From the data observed from the UV spectrometer, we were able to determine the sources of the unknown α-amylase proteins. Aspergillus oryza is a fungus that prefers acidic conditions and high temperature environments, it matches best with sample 1. Bacillus licheniformis is a bacterium that likes slightly acidic conditions and extreme temperature environments. This bacterium matches best with sample 2. The last is porcine pancreas. It’s α-amylase functions best at 370C and prefers a neutral pH environment. Porcine matches best with sample 3 from the data.

The second source was derived from the fungus Aspergillus Orzae, whose alpha amylase functions optimally under acidic conditions. The last source derived from the pancreas of the mammal porcine, whose alpha amylase’s optimal conditions are a temperature of 37 C and neutral pH.

By testing the affects temperature and pH had on Alpha amylase activity, we were able to determine the source of the protein from three different organisms.

Factors Effecting Enzyme FunctionMykel Mussatto and Pamela Young, St. Louis Community College, 2014

Enzymes are proteins that serve as catalyst for many biological reactions by reducing the activation energy required for these processes to occur. Enzymes allow the creation of chemical substances required for the biological functions of life such as lipids, carbohydrates, and other proteins. Shape is the primary factor determining the function of an enzyme.

All enzymes are comprised of amino acid chains whose arrangement and personal interactions create the coiled and folded subunits enzymes are comprised of. These interactions and arrangements produce a plethora of different three-dimensional shapes therefore allowing many unique enzyme forms and functions. Contained within each protein is an active site. This site is the location where proteins interact with substrate molecules to trigger a catalytic reaction.

Alpha-Amylase is the enzyme responsible for the hydrolysis of alpha linked polysaccharides especially glycogen and starch. These polysaccharides are broken down into the easily absorbed glucose responsible for providing energy for cellular processes and the disaccharide maltose. This ability makes alpha-amylase not only an important enzyme in digestive and nutrient uptake for many living organisms but also important in the commercial production of high-fructose corn syrup.

Though the primary function of alpha-amylase does not change from organism to organism its optimum range for activity varies greatly just as the biome of these creatures varies. As mentioned above enzyme form and function is greatly determined by its shape which can ultimately be affected by the conditions at which the enzyme is subjected to. Factors such as temperature and pH can have an adverse effect on the disulfide bonding amongst the polypeptides resulting in misshapen subunits. These misshapen subunits can render an enzyme unusable or greatly reduce its catalysitic function.

In this experiment our team explored the various factors that can have an ill effect on the hydrolysis ability of Alpha amylase amongst different kingdoms. For this experiment our team was supplied with alpha amylase derived from Bacillus Licheniformis, Aspergillus Orzae, and Porcine pancreas. The first source was derived from the bacterium Bacillus Licheniformis, whose Alpha amylase functions optimally at high temperatures and acidic pH.

INTRODUCTION RESULTS CONCLUSIONS

FIGURE 3: CAPTION AND FIGURE DESCRIPTION (SOURCE, YEAR).

REFERENCESImages:Alpha amylase Images: Protein Data Bankhttp://rcsb.org/pdb/home/home.doActivation energy: Scitable, nature educationhttp://www.nature.com/scitable/topicpage/protein-function-14123348Enzyme function: School work helperhttp://schoolworkhelper.net/factors-affecting-the-activity-of-catalase-and-amylase-lab-answers/UV Spectrometer setup:http://nanocomposix.com/pages/characterization-techniquesAspergillus Orzae: Environmental informationhttp://enfo.agt.bme.hu/drupal/en/gallery/9574Bacillus licheniformis: Nutri-systems international, inchttp://nsspo.com/p1/UP.htmPig: The guardian online news articlehttp://www.theguardian.com/world/2012/feb/15/pig-farmer-animal-cruelty-found-deadResults:tabulation of class data

The data collected shows obvious distinction between the three sources of alpha amylase. Sample 1 appears to has an optimal temperature range near 650C and functions best in acidic environments. These are conditions that match Aspergillus oryza. Sample 2 has a high temperature range in the 90’s and an optimal pH of 7, characteristics closely similar to Bacillus licheniformis. Sample 3 alpha amylase functions best at 370C and prefers a neutral pH environment. This is what is expected to see from porcine pancreas.

TEMPERATURE EFFECTS4˚C 25˚C 37˚C 65˚C 90˚C

A 0.135 0.148 0.229 0.458 0.434A 0.08 0.3 0.49 1.25 0.53A 0.35 0.69 1.02 1.69 1.03Average 0.186667 0.377333 0.577333 1.131667 0.664

B 0.012 0.117 0.135 0.503 0.743B 0.16 0.14 0.21 0.63 0.96Average 0.088 0.1285 0.173 0.5645 0.851

C 0.005 0.048 0.1 0.103 0.039C 0.07 0.53 0.86 0.63 0.33Average 0.0375 0.289 0.48 0.3665 0.1845

pH EFFECTS

pH 5 pH 6 pH 7 pH 8 pH 9

A 0.307 0.236 0.054 -0.11 -0.22

A 0.94 0.76 0.47 -0.04 -0.04

A 1.28 1.02 0.59 -0.07 -0.17

Average 0.839667 0.671667 0.370333 -0.07367 -0.14367

B -0.053 -0.17 0.111 -0.146 -0.158

B 0.01 -0.18 2.46 -0.18 -0.14

Average -0.0235 -0.1725 1.2835 -0.1635 -0.149

C -0.08 -0.05 0.24 -0.05 -0.07

C -0.08 0.01 3.80 -0.14 -0.08

Average -0.082 -0.0197 2.0192 -0.0927 0.07205

Bacillus licheniformis alpha-amylase Pig pancreatic alpha-amylaseAspergillus oryzae alpha-amylase

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METHODSTo identify the optimal conditions of the α-amylase enzyme, we incubated unknown samples of the alpha amylase enzyme in 1% starch solutions at various temperatures and pH environment ranges. After the incubation period the samples were then read using a UV spectrometer to determine the maltose concentration.A UV spectrometer is comprised of 4 basic parts; a light source, sample holder, detector, and prism that allow UV spectrometers to measure absorbency.

The concentration is determined by the amount of light absorbed by the sample when a beam of light is passed through it. The absorbency is determined by the amount of molecules within a sample that can interact with the light passing through. If a sample is intensely pigmented the absorbency reading will be high due to the large amount of molecules giving the sample it’s color. Inversely a lightly colored sample will have a very low absorbency because it lacks a large amount of molecules. The UV spectrometer calculates the % transmittance ratio by measuring the intensity of the light passing through a blank sample (usually made of water or the clear base liquid of the sample) and the actual sample.

ABSTRACT

Aspergillus oryza

Bacillus licheniformis