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

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Full Length Research Paper

Purification and characterization of -amylase from a newly isolated Aspergillus flavus F2Mbb
Nagwa M. Sidkey1; Maha A. Abo-Shadi2; Reham Balahmar3; Reham Sabry3; Ghadeer Badrany3
2 1

Botany & Microbiology Dept., Faculty of Science (Girls), Al-Azhar Univ., Egypt. Microbiology and Immunology Dept., Faculty of Pharmacy (Girls), Al-Azhar Univ., Egypt. 3 Biology 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 precipitation and 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 and incubation time. The enzyme exhibited maximum activity at 30C and pH 6.4 with the optimum starch concentration 15 mg/ml. Key words: -amylases; Production; Purification; Characterization, Aspergillus flavus F2Mbb; Saudi Arabia. INTRODUCTION -Amylases (EC3.2.1.1, 1,4-a-D-glucan-glucanohydrolyase) are extracellular enzymes which hydrolyze starch into a range of products such as glucose and maltose or specific malto-oligosaccharide or mixed maltooligosaccharides (Dey et al., 2002; Messaoud et al., 2004; Hashim et al., 2005). Although -amylases can be derived from several sources, such as plants, animals and microorganisms, the enzymes from microbial sources are preferred in industrial sector and a large number of them are available commercially (Crueger and Crueger, 1989; Kathiresan and Manivannan, 2006). Sources of amylases in bacteria, yeast and other fungi have been reported and their properties described by (Chi et al., 2007; Gupta et al., 2008; Liu and Xu, 2008). Due to their diversity, fungi have been recognized as a source of new enzymes with useful and/or novel characteristics (Bakri et al., 2009). Fungal amylases are used for hydrolyzing carbohydrate, protein and other constitutes of soy beans and wheat into peptides, amino acids, sugars and other low molecular weight compounds in soy sauce production. The enzyme is also used in the preparation of miso (bean paste), tofu (bean curd), sofu (Chinese cheese) and soymilk (Negi and Banerjee, 2009). These enzymes also have various applications in major areas of food processing, beverage production, animal nutrition, leather, paper and pulp, textiles, detergents, etc. With the advent of new frontiers in biotechnology, the spectrum of amylase applications has expanded into many new fields such 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 day because of its magnificent potentiality in the above mentioned industrial sectors taking into consideration that the properties of -amylases such as thermostability and pH profile should match the application (Karakas et al., 2010). Considering the industrial importance of amylases, we were focusing in our previous study (Sidkey et al., 2010) on the possibility of using different fermented enviro-agroindustrial wastes as very cheap and available substrates for obtaining microbial -amylases. That investigation has led to the selection and identification of a high amylase

*Corresponding author E-mail:m_a_shadi@hotmail.com

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producer one (A. flavus, F2Mbb) isolated from brown bread waste and capable of growing on different enviroagro-industrial wastes. In addition, some environmental and nutritional parameters affecting the biosynthesis of amylases from A. flavus, F2Mbb were studied. In completion to that work, the purpose of this investigation was purification and studying some parameters affecting the activity of the pure enzyme. MATERIALS & METHODS Production of A. flavus, F2Mbb -amylase under solid state fermentation (SSF) conditions To obtain the maximum amylase production, A. flavus, F2Mbb was grown under all the optimal conditions previously determined by Sidkey et al. (2010) as follows: incubation temperature, 37C; pH, 6.0 using a 0.2 M phosphate buffer; shaking condition at 200 rpm; 8 incubation period, 6 days; inoculum size of 13.010 spore/ml on Modified Czapek-Dox wheat bran agar medium (MCDWB) under SSF with a substrate concentration of 20% bran (instead of sucrose); chemical treatment of bran with 6 N phosphoric acid; carbon source, corn gluten; nitrogen source, NaNO3; amino acid, methionine; and a mixture of different salts (100 ppm MgSO4; 200 ppm KCl; 50 ppm K2HPO4 and 50 ppm NiSO4). Purification procedures of A. amylase flavus, F2Mbb -

