Abstract

Amylase is amongst most widely used enzymes in industries such as food, fermentation, starch processing, textile and paper. In the present investigation bacteria were isolated from different soil samples collected from different dump sites, in Molyko, Buea, screened for the production of amylase and their optimum growth tested. The isolate displaying maximum amylase activity on quantitation was selected. Characteristic feature of the strain indicates that it belongs to the genus Bacillus and will be later used for further characterization. Maximum yield of amylase was obtained after 48hrs of incubation. The optimum pH for enzyme activity was found to at pH 7.0 and the optimum temperature for activity was found to be at 400c.

Keywords: Amylase, starch hydrolysis, characterization, amylase activity.

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Enzymes are biological molecules, protein in nature needed in small amount to speed up the rate of a chemical reaction but not completely used up at the end of the reaction. Enzymes are vital to life. An amylase is an enzyme that catalyzes hydrolysis of starch sugars. Amylase is present in saliva of humans and some other mammals where it begins the chemical process of digestion. Foods that contain large amount of starch but little sugars such as they are chewed because amylase degrades some of the starch to sugar.

Starch degrading amylase enzymes are the most important in biotechnology industries with huge application in food, fermentation, textile and paper. They are degrading enzymes which breakdown the starch into sugars by acting on alpha -1, 4-glycosidic bonds

Amylases are obtained from different origins like plants, animal, fungal and bacteria. Microorganisms are used for industrial production due to the advantages such as cost effectiveness, consistency, less time, space required for production.

Many microorganisms are able to produce amylase including Bacillus spp., Lactobacillus, Escherichia proteus, Steptomycessp, Pseudomnas Sp.

For production of amylase for industrial use, isolation, characterization of new promising strains in a continuous process. In the present day study, we report isolation, screening and characterization of new promising strains in continuous process. In the present day study, we report the isolation, screening and characterization of amylase producing bacterial from soil sample of different dump sites. Production conditions were optimized (temperature, pH) to achieve high enzyme production and better enzyme activity.

1.2 LITERATURE REVIEW

1.2.1 Information about Bacillus bacteria

Bacillus is a genus of gram positive and rod shaped bacteria, a member of the phylum Firmicutes with 266 named species. The term is also used to described the shape of certain bacteria to which the genus belongs.

1.2.2 Techniques to isolate bacteria

• Steaking for isolation on an agar plate
• The pour plate method

1.2.3 How to extract enzyme from bacteria

Enzymes are generally extracted from cells of microorganisms by either allowing the culture broth to stand and wait the liberation of the enzymes affected by the autolysis of the cell walls accelerating the lysis of the cells with the employment of lysis-promoting-agent such as sodium dodecyl sulphate and then isolating the enzymes from the cells

The alternative methods which have been purposed are the methods involving mechanical destruction of the cell walls either by trituration, ultra-sonication or the French press method and freezing and thawing method as well as the method of destroying the cell walls enzymatically with the employment of lysozymes.

These methods are not easily applicable for Industrial practice and moreover high efficiency cannot be realized with any of the above method.

1.2.4 History of amylase.

The history of amylase began in 1811 when the first starch degrading enzyme was discovered by Kirchhoff in wheat and lard when the foundation for the discovery and research on amylase. The X-amylase named by Kuhn in 1925 because the hydrolysis products are in alpha configuration. In 1930, Ohisson discovered another amylase -manose, he named it -amylase. Crystal structures was established using 3Å reduction structure which was further improved to a 1.5Å resolution of X-amylase. The 3 dimensional crystal structure of each form where determined in the 1990 and found to be effectively identical.

As diastase amylase was first enzyme to be discovered and isolated by Anselme Payen, 1833. Interestingly the first enzyme produced industrially was an amylase from a fungal source in 1894 which was used an amylase from a fungal source in 1894 which was used as a pharmaceutical aid for the treatment of digestive disorders. Boidin and effront, 1917 were the first to use Bacillus. Subtilis and Bacillus mesentericus for the production of X-amylases on commercial scale using large fermentors in submergermed fermentation. Employment of bacterial cultures for the production of commercial enzyme was pioneered by them and accepted as an industrial practice throughout the world for the production of bacterial x- amylases. Prior to the developments, fungal amylases were extensively produced in the united states by the SSF techniques as pioneered by Takamine.

1.2.5 Classification of Amylases.

Enzymes belonging to amylases, enoamylases and exoamylases are able to hydrolyse starch. These enzymes are classified according to the manner in which the glycosidic bond is attacked.

The starch degrading enzymes are found on the numerous glycoside hydrolase families 13 (GH. 13 families).

1.2.5.1 α- amylase (EC 3.2.1.1)

Endoamylases are able to cleave α, 1-4 glycosidic bonds present in the inner part of the amylase or amylopecton.

