Detection of Alpha Amylase Activity From Bacillus Species Isolated From Soil Salihu Yahuza M.sc Genetics and Bioengineering Fatih University, Istanbul - Turkey

ISLOATION AND IDENTIFICATION OF SOIL BACTERIA CAPABLE OF PRODUCING ALPHA-AMYLASE

Salihu Yahuza

M.Sc Genetics and Bioengineering

Fatih University, Istanbul-Turkey

Outlines1. Introduction Background Aim(s) and Objectives Scope and Limitation 2. Methodology 3. Results 4. Conclusion 5. References

1. Introduction 1.1 Background:

Microorganisms have become increasingly important as producers of industrial enzymes.

Because of their biochemical diversity and the ease with which enzyme concentrations may be increased by environmental and genetic manipulation, attempts are now being made to replace enzymes, which traditionally have been isolated from complex eukaryotes.

Starch degrading amylolytic enzymes play a significant roles in biotechnology industries with huge applications in food, fermentation, textile and paper industries.

Contd.A vast number of organisms live in the soil, so great are microflora number that they dominate the biomass in spite of the minute size of each organisms.

Together with 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.

Contd.The ability of bacteria to degrade a variety of organic compounds is remarkable.

Highly specialized groups of microorganisms play important roles in the mineralization of specific classes of organic compounds.

Which enzyme is alpha-amylase?Alpha-amylase is an enzyme that belongs to a family of endo-amylases that has ability to catalyse the initial hydrolysis of starch into shorter oligosaccharides through the cleavage of alpha-D-(1-4) glycosidic bonds.

Oligosaccharides with varying length with an alpha-configuration and alpha-limit dextrins are the end-products of alpha-amylase action which constitutes a mixture of maltose, maltotriose and branched oligosaccharides of 6-8 glucose units that contain both alpha-1,4 and alpha-1.6 linkages.

Contd.Other amylolytic enzymes participate in the break down of starch but alpha-amylase play more important role in the initiation of this process.

Alpha-amylase are produced by plants, animals and microorganisms.

In humans, the enzymes are secreted in pancreas and salivary glands.

Structure of alpha-amylase

Bacillus Bacillus belongs to a diverse group of bacteria that are able to colonize a variety of habitats ranging from soil and insects to humans.

They constitute several species that synthesize important antibiotics and industrial enzymes of which alpha-amylase is inclusive.

They are rod-shaped, Gram-positive, sporulating, aerobes or facultative anaerobes that are mostly saprophytes.

Contd.Spores produced by Bacillus are resistant to heat, cold, radiation, dessication and disinfectants.

They exhibit an array of physiologic abilities that allow them to live in a wide range of habitats including many extreme habitats such as desert sands, hot springs and arctic soils.

Researches have shown that alpha-amylase can be produced by several species of microbes, but for commercial applications, the enzyme is mainly derived from the genus Bacillus

Contd.Alpha-amylases produced by B. licheniformis, B. stearothermophilus B. amyloliquefaciens find potential applications in a number of industrial processes like food, textiles and paper industries.

Contd.1.2aim(s) and objectivesAim:The study is aimed at isolating and identifying bacteria from different soil environments that are responsible for the production of alpha-amylase.

Contd.Objectives:Isolation and identification.

Phylogenetic analysis.

Enzyme activity assay.

Effects of pH and temperature determination.

Contd.1.3Scope and Limitation Scope:This research is based on isolation and identification of bacteria from different soil environments that show alpha-amylase activity when grown in the culture media.

Therefore, samples that showed positive response were stocked and subjected to enzyme activity assay.

Contd.Limitations:Purification of the enzyme.

Gene cloning.

2. Methodology Sample collection.

Media preparation.

Grams staining.

16s ribosomal RNA sequencing.

Enzyme activity assay.

Contd.Sample collection:

Soil samples were collected in a clean, dried, sterile containers from different environments as follows;Behind E-block and car-pack, Fatih university, Istanbul.Buyucekmece lake, Istanbul.Agricultural area, Bursa.

Soil containing humus was collected from these areas because it is expected to be a source of nutrients that supports the growth of bacteria.