Sephadex G-200 column (Pharmacia, Sweden, 2.5 x 45 cm) pre-equilibrated with 0.2 M phosphate buffer (pH 6.0), and eluted with the same buffer containing 0.2 M NaCl according to the method of El-Safey and Ammar (2002). Active fractions exhibiting amylolytic activity were collected, combined and assayed for both -amylase activity and protein content. Enzyme activity and protein estimation Protein content of the enzyme extracts was estimated by the method of Lowry et al. (1951) after preliminary precipitation with 50% trichloroacetic acid using bovine serum albumin (BSA) as the standard. While the -amylase activity was performed by the starch clearing zone technique adopted by El-Safey and Ammar (2002), the specific activity of the enzyme protein was determined as El-Safey and Ammar (2004) and expressed in terms of units/mg protein/ml according to the following equation: Specific activity = enzyme activity (U) / protein content (mg/ml). Determination of the molecular weight of the purified enzyme The molecular weight of the purified enzyme was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% (w/v) acrylamide gel that was performed as described by Laemmli (1970) and modified later by Studier (1973). Factors affecting -amylase activity The following parameters were investigated and amylase activity was assayed at the end of each incubation period: Enzyme concentrations: Serial dilutions were performed in terms of mg protein viz. 0.0075, 0.015, 0.03, 0.060 and 0.12 with the same substrate concentration (1% starch solution) with incubation at 37 C, 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 mixture was incubated for 1 hour at 37 C. Incubation temperature: The optimal incubation temperatures of the -amylase was determined by assaying 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 pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, and 7.6.

All experiments were carried out in triplicates and all purification procedures were carried out at 4 C as follows: Preparation of the crude enzyme: At the end of incubation period, the culture was centrifuged at 5000 rpm for 20 minutes at 4 C. The resulting culture supernatant was filtered through Whatmann No 1 filter paper and the filtrate used as crude enzyme solution. The obtained filtrate was then estimated for both protein content and amylolytic activity. Ammonium sulfate precipitation: Ammonium sulfate was added to the crude culture supernatant to different 20-100% saturations according to the method of Gomori (1955). The precipitate of crude enzyme was dissolved in a minimum volume of 0.2 M phosphate buffer (pH 6.0); and dialyzed overnight in a dialysis bag against the same phosphate buffer at pH 6.2 at 4 C. The obtained amylase enzyme preparation was concentrated against crystals of sucrose and was kept in the refrigerator at 4 C for further purification steps. Sephadex G-200 chromatography: The concentrated enzyme preparation was loaded onto

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Table 1. Ammonium sulfate fractionation pattern of -amylase produced by A. flavus F2Mbb.

Amm. sulfate concentration (%) 0.0 20 40 60 80 100

Enzyme activity (U) 12.7 12.7 14.7 177.8 158.4 87.0

Protein content (mg/ml) 0.48 0.62 0.62 0.88 0.96 0.82

Specific activity (U/mg protein/ml) 26.46 20.48 23.71 202.05 165.0 106.1

Purification fold 1 0.77 0.89 7.64 6.24 4.01

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

Incubation periods: The reaction mixture was incubated 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 at different starch concentration ranging from 1 to 30 mg/ml. The kinetic constants Km and Vmax were estimated from the Lineweaver-Burk plot by plotting reciprocal of substrate concentration versus velocity. RESULTS AND DISCUSSION Recently, interest and demand for enzymes with novel properties are very high in various industries and it leads to the discovery of various types of the amylases with unique properties. Each application of amylases requires properties with respect to specificity (Ashwini et al., 2011). Amylases from microbial sources, especially fungi (Aspergillus spp.), have gained much attention because of the availability and high productivity of fungi, which are also 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 was selected 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 sephadex G-200 column chromatography revealed that fractions (712) represent the most active fractions (Figure 1). Enzyme purification using 60% ammonium sulfate for precipitation and subsequent Sephadex G-200 gel filtration resulted in 15.74% recovery, specific activity of 4348 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 amylase secreted by Streptomyces aureofaciens 77 using a 50-70 % saturation ammonium sulfate and the amylase purification on Sephadex G-200 column chromatography resulted in an increase of purification up to 74 fold. Similarly, Sidkey et al. (1997) purified -amylase from A. flavus, S-7 by a process of ammonium sulfate precipitation at 80% saturation and sephadex G-200 column chromatography resulting in a purified enzyme with specific activity of 28.6 (units/mg protein/ml) and 6.7 purification folds. El-Safey and Ammar (2004) also