Chain. α- amylase is well known endoamylase. It is found in a wide varying of microorganisms, belonging to the Arch as well as the bacteria. The end products of amylase action are oligosaccharides with varying length with X centrifugation and α- limit dextrins, which constitute branch oligosaccharides.

α-amylasses are often divided into two cartegories according to the degree of hydrolysis of the substrate. Saccharifying α-amylase hydrolyze 50 to 60 % and liquefying α-amylases cleave about 30 to 40% of the glycosidic linkages of starch. The α-amylase breaks down, longchain carbohydrates by acting at random locations along the starch chain, ultimately yielding maltorriose and maltose from amylase, or maltose, glucose and “limit dextrin” from amylopectin. α-amylase tends to be faster acting than -amylase because it can act anywhere on the substrate. In human physiology both the salivary and pancreatic amylases are α-amylases and found in plants (adequately), Funi (ascomycetes and basidiomycetes) and Bacteria (Bacilus).

1.2.5.2 α-amylase (Ec 3.2.1.2)

Enzymes belonging to the second group, the exoamylases either exclusively cleave α, 1-4 glycosidic bonds such as α-amylase or cleave both α, 1-4 and α 1-6 glycosidic bonds like amyloglusidase or glucoamylase (Ec. 3.2.1.3). excoamylases acts on the external glucose residues of amylase or amylopectin and thus produce only glucose (glucoamylase and X-glycosidase) or maltose and  α-limit dextrin.  α-amylase and glucoamylase also convert the anomeric configuration of the liberated maltose from α to β. Glucoamylase and α-glucosidase differ in their substrate preference, α-glucosidase acts best on short malto oligosaccharides and liberates glucose with α-configuration while glucoamylase hydrolyses long-chain polysaccharides best β-amylases and glucoamylases have also been found in a large variety of microorganisms.

1.2.5.3 γ-amylase (EC 3.2.1.2)

γ amylase cleaves α (1,6) glycosidic linkages. In addition to cleaving the last α (1.4) glycosidic linkages at the non-reducing end of amylase and amylopectin, yielding glucose.

Unlike the other forms of amylase, γ amylase is most efficient in acidic environment and has an optimumpH of 3.

1.2.6 Sources of amylase

α-amylase – Animals, plants, microbes

β-amylase – Plants, microbes

γ-amylase – Animals, microbes

1.2.7 Industrial application of amylase

1.2.7.1 Textile industry.

Textile industries are extensively using alpha amylases to hydrolyse and solubilize the starch which is then washed out of the cloth for increasing the stiffness of the finished product. Fabrics are sized with starch alpha amylase is used as a deciding agent for removing starch from the grey cloth before its further processing of bleaching and dyeing.

1.2.7.2 Sugar and glucose industries.

Alpha amylase plays a very important role in the production of starch conversion products of low ferment ability. The presence of starch and other polysaccharides in sugar cane creates problem throughout the sugar manufacturing which is minimized by the action alpha amylase.

The high quality product depends upon the efficiency of the enzyme which leads to low production, cost for the starch processor has increased.

Many industries use alpha amylases for the production of glucose. Enzyme hydrolyse the starch and convert it into glucose. They hydrolyse α-1,4 glycosidic linkage in the starch polymer in a random manner to yield glucose and maltose. Therefore, alpha amylase is extensively used in many industries for the production of glucose and also used in water soluble dextrin.

1.2.7.3 Alcohol industry

Alpha amylases convert starch into fermentable sugars. Starches such as grain, potatoes are used as raw materials that helps to manufacture ethyl alcohol. In the presence of amylases, the starch is first converted into fermentable sugars. The use of bacterial enzymes partly replace malt in brewing industries thus making the process more economically significant. Alpha amylase can also carry out the reaction of alcoholysis by using methanol as a substrate.

1.3 Rational of study

I carried out this project in other

1. To produce amylase which can be used for industrial purposes
2. To use in paper, textile, chocolate and alcohol industries
3. To produced amylase which can be administered to child to degrade starch in their food to glucose since their food are mostly made from cereals.
4. To actually confirm on the fact that the bacterials has a great enzyme activity.
5. To know which soil type contains bacterials that has amylase activity
6. To isolate amylase this can be used in the treatment of processing waste water.
7. To also get my project into conclusion

1.4 Objectives

1.4.1 General Objectives

The screening and Isolation of amylase producing bacteria.

1.4.2 Specific Objectives

– Isolation of bacteria

Screening and Isolation of bacteria with amylase activity.

1.5 Hypothesis

1.5.1 Null hypothesis:      Bacteria have amylase activity.

1.5.2 Alternative hypothesis:  Bacteria does not have amylase activity.