Partial purification and characterization of α-amylase produced by Aspergillus oryzae using spent-brewing grains

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1 Indian Journal of Biotechnology Vol. 4, July 2005, pp Partial purification and characterization of α-amylase produced by Aspergillus oryzae using spent-brewing grains Anil Kumar Patel 1, K Madhavan Nampoothiri 1, Sumitra Ramachandran 1 George Szakacs 2 and Ashok Pandey 1* 1 Biotechnology Division, Regional Research Laboratory (CSIR), Thiruvananthapuram , India 2 Department of Agricultural Chemical Technology, Technical University of Budapest, 1111 Budapest, Hungary Received 23 December 2003; revised 13 July 2004; accepted 25 July 2004 Solid-state fermentation (SSF) was carried out to produce α-amylase from Aspergillus oryzae (IFO 30103) using spent brewing grains (SBG) as substrate. A maximum of U/gds amylase activity was obtained after 48 h of fermentation. The extracted enzyme was subjected to partial purification by ammonium sulphate fractionation. Maximum specific activity was obtained with 40-70% fraction. SDS-PAGE of the corresponding sample revealed an approximate 66 kda band, which was confirmed by activity staining as α-amylase. It was optimally active at ph 5 and 50 C by using 1% starch as substrate concentration. The partially purified α-amylase loses activity rapidly above 50 o C but it can be retained in the presence of Ca 2+. Presence of Mn 2+ and Fe 2+ enhanced the enzyme activity and is almost doubled in presence of Mn 2+. However, in the presence of Hg 2+ and Cu 2+, the activity is reduced. Hg 2+ reduced the enzyme activity by half. Keywords: α-amylase, Aspergillus oryzae, solid-state fermentation, spent brewing grains IPC Code: Int. Cl. 7 A01N63/02; C12N9/30; C12R1: 69 Introduction Various enzymes with different specificities are involved in the hydrolysis of starch. α-amylase (endo 1,4,α-D-glucan glucohydorlase, EC ) is an extracellular enzyme that randomly cleaves the 1,4-α-D-glucosidic linkages between adjacent glucose units in the linear amylose chain. Although amylases can be obtained from several sources, such as plants, animals and microorganisms, the enzymes from microbial sources generally meet industrial demand 1. Amylases find potential application in a number of industrial processes such as in food, textile and paper industries. Microbial amylases have successfully replaced chemical hydrolysis of starch in starch processing industries. Fungal α-amylase is preferred for use in formulations for human or animal consumption involving applications under acidic conditions and around 37 C 2. Similarly, due to its biocompatibility, fungal amylases are preferred in baking and food processing. With the advent of new frontiers in biotechnology, the spectrum of amylase application has expanded into many other fields, such as clinical, medical and analytical chemistry 1. *Author for correspondence: Tel: ; Fax: ashokpandey56@yahoo.co.in During the last decade, an increased attention was paid to use various agro industrial wastes for value addition using solid-state fermentation (SSF) by filamentous fungi as the major conversion technology 3 7. Spent brewing grain (SBG) consists of the residual seed hull and fiber of barley after the malting process of the brewing industry. The potential of SBG to be used as a substrate for the production of enzymes including cellulase and xylanase under SSF has been analysed 8. The filamentous fungus, Aspergillus oryzae is extensively used to produce several industrial enzymes including amylases 9. This paper deals with partial purification and biochemical characterisation of an α-amylase produced by a mesophilic mould, Aspergillus oryzae (IFO 30103) by SSF using SBG as substrate. Materials and Methods Microorganism and Maintenance A fungal strain of Aspergilus oryzae (IFO-30103) was used for the present study. Potato-dextrose-agar (PDA) was used for growing and maintaining the culture. The fully sporulated slants were obtained after 8 days of incubation of the newly streaked slants at 30 C and were stored at 4 o C for further use.

