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Production of Pectinase by Aspergillus Niger Cultured in Solid State Media

Nazneen Akhter
Department of Biotechnology and Genetic Engineering, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh.

M. Alam Morshed
Department of Biotechnology and Genetic Engineering, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh; Department of Pharmacy, North South University, Bashundhara, Dhaka-1229, Bangladesh.

Azim Uddin
Department of Pharmacy, North South University, Bashundhara, Dhaka-1229, Bangladesh.

Feroza Begum
Bangladesh Industrial Microbiology Laboratory, IFST, BCSIR, Dhaka, Bangladesh.

Tipu Sultan
Department of Biotechnology and Genetic Engineering, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh.

Abul Kalam Azad
Department of Biotechnology and Genetic Engineering, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh.

Abstract/ Executive Summary:

Solid-state fermentation was carried out with 7 fungal strains, obtained from different sources. Among 7 isolates of Aspergillus niger, IM-6 was found as an effective pectinase producer. Maximum enzymatic activity (142.44U/gm) was observed after 7 days incubation at 40^°C temperature in a 750 ml conical flask. In this study 1.69% (NH₄)₂SO₄ was used as a nitrogen source, although peptone as a nitrogen source showed better results use of peptone was not cost-effective. As a substrate, wheat bran and potato starch showed good results (85.54U/gm) in solid-state culture. The addition of 9.68% pectin was found to increase the enzyme production as 116.57U/gm. Pectinase production was optimum in 60% moisture (98.34U/gm). Aeration showed positive effects on pectinase production (136.86U/gm) at 750 ml flask than 1000 ml flask. Thus the wild strain Aspergillus niger IM-6 has outstanding pectinase producing capability at 40°C in 60% initial moisture content for 7 days of incubation in solid-state fermentation.

Keywords: Pectinase, Pectin, Aspergillus. Solid state fermentation.

Location: Microbiology Section, IFST, BCSIR, Dhaka, Bangladesh.

Start Date:

End Date:

Program Area: Pest Management

Commodity: Fungal Disease

Objectives:

To clarify the specific fungal strain with the best enzyme (pectinase) production activity.

Methodology:

Microorganisms- The pectinase production was carried out with seven different strains of fungi namely P1, P2, P3, P4, P5, P6, P7. Five fungal strains (P1–P5) were supplied from the Industrial Microbiology Section, IFST, BCSIR, Dhaka, Bangladesh. Two strains (P6 and P7) were collected respectively from spoilage wood apple (Aegle marmelos) and bread. Pure cultures were maintained in PDA media at 4^°C and were subcultured at 30 days interval. Screening of best isolates- Seven strains of fungi were used for the production of pectinase. The enzyme activity was measured by the method of Stiles et al., (1926). The extensive screening was carried out by measuring glucose and pectinase activity. On the basis of the screening program, Aspergillus niger IM-6 was selected for further experiments. Culture condition for pectinase production- For the production of pectinase solid state fermentation was performed. To select the optimum growth condition for maximum enzyme production, following parameters was studied stepwise using 14 gm wheat bran and 6 gm rice husk for the solid state fermentation process. Fungus, Aspergillus niger IM-6 was inoculated in PDA media in test tube and these inoculums were inoculated at 30^°C for 4 days to produce enough mature spore. The fungul spores from 4 days old culture were suspended in 4 ml of sterile water. The fungal suspension was then scrubbed with loop and shaken gently to make homogeneous suspension. This suspension was used as inoculum and was inoculated in solid state medium. To determine the effect of temperature on enzyme production, solid substrates with inoculum were incubated at temperatures ranging from 30^°C to 50^°C. To find the moisture content suitable for the production of maximum enzyme, 50, 60, 70, and 80% tap water was added with solid substrate. The cultures were incubated at different days (up to 9th days) to identify the correct incubation period for the maximum enzyme production. Various concentration of pectin was used as carbon sources. The cultures were incubated with different kinds of nitrogen sources such as urea, peptone yeast extract, (NH₄)₂SO₄ for the production of pectinase. Extraction of culture filtrate- Ten-gram koji (the fermented ingredient of solid medium is called koji) and 0.5 gm NaCl were in 100 ml of distilled water. It was stirred for 10 minutes and then kept stand for one night in the refrigerator. It was then centrifuged at 4000 rpm for 15 minutes and filtered through Whatman filter paper and volume was adjusted. This filtrate was used as crude enzyme for assay of pectinase activity and reducing sugar. The extracted solution was taken in an Erlenmeyer flask plagued with cotton and preserved at 4^°C with one drop of toluene. Glucose estimation and pectinase assay- The reducing sugar of the extracellular enzyme was determined according to Stiles et al., 1926. pectinase was measured as follows: 2ml pectin solution, 1 ml distilled water, 1 ml acetate buffer (0.05 M, P^H 4.0) was incubated at 40^°C in water bath for 10 minutes then 1 ml enzyme solution was added and kept it for 60 minutes and the increase of reducing sugar was estimated by the usual method. One unite of pectinase is defined as1µ mol reducing sugar liberated per minute under assay condition.

Source of Information: International Journal of Biosciences; Vol. 1, No. 1, p. 33-42, 2011

Article Link (Online Journal): http://www.innspub.net

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It can be concluded that the wild strain Aspergillus niger IM-6 has outstanding pectinase-producing capability at 40^°C in 60% initial moisture content for 7 days of incubation in solid-state fermentation. This could be highly beneficial for the production of microbial enzymes, pectinase, from lignocellulosic materials in the food and beverage based biotechnological industries

Knowledge Management: Journal