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Bachelor Thesis Project |
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It's unrelatable in this era to not have a digital copy of a bachelor dissertation, but that's how it has been for me since I graduated long back in 2009. This was when students at the university were supposed to submit only a printed version. When I finished my master's thesis this year, I realized I might need to write a bit about my bachelor's thesis project as well somewhere. And that's what this blog post is mainly about. My actual bachelor thesis dissertation/report was around 80-85 pages as I recall, and this post is mainly a gist of the report based on my memory in combination with a standard scientific protocol -- luckily, I found a digital copy of the protocol that my group and I had followed during the project.
Thesis title: Extraction, purification, and estimation of specific activity of microbial amylase
Timeline: Summer 2009
Amylases are starch- and glycogen-breaking enzymes that are produced by many different kinds of organisms. From bacteria to fungi, plants and humans, amylase production is done by all. Specifically, bacteria and fungi produce amylase and then releases it outside their cell membranes to conduct digestion extracellularly. By breaking insoluble starch using amylases, they can absorb the remaining soluble parts such as glucose or maltose through their cell wall pores.
There are three different types of amylases that are produced by bacterial and fungal cells and are divided in line with their ability to break down starch molecules. Most microbial amylases contain a mixture of these different types and they are:
- Alpha amylases: break down the sugar bonds at random and thereby reduce the viscosity of starch
- Beta amylases: break down glucos-glucose bonds by remocing two glucose units at a time and produce maltose
- Amyloglucosidases: break successive bonds from non-reducing end of straight chain and produce glucose
For an industrial level production of amylases, bacteria and fungi are cultured and harvested on a large scale -- mainly these species: Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquifaciens and Aspergillus niger. These amylases are often used for corn syrup preparation, as detergent additives, starch saccharification in alcohol production, and brewing.
Amylase-producing microbes can be sourced and isolated in a few different ways, getting them from the moist debris collected from the top soil in a nearby park was convenient. 100 grams of the collected soil sample was mixed with 90 mL sterile distilled water, which was then further diluted by adding 10 mL of this mix into another fresh breaker of 90 mL sterile distilled water. This dilution was then used to inoculate multiple agar plates for respective bacterial and fungal growth.
For growing bacterial culture, nutrient agar plates with 1% weight-by-volume soluble starch concentration were inoculated using 0.1 mL of the dilution mix and then incubated at 30°C for 24 hours.
For growing fungal culture, 0.1 mL of dilution was used to inoculate potato dextrose agar plates fortified with 0.1 mg/mL streptomycin sulfate, in order to avoid any bacterial growth. Inoculated plates were then incubated at room temperature for 72 hours.
It was important that the producers chosen for amylase extraction were relevant as many a times there could be certain strains that produce starch and thus needed to be avoided for the following processes. Therefore, starch-producing bacterial colonies were identified first by means of visible clearing around them, and then by staining the whole culture using Gram's iodine. Portions of the culture that stained black were avoided so as to pick only the amylase producers.
Using a sterilized dissecting needle, fresh agar plates for both bacterial and fungal cultures were streaked in the same way as done previously in the first round. Newly inoculated plates were then incubated at respective requisite temperature and for the respective durations (24 hours for bacteria and 72 hours for fungi). Once the incubation periods were over, cultures were stored in a referigerator until the next usage.
For allowing the cultures to produce amylase, similar inoculation and incubation procedures were followed again but this time in agar slants in flasks instead of agar plates in petridishes. Incubation for fungal growth at this stage was conducted with intermittent shaking.
For the extraction of amylase from fungal culture slants, mycelium was removed and the remaining part was filtered using Whittmann number 1 paper. The filtrate was used as the crude extract containing fungal amylases.
For the extraction from bacterial culture slants, a high-speed referigerated centrifuge was used. The bacterial culture from the slants was poured into the centrifuge tubes and spun for about 20 minutes at 5000 rpm. Supernatant was used as the crude extract containing bacterial amylases.
What my group and I actually observed and measured during the project is no longer accessible to me. However, this section explains how we obtained and analysed the final results experimentally and empirically.
Amylase extracts from both fungal and bacterial slant cultures were collected in separate test tubes and mixed with 1 mL of 1% soluble starch in citrate-phosphate buffer with a pH of 6.5. Amylases typically function in a neutral to slightly acidic environment and this mix was to allow for the enzyme to remain active and continue to break down starch for next stage of specific activity measurements. The immediate next step after mixing enzyme extracts with buffer and starch was to incubate the mix in a water bath for 40°C for 30 minutes. This allowed for an optimal temperature for extracted amylases to break down the starch.
Comparative measurement was conducted by means of a blank with same amount of enzyme mix, with enzymatic activity inactivated by boiling the enzyme extracts for 20 minutes before adding to the starch and buffer mix.
Enzymatic reaction in the experimental tubes were stopped by adding 2mL of dinitrosalicylic acid (DNS) reagent, followed by 5 minutes of boiling. Use of DNS allowed for the formation of a colored compound on reaction with reducing sugars in the solution, while boiling denatured the enzyme. On cooling, 20mL of distilled water was added to the colored solution and a spectrophotometer was used to measure its absorbance levels based on its color intensity at 540 nm -- as the intensity of its color and thus the absorbance of light are directly proportional to the amount of reducing sugars present in the solution. A pre-prepared glucose concentration calibration curve was used to find the equivalents based on absorbance levels estimated using the spectrophotometer.
Once the equivalent concentrations of reducing sugars for all experimental tubes were estimated, specific activity was calcuated by dividing the total amount of reducing sugars (glucose) produced by the product of incubation bath time (30 minutes) for enzymatic reaction and amount of enzyme.
Toye Ekunsaumi, Laboratory production and assay of amylase by fungi and bacteria.UWWashington county. Vihinen, M. and Mantsala, P. (1989). Characterization of a thermostable Bacillus stearothermophilus a-amylase. Biotechnol Appl Biochem 12, 427–435