2 PATEL et al: PARTIAL PURIFICATION & CHARACTERIZATION OF α-amylase PRODUCED BY A. ORYZAE 337 Preparation of Inoculum Ten ml of distilled water containing 0.1% tween-80 was transferred to a fully sporulated PDA slant culture. The spores were dislodged using an inoculating needle under aseptic conditions. This spore suspension was appropriately diluted for the required density of spores. The total viable spore number in PDA slant was determined by colony count technique after serial dilution. Unless otherwise mentioned, an inoculum of spores/ml was used. SSF SBG obtained from Hungary was used as the substrate for SSF. Five grams of SBG was added to 250 ml Erlenmeyer flask and was adequately moistened with a salt solution having the composition: KH 2 PO 4, 5; NH 4 NO 3, 5; Nacl, 1; and MgSO 4, 1 g/l. The contents were thoroughly mixed and sterilized by autoclaving at 121 o C for 20 min. The sterilized solid medium was inoculated with one ml spore suspension of A. oryzae, mixed uniformly and incubated at 30 C. Enzyme Extraction At the end of fermentation, the fermented solid mass was mixed with 50 ml distilled water containing 0.1% Tween-80 and agitated on a rotary shaker for 30 min. Whole contents were filtered though muslin cloth. The reside was again treated with 50 ml distilled water in the same way and filtered. The filtrates were pooled together and centrifuged at 8000 rpm for 10 min and the clear supernatant was used as the crude enzyme source. Enzyme Assay α-amylase activity was determined as described by Okolo et al 10. The reaction mixture (2 ml) contained of 1.25 ml of 1% soluble starch solution as the substrate, 0.25 ml of 0.1 M sodium acetate buffer (ph 5), 0.25 ml distilled water and 0.25 ml of adequately diluted crude enzyme sample. After 10 min of incubation at 50 o C, the liberated reducing sugars (glucose equivalent) were estimated by Dinitrosalicylic acid (DNS) method of Miller 11. The soluble protein of different enzyme samples was analyzed by using Folins Ciocalteu reagent as described by Lowry et al 12. Production, Partial Purification and Biochemical Characterization The time course study was carried out to monitor the production pattern of α-amylase by A. oryzae. SSF was carried out as previously described. Initial moisture content of the substrate was 70%. It was inoculated with 1 ml spore suspension ( spores/ml) and incubated at 30 o C. Samples were withdrawn every 24 h and the crude enzyme extract was checked for α-amylase activity and total soluble protein content. Partial purification of crude enzyme sample was achieved by fractionation using (NH 4 ) 2 SO 4. The crude sample was fractionated into thee, 0-40, and 70-90% based on the saturation of ammonium sulphate. The thee fractions were separately dissolved in minimum amount of 0.1 M acetate buffer (ph 5) and dialysed overnight against the same buffer. The purified fractions were assayed individually for total soluble proteins and α-amylase activity. SDS-PAGE on a 12% w/v acrylamide gel was performed for the determination of approximate molecular weight in accordance with the procedure of LaemmLi et al. 13 α-amylase activity was also localized after electrophoresis by immersing the gel for 1 h in a solution of 1% soluble starch in 0.1 M acetate buffer (ph 5) at room temperature. The gel was then kept in the same buffer for 10 min without soluble starch and stained with iodine solution (0.05 g iodine and 0.5 g KI in 100 ml) for 5 min. To determine the optimum concentration of substrate for maximum enzyme activity, starch solutions of various concentrations (0.25, 0.5, 0.75, 1.0, 1.5, 2.5 and 3%) were used for the assay and enzyme concentration remained constant. Similarly, to find out the optimum enzyme concentration for maximum α-amylase activity, various enzyme concentrations [0.25 ml (2 mg), 0.50 ml (4 mg), 0.75 ml (6 mg), 1 ml (8 mg) and 1.25 ml (10 mg)] were used for the assay and substrate (starch) concentration remained constant. To determine the optimum incubation temperature for maximum α- amylase activity, the reaction was carried out at various incubation temperatures (35, 40, 50, 55 and 60 o C) for 10 min, optimum enzyme and substrate concentration were used for assay. In order to find out optimum ph for maximum enzyme activity the reaction was carried out at different ph using 0.1 M acetate buffer of varying ph value (3.6, 4, 4.4, 4.8, 5, 5.2 and 5.6). To study the effect of metal ions on α-amylase activity, a reaction mixture containing 0.25 ml (2 mg) of enzyme solution, 1.25 ml of 1% starch solution and 0.25 ml of 1 mm metal ions solution was incubated for 10 min at 50 o C, as mentioned

