EFFECT OF DIFFERENT CARBON SOURCES ON THE FERMENTATIVE PRODUCTION OF POLYGALACTURONASE BY SSF USING

e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science Volume :02/Issue :10/October -2020

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EFFECT OF DIFFERENT CARBON 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 and implementing various physico-chemical factors, it was observed that use of different carbon 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 carbon sources in different combinations used were glucose, pectin, polygalacturonic acid, orange bagasse, xylan, CM cellulose and sucrose. These carbon sources were used individually with wheat bran as the main SSF substrate. The stimulating action of glucose when used along with pectin, PGA and orange bagasse proved that glucose has an important role in the formulation of media for SSF processes for these enzymes.

During this study it was found that wheat bran and glucose in combination with either pectin, PGA or orange bagasse proves to be stimulatory for the production of polygalacturonase enzyme in Aspergillus tubingensis.

Keywords: polygalacturonase, solid state fermentation, Aspergillus tubingensis, pectinase, carbon sources, polygalacturonic acid, pectin, orange bagasse.

I.

INTRODUCTION

For obtaining a better output from the pectinolytic fungus, Aspergillus tubingensis, it has to be cultivated and maintained in the best possible way by providing them the basic 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 carbon sources for formaulation of fermentation media for this experimental set are Glucose, Sucrose, Pectin, Polygalacturonic acid, Orange bagasse, Xylan, CM Cellulose and various combinations from them. 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

Polygalacturonase production by Aspergillus tubingensis in solid state fermentation medium using different carbon sources was determined. The various carbon sources in different combinations used were glucose, pectin, polygalacturonic acid, orange bagasse, xylan, CM cellulose and sucrose. These carbon sources were used individually with wheat bran as the main SSF substrate. They were also used in combination with wheat bran and glucose, since glucose is one of the inducers for the better production of enzyme as well as for the fungal growth. Each of the above carbon sources either used individually or in combination with glucose was added in 1.5% concentration for each of the experimental sets.

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 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 spores / gram dry matter and incubated at 28°C for 3-5 days.

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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 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 tartarate 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 colour 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.

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III.

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RESULTS AND DISCUSSION

Effect of various carbon sources on the production of polygalacturonase by Aspergillus tubingensis was studied.

The various carbon sources incorporated in the medium included various combinations of glucose, pectin, polygalacturonase, orange bagasse, CM cellulose, xylan and sucrose.

Study was conducted using glucose as the sole source of carbon. While in another study pectin was used as the sole carbon source.

Table-1 Effect of different carbon sources on polygalacturonase production by Aspergillus tubingensis No.

Carbon Source (1.5%)

Polygalacturonase Activity ( U/gds)

1

Wheat bran + Glucose

15.080  0.262

2

Wheat bran + Pectin

13.780  0.576

3

Wheat bran + PGA

16.113  0.369

4

Wheat bran + Orange bagasse

20.640  0.544

5

Wheat bran + Xylan

8.730  0.591

6

Wheat bran + CM Cellulose

11.647  0.479

7

Wheat bran + Glucose + Pectin

35.120  0.262

8

Wheat bran + Glucose + PGA

37.577  0.580

9

Wheat bran + Glucose + Orange bagasse

42.017  0.301

10

Wheat bran + Glucose + Xylan

14.083  0.169

11

Wheat bran + Glucose + Sucrose

2.883  0.280

12

Wheat bran + Glucose + CM Cellulose

6.150  0.334

Simultaneous experiments were carried out testing the role of different combinations of various carbon sources as the inducer. The inducers added in the medium were at a concentration of 1.5 gm/100 ml as mentioned in the original medium prepared by Maldonado and (Strasser de Saad, 1998). Glucose was used in the medium at a concentration of 500 mg/100 ml. Initial studies were conducted where the concentration of glucose was varied from 250, 500 and 750 mg/ 100 ml water, but 500mg/ml of glucose showed the best activity of polygalacturonase when tested along with different carbon sources such as pectin, PGA and orange bagasse.

Wheat bran used to be the main solid substrate for the fermentation process.