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

Precipitation steps Cell free filtrate (NH4)2SO4 precipitation 60% Dialysis against sucrose Sephadex G-200 (fractions 7- 12)

Volume 500 100 4.0 1

Total activity (unit/ml) 6350 17780 2252 1000

Total protein (mg/ml) 240 88.0 7.6 0.23

Specific activity (Unit/mg-1 protein) 27 202 296 4348

Purification folds 1 7.5 10.9 161

Recovery (%) 100 280 35.5 15.74

Figure 2. SDS-PAGE of pure A. flavus, F2 Mbb -amylase.(1) Molecular size marker. (2) Enzyme after Sephadex G-200 chromatography.

purified -amylase using ammonium sulfate precipitation resulted in specific activity of 3670.51 (units/mg protein/ml) and purification folds 5.66 times, dialysis against 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 of 6471.6 (units/mg protein/ml) with purification folds 9.97 times. In a recent study, by Nouadri et al. (2010) amylase purified by ammonium sulfate precipitation, dialysis, sephadex G-100 and DEAESepharose CL-6B column chromatography resulted in an enzyme with specific activity of (154.2 units/ml/mg protein) with (38.5 folds) purification . The purified enzyme in our work (the most active fractions 7-12 after collection and concentration) was then subjected to SDS-PAGE for molecular weight determination. From the gel (Figure 2), it could be predicted that the relative molecular weight of fungal amylase was ~ 56 kDa. This result is supported by Vihinen and Mantsala (1989) who found that molecular weights of microbial -amylases are usually 50-60 kDa as shown directly by analysis of cloned -amylase genes and deduced amino acid sequences. This observation also corroborates that reported by Khoo et al. (1994) who purified -amylase enzyme produced by A. flavus using ammonium sulfate precipitation and ion-exchange chromatography and

found that the enzyme is homogeneous on SDS-PAGE with a molecular weight of 52.5 +/- 2.5 kDa. With regards to the findings of other workers, the enzyme obtained by Liu and Xu (2008) showed a molecular weight of 56 kDa by SDS-PAGE after purification using ammonium sulfate precipitation, ion exchange and gel filtration chromatography from a newly isolated Bacillus sp.YX-1. Chakraborty et al. (2009) found that SDS-PAGE and zymogram activity staining of their -amylase enzyme from strain marine Streptomyces sp. D1 showed a single band equal to molecular weight of 66 kDa. Abou-Zeid (1997) found that the molecular weight of the A. flavus -amylase was approximately 75 +/- 3 kDa by SDS-PAGE. More recently, Varalakshmi et al. (2009) found that partial purification of the alpha-amylase obtained from A. niger JGI 24 using ammonium sulfate fractionation results in enzyme with a molecular weight of 43 kDa by SDS-PAGE. Prakash et al. (2009) purified two kinds of amylase activity, designated amylase I and amylase II, from culture filtrates to homogeneity with molecular masses of 72 and 62 kDa, respectively from halophilic bacterial strain Chromohalobacter sp. TVSP 101. Analyses of enzyme from Penicillium camemberti PL21 in study of Nouadri et al. (2010) by SDS-PAGE electrophoresis revealed one band of molecular mass of 60.5 kDa. Recently, Kikani and Singh (2011) had

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30
-amylase activity (U/mg)

20 10 0 0.0075 0.015 0.03 -amylase conc. (mg/ml) 0.06 0.12

Figure 3. Effect of enzyme concentrations on the activity of the purified A. flavus F2Mbb -amylase

Figure 4. Effect of different concentrations of starch substrate on the activity of the purified A. flavus F2Mbb amylase.

