Temperature effectson bacteria (Bacillus licheniformis) and fungi (Aspergillusoryzae)

The intention of this experiment was to find out different temperature effects (0-85) degree Celsius on bacteria (Bacillus licheniformis) and fungi (Aspergillusoryzae) amylase. In-fact this test was carried out to investigate specified specimens’ optimal temperature and the variance between them. Six replicated groups conducted the test in the laboratory. Amylases were tested randomly to eliminate any probable bias. The total number of test tubes used were sixteen where eight used for testing amylase and the remaining eight used for testing starch. The chemical solution employed for coloring was iodine. Acclimation duration was recorded after every test tube was located into the right bath temperature. Experimental results from the entire replicate groups indicated that the 25 degree Celsius was the maximum temperature for fungal amylase as well as bacterial amylase. Though other group could find that the maximum temperature for fungal amylase was 55degree Celsius and the maximum temperature of bacterial amylase being 85 degrees Celsius, it was detected that some groups may have been involved in numerous errors on the finalization part of experiment and as could be alluded, starch solution might have missed the required pumping. Furthermore, there is a possibility that bath temperatures might have decreased in the process of the experiment leading to a poor outcome at the end of the test. The significance of the test was just to impact the knowledge of enzymes and various areas where they have been widely used in mass production of goods to the learners. Enzymes are very important biological components because they enhance biochemical reactions in the body of a living organism

Effects of Temperature on Enzymes

Introduction

               In numerous biological studies, one of the major biological catalysts that has been mentioned and demonstrated many times is an enzyme.  Substrates are the molecule upon which enzymes could respond and react. In most of the research studies, it has been confirmed that enzymes have the ability to convert substrate into various smaller particles called products. They enhance biochemical reactions via a contact action criterion. According to Whitehurst and Van (2009), the site of an enzyme which is active is usually alone, permitting only substrate that can accommodate active site follow enzyme in a protocol termed as cardinal theoretical account and cardinal. Some researcher like Reece, Urry, Cain, Wasserman, Minorsky, & Jackson, (2011) holds a wide explanation of different features of enzymes, they further allude that the characteristics of enzymes in numerous biological system has provided a platform to which organisms can meet the threshold of their energy demand in a system of various components. In a living organisms the operations of enzymes are through chemical reactions activation energy lowering permitting individual organisms to digest nutrition sources into smaller particles essential in operations of cells.

               Amylase is one of the examples of an enzyme. This kind of an enzyme is generated by most of living organisms which has starch catabolism and therefore, permitting living organisms which generates enzyme to obtain nutrition from different part of vegetation. We have different types of amylases including fungus amylase and bacteria amylase. Aspergillusoryzae generates an alpha amylase called fungal amylase. Its availability may be through powder formulation or as liquid. Fungal amylase sides which are active entails hemi-cellulose, protease and beta-glucanase. Fungal amylase has been widely applied in cleaning compounds, starch modification which is commercial, animal feeds, pretreatment fermentation and textile processing. Bacillus licheniformis generates an alpha amylase called bacterial amylase. The characteristics of alpha amylases like pH profile, Ca-independence, thermo-stability and pH stability are significant in improving the process of fermentation.

               Multiple factors affect the functioning of an active enzyme. These factors entails; pH range that is from six to eight, variation in temperatures for instance low temperatures and higher temperatures causes the denaturing of active sites, concentration of substrate in that when the limit has been reached there is no further change in reactions and activators and allosteric inhibitors. Amylase enzymes has a wider spectrum of locations in the structure of a living organism. They are available from musculus contraction coevals to signal transduction. Besides aiding the digestion process by organizing smaller particles to a form of more absorption in mammal, enzymes digest starch particles (Willems, Lelimousin, Koldsø, & Sansom, 2016). Conversely, enzymes ability is to digest starch and the repercussion of the temperature in this experiment will be critically reviewed. The affirmation of temperature consequences will be well indicated by the results from the test of fungus amylase and bacterial amylase

Methods

               The test should be carried out one group per session for the essence of using bacterial and fungus amylase. Starch contact activity was controlled by exploiting iodine attempt that changes to blue-black from Xanthus if amylase is available.

