Extracellular Amylase Activity from Endophytic Streptomyces griseoflavus P4

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1 346 Chiang Mai J. Sci. 2012; 39(2) Chiang Mai J. Sci. 2012; 39(2) : Short Communication Extracellular Amylase Activity from Endophytic Streptomyces griseoflavus P4 Julaluck Tang-um and Hataichanoke Niamsup* Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. *Author for correspondence; hataichanoke.n@cmu.ac.th Received: 8 August 2011 Accepted: 16 November 2011 ABSTRACT Amylase enzymes are currently used to accelerate starch degradation in many industrial processes. Streptomyces sp., an endophytic actinomycete, is well known as a potential source of hydrolytic enzymes, antimicrobial agents, and many secondary metabolites. Previously, an endophytic Streptomyces griseoflavus P4 was isolated from sweet pea root and identified by 16S rrna sequence analysis. Using a plate assay, the S. griseoflavus P4 was found to be capable of producing amylase enzymes. The maximum amylase activity was 1.66±0.07 U/mL on the 7 th day of growth, while the total biomass was 0.085±0.005 g of dry pellet in 100 ml medium. In this work, the P4 strain exhibited a specific activity of U/mg protein. Amylase production was found to be growthassociated and started in a log phase. Maximum enzyme production was reached during the stationary phase. The optimal conditions for activity were found to be 40 degrees C and a ph of 7. Keywords: Endophyte, actinomycete, streptomycete, amylolytic activity, starch 1. INTRODUCTION Amylases are some of the most important enzymes used in the modern biotechnological industry, especially in starch hydrolysis processes. They are employed in the food, feed, fermentation, and detergent industries. They are classified into -amylase (EC ), -amylase (EC ), and glucoamylase (EC ). The -amylase (endo 1, 4- -D-glucan glucohydrolase) randomly hydrolyzes -1, 4 glycosidic linkages in starch molecules, producing oligosaccharides of varying chain lengths with -configurations and -limited dextrins as products. These are further cleaved into glucose by exo-acting amylases and other amylolytic enzymes. The -amylase can attack the -1, 4 linkages from the nonreducing ends of the starch molecules, giving maltose units. These enzymes are used in the production of high maltose syrup from starch in combination with pullulanase. Maltose covers a wide range of application in the food and pharmaceutical industries [1-3].

2 Chiang Mai J. Sci. 2012; 39(2) 347 The occurrence of amylase in actinomycetes has been commonly observed in Nocardia and Streptomyces [4], however, the genus Streptomyces was not previously considered to be a potential source of amylolytic enzymes. The most frequently used amylases are -amylases obtained from Bacillus sp. such as B. subtilis [5], and also from mesophilic and thermophilic fungi, such as Aspergillus sp. [6]. The exploration of new bacteria and fungi as sources for potent starch converting enzymes can be beneficial. Endophytic Streptomyces griseoflavus P4 previously isolated from the sweet pea root was, thus, investigated for its capacity to produce amylase in order to be an alternative enzyme source. 2. MATERIALS AND METHODS Bacterial strain and growth conditions The P4 strain was isolated from the sweet pea root and identified as Streptomyces griseoflavus P4 by 16S rrna sequence analysis [7]. Plate assay for amylase production The ability of S. griseoflavus P4 to produce amylase was determined by the plate agar method [8]. The isolate was inoculated on an agar medium containing (g/l): 20.0 soluble starch; 0.5 yeast extract; 1.0 (NH 4 ) 2 SO 4 ; 0.3 MgSO 4 7H 2 O; 1.36 KH 2 PO 4 ; 15.0 agar. The ph was adjusted to 7.0. The substrate, 2% soluble starch, was used as the carbon source. S. griseoflavus P4 was incubated for 14 days at 30 C. After incubation, 1% iodine in 2% potassium iodide was added to the plate. The colonies which were surrounded by a clear zone were considered to be positive for amylase production. Assay of amylase activity S. griseoflavus P4 was grown aerobically in 100 ml of IMA-2 liquid culture, consisting of 5 g glucose, 5 g soluble starch, 1 g beef extract, 1 g yeast extract, 2 g NZ-case, 2 g NaCl and 1 g CaCO 3 per liter, for 7 days at 30 C under a shaking condition at 160 rpm. 5 ml of pre-culture (0.03 g dry weight/ 100 ml culture) was inoculated into amylase production medium supplemented with 2% soluble starch as the substrate and incubated on a rotary shaker at a speed of 160 rpm, 30 C. The culture filtrate containing amylase was collected by filtration every 2 days for 9 days to evaluate amylase activity and protein content. In addition, the cell pellet was measured for biomass production as described below. The experiments were performed in duplicates. Amylase activity was assayed using the dinitrosalicylic acid (DNS) method [9]. The reaction mixture containing 0.3 ml of 1% soluble starch in 0.1 M citrate phosphate buffer (ph 6.5) and 0.05 ml culture filtrate was incubated in water bath at 37 C for 30 min. 2 ml of DNS reagent were added to stop the reaction, and the mixture was boiled at 100 C for 15 min. After cooling, the reaction mixture was diluted with 4 ml distilled water, and the absorbance was measured at 540 nm (Spectrophotometer Model 6400, Jenway, UK). The assay was repeated in triplicate. One unit (U) of amylase activity was defined as the amount of enzyme necessary to liberate reducing sugars equivalent to 1 μmol of glucose per minute, at 37 C [9]. Protein content was determined by the Bradford dye binding method, using Bovine Serum Albumin (BSA) as standard. Biomass production The mycelial mass was harvested by filtration and washed three times with distilled water followed by drying in an oven at 100 C to measure dry biomass weight which is expressed in terms of g dry mass per 100 ml culture medium.

