Research paper

Research Paper

February 27

2011Abstract:The present study covers the isolation of fungi Aspergillus niger from garden soil of government science collage, K.K.Shastri campus, maninager, Ahmedabad. The fungi was identified using lactophenol cotton blue staining method and was tested in starch hydrolysis agar medium for its amylase enzyme production; a highly demanded industrial enzyme in various sectors such as food, pharmaceuticals, textiles, detergents,etc. It also includes fermentative production of Amylase enzyme in submerged aerobic fermentation process using the isolate. The effect of different carbon source, nitrogen compound and physico-chemical conditions like temperature, pH and incubation periods were studied for derivation of amylase enzyme.

Key words: Aspergillus niger, amylase enzyme, submerged aerobic fermentation

Isolation of Aspergillus niger from soil and production of Amylase enzyme

ACKNOWLEDGEMENT

We take this opportunity with much pleasure to thank all the people who have helped us through the course of our journey towards producing this research paper.

We are greatly indepted to express our gratitude to the Almighty for such strength through the journey on path that leads to success.

We are thankful to our principal Dr.Y.M. Dalal and our Head of the Department Dr. B.N. Yagnik for their wholehearted support and cooperation in producing the subject matter. We hope you would undoubtedly find the matter interesting and informative as well. We are sincerely thankful to our supervisor, Miss. Tallika Patel for her guidance, help and motivation. Apart from the subject of our research, we learnt a lot from her, which we are sure, will be useful in different stages of our lives. Without her valuable guidance and time spent behind us, it would have been difficult to keep up the constant high spirit of work.

We would like to express gratitude to the other faculty members Miss. Chinki Soni, Miss. Deepmala Lawani, Mrs. Pratima sharma, Mr. Omkar Panchal, Mr. Chirag Shah, Mr. Manan thakker and Mr. Arpan Bhatt for many helpful comments. My sincere gratitude also goes to all those who instructed and taught me through the year. We also like to express our special thank to our lab assistant Mr. Ramesh Thoda, without whom support the study could not be completed fairly.

We are especially grateful to our colleagues for their assistance, criticisms and useful insights. We are thankful to all the other fellow students (our seniors) of Government Science College (khokhara) with whom we share tons of fond memories. We would like to acknowledge the support and encouragement of all our friends.

Finally, this research project would not have been possible without the confidence, endurance and support of our families. Our families have always been a source of inspiration and encouragement. We wish to thank our parents whose love, teachings and support have brought us this far. We wish to thank our brothers and sisters for their affection, understanding and patience.

Last but not the least thanks to all readers for their keen interest in our work.

Authers:

Khushboo. Y. Sheth

Yesha. A. Patel

Deval. P. Vyas

INTRODUCTION

Isolation of Aspergillus niger from garden soil for the production of amylase enzyme and effect of various parameters on enzyme production is the main aim of this study. Enzymes are protein catalysts synthesized by living systems and are important in synthetic as well as degradative process. Amylases are starch-degrading enzymes that catalyze the hydrolysis of internal a-1,4-O-glycosidic bonds in polysaccharides such as starch in to simple sugar (glucose & maltose). It is produced by a variety of living organisms ranging from bacteria, fungi to plants and humans [14, 15].

Enzymes are catalysts of biological processes. They are synthesized in cells by the normal machinery of protein synthesis. The structure of any given enzyme is encoded by a structural gene, whose DNA base sequence is transcribed into a messenger RNA, and the mRNA us translated from its triplet code into the amino acid sequence of the desired protein by the ribosome and associated factors [54,55]. The enzyme then folds spontaneously into its active conformation. Posttranslational modifications may be required to target an enzyme to its ultimate intracellular or extracellular location.

Enzymes (including amylases) are the primary metabolites of microorganisms. Microbial production of primary metabolites contributes significantly to the quality of life. Through fermentation, microorganisms growing on inexpensive carbon sources can produce valuable products such as enzymes, amino acids, nucleotides, organic acids, and vitamins which can be added to food to enhance its flavor or increase its nutritive value.

