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European Journal of Applied Sciences – Vol. 9, No. 5

Publication Date: October 25, 2021

DOI:10.14738/aivp.95.11036. Agwu, C. H. (2021). Molecular Screening and Production of α-amylase from Fungal sp. European Journal of Applied Sciences, 9(5).

365-379.

Services for Science and Education – United Kingdom

Molecular Screening and Production of α-amylase from Fungal sp

Agwu Cletus H.

National agency for Food and Drug Administration and Control

Enugu division

ABSTRACT

α-amylase producing Aspergillus sp. was isolated from decaying bread collected

from bakery site located within Enugu metropolis, Enugu state. Standard

microbiology, biochemical and molecular techniques (18s DNA sequencing) were

used for confirmation of the fungal strain as Aspergillus tamari.. p-NPG infused

nutrient broth confirmed Aspergillus tamarrii as prolific producer of α-amylase

with rapid yellow colouration after forty eight hours of incubation. Solid state

fermentation on rice bran matrix (SSF) system was used for the enzyme production.

Cork borer of 2mm diameter was used throughout the production and optimization

studies. Carbon sources including: Starch, wheatbran and sugarcane baggase were

optimized, starch was found suitable for the protein production with highest α- amylase activity (91.71 μmol/min). Among the nitrogen sources optimized,

peptone was found optimal for α-amylase production with activity of 90.34

μmol/min. pH 6.0 was found the best for the enzyme production. Effect of

incubation period on the enzyme production showed the 4th day of fermentation as

the peak day for α-amylase production from Aspergillus tamari. The results from

this study have shown that Aspergillus tamari among other fungal isolates from

mould bread collected from bakery sites in Enugu metropolis has the potential for

α-amylase production in a commercial scale for both industrial and clinical

applications.

Key words: 18s rDNA, α-amylase, fermentation, Optimization, Aspergillus tamarii T5

INTRODUCTION

α-amylase (1, 4-α-D-glucano hydrolase; E.C 3.2.1.1) is a ubiquitous enzyme responsible for

random hydrolysis of α 1-4, α1-6 glycosidic bonds in polysaccharides. Generally, the amylase

family (clan GH-H glycoside hydrolase) is the largest family of the glycoside hydrolase,

transferases and isomerases comprising nearly thirty different enzymes specificities

(Vijayaraghavan et al., 2011). As described by Vander Maarel et al. (2002) the family of the

amylase is categorized into four sub groups based on their specificity of their actions and they

include: endo, exo, debranching and transferase amylases. Verily α-amylase is widely

distributed in plants, animals and microbes, where they play important role of carbohydrate

metabolism (Swetha et al., 2006). Amylases from plants and animals suffer many demerits such

as: enzyme loads, doubling in production and ease of separations. Microbial amylases are

among the most important hydrolytic enzymes and have been studied extensively (Singh et al.,

2014). Their ease of separations, short doubling time and much know how technicality in

culturing and production makes much very indispensable for vast applications (clinical,

industrial and biotechnological bias fields). α- amylases have been reported to be produced by

a number of fungi including the family of Basidomycete, Ascomycete and the Deutromycetes

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European Journal of Applied Sciences (EJAS) Vol. 9, Issue 5, October-2021

Services for Science and Education – United Kingdom

(Balkan and Ertan, 2005, Nwagu and Okolo, 2011and Mohammed et al., 2007). Amylase family

represents one of the three largest groups of industrial enzymes and accounts for

approximately 25–33% of the world enzyme market (Van der maarel et al., 2002). They are

very indispensable in food industries necessarily for starch hydrolysis, clinical for replacement

therapy (case of metabolisms and in-born errors), chemical industries e.g detergent, pulp and

paper making etc (Mohammed et al., 2007). Search for novel strains of organisms with unique

ability for production of amylase has been of great paramount for much advanced

biotechnological relevance of the enzyme. Most applications of amylase occur at high

temperature and lower pH which are the conditions for starch hydrolysis and liquefaction.

((Goyal et al., 2005; Silva et al., 2005). Most α- amylases are seen denatured at these given

physiologic conditions and thus decreases their indispensability; however, novel strains of

filamentous fungi especially those of the basidomycetes and zygomycete families are of thermal

tolerant and can withstand relatively low pH condition. Production of α- amylases from these

organisms is believed to meet the desired condition required for industrial need of the enzyme.

The present study identifies some fungal strains using molecular techniques with unique

properties for production of α- amylases with promising enzymatic characteristics.

MATERIAL AND METHODS

All chemicals/reagents used in the present study are of analytical grade and are products of

Sigma-Aldrich, Bristol and May and baker. The analytical equipments are in good working

conditions and always calibrated at each use.

Sample Collection

Bread from bakery site was collected within the axis of Enugu metropolis in a clean plastic

container. Thereafter it was transported to the laboratory where it was placed in an anaerobic

jar for microbial infestations and decay.

Isolation and Identification of Fungal Strains from the Decayed Bread

Strains of fungi isolates were isolated from the decayed bread in a prepared nutrient broth

using standard microbiology (culturing and microscopy mounting) and biochemical (sugar

fermentations) techniques as described by Ezeonu et al.(2013).

Screening of Fungi isolates. for α-Amylase Production

Identified fungi isolates were screened for α-amylase producing ability using potato dextrose

broth supplemented with 2mM p-NPG as described by Gheytanchi et al. (2010). The inoculated

culture broth was incubated at 37°C for 3 days.

Molecular Identification of Aspergillus sp.

Genomic DNA (gDNA) from the selected isolate with high potentials of α-amylase production

was obtained using the QIA amp DNA Mini Kit. The 18S rDNA gene was amplified by RT-PCR

(the conditions for the amplification stated below) using the forward (5'-

GGTTTGATCATGGTCAG-3') and reverse (5'-AGTTACCTTGTTACGACT-3') primers. The

amplified DNA sequence was compared to the Gen Bank database of National Center for

Biotechnology Information (NCBI) using the BLAST program (Kumar et al., 2016).