Production of amylase enzyme by Aspergillus niger using Rice bran and potato skin

 

Sangeetha, S. and  Anuradha, R.*

PG and Research Department of Biochemistry, S.T.E.T. Women’s College, Sundarakkottai, Mannargudi -614016, Tamilnadu, India.

 

ABSTRACT:

The present study was carried out to determine the production of the amylase enzyme from Aspergillus niger  under submerged fermentation method. Amylase containing protein content was estimated. Effect of Ph, temperature and substrate concentration of amylase activity were also monitered during amylase production. The enzyme was active at pH 4 and 40°C.  A maximum amylase production 15.20 IU/ml in rice bran and 13.05 IU/ml in potato skin waste medium was obtained at 48 hours under optimum conditions.

 

KEYWORDS: Aspergillus niger, rice bran, potato skin, pH, temperature, protein

 

INTRODUCTION:

The amylases are being exploited due to their low cost of extractions, thermal and pH stability substrate specificity and activity in organic solvents.The cheif products of commercial amylase are aspergillus niger, candida cylindralea, rhizopus japonius, rhizopus oryzae (Gog Fredson et al., 1998). In recent years, the potential of using mirco organism as biotechnological sources of industrially relevant enzymes has stimulated interest in the exploration of extra cellular enzymatic activity in several microorganisms (Akpam et al., 1999).

 

Amylase is also secreted by the acines cells of the pancreas and is measured blood Samples. It is elevated in pacreatitis, trauma to the bower and mumps infection. But the enzymes from fungi generally meet industrial demand in low cost. studies on conditions for the production of extra cellular amylase by aspergillus niger show variation  among different strains but the requirements for carbon source such as malt extract peptone, yeast extract, ammonium chloride, tryptone and various salts such as KCl, MgSo4,FeSo4.

Some inducers are also produce amylase enzyme such as coconut oil or, gingelly oil (Kudo et al.,1995). Amylase enzyme breaks down starch molecules in to smaller sugars, including bacillus subtilis and aspergillus niger produce large amounts of amylase diluting their normal cycles of growth. The enzyme can also be used to produce gasohol, with corn starch as the starting material and may spot remover contain amylase to breakdown the starches in plant material that soil clothing (Keithweller et al., 1995)

 

MATERIALS AND METHODS:

Sample collection

Rice bran were produced from rice mill, Mannargudi . The substrates were ground  in to coarse powder with a blender. Cheaply available natural waste namely rice bran were procured from rice mill soaked in 50 ml (5.5g waste ) distilled water and autoclaved at 121°C for 15 minutes. After cooling the liquid content of the rice bran was squeezed  out using a muslin cloth by a hand mill. This extract was used as substrate. Various sugar such as glucose, fructose, Starch,  mannitol, lactose (1%) are added to this extract .The initial PH of the medium was maintained at pH7

 

 

Microorganism used

Strains of  aspergillus niger are used in the commercial production of citric, gluconic and gallic acids. Almost all of the commercial citric acids is produced by Aspergillus niger growing sucrose solutions. The organisms are a source of amylase and pectinolytic enzymes.

 

Fermentation  process (Shake flask culture method)

After preparation of the production media 10% inoculums was transferred to the production medium and incubated at 28°C on a rotary shaker at 150rpm for 72 hrs. The mycelium was separated by filtration was used for the enzyme assay.

 

Estimation of total soluble sugar

The concentration of pentose, hexose, disaccharide, including sucrose, lactose, maltose and hexuoric acid present freely or polysaccharide can be estimated by anthrone method. 10keto 9, 10-hydro anthrone reduction produce of anthroquinine react by condensing with carbohydrate furfural and a blue colour is found in a concentrated solution.

 

Estimation of protein (Lowry’s method, 1951)

The protein content of enzyme was estimated by Lowry’s method .protein reacts with copper at an alkaline PH to form copper protein complex. This complex, when treated with phosphotungestic (Folin’s reagent or phenol reagent) forms blue which is measured at 640nm in a spectrophotometer.

 

Effect of PH and Temperature

Effect of pH on amylase activity were determined by incubating the reaction mixture at PH values ranging from 3-7.(3,4,5,6,7).Effect of temperature on amylase activity was determined by incubating the reaction mixture at temperature ranging from 25°C-65°C (25,35,45,55,65).

 

RESULTS AND DISCUSSION

In the present investigation on the amylase production in submerged fermentation of potato skin waste and rice bran medium was carried out. Effect of PH, effect of temperature, estimation of total sugar and total soluble protein were analyzed.

 

Effect of PH and temperature of amylase activity and stability were monitored during amylase production. Aspergillus niger produce black colour colonies on the rosebengal  Chloremphenicol agar plates . Macroscopic character are important in subgenic classification. Aspergillus niger produce black colour spore on the rosebengal chloramphenicol agar plates. Aspergillus niger is an anemographic genus that produce sexual spores (conidia, cardio spores) on a specialized structure which is a characteristic of genus called aspergillum. Aspergillus niger gives a direct visual indication of starch hydrolysis. It requires no flooding, no prior replication of colonies on slants the zones are very sharp and contract sharply with  the blue black ground.

