Isolation, Purification and Characterization of Free and Immobilized Alpha-amylase From Bacillus Licheniformis

Isolation, Purification and Characterization of Free and Immobilized Alpha-amylase From Bacillus Licheniformis

CHAPTER ONE

Aim

To isolate, purify and characterize free and immobilized alpha-amylase from Bacillusmlicheniformis.

Objectives

Isolation of the enzyme alpha-amylase in fermentation media from the cell of the Bacteria isolate of Bacillus licheniformis.
Purification of alpha-amylase obtained from the Bacillus licheniformis
Immobilization of the enzyme by entrapment in calcium alginate beads.
Characterization of the free and immobilized alpha amylase.

CHAPTER TWO

Literature Review

Enzymes

An enzyme is a biological catalyst which alters the rate of chemical reactions usually by speeding it up. An enzyme is a protein molecule with a specific shape to accommodate reactant molecules. One enzyme molecule can catalyze 10 million reactions in a single second.

Enzyme works at relatively low temperature (usually about 40⁰C) thereby saving energy and saving money. The exact function of enzymes is determined by their structure, which is a combination of the sequence of amino acids from which they are build up. The sequence will result in a certain three-dimensional structure that may be stabilized by moieties other than amino acids that are added to the protein backbone. Their complete structure determines their function. Factors that modify enzyme structure will often also affect the function. (Bugg, 2004).

Uses of Enzymes in the Industries

Enzymes are used in the industries in many areas. They are starting to replace petroleum based solvents used to make vegetable seed oil, replacing harsh acids used in the production of glucose products such as corn syrup, replacing sulphides used in the tanning industry and many other aspects. Today, enzymes are helping industry to manufacture products used by society in a way that is less harmful to the environment.

Some of the uses of enzymes in the industries are highlighted below.

Amylases is use in the production of high fructose corn syrup by converting starch to sugars. Trypsin is used for primary food processing for babies because it can predigest baby foods. Papain is used for meat tendering (Nagodawithana and Reed, 1993).

Amylase is used to split polysaccharides from malt during mash process. Proteases help to remove remaining protein from yeasts, which make beer clearer and easily filtrated. Glucose isomerase is used to convert glucose into fructose in production of high fructose syrups from starchy materials. Fungal enzymes are used to convert starch to glucose (Julio and Andrew 2007).

Amylases are used to degrade starch to lower viscosity thereby aiding sizing and coating of paper. Xylanases are used to reduce bleach required for decolorizing. Cellulases are used to smooth fibers, enhance water drainage and to promote ink removal. Amylases are used in detergents for machine dish washing to remove resistant starch residues Cellulases are used in biological fabric conditioners. Alpha-amylases are used to improve bread quality and to increase shelf life. Xylanases decrease the water absorption and thus reduces the amount of water needed in baking which leads to more stable dough. Proteinases are used to improve dough-handling properties. (Julio and Andrew 2007).

Microorganisms

A microorganism or microbe is a microscopic organism, which may be a single cell or multi-cellular organisms. The study of microorganisms is called microbiology. This subject began with Anlon Van Leeuwenhoek‟s discovery of microorganisms in 1675, using a microscope of his own design. Microorganisms are very diverse; they include all of the prokaryotes, namely the bacteria and archaea, and various forms of eukaryotes, comprising the protozoa, fungi, algae, microscopic plants (green algae) and animals such as rotifers and planarians. Some microbiologists also classify viruses as microorganisms, but others considered these as non-living. Most microorganisms are microscopic but there are some like Thiomargarita namibienses, which are macroscopic and visible to the naked eye.

Microorganisms live in every part of the biosphere including soil, hot springs, on the ocean floor, high in the atmosphere and deep inside rocks within the earth‟s crust. Microorganisms are crucial to nutrient cycling in ecosystems because they act as decomposers. Some of them can fix nitrogen, they are a vital part of the nitrogen cycle, and recent studies indicate that airborne microbes may play a role in precipitation and weather (Madigan and Martinko, 2006).

