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نسخة Determination of alpha amylase

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9 views7 pages

نسخة Determination of alpha amylase

Gh

Uploaded by

An An
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Republic of Iraq

The Ministry of Higher Education &


Scientific Research
Al-Safwa University College

Determination of alpha amylase

By Students:‫ھﺒﮫ ﷲ ﺣﯿﺪر ﻋﺒﺪ ﻋﻮن‬

Supervised by:

2020 – 2021

Karbala
Abstract
Background: Alpha-amylase is one of the industrial enzymes that hydrolyze starch
molecules into polymers composed of glucose units. The enzyme has potential
application in a wide number of industrial processes such as food, textile, paper,
detergent, fermentation and pharmaceutical industries. Alpha-amylase can be
produced by microorganisms, plants or animals. Aim: The aim of this study is to
detect the activity of alpha-amylase from bacteria isolated from soil environment.
Method: Soil samples were inoculated onto the media that are rich in nutrient that
favour the growth of the bacteria and incubated for 24 hours at 37 o C after which
the bacterial growth was detected in form of colonies. In this study, bacterial
species belonging to the genus Bacillus were identified through phylogenetic
analysis using 16s-ribosomal RNA sequencing for detection of the enzymatic
activity. Effects of pH and temperature on the enzymatic activity were observed
using DNS activity assay method. Results: Positive response to alpha-amylase
activity by the soil bacteria was observed by the formation of clear zone of
inhibition shown by the colonies on the petri plates. Conclusions: The optimal pH
and temperature activities showed that the bacteria exhibit enzymatic activity at
mesophilic temperature and acidophilic or alkalophilic pH.

Introduction

Microorganisms have become increasingly important producers of industrial

enzymes. In view of their


biochemical diversity and the ease with which enzymes concentrations may be
increased by environmental and
genetic manipulation, several attempts are now being made to replace enzymes,
which traditionally have been
isolated from complex eukaryotes. Amylolytic enzymes capable of degrading
starch are most important in
biotechnology industries with large application in food, fermentation, textile and
paper industries [1].

Amylases can be obtained from several sources such as plants, animals and

microorganisms [2]. The


microbial source of amylase is preferred to other sources of its plasticity and vast
abundance. Amylase obtained
from microorganisms has almost surpassed the synthetic sources in different
industries [1]. Amylolytic enzymes
are widely distributed in bacteria and fungi. They are categorized into exo-acting,
endo-acting and debranching
enzymes. Among the amylases, beta-amylase is exo-acting, where alpha-amylase
is endo-acting enzyme.

A vast number of organisms live in the soil [3], so great are microfloral number

that dominate the


biomass in spite of the minute size of each organism [4]. Together with
the earthworms, the microflora
monopolizes the metabolic activity in the soils. It is estimated that 60-80% of
the soil metabolism is due to
microflora. Not only do they destroy plant residues but they function in the
digestive tracts of animals and
eventually decompose the dead bodies of all organisms.

Alpha-amylases are hydrolytic enzymes that are widespread in nature being founin

microorganisms,
plants and animals [5]. They are among the most important in present day
biotechnology. The enzyme has found
numerous applications in commercial processes including thinning and
liquefaction of starch alcohol, brewing
and sugar industries [6].

The genus Bacillus produces a large variety of extracellular enzymes of which

amylases and proteases


are of significant industrial importance. Najafi et al [7] showed that the optimum
pH and temperature of the
o
alpha-amylase from Bacillus subtilus were 6.0 and 55 C respectively. The end
products of soluble starch were
glucose (70-75%) and maltose (20-25%).

Demands for novel amylases worldwide are increasing day to day as these

enzymes application spectra


are spreading in various industrial sectors [8]. This class of industrial enzyme
constitutes approximately 25% of
the enzyme market covering many industrial processes [9].
This study is aimed at detecting alpha-amylase activity produced by some soil

bacteria. The activity of


the bacterial enzyme was detected at different pH and temperatures after which
the optimal activities were
obtained. The objectives of this study include: (1) Isolation and identification of
the soil bacteria. (2) Gram’s
staining characteristics of the bacteria. (3) 16s ribosomal sequencing of the alpha-
amylase producing bacteria.

(4) Enzymatic activity assay of the bacteria

Material and Method


2.1 Study Site, Sample Collection and Media Preparation

This study was conducted in Fatih University Istanbul, Turkey. The soil samples

were collected in
clean dry sterile containers from different soil environments as follows: Behind E-
block and car-pack of Fatih
university, Istanbul; Buyucekmece Lake, Istanbul and Agricultural land of Bursa
all in Turkey.

Upon soil samples collection, the culture media were prepared and the soil was

inoculated onto the


plates containing the media that support the growth of the bacteria. The plates
o
were put in an incubator at 37 C
for 24 hours after which the bacterial growths were detected in form of colonies.
Similarly, the bacterial samples
that showed positive response to alpha-amylase were grown in LB-medium and
o
placed in an incubator at 37 C
for 24 hours for the bacterial cell cultivation.
2.2 Preparation of Media and
Regents 2.2.1 Alpha-amylase media

Two alpha-amylase media were prepared for the growth of soil bacteria. The

media were enriched with


the starch as the substrate for alpha-amylase enzyme (table 2.1). Following the
mixture of the chemicals, the
o
media were autoclaved at 121 C for 15 minutes using autoclave (Nuve OT4060
steam sterilizer (Turkey)). They
were poured on to the clean and dry Petri plates and allowed to cool after which
they solidified. The plate
o
containing the media were sealed with stretch film and stored at 4 C before use.
The preparation of the media
was performed under aseptic conditions.

2.2.2 LB-Medium

For the preparation of 500Ml LB-broth medium, 10g powder was suspended

in 500ml of distilled
o o
water. The media were autoclaved at 121 C for 15 minutes, then cooled to 50 C
o
and stored at 4 C before use.

2.3 Preparation of Buffers and


Solutions 2.3.1 Phosphate buffer

Reagents used

Monobasic sodium phosphate: 2.78g of sodium dihydrogen phosphate was

dissolved in 100ml
distilled water (Merck, Germany).
Dibasic sodium phosphate (0.2M): 5.3g of disodium hydrogen phosphate (Sigma-

Aldrich, Germany)
was dissolved in 100ml distilled water

Result
Biochemical Activation of the Colonies

Following the overnight incubation of the culture media, the bacteria were able to

degrade the media


there by producing a clear zone of inhibition. The larger the zone of inhibition, the
more active is the bacteria!
The diameter of the zone of inhibition and that of the colonies were measure

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