Fermentative extraction of Amylase using waste Biomaterials as Substrate

S. Sharmila*, S. Preetha, E. Kowsalya, R. Kamalambigeswari, L. Jeyanthi Rebecca

Dept. of Industrial Biotechnology, Bharath Institute of Higher Education and Research,

Chennai, Tamil Nadu, India – 600073.

*Corresponding Author E-mail: sharu312@gmail.com

ABSTRACT:

Enzymes are biological molecules that significantly speed up the rate of virtually all of the chemical reactions that takes place within the cells. They are vital for life and serve as a wide range of important functions in the body. Solid state fermentation holds a high potential for the production of enzyme amylase using Aspergillus niger. In this work, different solid substrates such as groundnut shells, coconut coir and Palmyra sprout peels were used for the production of amylase as they are very cheap and also easily available raw materials. Then the maximum enzyme activities were analysed. Results showed that the enzyme activity of for which palmyra sprout peel was used as substrate had maximum activity in both crude sample (0.63µmol/ml.min) and in partially purified sample (1.42µmol/ml.min) and activity was found to be less for groundnut shell as substrate (crude sample 0.36µmol/ml.min) and in (treated sample 0.26µmol/ml.min) and also the specific activity was found to be maximum in palmyra sprout peel (29.2U/mg of protein) and less in groundnut shell (8.6U/mg of protein).

KEYWORDS: Solid substrate fermentation, Aspergillus niger, groundnut shells, coconut coir and Palmyra sprout peels.

INTRODUCTION:

Enzymes are biocatalyst that can speed up the reactions. These are commercially used in many industries such as detergents, food, pharmaceutical, diagnostic and fine chemical industries. Many plant¹, marine algae^2,microbes^3-12 have been studied for their enzyme production. Amylase was the first enzyme to be discovered and isolated by Anselme Payen in 1833. These are found to be most important enzymes for their specific use in the conversion of starch. Amylases accounts for about 30% of the world’s enzyme production³. Plants and some bacteria also produce amylase. In food industry amylase have a wide applications, such as the production of glucose syrups, high fructose corn syrups, maltose syrup, reduction of viscosity of sugar syrups, reduction of turbidity to produce clarified fruit juice for longer shelf-life, solubilization and saccharification of starch in the brewing industry. It has remarkable use in baking industry.

They uses amylases to delay the staling of bread and other baked products. In the paper industry, amylase is used for the reduction of starch viscosity to achieve the appropriate coating of paper. Amylase is also used in the textile industry for sizing of textile fibers, and is used as a digestive aid in the pharmaceutical industry. Amylases are mainly used in the starch processing industries for the hydrolysis of polysaccharides like starch into simple sugars^13-15. Amylase has been derived from several fungi, yeasts and bacteria. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors¹⁶. Amylase is produced by both Solid substrate (SSF) as well as submerged fermentation (SmF). SmF has been traditionally used for the production of industrially important enzymes because of the ease of control of different parameters such as pH, temperature, aeration and oxygen transfer and moisture^17,18. In this study, amylase was produced by solid substrate fermentation suing different waste materials and was purified.

MATERIALS AND METHODS:

Collection of samples:

Different samples such as groundnut shells, coconut coir and palmyra sprout peels were collected. Then it was cleaned well and dried under sunlight. After complete drying it was ground well using mixer and stored.

Preparation of pure culture:

Pure potato dextrose broth (PDB) was prepared and A.niger was inoculated and kept for growth and pure colonies were isolated and cultured in freshly prepared potato dextrose broth.

Fermentation process:

A known amount of three powdered substrates were weighed and taken in 3 different conical. Then it was moistened with 2% of dextrose solution. After that 2ml of culture was inoculated to the solid substrate and kept 48 hrs for fermentation.

Preparation of phosphate buffer:

Phosphate buffer was prepared for pH 5.7 as per the standard procedure.

Extraction of amylase:

After fermentation was completed, 100ml of phosphate buffer was added to each conical flask and allowed to stand for 2 hrs. Then it was filtered using filter paper.

