SO4precipitation60%

100 17780 88.0 202 7.5 280

Dialysis againstsucrose

4.0 2252 7.6 296 10.9 35.5

SephadexG-200

(fractions 7- 12)1 1000 0.23 4348 161 15.74


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0

10

20

30

0.0075 0.015 0.03 0.06 0.12

-amylaseactivity

(U/mg)

-amylase conc. (mg/ml) Figure 3. Effect of enzyme concentrations on the activity of the purified A. flavusF2Mbb -amylase

Figure 4. Effect of different concentrations of starch substrate on the activity of the purified A. flavusF2Mbb -amylase.

0

10

20

30

40

10 20 30 40 50 60

-amylaseactivity(U/mg)

Incubation temp.

Figure 5. Effect of different incubation temperatures on the activity of the purified A. flavus, F2Mbb -amylase

obtained purified -amylase from Bacillus

amyloliquifaciens TSWK1-1 with a molecular weight of43kDa after partial purification using ammonium sulfatefractionation followed by dialysis.

Regarding different factors affecting the pure A.flavus, F2Mbb -amylase, there was a continuousincrease in enzyme activity with the correspondingincrease in its concentration (Figure 3). Abd El-Rahman(1990), El-Safey (1994), Sidkey et al. (1997), El-Safeyand Ammar (2004) and Nouadri et al. (2010) previouslyreported the same observation which is in completeaccordance with the general behavior of most enzymes.In the present investigation,the maximum -amylase

activity was attained at the least substrate (starch

concentration 15 mg/ml (Figure 4). Other investigators asMoustafa (2002) found that 1% , 4% starch solution gavethe highest -amylase activity in case of T. lanuginosusF4 and S. moniliformisB7, respectively. Additionally, theoptimum concentration of soluble starch for -amylaseactivity was 1.67% (Kuiper et al. 1978), between 2-3%(Abd El-Rahman, 1990), 0.2% from A. flavus vacoluminaris (El-Safey and Ammar, 2004), 0.1% from Aflavus (Sidkey et al., 1997), and 1% from PenicilliumcamembertiPL21 (Nouadri et al., 2010).

Also, temperature was found to play a significant role inthe activity of the produced -amylase. Figure 5 showed


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Sidkey et al. 101

0

10

20

30

40

50

60

6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6

-amy

laseactivity(U/mg)

pH

Figure 6. Effect of different pH values on the activity of the purified A. flavus, F2Mbb -amylase.

0

10

20

30

40

50

60

6 6.2 6.4 6.6 6.8 7 7.2

-amylaseac

tivity(U/mg)

Incubation time (hours)

Figure 7. Effect of different incubation time on the activity of the purified A. flavus, F2Mbb -amylase

the optimum activity was at 30C and with a decliningtrend showing its least activity at 60 C. Manyinvestigators have studied this parameter and results ofsome of them were in complete accordance to our result.

The optimum temperature of the purified -amylase was30C from each of A. flavus(Abou-Zeid, 1997), A. nigerJGI 24 (Varalakshmi et al., 2009), Penicillium camemberti

PL21 (Nouadri et al., 2010); 35 C from A. flavus varcoluminaris (El-Safey and Ammar, 2004); 40 C from A.nigerNRRL-337 (Mahmoud et al., 1978) and 45 C fromstrain marine Streptomyces sp. D1 (Chakraborty et al.,2009).

The purified A. flavus, F2Mbb -amylase exhibitedmaximum activity at pH 6.4 of phosphate buffer and gave52.48 U, above or below this pH a sharp decrease in theactivity was noticed as shown in Figure 6.

In comparison with our result, maximum-amylases

activity was obtained at pH 7.2 of phosphate buffer andpH 6.0 of citrate buffer for T. lanuginosus, F4 (Moustafa,2002), pH 7.0 for A. flavus(Abou-Zeid, 1997), pH 6.0 forA. flavus(Khoo et al., 1994; Sidkey et al., 1997), pH 6.2for A. flavusvar columinaris(El-Safey and Ammar, 2004),pH 6 from A. flavus(Sidkey et al., 1997),pH 4.3 from A.niger NRRL-337 (Mahmoud et al., 1978). On the otherhand, Varalakshmi et al. (2009) found that pH 9.5resulted in maximum enzyme activity from A. niger JGI24.

Regarding the effect of different time intervals, resultsrevealed that the enzyme activity was increased by

increasing time intervals as shown in figure 7. This resuwas in full agreement with El-Safey (1994) and Sidkey eal. (1997). El-Safey and Ammar, (2004) found theoptimum incubation period for -amylase activity

obtained from A. flavusvar columinariswas at 30 h.Enzyme kinetics, Km and Vmax are significancoefficients in guiding scientific research and engineeringdesign. The more firmly the enzyme binds to itssubstrate, the smaller will be the value of Km. MoreoverKm is independent of enzyme concentration and is a truecharacteristic of the enzyme under defined conditions otemperature, pH, etc (Negi and Banerjee 2009).

From the Lineweaver-Burk plot of the reciprocal oinitial velocities and substrate concentrations (Figure 8)The Km and Vmax values were 0.5 mg/ml and 17.78mg/ml/min at 30C and pH 6.4 with 0.2 M phosphatebuffer. Higher Vmax and lower Km had confirmed theefficiency of this enzyme for diverse applications. This Kmvalue is near to Km value (0.6 mg/ml) of -amylase fromBacillus amyloliquifacienceTSWK1-1 (Kikani and Singh2011) and Km (0.68 mg/ml) from T. lanuginosus (Quanget al., 2002). With regard to other investigators, Km valueof -amylase was 0.92 mg/ml produced from bothBacillus sphaericus(Al-Qodah et al., 2007) & Penicilliumcamemberti PL21 (Nouadri et al., 2010). On the otherhand, Vmax of glucoamylase for starch by Aspergillusawamori:nakazawa MTCC 6652 was calculated as 56.18mg/ml/min and Km as 9.79 mg/ml (Negi and Banerjee2009).


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Figure 8. Lineweaver-Burk plot of the reciprocal of initial velocities and starch concentration for determination ofKm value of A. flavus, F2Mbb -amylase.

ACKNOWLEDGEMENT

This study was supported by grant No. 145/428 fromDeanship of scientific Research, Taibah University, KSA,We are thankful for their financial support.

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