IMPACT OF DIFFERENT NITROGEN SOURCES ON THE FERMENTATIVE PRODUCTION OF POLYGALACTURONASE BY SSF USIN

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IMPACT OF DIFFERENT NITROGEN SOURCES ON THE FERMENTATIVE PRODUCTION OF POLYGALACTURONASE BY SSF USING Aspergillus tubingensis Dr. Viral N. Patel *1, Dr. Samir C. Parikh*2 *1Department

of Microbiology, Smt. L. M. Shah Science College, Radhanpur, Gujarat, India.

*2 Department

of Microbiology, Smt. S. M. Panchal Science College, Talod, Gujarat, India.

ABSTRACT During the study of polygalacturonase production using various physico-chemical factors, it was observed that use of different nitrogen sources in the SSF media can help find the best substrate combination that can optimize the ability of the fungus for the production of polygalacturonase in standard conditions. The various nitrogen sources used were urea, yeast extract, casein, tryptone, ammonium sulphate and peptone. Ammonium suphate and urea proves to be the best possible nitrogen sources for the production of polygalacturonase in SSF medium using Aspergillus tubingensis.

Keywords: Polygalacturonase, Solid state fermentation, Aspergillus tubingensis, pectinase.

I.

INTRODUCTION

For achieving better production of the enzyme from the pectinolytic fungus, Aspergillus tubingensis, it has to be maintained and cultivated in the best possible way by providing them the basic nutritional reԛuirements including sources of energy, carbon, nitrogen, mineral elements, vitamins, water and oxygen, if aerobic (Stanbury et al., 1997).

The term fermentation is used mostly for the production of microbial metabolites including enzymes, organic acids, antibiotics, etc. The precise formulation of the fermentation media is very important. The main objective of this experimental work is to check the impact of various physical and chemical components of the medium to find out the activators and inhibitors of pectinases, especially polygalacturonase from Aspergillus tubingensis.

The nitrogen sources for formaulation of fermentation media for this experimental set are urea, yeast extract, casein, tryptone, ammonium sulphate and peptone. Wheat bran was used as the main supporting substrate for this experimental work due to its granular particle size and appearance that can help in supporting fungal growth and metabolism which are very vital for the production of enzyme.

II.

MATERIALS AND METHODS

Pectinase production by Aspergillus tubingensis was comprehensively analyzed for the effect of different nitrogen sources under laboratory conditions. Urea, however serves as a control since it is already present in the medium in 0.3% concentration (Maldonado and Strasser de Saad, 1998). So urea was replaced by 0.3% concentration each of yeast extract, casein, tryptone, ammonium sulphate and peptone respectively, for each of the experimental sets. Various nitrogen sources were incorporated in the medium for selecting the ideal one for better production of polygalacturonase. Urea, yeast extract, casein, tryptone and peptone were used as nitrogen sources.

Production of Pectinolytic Enzymes in SSF: 10 grams of the pretreated and dried wheat bran was taken in a 100 ml Erlenmeyer flask or even using sterile glass petri plate and it was then moistened with mineral salt solution to keep the moisture content at a level of 70% (Acuna-Arguelles et al., 1995). Then all the flasks were autoclaved at 120°C at 10 lbs. for 15 minutes in order to prevent degradation of pectin. The flasks were then cooled and inoculated with 2.0 X 107 Aspergillus tubingensis spores/ gram dry matter and incubated at 28°C for 3-5 days.

Recovery of Pectic Enzyme: Polygalacturonase: After the fermentation period of 72 hours, the fermented media was extracted with 40 ml of 0.05 M Sodium acetate buffer (pH- 5.0). For the extraction process, the flasks were shaken at 150 rpm for 30 minutes at 25° C and kept for one hour before they were filtered through muslin cloth. The extract was then centrifuged at 8,000

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rpm in a centrifuge (REMI Research Centrifuge, Model No.: R-24) and the supernatant was then filtered through Whatman No.1 filter paper adjusted into a glass filtration assembly to remove the fungal spores completely. The filtrate was then used for enzymatic assays. The activity of polygalacturonase enzyme is mentioned as U/gds (Units/gram dry substrate)

Assay of Polygalacturonase Activity: The activity of Polygalacturonase was measured by determining the amount of reducing substances released according to the method outlined by Nelson (1944) and Somogyi (1952).