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Polygalacturonase Activity (U/gds)

45

Enzyme activity (U/gds)

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40 35 30 25 20 15 10 5 0 A

B

C

D

E F G H Carbon Source (1.5%)

I

J

K

L

Graph-1: Effect of different carbon sources on polygalacturonase production by Aspergillus tubingensis Where, ABCDEFGHIJKL-

Wheat bran + Glucose Wheat bran + Pectin Wheat bran + Polygalacturonic Acid (PGA) Wheat bran + Orange bagasse Wheat bran + Xylan Wheat bran + CM Cellulose Wheat bran + Glucose + Pectin Wheat bran + Glucose + PGA Wheat bran + Glucose + Orange bagasse Wheat bran + Glucose + Xylan Wheat bran + Glucose + Sucrose Wheat bran + Glucose + CM Cellulose

For polygalacturonase, maximum activity was observed in case of wheat bran + glucose + orange bagasse which was 42.017 U/gds where as in case of wheat bran + glucose + polygalacturonic acid, the activity of polygalacturonase was 37.577 U/gds. Supplementing glucose and pectin in wheat bran also showed a remarkable increase in the polygalacturonase activity of 35.120 U/gds. Use of CM cellulose and xylan, either alone with wheat bran or including glucose as an inducer did not show any increase in the polygalacturonase activity by Aspergillus tubingensis. The best combination of carbon sources as per experiment is wheat bran + glucose + orange bagasse that exhibited the highest polygalacturonase activity of all.

IV.

CONCLUSION

During solid state fermentation, the presence of glucose does not inhibit pectinase production, since solid state fermentation is less affected by catabolic repression. This may be one of the reasons for the better production of polygalacturonase by fungi when used for fermentative production using glucose as one of the carbon sources along with some others such as pectin, PGA and orange bagasse. But at concentrations higher than 500 mg/ 100 ml, the production of pectinases was repressed. Similar studies indicating repression of pectinase production due to very high initial glucose concentration in solid state fermentation media were observed by (Solis-Pereyra et al., 1996). According to them, the high initial concentration of glucose favors fungal growth and ultimately lowers pH of the media due to accumulation of acidic waste in very high amount. Catabolic

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repression may be another reason for the repression of polygalacturonase production at higher initial glucose level.

(Shastri et al., 1988) observed that pectin, galacturonic acid, fructose and mannose induced polygalacturonase in Geotrichum candidum. Macfarlane et al., (1990) found that galactose and starch induces the production of polygalacturonase in Bacteroides ovatus. The present observations indicate the constitutive nature of pectinases and also the stimulating capacity of different carbon sources for the polygalacturonase production that varies from fungal strain to strain. The stimulating action of glucose when used along with pectin, PGA and orange bagasse proved that glucose has an important role in the formulation of media for SSF processes for these enzymes.

The GRAS (Generally Regarded As Safe) status of the Aspergillus species as well as the constitutive nature of polygalacturonase from Aspergillus tubingensis makes it one of the important findings to use it for commercial production of the enzyme. Because the constitutive enzymes are produced at a relatively constant rate irrespective of the presence of substrate in the fermentation medium. However, the rate of synthesis of such enzymes tends to increase several folds when a suitable carbon source is used as an inducer (Ward, 1989).

V.

REFERENCES

[1]

Stanbury, Peter F., Allan Whitaker, and Stephen J. Hall. Principles of fermentation technology. Elsevier, 2013.

[2] AcuĂąa-ArgĂźelles, 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.

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

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

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

[6] Maldonado, M. C., and AM Strasser De Saad. "Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems." Journal of Industrial Microbiology and Biotechnology 20.1 (1998): 34-38.

[7] Solis-Pereyra, S., et al. "Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations." World Journal of Microbiology and Biotechnology 12.3 (1996): 257-260.

[8] Shastri, P. N., M. Patil, and N. V. Shastri. "Production, purification and properties of Geotrichum candidum polygalacturonase: regulation of production by pyruvate." Indian journal of biochemistry & biophysics 25.4 (1988): 331.

[9] Macfarlane, G. T., et al. "Effect of different carbohydrates on growth, polysaccharidase and glycosidase production by Bacteroides ovatus, in batch and continuous culture." Journal of Applied Bacteriology 68.2 (1990): 179-187.

[10] Ward, Owen P. "Fermentation biotechnology: principles, processes and products." (1989).

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

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