3 338 INDIAN J BIOTECHNOL, JULY 2005 earlier 10 for α-amylase assay. In order to study the effect of Ca 2 + on α-amylase activity/stability, the reaction was carried out at 50, 60, 65 and 70 C with 0.25 ml of 10 mm CaCl 2 and without CaCl 2. In all the above experiments the enzyme activity was calculated as the average of thee independent sets of experiments and the standard deviation in all cases was negligible. Table 1 Partial purification of α-amylase by ammonium sulphate fractionation Sample Crude Enzyme 40-70% fraction Volume (ml) Protein (mg) Activity (U) Sp.activity (U/mg protein) fold Results and Discussion A. oryzae grew well on SBG and showed a good amount of α-amylase production, as it is evident from Fig.1. After 24 h, 817 U/gds α-amylase was produced which, increased to a maximum of 11,296 U/gds in 48 h of fermentation. Incubation beyond this period resulted in the decrease of enzyme activity. Partial Purification of α-amylase The results of partial purification studies are summarized in Table 1. With 40-70% ammonim sulphate fraction, 7.14-fold increase in the enzyme activity was obtained. Fig. 2 shows a 12% SDS- PAGE of partially purified α-amylase produced by SSF. A thick band of approximately 66 kda was obtained. Activity Staining of α-amylase Activity staining of α-amylase was performed stepwise as mentioned earlier. Enzyme activity was visible as a pale yellow band in the dark coloured gel. The band approximate to 66 kda showed a pale yellow colour thus confirmed that the band belongs to α-amylase. It is worth to mention that Chakraborty et al 14 noticed the purified α-amylase of Bacillus stearothermophilus as a monomeric protein with a molar mass of 64 kda. Similarly Lefugi et al 15 reported the molecular weight of the α-amylase from the yeast Cryptococcus sp. S-2 as 66 kda. However, the molecular weight of A. oryzae α-amylase (Takaamylase A) was estimated to be 51, by the combined use of high-pressure silica gel (TSK-GEL G3000SW) chomatography and the low angle laser light scattering technique 16. Enzyme Characterization Effect of substrate concentration on α-amylase activity Enzyme activity was evaluated with different substrate (soluble starch) concentrations as mentioned in Materials and Methods. As per the data presented in Fig. 3, maximum activity was 36.13U/mg protein with 1% starch as the substrate concentration. Fig.1 α-amylase production by A. oryzae using SBG as substrate for SFS Fig. 2 SDS-PAGE showing the partially purified α-amylase Effect of enzyme concentration on α-amylase activity Enzyme concentration influenced the α-amylase activity. Data presented in Fig. 4 showed that by using 0.25 ml (2 mg) of enzyme, the specific activity was maximum (36.88 U/mg protein). With further increase in enzyme concentration there was a sharp decline in enzyme activity. As per the concentration and total volume of substrate, enzyme concentration should be specific for attaininig maximum activity. Effect of incubation temperature on α-amylase activity The results presented in Fig. 5 suggest that for maximum enzyme activity, incubation temperature

4 PATEL et al: PARTIAL PURIFICATION & CHARACTERIZATION OF α-amylase PRODUCED BY A. ORYZAE 339 should be specific. Maximum activity (51.47 U/mg protein) was obtained at 50 o C. So it is concluded that enzyme catalyses the reaction at specific temperature and below this temperature it may be less active. Fig. 3 Effect of substrate (starch) concentration on α-amylase activity Effect of ph on α-amylase activity Optimum ph is required for maximum enzyme activity. α-amylase is an acidic enzyme, in their catalytic mechanism oxido-reduction reaction is involved, for this particular reaction, H + concentration should be optimum for the proper catalysis. Enzyme reaction was carried out at different ph. Results showed that the maximum specific activity (48.91 U/mg protein) was obtained at ph-5 (Fig. 6). Effect of metal ions on α-amylase activity Study of the different metal ions, that influence α-amylase activity, indicated that the enzyme was considerably activated by Mn 2+ and Fe 2+ ions and inhibited by Hg 2+ and Cu 2+ ions (Table 2). The stimulatory effects of Mn 2+ and Fe 2+ were also reported by Selvakumar et al 17 in the case of glucoamylase produced by A. niger. The inhibitory effect of Hg 2+ has also been reported on glucoamylase produced by A. awamori 18. Effect of Ca 2+ on α-amylase stability It has generally been reported that partially purified α-amylase, particularly those of fungal origin, lose Fig. 4 Effect of enzyme concentration on α-amylase activity Fig. 6 Effect of ph on α-amylase activity Table 2 Effect of metal ions on α-amylase activity Fig. 5 Effect of temperature on α-amylase activity Metal ions α-amylase sp. Activity (IU/mg protein) Control Mn Fe Mg Ag Cu Hg Na K