Definition of Unit Activity: One unit of polygalacturonase activity is defined as the amount of enzyme that converts substrate into one micro mole of galacturonic acid/ml/minute under the standard assay conditions.

Reagents: 0.2 M Tris acetate buffer (pH 4.5), 0.01 M CaCl2, Polygalacturonic Acid (Sigma Aldrich) 1%, Alkaline copper tartarate reagent (Solution A: Dissolved 2.5 gm of anhydrous Sodium carbonate, 2 gm of Sodium bicarbonate, 2.5 gm of Potassium sodium tartarate and 20 gm of anhydrous Sodium sulphate in 80 ml of water and made up the volume to 100 ml.; Solution B: Dissolved 15 gm Copper sulphate in a small volume of distilled water. Added one drop of sulfuric acid and made up the volume to 100 ml; Prepare fresh and mixed 4 ml of solution B and 96 ml of solution A before use.), Arsenomolybdate solution (Dissolve 2.5 gm Ammonium molybdate in 45 ml water. Added 2.5 ml Sulfuric acid and mixed well. Then add 0.3 gm of Sodium hydrogen arsenate dissolved in 25 ml water. Mixed well and incubated at 37°C for 24-48 hrs.), Polygalacturonate Standard Solution: (Galacturonic acid monohydrate (Fluka) 1.0%) Procedure: 1. The assay mixture was prepared with the following components: • 0.2 ml enzyme • 0.2 ml 0.2 M tris-acetate buffer (pH 4.5) • 0.1 ml 0.01 M CaCl2 • 0.5 ml of 1.0% solution of polygalacturonic acid (PGA) 2. Prepared a blank for each sample by boiling the reaction mixture before the addition of the substrate.

3. Incubated at 37°C for 1 hour.

4. Stopped the reaction by heating at 100°C for 3 minutes.

5. 0.5 ml of the solution mixture was taken and analyzed for reducing sugars by Nelson-Somogyi Method.

6. Made up the volume in both sample and standard tubes to 2 ml with distilled water.

7. Pipetted out 2.0 ml of distilled water in a separate tube to set up a blank.

8. Added 1.0 ml of Alkaline copper tartarated reagent and kept for 10 minutes.

9. Cooled the tubes and added 1.0 ml of arsenomolybdate reagent to each of the tubes.

10. Made up the volume in each tube to 10 ml with distilled water.

11. Read the absorbance of blue color at 620 nm after 10 minutes in a Spectrophotometer. (Ramchandran, Sandhya 2005) All the experiments were conducted in triplicate and the mean values of all the sets of observations were taken for evaluation of the experimental results.

III.

RESULTS AND DISCUSSION

Nitrogen is one of the important nutrient sources in solid state fermentation process. Many solid substrates can be supplemented with soluble sources of nitrogen during preparation of the substrate for SSF processes.

Aspergillus tubingensis was cultured under various nitrogen sources for studying their impact on polygalacturonase production under solid state fermentation. The various nitrogen sources used included urea, yeast extract, casein, tryptone, ammonium sulphate and peptone.

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Since urea was one of the nitrogen sources that showed remarkable increase in the production of polygalacturonase (Maldonado and Strasser de Saad, 1998), it was used as a control in the medium. The concentration of nitrogen source in the medium was kept at 0.3%.

Table-1 Effect of different nitrogen sources on polygalacturonase production by Aspergillus tubingensis Polygalacturonase Activity No.

Nitrogen Source (0.3%)

( U/gds)

1

Urea (Control)

24.790  0.456

2

Yeast Extract

5.043  0.248

3

Casein

2.250  0.210

4

Tryptone

1.387  0.146

5

Ammonium Sulphate

27.253  0.359

6

Peptone

1.050  0.061

It was observed that (NH4)2S04 was one of the best nitrogen sources used in the experiment that exhibited 27.253 U/gds polygalacturonase activity while urea as a control showed 24.790 U/gds polygalacturonase activity.