Partial purification of enzyme:

The crude extract solution was taken and it was purified by adding 70% ammonium sulphate. Then it was kept for centrifugation at 10,000rpm for 10 mins. After centrifugation pellet was collected and resuspended with buffer.

Pretreatment of membrane:

Before dialysis, membrane was underwent pretreatment process by soaking it in distilled water for 15 mins and then soaked in 10ml of sodium bicarbonate solution for 30 mins at 80°C. Then it was kept in 10ml of EDTA for 30 mins. Finally it was soaked in distilled water for 30 mins at 80°C with constant stirring.

Dialysis:

Dialysis was performed to purify the enzyme. After pretreatment, both the ends of the membrane was tied tightly. Then the sample was transferred into the membrane and was kept in the beaker containing phosphate buffer and was stirred with magnetic stirrer for 2 hrs.

Enzyme activity:

200µl of sample and 200µl of 1% starch solution was added and kept for incubation for 30 mins. Then 0.5 ml of DNS was added and heated in water bath for 15 mins. After cooling, 0.5 ml of Rochelle salt was added. Then OD was noted down at 620 nm.

Estimation of protein content:

Protein content of samples was analyzed by standard Bradford method¹⁹.

RESULTS AND DISCUSSION:

In this work, different types of waste such as groundnut shells, coconut coir and palmyra sprout peels (Fig.1, 2 and 3) were used as a solid substrate for fermentation (Fig.4). Microorganism Aspergillus niger was used as a inoculum for the production of amylase. After fermentation crude enzyme (Fig.5) was extracted and partially purified (Fig.6).

Fig 1: Coconut coir

Fig 2: Peels of Palmyra sprout

Fig 3: Groundnut shell powder

Fig 4: Solid substrate fermentation

Fig 5: Crude enzyme

Fig.6 Purified enzyme

The extracted amylase activity was analyzed for crude as well as for purified. Results showed that the amylase activity was more in which palmyra sprout peel was used as a substrate (1.42mol/min) for both crude and purified enzyme. Less activity was found in groundnut shell powder as substrate (0.26µmol/min) (Table.1). In Dibyangana Raul et al., research showed that less specific activity (13.19µmol/mg.min)²⁰, where as in this palmyra sprout peel substrate showed higher activity (29.2 U/mg.min) than that. Coconut coir substrate also showed good enzyme activity (14 U/mg.min) (Table.2).

Table 1: Enzyme Activity of Crude and purified amylase

S. No	Substrate	Crude enzyme		Purified enzyme
S. No	Substrate	Glucose concentration (µmol/ml)	Amylase activity (µmol/min)	Glucose concentration (µmol/ml)	Amylase activity (µmol/min)
1	Coconut coir	2.7	0.45	2.5	0.42
2	Groundnut shell	2.2	0.36	1.6	0.26
3	Palmyra sprout peel	3.8	0.63	8.8	1.42

Table.2 specific activity of crude and purified amylase

S. No	Substrate	Crude enzyme		Purified enzyme
S. No	Substrate	Protein concentration (mg/ml)	Specific activity (U/mg) or (µmol/min.mg)	Protein concentration (mg/ml)	Specific activity (U/mg) or (µmol/min.mg)
1	Coconut coir	0.07	6.43	0.03	14
2	Groundnut shell	0.11	3.27	0.03	8.6
3	Palmyra sprout peel	0.13	4.85	0.05	29.2

CONCLUSION:

An Amylase is an enzyme that plays a vital role in the hydrolysis of starch into sugar. This work showed that using of different waste biomaterial such as groundnut shell, palmyra sprout peel and coconut coir was used as substrate for the production of amylase enzyme. Results showed that palmyra sprout powder substrate produce best enzyme activity among all. In future, further optimization may be carried out to increase the efficiency of amylase enzyme.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 09.06.2020 Modified on 20.08.2020

Research J. Pharm. and Tech. 2021; 14(9):4580-4582.

DOI: 10.52711/0974-360X.2021.00796