5 340 INDIAN J BIOTECHNOL, JULY 2005 References Fig. 7 Effect of Ca 2+ on α-amylase activity activity rapidly above 50 o C but the activity could be retained in the presence of calcium. α-amylase does not contain co-enzyme but they are calcium metalloenzyme with at least one atom of this metal per molecule of the enzyme. Calcium free amylases are very susceptible to denaturation by acid, heat, and urea and readily degraded by protease. Fig. 7 shows effect of CaCl 2 on α-amylase stability. At 50 o C, the α-amylase specific activity was U/mg protein. At higher temperatures 60, 65 and 70 o C, the activities were 20.93,12.10 and U/mg protein, respectively. On the other hand, when the reaction was carried out at 65 C in the presence of 10mM CaCl 2 (0.25 ml) in the reaction mixture, the activity was even better than 50 C. It has been reported 19 that the pancreatic α-amylase is a single polypeptide chain of about 475 residues and has two SH groups and four disulfide bridges and it contains a tightly bound Ca 2+. Levitzki and Steer 20 reported on a binding site for Cl -2, which effects a conformational change that enhances activity. D Amico et al 21 reported the presence of amino acid residues Arg, 195; Asn, 298 and Arg/Lys, 337 forming the chloride-binding site of several specialized α- amylases. It has been established that Ca 2+ and Cl -2 ions are essential for the stability of most of the α- 22, 23. Amylases Acknowledgement This work was financially supported by the Department of Science and Technology, Government of India and OMFB, Ministry of Science and Technology, Hungary, though Indo-Hungarian bilateral collaborative research project. 1 Pandey A, Nigam P, Soccol C R, Soccol V T, Sing D & Mohan R, Advances in microbial amylases, Biotechnol Appl Biochem, 31 (2000) Karuki T & Imanaka T, The concept of the α-amylase family: Structural similarity and common catalytic mechanisms, J Biosci Bioeng, 87 (1995) Pandey A, Nigam P, Selvakumar P & Soccol C R, Solid state fermentation for the production of industrial enzymes, Curr Sci, 77 (1999) Pandey A, Soccol C R & Mitchell D, New developments in solid state fermentation, Process Biochem, 35 (2000), Pandey A, Soccol C R, Nigam P, Brand D, Mohan R & Roussoss S, Biotechnological potential of agro-industrial residues, Part 11. Cassava bagasse, Bioresour Technol, 81 (2000) Pandey A, Soccol C R, Nigam P, Brand D, Mohan R & Roussos S, Biotechnological potential of coffee pulp and coffee husk for bioprocesses, Biochem Eng J, 6 (2000) Pandey A, Soccol C R, Rodriguez-Leon J & Nigam, P. Solid state fermentation in biotechnology (Asia Tech Publishers, Delhi) 2001, Sim T S & Oh J C S, Spent brewing grains as substrate for the production of cellulase by Trichoderma reesei QM 9414, J Ind Microbiol, 5 (1990) Barbesgaard P, Heldt-Hansen H P & Didierichan B, Minireview on the safety of Aspergillus oryzae, Appl Microbiol Biotechnol, 36 (1992) Okolo B N, Ezeogu L I & Mba C N, Production of raw starch digesting amylase by Aspergillus niger grown on native starch sources, J Sci Food Agric, 69 (1995) Miller G L, Use of Dinitro salicylic acid reagent for determination of reducing sugar, Anal Chem, 31 (1959) Lowry O H., Rosebrough N J, Farr A L & Randall R.J, Protein measurement with the Folin-Phenol reagents, J Biol Chem, 48 (1951) LaemmLi U K, Cleavage of structural protein during the assembly of the head of bacteriophage T4, Nature (Lond), 227 (1970) Chakraborty K, Bhattacharya B K & Sen S K, Purification and characterisation of a thermostable α-amylase from Bacillus stearothermophilus, Folia Micribiol, 45 (2000) Lefugi H, Chino M, Kato M & Limura Y, Raw starch digesting and thermostable α-amylase from the yeast Cryptococcus sp. S-2: Purification, characterisation, cloning and sequencing, Biochem J, 318 (1996) Takagi T, Confirmation of molecular weight of Aspergillus oryzae α-amylase using the low angle laser light scattering technique in combination with high-pressure silica gel chomatography, J Biochem, 89 (1981) Selvakumar P, Ashakumary L, Hellen A & Pandey A, Purification and characterisation of glucoamylase produced by Aspergillus niger in solid-state fermentation, Lett Appl Microbiol, 23 (1996)

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