The use of all the other nitrogen sources including yeast extract, casein, tryptone and peptone showed considerable lower polygalacturonase activity.

Polygalacturonase Activity (U/gds) Enzyme activity (U/gds)

30 25 20 15 10 5 0 Urea (Control) Yeast Extract

Casein

Tryptone

Ammonium Sulphate

Peptone

Nitrogen Source (0.3%) Graph-1: Effect of different nitrogen sources on polygalacturonase production by Aspergillus tubingensis Most of the industrially useful microorganisms use inorganic or organic sources of nitrogen. Ammonia gas, ammonia salts and nitrates are some of the important inorganic nitrogen sources (Hunter, 1972). Supplements

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of amino acids, urea and proteins are considered to be the vital organic nitrogen sources for fermenting microorganisms.

In the present study, Urea and ammonium sulphate were used as the nitrogen sources in the control set of experiment. Urea was substituted for by yeast extract, casein, tryptone, ammonium sulphate and peptone in the culture media for Aspergillus tubingensis for the production of polygalacturonase. When ammonium sulphate was used as a sole source of nitrogen, the fungi showed the best possible production of polygalacturonase.

IV.

CONCLUSION

The inhibitory effect observed in case of other nitrogen sources may be due to the imbalance of carbon and nitrogen sources in the medium. As a result, it may also affect the pH control of the fermentation medium.

Similar results were observed in case of Geotrichum candidum (Shastri et al., 1988). Incorporation of urea as a nitrogen source in the medium increased the production of polygalacturonase was also supported by the findings of Gupta et al., (1997). The sources of nitrogen can also play a vital role in affecting the pH changes in the substrate during the process of fermentation. The incorporation of urea and ammonium sulphate can be used to control pH changes during the fermentation (Prior et al., 1992; Torrado et al., 1998). The present study is best supported by the findings of Gokhale et al., (1992) that reported the ability of urea to prevent the drop in pH in an unbuffered fermentation medium.

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REFERENCES

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Acuña-Argüelles, M. E., et al. "Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid-state fermentation." Applied microbiology and biotechnology 43.5 (1995): 808-814.

Nelson, Norton. "A photometric adaptation of the Somogyi method for the determination of glucose." J. biol. Chem 153.2 (1944): 375-380.

Somogyi, Michael. "Notes on sugar determination." Journal of biological chemistry 195 (1952): 19-23.

Ramachandran, Sandhya. "Isolation, purification and characterization of pectinase from penicillium citrium." (2005).

Hunter, R.I.E. (1972). Relationship of plant constituents to seedling diseases. In: Proc. Beltwide Cotton Rhizoctonia solani, Kuihn und den Einfliss einiger Prod. Res. Conf., 10-12 January, Memphis, Tennessee.p:86-87.

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Gupta, P., Dhillon, S., Choudhary, K. and Singh, R. (1997) Production and Characterization of an Extracellular Polygalacturonase from Penicillium sp., Indian Jour. Microbiol. 37:189-192.

Prior, B.A., Du Preez, J.C. and Rein, P.W. (1992) In: Solid substrate cultivation. Doelle, H.W., Mitchell, D.A. and Rolz, C.E. (Eds.) Elsevier, London.

Torrado, A., Gonzalez, M., & Murado, M. A. (1998). pH regulation in solid state culture through the initial ratio between oxidized and reduced sources of nitrogen. A model applicable to the amylase production by Aspergillus oryzae, Biotechnology techniques, 12 (5): 411-415.

Gokhale, D.V., Patil, S.G. & Bastawade, K.B. (1992) Protection of Aspergillus niger cellulases by urea during growth on glucose or glycerol supplemented media. Applied Biochemistry and Biotechnology 37: 11–17.

Patel, V.N. (2015) Production, Characterization and Scale up of Fungal Pectinase [Ph.D. Thesis, Hemchandracharya North Gujarat University, Patan]

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