Amylases from plant and microbial sources have been employed for centuries as food additives. Barley amylases have been used in the brewing industry. Fungal amylases have been widely used for the preparation of oriental foods. In spite of the wide distribution of amylases, microbial sources, namely fungal and bacterial amylases, are used for the industrial production due to advantages such as cost effectiveness, consistency, less time and space required for production and ease of process modification and optimization [4]. Due to the increasing demand for these enzymes in various industries, there is enormous interest in developing enzymes with better properties such as raw starch degrading amylases suitable for industrial applications and their cost effective production techniques.

Nowadays the new potential of using microorganism as biotechnological sources of industrially relevant enzymes has stimulated renewed interest in the exploration of extra cellular enzymatic activity in several microorganisms. Although many microorganisms produce this enzyme, the ones most commonly used for their industrial production are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and Aspergillus niger. Among mold species producing high levels of amylase, Aspergillus niger is used for commercial production of alpha amylase. This enzyme is an extra–cellular enzyme and therefore can be easily separated from the cell mass.

Fermentation can be defined as aerobic or anaerobic oxidation of substrate to form the product of high value using microorganisms. To meet the demand of industries, low-cost medium is required for the production of a-amylase. Both 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.

For amylase production, liquid medium was used by shake flask method carried out at laboratory scale. Shake flask method is best suited at laboratory scale as maximum product formation can be obtained at low cost and a large number of experimental values can be evaluated in minimum time, with low risk of contamination and lesser chances of media and material wastage. Also, optimum conditions for maximum enzyme production can be set for production at industrial scale.

Protein estimation in any sample can be carried out by following methods:

(1) Biuret method (2) Folin Lowry’s method

Most protein estimation techniques use Bovine Serum Albumin (BSA) universally as a standard protein, because of its low cost, high purity and ready availability. Folin Lowry’s method is sensitive down to about 10µg/mL and is probably the most widely used protein assay. In this method, the incubation time is very critical for a reproducible assay. The reaction is also dependent on pH and a working range of pH 9 to 10.5 is essential.

Purification processes in downstream processing after fermentation strongly depend on the market, processing cost, final quality, and available technology. Most enzymes are purified by chromatographic techniques after crude isolation by precipitation with ammonium sulphate and membrane separations [70].

REVIEW OF LITERATURE

Aspergillus niger:

Amylases:

Although amylases are widespread in nature, microbes serve as a preferred source of these enzymes because of their rapid growth, the limited space required for their cultivation and the ease with which they can be genetically manipulated to generate new enzymes with altered properties that are desirable for their various applications.

Amylases are one of the most important industrial enzymes that have a wide variety of applications ranging from conversion of starch to sugar syrups, to the production of cyclodextrins for the pharmaceutical industry. These enzymes account for about 30 % of the world’s enzyme production [2]. The amylase family can roughly be divided into two groups: The starch hydrolyzing enzymes and the starch modifying, or transglycosylating enzymes. The enzymatic hydrolysis is preferred to acid hydrolysis in starch processing industry due to a number of advantages such as specificity of the reaction, stability of the generated products, lower energy requirements and elimination of neutralization steps[3].

Amylases are an extracellular enzyme and are classified based on how they break down starch molecules

i. α-amylase (alpha-amylase) - Reduces the viscosity of starch by breaking down the bonds at random, therefore producing varied sized chains of glucose

ii. ß-amylase (Beta-amylase) - Breaks the glucose-glucose bonds down by removing two glucose units at a time, thereby producing maltose

iii. Amyloglucosidase (AMG) - Breaks successive bonds from the non-reducing end of the straight chain, producing glucose

Many microbial amylases usually contain a mixture of these amylases.

To prepare these extra cellular enzymes on a commercial scale, many attempts have been made to specify cultural conditions and to select superior strains of the fungus [5, 6]. Few attempts have been made to elucidate the control mechanism involved in the formation and secretion of the extra cellular enzymes. Among mold species producing high levels of amylase, Aspergillus niger is used for commercial production of alpha amylase.

MATERIALS

[A] REAGENTS:

Enzyme production:-

(1) Starch agar medium (gm/100ml):-

Soluble starch : 1.2

Beef extract : 0.3

Agar : 3.0

Distilled water : 100ml

pH : 7.5

Dissolve by heat beef extract and agar in 50ml of distilled water. Dissolve separately soluble starch in about 25ml of warm distilled water. Mix the above solution and adjust the final volume to 100ml. Autoclave for 15mins at 15 lbs pressure and at 121ºC.

