Purification And Properties Of Alpha Amylase Produced From Bacillus Subtilis Isolated From The Soil

Project and Seminar Material for Microbiology

Purification And Properties Of Alpha Amylase Produced From Bacillus Subtilis Isolated From The Soil


Thermophiles are organisms which are adapted to live at high temperatures. The enzymes from thermophiles find a number of commercial applications because of their thermostability and thermoactivity including in beer brewing. One of the most attractive attributes of thermophiles is that they produce enzymes capable of catalysing biochemical reactions at temperatures higher than those of mesophilic organisms. The objective of this study was to isolate and identify industrially relevant extracellular enzymes producing thermophilic bacteria from cassava processing site in Nsukka, Enugu State. In this present study, soil samples were collected and screened for thermophilic Bacillus strains with amylase activity and to examine the amylase heat tolerance potential.

This study was also aimed at determining the optimal culture conditions for growth and amylase production. The optimum temperature of amylase activity ranged between 50-60oC. The isolate was Gram positive, motile rod, bearing terminal endospore. Furthermore, the effects of incubation period, temperature, pH, different carbon and nitrogen sources, metal salts were investigated. The maximum enzyme production was found at 24 hours and pH 7. Corn starch (1.87U/mg protein) was found to be the best inducer followed by soluble starch (1.20U/mg protein). The effect of metal salts was investigated and it was found that calcium chloride increased enzyme production (1.97U/mg protein), while all the others exhibited inhibitory effects. These characteristics of Bacillus subtilis, suggests its promising nature with potential for further investigation in various biotechnological processes.

Chapter One

1.0 Introduction

The potential of using microorganisms as biotechnological sources of industrially relevant enzymes has stimulated interest in the exploration of extracellular enzymatic activity in several microorganisms (Abu et al., 2005). Microbial species exist in many environments like extremes of temperature, pH, chemical content and pressure. This existence of microbes is due to certain genetic and or physiological adaptations (Aguilar,1996). Thermophiles are the organisms which are adapted to grow optimally at high temperatures. The enzymes from thermophiles are known as thermophilic enzymes and these enzymes find a number of commercial applications because of their thermostability and thermoactivity. One of the most attractive attributes of thermophiles is that they produce enzymes capable of catalysing biochemical reactions at temperature higher than those of mesophilic organisms (Aguilar,1996).

Alteration in the amino acid composition of proteins bring about additional electrostatic interactions, formation of hydrogen and disulfide bonds, enhancement of hydrophobic interactions or compaction of the structure and there are only a few cysteine residues in thermophilic enzymes. Amylases are enzymes which utilize and hydrolyse starch and glycogen as substrate. On the basis of how amylase breakdown starch molecules and produce glucose, these are classified as α-Amylase (which breaks down the bonds at random manner), β-amylase (which acts on the glucose-glucose bonds and remove two glucose unit at a time and produce maltose and amyloglucosidase (AMG), β-Amylase breaks the non reducing end of the straight chain and produce glucose. Amylase producing industries need a temperature tolerating amylase-producing bacterial strains which are able to produce thermostable amylase enzyme, because temperature and pH controls are critical during some stages of production.

α-Amylase (endo 1,4-α D glucan glucohydrolase) is an extracellular enzyme which acts on substrate starch and degrades it into disaccharide and trisaccharide (Crabb and Mitchinson,1997). Amylase can be derived from various sources such as microorganisms (bacteria and fungi), plants and animals. Microbial production of amylase is more fruitful than that of other sources like plants or animals because microbial amylase is fast, cost efficient, easy and moderate for obtaining enzymes of desired characteristics (Leveque et al., 2000). In general, the enzymes get denatured and lose their activities at temperatures over 50-600C.

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The B.subtilis strain produced high levels of thermostable α-amylase with characteristics such as ability to grow under extreme temperatures, incubation periods and substrate concentration, suitable for application in starch processing and other food industries. Most of the alpha amylases are metalloenzymes, therefore supplementation of the medium with salts of certain metal ions, provided good growth of bacteria and thereby better enzyme production, an example is calcium salts. Alpha amylase is known to be a calcium metalloenzyme having at least one calcium ion associated with its molecule. The enhanced bacterial growth may be as a result of the increased availability of calcium ions. The production process can be commercialized after further optimization for enhanced enzyme production. It is already known that amylase having approximately 25% of enzyme market is one of the most useful enzyme known and has a great significance. This study reports biological production of thermostable α-amylase by soil bacteria B.subtilis isolated from cassava processing site. Enzyme production was found maximum in presence of 24 hour incubation period, temperature of 55oC and pH 7.0. At the optimum conditions, B.subtilis produced higher amount of amylase per litre of culture broth, this amount of enzyme was higher than before optimization. After due comparisons to other studies and research, we can conclude that moderate thermophilic amylase producing bacterial strains or thermophilic amylase producing bacterial strains contain thermostable amylase which degrade starch substrate and shows catalytic activity at high temperature. It is evident that the induction of alpha amylase requires substrates having alpha 1,4 glycosidic bond, including starch but glucose represses its production. The characteristic temperature, slightly acidic pH, short lag and initial stationary phase are novel features which could be exploited for production of alpha amylase in starch and other food industries.


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