 

The enzyme was active at PH 7 and 37°C, stable at pH 4 and 40°C. The enzyme used for removal of triglycerides in laundry (Kamahi et al., 2003). A maximum amylase production 15.20 IU/ml in rice bran and 13.05 IU/ml in potato skin waste medium was obtained at 48 hours under optimum conditions (Table 1). The total sugar concentration in rice bran. The production of amylase was monitored at 24, 48, 72 hours intervals were 9.5 mg/ml,10.03 mg/ml and 12.5mg/ml for rice bran and 7mg/ml, 8.74 mg/ml and 10.67 mg/ml for potato skin (Table 2). A prolonged incubation period beyond this period did not help to further increase in the enzyme yield (Nutan et al., 2001).

 

The amylase production was carried out in rice bran and potato skin. The protein content was mentioned at 24, 48 and 72 hrs intervals were 22.70 mg/ml, 24.30 mg/ml and 20.70 mg/ml and 20.15 mg/ml, 21.95mg/ml, 19.70 mg/ml respectively (Table 3).The protein content was increased up to 48 hrs after which it decreases.The maximum growth of Aspergillus niger was increased from 48 hrs to72 hrs (Table 4). The maintenance of favourable PH is very essential for successful production and amylase (Memahon et al., 1999). The maximum amylase production in rice bran and potato skin medium 14.03 IU/ml and  9.64 IU/ml was obtained when the pH of the medium was maintained at pH 5. The maximum amylase production 10.60 IU/ml for rice bran and 8.05 IU/ml for potato skin waste medium was maintained at temperature 35°C (Table 5 and Table 6). The maintenance and favourable temperature is essential for the production of amylase (Khoo et al., 1994).

In conclusion, the crude amylase of Aspergillus niger was selected for this study and is capable of hydrolyzing both cereal and tuber starches into glucose, which can then be used directly in the production of ethanol and fructose.

 

Table  1: Amylase production in submerged fermentation

S.

Time (hrs)

Amylase (IU/ml)

Potato ski   (IU/ml)

1

24

13.43

11.59

2

48

15.20

13.05

3

72

11.0

9.12

 

Table  2: Total sugar concentration by amylase production

S.No

Time(hrs)

Rice bran (mg/ml)

Potato skin (mg/ml)

1

24

9.5

7.00

2

48

10.63

8.74

3

72

12.5

10.67

 

Table  3: Total protein concentration by amylase production

S.No.

Time (hrs)

Protein content

Rice bran ( mg/ml)

Potato skin (mg/ml)

1

24

22.70

20.15

2

48

24.30

21.95

3

72

20.70

19.70

 

Table  4: Growth curve in rice bran potato skin waste medium

S.No.

Time (hrs)

Optical Density value Rice bran

Potato skin

1

0

0.75

0.69

2

12

7.00

6.00

3

24

13.02

10.02

4

48

16.00

13.06

5

72

20.04

17.95

 

Table 5: Effect of pH on amylase production

S.No.

PH range

Rice bran (IU/ml)

Potato skin (IU/ml)

1

4

6.46

6.00

2

5

14.03

9.64

3

6

13.12

8.95

4

7

10.43

7.12

5

8

12.06

8.05

 

Table  6: Effect of temperature on amylase production

S.No.

Temperature range °C

 Substrate (IU/ml)

Rice bran °C

Potato skin °C

1

25°C

13.86

10.56

2

35°C

10.60

8.05

3

45°C

8.95

7.43

4

55°C

7.54

7.10

5

65°C

6.42

6.95

 

ACKNOWLEDGEMENTS

The authors are grateful to the management of STET Women’s College, Mannargudi for their encouragement and support.

 

REFERENCES

1.       Akpam., Memahon., Nutan; Production of amylase byAspergillus niger in solid state fermentation. J.Microbiology.1999, 45 (5); 233-245.

2.       Gog Fredson, S.E; In Microbiol enzymes and biotechnology,  Applied science,1988,255- 273.

3.       Kamahi., mahadix., nutan; Production of Amylase by Bacillus Subtilis, in solid state fermentation, Journal of Biotechnology, 1993, 45(5); 23-25.

4.       Keithweller., Edwin,P., Janis Cristie; Journal Bacteriol Volume, 1995, 177(22), 6401-6410.

5.       Khoo., Hoosain., Nutan; Production of catalase in solid state fermentation, Journal of Biology, 1994, 53(6); 45-47.

6.       Kudo, N., Horinuchi,S., Edwin,P; Journal of bacterial Volume,1995, 177, 6401-6410.

7.       Lowry’s,D.H., Rose Brough, N.J; Farv, AI; Protein measurement with the folin phenolreagent, Biological chemistry, 1956, 193: 265-275.

8.       Nutan., Mahadik., Ulkas., Puntambekar., Kulbhushan.B., Bartawde., Jayant.M; Khire., and Diganbar.V., Gokhak; Production of acidic lipase by Aspergillus niger in solid state fermentation, Rev. Microbial volume, 2002, 25(2), 110-140.

 

Received on 16.02.2012

Modified on 09.03.2012

Accepted on 18.03.2012     

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Research J. Science and Tech.  4(2): March-April 2012: 59-61