Medium is used for detection of amylase production (starch hydrolysis) by organism from soil sample.

(2) Fermentation medium (gm/liter):-

KH2PO4 : 1.4

NH4NO3 : 10

KCl : 0.5

MgSO4.7H2O : 0.1

FeSO4.7H2O : 0.01

Soluble starch : 20

pH : 6.5

Distribute in 100 ml of medium into 250 ml Erlenmeyer flasks and sterilize by autoclaving at 121ºC for 15 minutes. Allow to cool down to room temperature.

(3) Culture of Aspergillus previously isolated from soil and identified by using standard method; lactophenol cotton blue staining method under microscope.

Enzyme purification:-

(1) Phosphate buffer (50mM, pH 7.2)(2) Ammonium sulphate

Enzyme assay:-

(1) Starch hydrolysis assay:-

Phosphate buffer (20mM, pH 7.0)

0.01N HCl

0.1% starch

0.01N I2 solution

Purified enzyme sample

(2) Protein estimation:-a. BSA solution: add 0.25g of BSA powder in 10mL distilled water to get 2.5mg/mL concentration

of BSA solution to be used as standard protein solution.b. Folin ciocalteau reagentc. Samples are diluted 10 times for protein estimation.

[B] INSTRUMENTS:

(1) Autoclave (from Sedko Laboratory Equipments)(2) pH meter (from Chemline, Model CL110)(3) Microwave Oven (IFB)(4) Incubator (from EIE Instrument Pvt. Ltd.)(5) Orbital shaker(6) Centrifuge (7) Digital photocolorimeter

[C] Others:

Whatman filter paper-1 (Qualitative Disk Filters, Sonar Axiva), Erlenmeyer flasks (Ambrosil), Pipettes, Petridishes, Spreader, Test tubes, Micropipettes, Tips.

PROCEDURE

(1) Isolation and identification :

Isolation of amylase producer from soil:-

The soil contains a rich deposit of both bacteria and fungi, which produce amylases. Starch hydrolyzing fungi or bacteria could be isolated from the soil, foods or could be purchased. Soil samples were collected from the garden of govt. sci. college, K.K Shastri campus, Khokhra, Maninager (E), Ahmedabad. The soil samples were collected in a sterile container and brought to the laboratory for further processing. Soil samples were serially diluted up to 10-3 dilutions by the serial dilution method [17] and spraded on starch agar plate in to which an antibiotic streptomycin is added after sterilization of medium to avoid bacterial contamination. The plates were incubated for 72h at room temperature.

Identification of Aspergillus niger strain producing amylase:-

After incubation mix culture of amylase producer fungi were obtained which were identified using standard method of fungal identification; lactophenol cotton blue staining method under microscope. The highest enzyme producer was identified by layering 1% of iodine solution on the agar plates and zone of clearance was observed for screening the fungi [16]. The identified fungal colony was again incubated in starch agar medium for 72h to get pure culture of Aspergillus niger.

Preservation of culture:-

The obtained pure culture of Aspergillus niger was preserved in starch agar slants covered with parafilm at lower temperature.

(2) Fermentative production :

Inoculum preparation:-

Pour 10 ml of sterile distilled water on the slant containing fungal spores. Scrape with a wire loop to loosen the spores. A standardized inoculum size of conidia (each ml of cells suspension contained 2.0 X 106 cells) was transferred from a stock culture in 250ml flask containing 100 ml of growth medium[18].

Submerged fermentation:-

Submerged fermentation was carried out in the Ehlenmeyer flasks by taking 100 ml of amylase production medium [19]; containing KH2PO4 (1.4 gm.L-1), NH4NO3 (10 gm.L-1), KCl (0.5 gm.L-1), MgSO4.7H2O (0.1 gm.L-1), FeSO4.7H2O (0.01 gm.L-1), Soluble starch (20 gm.L-1) and pH adjusted at 6.5. The flasks were incubated for 72 hrs at 28°C ± 2°C on a rotatory shaker at 150 rpm.