CHAPTER THREE

MATERIALS AND METHODS

Materials

Microorganism Sample

The microorganism sample Bacillus licheniformis which was isolated from Kaduna metropolis soil was obtained from the microbiology department of Ahmadu Bello University, Zaria.

Chemicals and Reagents

Coomasie Blue G-250, Sephadex G-25, DEAE-Sephadex, bacteriological peptone, dinitrosalicylic acid (DNS), sodium alginate were purchased from representatives of Sigma Chemical Co. St Louis, England. Sodium dihydrogen phosphate, hydrogen disodium phosphate, sodium chloride, maltose, starch, glycerol, copper(II) tetraoxosulphate (VI), mercury(II) chloride, manganese(II) chloride, cobalt(II) chloride, zinc chloride, aluminium chloride, tris, glycine and ammonium sulphate are of analytical grade.

CHAPTER FOUR

RESULTS

Incubation Time for Alpha-amylase Production

The production of alpha-amylase was lowest at 3h incubation period and this was 0.05 unit/mg/min while the maximum production was observed at 15h incubation period and this was 0.41 unit/mg/min. Further increase in the incubation period did not show any significant increase in enzyme production rather it was decreased. The enzyme production continue to decrease gradually until 24h incubation period where it was 0.06 unit/mg/min (Figure 4.1).

CHAPTER FIVE

DISCUSSION

The optimum incubation period for B. licheniformis for alpha amylase production was 15h, with an enzyme activity of 0.41unit/mg/min. Incubation beyond the optimum time course was followed by a decrease in the enzyme activity which gradually declined to 0.06 unit/mg/min after 24hr.

The decreased activity in the later phase of growth was probably due to the repression of the catabolite by the readily metabolizable substrate glucose (Lim et al., 1998). Catabolite repression is an important part of global control system of various bacteria and other micro-organisms. Catabolite repression allows bacteria to adapt quickly to a preffered carbon and energy source first. This is usually achieved through inhibition of synthesis of enzymes involved in catabolism of carbon sources other than the preffered one. Catabolite repression can be initiated by glucose and other carbon sources (Deutscher, 2008). Similar results have been reported in B. flovothermus after 24h (Tourney et al., 2007) and in B. amyloliquefaciens after 72h (Sarikaya and Gurgun, 2000).

CHAPTER SIX

SUMMARY AND CONCLUSIONS

Summary

The local strain of B.licheniformis used in this research work is a good producer of the enzyme alpha-amylase. The alpha-amylase produced by this bacterium is a thermostable alpha-amylase which can be used in the textile and starch processing industries.

The immobilization of this enzyme in calcium alginate beads increased its optimum temperature which will in turn affects its shelf life and this will help in the repeated use of the enzyme.

The immobilized alpha-amylase from the local strain of B.licheniformis used in this research work can also be used commercially as a replacement of free enzyme system because the immobilized enzyme has shown greater operational flexibility and higher enzymatic activity than the free enzyme.

Conclusions

Immobilization of alpha-amylase from B. licheniformisby entrapment in calcium alginate beads affects it‟s optimum temperature, pH and kinetics.
The bacteria, Bacillus licheniformisproduces a thermostable alpha-amylase with optimum temperature of 60⁰C and 70⁰C for the free and immobilized enzymes respectively and therefore can be used in starch processing and textile industries.
The immobilization of this enzyme in calcium alginate beads increased its optimum temperature and this will also increase its shelf life.
The enzyme can work best in neutral to alkaline pH and therefore can be useful in starch and textile industries.

RECOMMENDATIONS

Further studies should test the reusability of this alpha amylase immobilized by entrapment in calcium alginate beads.
There is a need to develop more thermotolerant and pH tolerant alpha amylases from the local strain of licheniformisused in this research work, modify it genetically to acquire desired properties in the enzyme.

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