Optimization And Production Of Fungal Amylases Using Aspergillus Oryzae

Optimization And Production Of Fungal Amylases Using Aspergillus Oryzae

Adharsh Rajasekar, RahulSribalaji Gandhiraj Dhamodharan

Aspergillus oryzae (NCIM 932) was grown in rice bran since its a good carbon and starch source which has the ability to produce amylase. The optical growth of the isolate was found to be at 26 hours. Optimum amylase activity in rice bran was expressed on the fifth day of incubation as 324.07 µmol/lit min. The optimum pH was found to be 6 since the enzyme activity was higher and stable. This investigation suggests a means of production of amylase using rice bran for industrial purposes.

Keywords: Aspergillus oryzae, rice bran, amylase, pH, optical growth, enzyme activity

1. 1. INTRODUCTION

       

      The genus Aspergillus includes over 185 species and about 20 species have so far been reported as causing opportunistic infections in man (Tortora et al., 2004; Prescott et al., 2005). Among these species, A.fumigatus is the most commonly isolated species followed by A. flavus and A. niger. A. clavatus, A.glancus, A. oryzae, A. terreus, A. ustus and A. versicolor are among the other species that are not commonly isolated and are considered as non.oppurtunistic pathogens (Brock DT, Madigan MT 1991; Brock et al., 1994). A. oryzae has attracted attention for their role in fermentation of oriental food products or industrial application of hydrolytic enzymes. Also the fungus is used in the production of rice vinegars. Within the range of carbohydrolases produced by this species, the majority has been considered as -amylase and glucoamylase1-3.

      -amylase(EC 3.2.1.1) has found its application in a range of industries including food, brewing, distilling industry, textile, paper pharmaceutical and bioconversion of solid waste etc.,(Adeniran AH et al., 2009). Amylases have been reported to be produced by plant, animal and microbial sources, although the microbial amylase production has been reported to be most effective. Solid state fermentation which has been reported to be a bit cheaper because of the enzyme extraction procedures (Alva S et al., 2007) is a ray of hope. In case of SSF, the cost of the substrate also plays a key role in deciding the cost of production. Agro industrial wastes have been reportedly used as good substrates for the effective cost cutting in the production of alpha amylases (Pandey et al., 1995) and are thus attracting researchers in using agro industrial waste as a substrate for alpha amylase production. Fungal species have been studied a lot for the production of alpha amylase (Pandey A, Nigam P et al., 2000 Adeniran AH et al., 2009)because

      of the low cost of substrates used for the production of alpha amylases. Thus the present study was designed in search of cheaper carbon sources for the production of alpha amylase enzyme by fungal strains.

   2. MATERIALS AND METHODS
      1. Culture conditions and preparation of inocula

          

         The pure culture (isolate) was obtained from NCIM; Pune was subcultured and maintained on Potato Dextrose agar plates. The fungus was further subcultured into test tubes of the same medium and incubated at 25°C. Seventy-two-hour-old culture of A. orzyae was used as inoculum. The culture was grown in a defined medium of the underlisted composition: MgSO4.7H20, K2HPO4, KH2PO4, L-cysteine, biotin, thiamine and FeSO4.7H20 with added carbon and nitrogen sources (Sigma). Conical flasks containing growth medium was inoculated with 1 ml of an aqueous spore suspension containing approximately 5×104 spores per ml of the isolate. Experimental and control flasks were incubated in the rotary shaker at 120rpm at 25°C for three days.

      2. Carbon and Nitrogen sources

         In this project, certain carbon and nitrogen sources of the growth medium were used. While the tryptone was used as nitrogen source of growth, the carbon source employed was starch. However, when the carbon source of growth was maltose, the nitrogen source employed was ammonium chloride.

      3. Rice as a source of carbon and growth medium

         Rice bran was bought at the main market, Tanjore, Tamilnadu, India. The rice bran was added to distilled water (1% w/v) and autoclaved at 15 Ib/in2 at 121°C. Conical flasks (250 ml) containing 100 ml of the rice medium was inoculated with 1 ml of an aqueous spore suspension containing approximately 5×104 spores per ml of the isolate. Experimental and control flasks were incubated without shaking at 250C. On a daily basis, the contents of each flask were filtered through glass fiber filter paper (Whatman GF/A). The protein content of the filtrates were determined using the method of Lowry et al. (1951).The filtrates were analyzed for

         amylase activity using the modified method of Xiao et al. (2006). The filtrates were used as crude preparation.

      4. Determination of pH

         The substrate along with the inoculate was subjected to various levels of pH ranging from 4- 8 to determine the optimum pH.

      5. Solid state fermentation

         Conical flask containing rice bran and the isolate were incubated at various temperatures ranging from 30-45°C for 3-7 days.

      6. Enzyme Assay

          

         Enzyme activity was defined in units. One unit of enzyme activity was defined as the amount of enzyme which produced 0.1% reduction in the blue colour of the starch-iodine complex.