Contd.Media preparation:Upon sample collection, alpha-amylase media were prepared.The media is rich in the following;

Medium 1

Ingredients Amounts in 1LNacl 0.4gPeptone 2.0gYeast extract1.0gStarch 10.0gNutrient agar20.0g

Medium 2Ingredients Amounts in 1LPeptone 3.0gKH2PO40.6gZnSO40.001gFeSO40.0005gMnSO40.05gStarch10.0gK2HPO40.4gMgSO40.5gNutrient agar20.g

Contd.Following the mixture of the chemicals, the media were autoclaved at 121 degree celcious for 15 minutes.

Upon autoclaving, the media were poured on to a clean dried petri plates and allowed to solidify.

After solidification of the prepare media, the soil samples were inoculated on to the plates.

The plates were incubated at 37 degree celcious for 24 hours.

Grams stainingMethod of differentiating bacterial species into two large group; Gram- positive and Gram- negative.Staining reagents include:Crystal violet (primary stain)Iodine (modant)Alcohol (decolourizer)Safranin (counterstain)

The smears of the bacterial colonies were fixed on a microscopic slides and Grams stain reagents were applied.The slides were observed microscopically under oil immersion lens (100 times magnification).

16s ribosomal RNA sequencingComponent of the 30s small prokaryotic ribosome subunit.It is 1.542kb (1542 nucleotide in length).

The genes coding for it are referred to as 16s r DNA and are used in constructing phylogenies.

Samples that showed positive response to alpha-amylase were sent MACROGEN (Korea) and REFGEN (Ankara) companies for the phylogenetic analysis.

Closely related bacteria that have a degree of agreement in the molecular structure of DNA, RNA and proteins were identified.

Enzyme activity AssayDNS method was employed for the alpha-amylase activity assay.

Following centrifugation of the bacterial cells, the supernatants were subjected to the assay.

Absorbance were sphectrophotometrically measured.

3. ResultsBiochemical activation of the colonies:

Contd.

Contd.

Contd.The clear zone of inhibition indicates positive response to alpha-amylase exhibited by the soil bacteria.

The diameter of the colonies and the zone of inhibition were measured.

The larger the zone of inhibition, the more active the bacteria.

Sample no.Colony diameterZone diameterEnzymatic indexA1.3.S2.17172.42A1.3.S2.26162.66A1.3.S2.35163.2A1.5.S3.27142A1.5.S3.27142

Contd.Grams stain result:

Contd.Phylogenetic result:

Contd.Activity assay result:

phTemperature (0C)Absorbance (nm)6800.2726370.2506250.2497800.2917370.2897250.2518800.23983706368250.262

4. Conclusion It can be concluded that alpha-amylase can be produced best by soil bacteria belonging to the genus Bacillus.

pH and temperature have influence on the enzymatic activity.

5. References Aiyer, P.V. (2004) Effect of C:N ratio on alpha -amylase production by Bacillus lichineformis. African Journal of Biotechnology; 3:519-522.

Brady, N.C. (1974) The nature and properties of soils, 8th eds. Macmillan Publishing Co., Inc. New York p111-129.

Giri, B.; Giang, P.H.; Kumari, R.; Prasad, R.; Varma, A. (2005) Microbial Diversity in Soils. Microorganisms in soils: Roles in Genesis and Functions. Soil Biology. 3. pp 19-55.

Octavio, L.F, Daniel J. R., Francislete R. M., Carlos Bloch Jr, Carlos P.S., and Maria F.G., (2000) Activity of wheat alpha-amylase inhibitors towards burchid alpha-amylases and structural explanation of observed specificities. Eur. J. Biochem. 267, 2166-2173.

Pandey A., Nigam P., Soccol V.T, Singh D., Mohan R (2000) Advance in microbial amylases. Biotechnol. Appl. Biochem. 31:135-152

Contd.Richele, D.W., Reinetta, M. B. and Lubbert, D. (1998) Engineering of factors determining alpha-amylase and cyclodextrin glycosyltransferase specificity in the cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EMI European Journal of Biochemistry Volume 253(3), pp598-605.

Rusell, E.W. (1977) Soil conditions and plant Growth 10th eds. Williams Clowes and sons Ltd. London. 150p.