-amylase activity (U/mg)

40 30 20 10 0 10 20 30 40 Incubation temp. 50 60

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 of 43kDa after partial purification using ammonium sulfate fractionation followed by dialysis. Regarding different factors affecting the pure A. flavus, F2Mbb -amylase, there was a continuous increase in enzyme activity with the corresponding increase in its concentration (Figure 3). Abd El-Rahman (1990), El-Safey (1994), Sidkey et al. (1997), El-Safey and Ammar (2004) and Nouadri et al. (2010) previously reported the same observation which is in complete accordance 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 as Moustafa (2002) found that 1% , 4% starch solution gave the highest -amylase activity in case of T. lanuginosus F4 and S. moniliformis B7, respectively. Additionally, the optimum concentration of soluble starch for -amylase activity was 1.67% (Kuiper et al. 1978), between 2-3% (Abd El-Rahman, 1990), 0.2% from A. flavus var columinaris (El-Safey and Ammar, 2004), 0.1% from A. flavus (Sidkey et al., 1997), and 1% from Penicillium camemberti PL21 (Nouadri et al., 2010). Also, temperature was found to play a significant role in the activity of the produced -amylase. Figure 5 showed

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60

-amylase activity (U/mg)

50 40 30 20 10 0 6 6.2 6.4 6.6 pH 6.8 7 7.2 7.4 7.6

Figure 6. Effect of different pH values on the activity of the purified A. flavus, F2 Mbb -amylase.

-amylase activity (U/mg)

60 50 40 30 20 10 0 6 6.2 6.4 6.6 Incubation time (hours) 6.8 7 7.2

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 declining trend showing its least activity at 60 C. Many investigators have studied this parameter and results of some of them were in complete accordance to our result. The optimum temperature of the purified -amylase was 30C from each of A. flavus (Abou-Zeid, 1997), A. niger JGI 24 (Varalakshmi et al., 2009), Penicillium camemberti PL21 (Nouadri et al., 2010); 35 C from A. flavus var columinaris (El-Safey and Ammar, 2004); 40 C from A. niger NRRL-337 (Mahmoud et al., 1978) and 45 C from strain marine Streptomyces sp. D1 (Chakraborty et al., 2009). The purified A. flavus, F2Mbb -amylase exhibited maximum activity at pH 6.4 of phosphate buffer and gave 52.48 U, above or below this pH a sharp decrease in the activity was noticed as shown in Figure 6. In comparison with our result, maximum -amylases activity was obtained at pH 7.2 of phosphate buffer and pH 6.0 of citrate buffer for T. lanuginosus, F4 (Moustafa, 2002), pH 7.0 for A. flavus (Abou-Zeid, 1997), pH 6.0 for A. flavus (Khoo et al., 1994; Sidkey et al., 1997), pH 6.2 for A. flavus var 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 other hand, Varalakshmi et al. (2009) found that pH 9.5 resulted in maximum enzyme activity from A. niger JGI 24. Regarding the effect of different time intervals, results revealed that the enzyme activity was increased by

increasing time intervals as shown in figure 7. This result was in full agreement with El-Safey (1994) and Sidkey et al. (1997). El-Safey and Ammar, (2004) found the optimum incubation period for -amylase activity obtained from A. flavus var columinaris was at 30 h. Enzyme kinetics, Km and Vmax are significant coefficients in guiding scientific research and engineering design. The more firmly the enzyme binds to its substrate, the smaller will be the value of Km. Moreover, Km is independent of enzyme concentration and is a true characteristic of the enzyme under defined conditions of temperature, pH, etc (Negi and Banerjee 2009). From the Lineweaver-Burk plot of the reciprocal of initial velocities and substrate concentrations (Figure 8), The Km and Vmax values were 0.5 mg/ml and 17.78 mg/ml/min at 30 C and pH 6.4 with 0.2 M phosphate buffer. Higher Vmax and lower Km had confirmed the efficiency of this enzyme for diverse applications. This Km value is near to Km value (0.6 mg/ml) of -amylase from Bacillus amyloliquifacience TSWK1-1 (Kikani and Singh, 2011) and Km (0.68 mg/ml) from T. lanuginosus (Quang et al., 2002). With regard to other investigators, Km value of -amylase was 0.92 mg/ml produced from both Bacillus sphaericus (Al-Qodah et al., 2007) & Penicillium camemberti PL21 (Nouadri et al., 2010). On the other hand, Vmax of glucoamylase for starch by Aspergillus awamori: nakazawa MTCC 6652 was calculated as 56.18 mg/ml/min and Km as 9.79 mg/ml (Negi and Banerjee, 2009).