(3) Extraction and purification of enzyme :

Extraction of enzyme:-

It is very easy to remove the fungal mycelium from the enzyme production medium. Pour the whole content of the flask containing the growing fungus through a funnel fitted with Whattman number 1 filter paper. The filtrate contains the crude amylase.

Purification (precipitation) of enzyme:-

The enzyme was precipitated from a clear supernatant at 4°C by adding solid ammonium sulphate to achieve 85% saturation. The ammonium sulphate was added slowly , keeping the solution in ice and the protein was allowed to precipitate by keeping it overnight at 4°C. The protein was separated by centrifugation at 2000 rpm for 30 minutes at 4°C, dissolved in minimum volume of phosphate buffer (50mM, pH 7.2) and used immediately for activity determination.

(4) Enzyme assay :

Enzyme activity was determined by DNS method described by using starch as the substrate[21]. The reaction mixture contained the following in a total volume of 8ml: 5ml of phosphate buffer (pH 7.0), 2ml 0.1% starch solution, 1ml of active enzyme in test flask and 1ml of inactive enzyme in both the controls (starch control and enzyme control). Incubate all 3 flasks for 10mins at room temperature (25-30°C) in dark. After incubation period, inactivate the enzyme activity by adding 2ml of 0.01N HCl. Add 4ml of 0.01N I2 solution in each flask and makeup the volume up to 100ml using distilled water. Take the absorbance at 580nm using digital photocolorimeter and phosphate buffer (pH 7.0) as blank (table: 2).

The enzyme activity was expressed in number of units. 1 unit of enzyme was defined as the amount of enzyme (protein) in milligram required for hydrolysis of starch to produce a millimolar of reducing sugar (glucose) in one hour under assay conditions. The specific activity was defined as number of units per gram protein.

Reagent Starch control Enzyme control Test flask

Phosphate buffer 5.0ml 5.0ml 5.0ml

0.1%starch - 2.0ml 2.0ml

Active enzyme - - 1.0ml

Inactive enzyme 1.0ml 1.0ml -

Incubate for 15mins at room temperature

0.01N HCl 2.0ml 2.0ml 2.0ml

0.01N I2 solution 4.0ml 4.0ml 4.0ml

Make up the volume up to 100ml

O.D at 580nm

Enzyme assay: To check the enzyme activity as unit per liter. Table: 1

(5) Protein estimation :

Folin Lowry’s method:-

The protein content of enzyme solutions extracted was estimated by Folin Lowry’s method. In this method, standard graph was prepared by estimation using standard protein solution (BSA solution= 2.5mg/mL concentration). Protein estimation was carried out according to following assay system. Samples were diluted 10 times for estimation (table 1).

Sr.no.

Aliquot protein solution

(mL)

Concentration

(μg)Distilled

water (mL)Alkaline copper

solution(mL)

Incubate at room

temperature for 15

minutes

Folin ciocalteau

reagent(mL)

Allow it to react at room

temperature for 30

minutes

Blank 0 0 1 5 0.5

1 0.2 40 0.8 5 0.5

2 0.4 80 0.6 5 0.5

3 0.6 120 0.4 5 0.5

4 0.8 160 0.2 5 0.5

5 1.0 200 0 5 0.5

S1 0.5 0.5 5 0.5

S2 0.5 0.5 5 0.5

S3 0.5 0.5 5 0.5

S4 0.5 0.5 5 0.5

S5 0.5 0.5 5 0.5

Table 1: Protein estimation by Folin Lowry’s method for different substrate concentration

Parameters controlling amylase production

(1) Various carbon sources:-

Flasks containing production media was supplemented with different carbon sources, viz: glucose, sucrose, starch, fructose, sorbitol, xylose, galactose and dextrin. The influence of these carbon sources were tested at different concentrations (0.5 to 2.0%). Starch, Sucrose and dextrin and galactose were good carbon sources for amylase production. Xylose, fructose and sorbitol could be considered as moderate source. Starch was recorded to be the best carbon source for production of alpha amylase from cells of Aspergillus niger (Table 1).

