PURIFICATION AND CHARACTERIZATION OF LEVASUCRASE FROM A BACTERIA ISOLATED FROM KITEEZI (UGANDA)

KAGGWA JOHN BASABOSE
05/U/3229
(BIOCHEMISTRY AND BOTANY 2005-08)

SUPERVISOR; Dr. J. HAWUMBA

2007
INTRODUCTION
Microorganisms including bacteria produce a number of extracellular enzymes into their environments:-
Of these enzymes, are the Carbohydrases, such as Amylases, Cellulases, Chitinase Xylanases, and among others, are the Sucrases. (Seeley, H. W, et al., 1991; Van Hijum, et al., 2004)

Advantages of Levansucrase enzyme and its product (Levan) to producing Bacteria.
To pathogenic strains (for example Actinomyce viscosus), Levan is used to trigger immunological injury and mutagenesis to human immune defense B-lymphocytes., a Levan-Levansucrase system aggregates the pathogens in the plaque. (Ray, et al, 1995)

OBJECTIVES
4.1 Major objective

§ Purification and characterization of a levansucrase from thermo-tolerant alkalophile isolated from Kiteezi landfill.

4.2 Specific objectives

ü Produce the enzyme.
ü Purify the enzyme using ion-exchange chromatography.
ü Determine the molecular weight of the purified levansucrase by gel filtration, SDS-PAGE chromatography and PI techniques.
ü Determine the maximum enzyme activity under various conditions of temperature, pH (or ionic-strength).
ü Determine Km and Vmax of the enzyme
ü Determonstrate levan production using purified levansucrase.

PROBLEM STATEMENT

Many Mesophilic reported Levansucrases act at low Temps <50oC and pH <5-to-6.5 these produce relatively unstable short chain levan. This project will therefore describe a thermo-active / stable and alkalophilic Levansucrase, thought capable of both Sucrose Hydrolysis and Transfructosylation producing stable and long chain levan. The purification and characterization of this enzyme and its ability to produce Levan at high Temps >50oC and pH, needs an urgent description, due to increasing Bio- requirements for thermo-stable products such as food ingredients, medical supplements, and Bio-degradable plastics

JUSTIFICATION

Production and utilization of microbial fructans from thermo-active Levansucrase transformation of sucrose is an important object for the sugar, food, medical, Research and other industries. Non-digestible homo- and hetero-Oligo fructosaccharides and bio-polymers (Levans) have beneficial effects on humans and animals such as their use in natural low-calorie sweeteners, Biopolymers, anti-tumor and anti-inflammatory agents.

MATERIALS AND METHODS

Sucrose and other chemicals to be used in preparation of the complex culture medium will be used.
Source of the enzyme; from a sample bacteria of a thermo-tolerant, alkalophile already isolate from Kiteezi.
Enzyme production, The bacterial cultures will be incubated at 55oC, and shaking over night and 72hrs resp. Cells will be removed by centrifugation at 10000xg, and the cell free supernatant used for enzyme purification.
Enzyme purification. Cold ethanol (4oC) will be added to the supernatant, and the mixture centrifuged at 10000xg, pooled and dialyzed against water at 4oC.

MATERIALS AND METHODS CONTINUED 2;

The dialyzed solution will be passed through a DEAE-cellulose column, pool the active fractions and dialyzed against distilled water and then run in a Bio-Gel P-100 column, Again active fractions will be pooled and applied to two successive Bio-Gel A1.5 columns
Purified Levansucrase molecular mass, will be determined using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) (Weber and Osborn, 1975).
Protein content measurements will be done spectrophotometrically using Lowry’s method.

MATERIALS AND METHODS CONTINUED 3;

Enzyme activity, will be determined as sucrose hydrolysis activity, to define One unit of Levansucrase that produces 1 μmol of glucose per minute under standard conditions.
Enzyme activity relation ship with pH and Temp shall be determined by varying pH (4.0 – 12.0) and Temp (room temp – 100oC).
Enzyme Kinetics shall be done by determining both the Km and Vmax