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

ACKNOWLEDGEMENT This study was supported by grant No. 145/428 from Deanship of scientific Research, Taibah University, KSA, We are thankful for their financial support.
REFERENCES Abd El-Rahman EM (1990). Studies on some thermophilic bacterial strains. Ph.D. Thesis. Al-Azhar Univ., Fac. of Sci., Bot. Dept., Cairo, Egypt. Abou-Zeid AM (1997). Production, purification and characterization of an extracellular alpha-amylase enzyme isolated from Aspergillus flavus. Microbios. 89(358): 55-66. Al-Qodah Z, Daghstani H, Geopel P, Lafi W (2007). Determination of kinetic parameters of -amylase producing thermophile Bacillus sphaericus. Afri. J. Biotechnol. 6: 699-706. Ashwini K, Gaurav K, Karthik L, Bhaskara KV (2011). Optimization, production and partial purification of extracellular -amylase from Bacillus sp. Marini. Archiv. Appl. Sci. Res. 3 (1): 33-42 Bakri Y, Magali M, Thonart P (2009). Isolation and identification of a new fungal strain for amylase biosynthesis. Pol. J. Microbiol. 58(3): 269-73. Chakraborty S, Khopade A, Kokare C, Mahadik K, Chopade B (2009). Isolation and characterization of novel -amylase from marine Streptomyces sp. D1. J. Molecular Catal. B: Enzym. 58: 1723. Chi HLZ, Wang X, Duan X, Ma L, Gao L (2007). Purification and characterization of extracellular amylase from the marine yeast Aureobasidium pollulans N13d and its raw potato starch digestion. Enzyme Microb. Technol. 40: 1006-1012. Crueger W, Crueger A (1989). Industrial Microbiology, Sinauer Association, Sundreland, MA, pp. 189-218. Dey G, Palit S, Banerjee R, Maiti BR (2002). Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS313. J. Ind. Microbiol. Biotechnol. 28: 193 200. El-Safey EM (1994). Production of microbial -amylase(s) under solid state incubation conditions in the open air. M.Sc. Thesis, Bot. and Microbiol. Dept., Fac. Of Sci., Al-Azhar Univ., Cairo, Egypt. El-Safey EM, Ammar MS (2002). -amylase production using Nile Hyacinth under solid-state fermentation (SSF) conditions. Int. Conf. for Develop. and the Environ. In the Arab world march 26-28, pp. 101-113, Assuit Univ. Center for Environmental studies, Assiut, Egypt. El-Safey EM, Ammar MS (2004). Purification and Characterization of amylase isolated from Aspergillus falvus var. columnaris. Ass. Univ. Bull. Environ. Res. 7 (1): 93-100. Gomori G (1955). Preparation of buffer for use in enzyme active studies. In Methods in Enzymology Vol. I, Colwick SP and Kaplan NO (eds.), Academic press. Inc. pub., New York. Gupta A, Gupta VK, Modi DR, Yadava LP (2008). Production and characterization of a-amylase from Aspergillus niger . Biotechnol. 1: 1-6. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B (2003).