3 348 Chiang Mai J. Sci. 2012; 39(2) Optimization of amylase activity. In order to find optimum ph and temperature conditions for activity, crude amylase was isolated from S. griseoflavus P4 as a cell-free filtrate from the P4 culture in the stationary phase. The amylase activity was measured at 37 C in buffers of various phs. phs conditions of 5.0, 5.5, 6.0, used 0.1 M citric acid-sodium citrate buffer; 6.5, 7.0, used 0.1 M citrate phosphate buffer; 7.5, 8.0, 8.5, and 9.0 used 0.1 M Tris-Cl buffer. In another set of experiments, the activity was evaluated in 0.1 M citrate phosphate buffer at ph 6.5 and incubated at 30, 37, 40, 45, 50, 55, and 60 C. Other parameters and methods were the same as described above. 3. RESULTS AND DISCUSSION Plate determination of amylase produced from S. griseoflavus P4 Plate agar results showed that the P4 strain could produce amylolytic enzymes as indicated by the transparent zone around the colony after iodine addition (data not shown). This simple plate method has been demonstrated to be useful in preliminary screenings of amylase producing bacteria and is in agreement with previous reports. Amylase activity during each growth phase Results shown in Figure 1 indicate that growth and amylase production patterns were closely associated. Amylase production of the P4 strain initiated in a logarithmic growth phase and maximum enzyme activity was observed during the stationary phase (5-9 days) of growth, which is in agreement with the study by Devi et al. [9]. Chakraborty et al. [10], however, reported that amylolytic enzyme production of Streptomyces sp. D1 was independent of growth phase, reaching maximal activity at late stationary phase. The maximum amylase activity of S. Figure 1: Growth and amylase production of S. griseoflavus P4 in starch broth medium. Amylase activity and cell biomass production are represented by solid and dashed lines with open squares and with solid circles, respectively. griseoflavus P4 on the 7 th day growth was 1.66± U/mL in 0.085±0.005 g dry weight per 100 ml culture and the maximum biomass production was reached at 120 h after incubation. On the contrary, Norcardiopsis sp. [4] and S. gulbargensis sp. DAS 131 [11] showed the maximum biomass production at the end of the logarithmic phase, at 72 h and 48 h after incubation, respectively. It follows that S. griseoflavus P4 is a slower growing bacterium as compared to the other species. Stamford et al. [12] reported that Streptosorangium sp., an endophytic actinomycete isolated from maize leaves, produced a maximum glucoamylase activity equivalent to 41.4 U/mL. Champreda et al. [2] reported a much lower -amylase activity of 0.312±0.020 U/mL by Fusicoccum sp. BCC4124, an endophytic fungus. The specific activity of the crude amylase from P4 strain was calculated to be U/mg protein, similar to a specific activity of U/ mg protein reported for partially purified amylase enzyme from Streptomyces strain D1 [10]. It appears that although the strain P4 exhibits lower specific activity than

4 Chiang Mai J. Sci. 2012; 39(2) 349 Streptosorangium sp., the P4 strain s specific activity is comparable to that of Streptomyces strain D1, and much higher than that of Fusicoccum sp. BCC4124. Optimization of amylase activity In order to elucidate the optimal conditions for amylase activity, the assay was performed at different phs and temperatures. As shown in Figure 2, extracellular amylase from S. griseoflavus P4 exhibited maximal activity at 40 C, while the ph of 7.0 was optimal (Figure 3). An optimum ph of 6.0 and temperatures of Figure 2: Amylase activity of S. griseoflavas P4 at different temperatures in a buffer of ph 6.5. Figure 3: Amylase activity of S. griseoflavas P4 at different phs when incubated at 37 C. 50 to 60 C were observed for amylases from Streptomyces sp. isolated in a milling factory [13]. In contrast, -amylases from haloalkaiphilic Streptomyces strain D1 and alkalithermotolerant S. gulbargenesis DAS131 exhibited the highest activity at a ph of 9.0 and 45 C [10,11]. 4. CONCLUSIONS This study presented the amylase production pattern of the S. griseoflavus P4 which was shown to be growth-associated. It was found that the P4 could produce amylase enzyme beginning in a log phase and reaching maximum production during the stationary phase of growth. The optimum ph and temperature conditions for the amylase activity were at ph 7 and 40 C. The reported enzyme may have wide industrial application. ACKNOWLEDGEMENTS This work was financially supported by the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Lanna Products Company, and the Graduate School of Chiang Mai University, Thailand. We would like to acknowledge Asst. Prof. Dr. Ampan Bhromsiri for providing endophytic S. griseoflavus P4. REFERENCES [1] Shambe T., and Ejembi O., Production of amylase and cellulase: degradation of starch and carboxymethylcellulose by extracellular enzymes from four fungal species, Enz. Microb. Tech., 1986; 9: [2] Champreda, V., Kanokratana, P., Sripang, R., Tanapongpipat, S., and Eurwilaichitr, L., Purification, biochemical charac- terization, and gene cloning of a new extracellular thermotolerant and glucose tolerant maltooligosaccharide-forming amylase

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