(2) Various nitrogen sources:-

The availability of the nitrogen source is the major controlling factor in the final biomass yield. Effect of different nitrogen sources on the production of alpha amylase was studied, it was observed that casein and gelatin caused poor enzyme production. Peptone supported maximum production of enzyme whereas urea produced considerable amount of alpha amylase from cells of Aspergillus niger. The optimum concentration of peptone was 0.03% (Figure 5).

(3) Incubation temperature:-

The influence of temperature on amylase production is related to the growth of the organism. Hence, the optimum temperature depends on whether the culture is mesophilic or thermophilic. Here we used the mesophilic strain of Aspergillus niger from soil and therefore assay of enzyme production was carried out at various temperature ranges 20 to 50°C for 24 hrs. It was found that cells of Aspergillus niger showed considerable amount of growth at 20°C but there was less enzyme production. However, the optimum temperature for enzyme production was 30°C for cells of A. niger (Figure 2).

(4) Different pH values:-

pH is one of the important factors that determine the growth and morphology of microorganisms as they are sensitive to the concentration of hydrogen ions present in the medium. pH is known to affect the synthesis and secretion of alpha amylase just like its stability (49). Fungi of Aspergillus sp. were found to give significant yields of alpha amylase at pH 5.0–6.0 in SmF (38,50,51). The initial pH of medium was adjusted to variable pH range by adding the 0.1N HCl. Enzyme purified was tested in the pH range (pH 3 to 8). The production of alpha amylase was found to be the best at pH 5.0 (figure 3).

(5) Incubation period:-

The alpha amylase activity was determined after every 24 hours of incubation in order to determine the optimum incubation period for maximum production of extra cellular alpha amylase. The enzyme production however, started after 24 hours of inoculation and showed maximum production on 5th day of incubation period for cells of Aspergillus niger (Figure 4).

(6) Use of surfactants:-

Surfactants in the fermentation medium are known to increase the secretion of proteins by increasing cell membrane permeability. Therefore, addition of these surfactants is used for the production of extracellular enzymes. The detergent Tween-80, Triton X-100 and SDS favored more amylase production in culture media. Tween-80 at 0.02% caused maximum enhancement where as Triton X-100 and SDS increased amylase activity at 0.002% and 0.0002% respectively for cells of A. niger (Table 2).

RESULT

Enzyme assay

BlankStarch control

OD1

Enzyme controlOD2

TestOD3

O.D at 580nm 0.00 0.05 1.50 1.33

Calculation for enzyme activity:-

V(actuvuty) = [E0-Et/Et]*A*(1/T)*(1/v) *1000

Where,E0 = OD2-OD1; 1.50-0.05 = 1.45Et = OD3-OD1; 1.33-0.05 = 1.28A = 12.35 (constant for amylase)T = time of incubation; 15minsV = volume of starch; 2ml

So, V(actuvuty) = [1.45-1.28/1.28]*12.35*(1/15)*0.5*1000 So, V(actuvuty) = 49.21 Unit per liter

From the above table and calculation the result obtained for enzyme activity under normal lab condition is 49.21 Unit per liter.

Protein estimation

Effect of temperature on extra cellular enzyme production

Table 4. Effect of various surfactants of various concentrations.

Table 3. Effect of various carbon sources of variousConcentrations.

detergents Concentration of surfactant

Enzyme activity unit/cell

Tween 800.2

0.020.002

0.0002

42.8

55.6945.66

44.3

TritonX-1000.2

0.020.002

0.0002

50.2

46.259.88

50.1

SDS0.2

0.020.002

0.0002

41.85

42.845.66

55.69

Carbon source

concentration Enzyme activity unit/cell

sucrose0.5

1.0

2.0

44.4

34.6

27.2

starch0.5

1.0

2.0

48.8

36.8

28.8

glucose0.5

1.0

2.0

10.44

7.52

1.88

dextrin0.5

1.0

2.0

42.6

33.5

26.6

fructose0.5

1.0

2.0

37.5

30.4

24.2

sorbitol0.5

1.0

2.0

32.2

28.5

22.8

xylose0.5

1.0

2.0

35.5

29.6

23.6

galactose0.5

1.0

2.0

41.4

31.2

25.5

Effect of pH on extracellular enzyme production

Effect of incubation period on extracellular enzyme production

Effect of various nitrogen sources at 0.03% concentration

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