Microbial -amylases: a biotechnological perspective. Process Biochem. 38: 1599-1616. Hashim SO, Delgado OD, Martnez MA, Kaul RH, Mulaa FJ, Mattiasson B (2005). Alkaline active maltohexaose-forming aamylase from Bacillus halodurans LBK 34. Enzyme Microb. Technol. 36, 139-146. Ibrahim AN, Ahmed FH, Ibrahim MMK, Arafa MAI (1990). Precipitation and purification of amylase enzyme produced by Streptomyces aureofaciens 77. Archiv. Pharma. Res. 13 (1): 28-32. Karakas B, Inan M, Certel M (2010). Expression and characterization of Bacillus subtilis PY22 -amylase in Pichia pastoris . J Molecular Catalysis B: Enzym. 64: 129-134. Kathiresan K, Manivannan S (2006). -Amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. Afr. J Biotechnol. 5 (10): 829-832. Khoo SL, Amirul AA, Kamaruzaman M, Nazalan N, Azizan MN (1994). Purification and characterization of alpha-amylase from Aspergillus flavus. Folia Microbiol (Praha). 39 (5): 392-8. Kikani BA, Singh SP (2011). Single step purification and characterization of a thermostable and calcium independent amylase from Bacillus amyloliquifaciens TSWK1-1 isolated from Tulsi Shyam hot spring reservoir, Gujarat (India). International Journal of Biological Macromolecules (in press). Kuiper J, Roels JA, Zuidwedg MHJ (1978). Flow through viscometer for use in the automated determination of hydrolytic activities. Application in protease, amylase and pectinase assay. The Netherlands Anal. Biochem. 90: 192-203. Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227: 680685. Liu XD, Xu Y (2008). A novel raw starch digesting -amylase from a newy isolated Bacillus sp. YX-1: Purification and characterization. Biores. Technol. 99: 4315-4320. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the folin-phenol reagent. J. Biol. Chem. 193; 265275. Mahmoud SA, Abdel-Hafez AM, Mashhoor WA, Refaat AA (1978). Utilization of industrial and agricultural by-products for fungal amylase production. Zentralbl Bakteriol Naturwiss. 133(2):115-20. Messaoud EB, Ali MB, Elleuch N, Masmoudi NF, Bejar S (2004). Purification and properties of a maltoheptaoseand maltohexaoseforming amylase produced by Bacillus subtilis US116. Enzyme Microb. Technol. 34: 662666. Moreira FG, de Lima FA, Pedrinho SRF, Lenartovics V, de Souza CGM, Peralta RM (1999). Production of amylases by Aspergillus tamari. Rev. Microbiol. 30: 157-162. Moreira FG, Lenartovics V, de Souza CGM, Ramos EP, Peralta RM (2001). The use of -methyl-D-glucoside, a synthetic analogue of maltose, as inducer of amylase by Aspergillus sp. In solid-state and submerged fermentations. Braz. J. Microbiol. 32: 15-19. Moustafa OA (2002). Thermostable alpha-amylase(s) from irradiated microbial origin utilizing agricultural and environmental wastes under solid state fermentation conditions. M.Sc. thesis, Botany Dept. (Girls), Fac. Of Sci., Al-Azhar Univ. Negi S, Banerjee R (2009). Characterization of amylase and protease produced by Aspergillus awamori in a single bioreactor. Food Res. Intern. 42: 443-448 Nouadri T, Meraihi Z, Shahrazed D, Leila B (2010). Purification and

Sidkey et al. 103

characterization of the -amylase isolated from Penicillium camemberti PL21. Afric. J. Biochem. Res. 4(6): 155-162. Pandey A, Nigam P, Soccol CR, Soccol VT, Singh D, Mohan R. (2000). Advances in microbial amylases. Biotechnol. Appl. Biochem. 31: 135-152. Pandey A, Selvakumar P, Soccol CR, Nigam P (1999). Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77: 149-162. Prakash B, Vidyasagar M, Madhukumar MS, Muralikrishna G, Sreeramulu K. (2009). Production, purification, and characterization of two extremely halotolerant, thermostable, and alkali-stable aamylases from Chromohalobacter sp. TVSP 101. Proc. Biochem. 44: 210-215. Quang DN, Judit MRS, Marc C, Stals I, Hoschke A (2002). Purification and characterizations of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosus strain ATCC 34626. Enzyme Microb. Technol. 31: 345-352. Sidkey NM, Abo-Shadi MA, Al-Mutrafy AM, Sefergy F, Al-Reheily N (2010). Screening of Microorganisms Isolated from some EnviroAgro-Industrial Wastes in Saudi Arabia for Amylase Production. J Americ. Sci. 6 (10): 926-939.

Sidkey NM, Shash SM, Ammar MS (1997). Partial purification and characterization of -amylase biosynthesized by A. flavus , S-7 allowed to grow on hyacinth water homogenate . Al-Azhar Bull. Sci. 8(2): 545-561. Studier FW (1973). Analysis of bacteriophage T7 early RNAs and proteins of slab gels. J. Mol. Biol. 79: 237-248. Varalakshmi KN, Kumudini BS, Nandini BN, Solomon J, Suhas R, Mahesh B, Kavitha AP (2009). Production and characterization of amylase from Aspergillus niger JGI 24 isolated in Bangalore. Polish J Microbiol. 58 (1): 29-36. Vihinen M, Mantsala P (1989). Microbial amylolytic enzymes. Crit Rev Biochem Mol Biol . 24: 329-418.