   3. RESULTS
      

      pH	4	5	6	7	8
      Enzyme Activity( µmol/litmin)	338.46	346.23	358.51	342.59	336.11

      Enzyme Activity( µmol/litmin)

      Enzyme Activity( µmol/litmin)

       

      Table 1: Optimization of pH for enzyme activity

      360

      355

      350

      345

      340

      335

      330

      0

      2

      4

      6

      8

      10

      360

      355

      350

      345

      340

      335

      330

      0

      2

      4

      6

      8

      10

      pH

      pH

       

      Figure 1: optimization of pH for enzyme activity

      Temperature(°C)	30	35	40	45
      Enzyme Activity( µmol/litmin)	320.04	356.48	310.18	301.18

      Enzyme Activity( µmol/litmin

      Enzyme Activity( µmol/litmin

       

      Table 2: Optimization of Temperature for enzymatic activity

      360

      350

      340

      330

      320

      310

      300

      290

      0

      10

      20

      30

      40

      50

      360

      350

      340

      330

      320

      310

      300

      290

      0

      10

      20

      30

      40

      50

      Temperature (°C)

      Temperature (°C)

       

       

      Figure 2: Optimization of Temperature for enzymatic activity

      Days	3	4	5	6	7
      Enzyme Activity( µmol/litmin)	296.29	305.55	324.07	310.18	296.29

      Table 3: SSF performed from days 3-7 for enzymatic activity

      Enzymatic activity(µmol/litmin)

      Enzymatic activity(µmol/litmin)

       

      330

      325

      320

      315

      310

      305

      300

      295

      290

      0 1 2 3 4 5 6 7 8

      Days

      Figure 3: SSF performed from days 3- 7 for enzymatic activity

DISCUSSION

Amylase was produced by A. oryzae in growth medium containing rice as carbon and growth source at 35°C. Production of activity was optimum on the fifth day. Conditions of growth of the organism seemed viable for amylase production. Enzymes occur in every living cell, hence in all microorganisms. Each single strain of organism produces a large number of enzymes, hydrolyzing, oxidizing or reducing, and metabolic in nature (Prescott et al., 2005). Bacterial -amylases are produced at a much wider range of temperature. Bacillus amyloliquefaciens, B. subtilis, B. licheniformis and B. stearothermophilus are among the most commonly used Bacillus sp. reported to produce – amylase at temperatures 37-60°C (Mielenz, 1983). Temperature plays an important role in terms of amylase production and organism growth. Hence, the optimum temperature depends on whether the culture is mesophilic, thermophilic or psycrophilic. Among the fungi, most amylase production studies have been done with mesophilic fungi within the temperature range of 2537°C (Francis et al., 2003). The carbon sources (starch and maltose) employed in this investigation, incorporated into the growth medium, induced amylase production by A. oryzae. Bread, starch and maltose, sucrose, lactose, glucose and galactose as carbon sources with potassium nitrate as nitrogen source supported growth and -amylase production by Lasiodiplodia theobromae (Prescott et al., 2005).An extremely important use for fungal amylases is in conversion of partially acid hydrolyzed starch to sweet syrups. Acid hydrolysis is a random action whereas enzymic hydrolysis is a patterned one. To meet the demand of industries, low-cost medium is required for the production of –

amylase. Both solid state fermentation (SSF) and submerged fermentation (SmF) could be used for the production of -amylases, although traditionally these have been obtained from submerged cultures because of ease of handling and greater control of environmental factors such as temperature and pH. SSF has been used for long to convert moist agricultural polymeric substrates such as wheat, rice, soy, cassava, etc. into fermented food products including industrial enzymes (Pandey et al., 1995). Conclusively, amylase can be produced for industrial purpose from A. oryzae grown in rice bran and the defined synthetic growth medium with starch as carbon source and tryptone as nitrogen source.

1. REFERENCES

Adeniran AH and Abiose SH, Amylolytic potentiality of fungi isolated from some Nigerian agricultural wastes. African Journal of Biotechnology, 2009, 8 (4): 667-672.

Alva S, Anupama J, Savla J, Chiu YY, Vyshali P, Shruti M, Yogeetha BS, Bhavya D, Purvi J, Ruchi K, Kumudini BS and Varalakshmi KN, Production and characterization of fungal amylase enzyme isolated from Aspergillus sp. JGI 12 in solid state culture, African Journal of Biotechnology, 2007, 6 (5): 576-581.

Brock DT, Madigan MT (1991). Biology of Microorganisms. Prentice-Hall International Inc. 900pp.

Brock DT, Madigan MT, Martinko JM, Parker J (1994). Biology of Microorganisms. Prentice- Hall, Inc. Eaglewood Cliffs, New Jersey. Pp 528-530.

Francis F, Sabu A, Nampoothiri KM, Ramachandran S, Ghosh S, Szakacs G, Pandey A (2003).

Use of response surface methodology

for optimizing process parameters for the production of -amylase by Aspergillus oryzae, Biochem. Eng. J. 15: 107115.

Liang XQ, Holbrook CC, Lynch RE, Guo BZ (2005). Beta-1,3- glucanase activity in peanut seed (Arachis hypogaea) is induced by inoculation with Aspergillus flavus and copurifies with a conglutin-like protein. Phytopathology 95: 506-511.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193:265-275.

Mielenz JR (1983). Bacillus stearothermophilus contains a plasmidborne gene for -amylase.

Proc. Natl. Acad. Sci. 80: 59755979.

Pandey A, Ashakumary L, Selvakumar P (1995). Copra waste- A novel substrate for solid-state fermentation. Biores. Technol. 51: 217220.

Pandey A, Nigam P, Soccol CR, Soccol, VT, Singh D, Mohan R, Advances in microbial amylases. Biotechnol. Appl. Biochem, 2000, 31: 135152.

Prescott LM, Harley JR, Klein DA (2005). Microbiology. McGraw Hill, New York. 992pp. Tortora GJ, Funke BR, Case CL (2004). Microbiology: An Introduction. Pearson Education Inc. 898pp.

Willey JM, Linda MS, Woolverton CJ (2008). Prescott, Harley and Kleins Microbiology. McGraw Hill Companies Inc. 1088pp.

Xiao Z, Storms R, Tsang A (2006). A quantitative starch-iodine method for measuring alpha- amylase and glucoamylase activities. Analytical Biochemistry 351(1): 146-148.