Experimental setup

               The paper was located under spot plate as indicated by the figure 4.5 provided, and across the top temperature was labeled (0, 40, 60, 95) degree Celsius together with the side times 0, 2, 4, 6, 8. 10 minutes. Testing tubes which were specified for the test and earlier had been identified were then obtained and every marked with a distinct temperature. Enymes were represented by the different identities and marks where Enzyme source B was the bacteria and F was representing fungi combined with group number were labeled. Another four testing tubes obtained and labeled with a different temperature, enzyme source B or F, group number and letter S which represented a starch solution. Finally, 5ml starch solution of 1.5% concentration was then added into every tubing used for trial marked S.

Effect on temperature on activity of amylase.

               1ml of amylase was added into every tube used for trial and didn’t have starch. Amylase solution was placed into the bacterial amylase designated test tubes in the event of being first assigned to deal with bacterial. The amylase solution was placed into the bacterial amylase designated test tubes in the event of starting with fungi. The entire four testing tubes for containing starch and the all four trial tubes containing amylase were located into their corresponding temperatures. Trial tubes were then entirely left to equilibrate for five minutes in their corresponding temperatures. Two to three drops of iodine then added to the zero-minute row. At the end of equilibrate process, without removal of tubes from the water bath, few drops of starch were then transferred from each temperature treatment to the first row of spot plate corresponding to zero time minutes. A separate transfer pipette was used for transfer in every treatment to avoid contaminations. Each transfer pipette was then labeled with the actual temperature so that they could be reused in each time interval. Starch solution was then added into the tube containing amylase within every temperature treatment.

               Setting of time was done at an interval of 2min with regards to the amylase drops. 2-3 additional droplets of a given chemical solution referred to as iodine was then added to every well at the 2min row, this was achieved repeatedly before immediate starch amylase transfer to the spot plate. At a particular period of 2min, accurate transfer pipette for every temperature was applied to eliminate fewer droplets of starch amylase mixture from every testing tube. 2-3 droplets of the following mixture located at the second raw where time interval was equivalent to 2min on the spot plate under corresponding temperature. Finally, the color changes were noted and observations recorded table 4.3 (fungal amylase) or 4.4(bacterial amylase) depending on the groups amylase source.

Results

Table 1: Class Average and standard deviation for fungal Amylase activity

Temperature in degree Celsius

0

25

55

85

Average

4.54166667

3.91666667

3.36111111

3.58333333

Standard deviation

0.61382873

0.70203785

1.04615699

0.89042526

This is the average data computed after all classes had acquired their information. This indicates color change with corresponding temperature.

Figure 1: class average fungal amylase action graphical representation.

Outcome from figure one as shown in the graph, indicates that fungal amylase optimal temperature is 25 degrees Celsius.

Table 2: Class Average and standard deviation for bacterial amylase activity

Temperature in degree Celsius

0

25

55

85

Average

4.09

4.24

3.4

3.6

Standard deviation

0.456

0.444

0.39

0.410

This is the average data computed after all classes had acquired their information about bacterial amylase. This indicates color change with corresponding temperature.

Figure 2: class average bacterial amylase action graphical representation.

This graph shows clear that the optimal temperature for bacterial amylase is 55 degrees Celsius

Fungal amylase statistics per class.

Table 1.1 data after 0min

Temp (°C)

0

25

55

85

Time (min)

Group 1

4.5

4.5

4.5

4.5

Group 2

4

4

5

5

Group 3

5

5

5

5

Group 4

5

2

4

5

Group 5

4

4

5

5

Group 6

5

4

5

3

SD

0.49159604

1.02062073

0.41833001

0.80104099

0 minutes                                   Mean ±

4.58333333

3.91666667

4.75

4.58333333

Table 1.2 data after 2min

Group 1

4.5

4.5

3

4

Group 2

4

4

3

3

Group 3

5

3

3

4

Group 4

5

5

3

4.5

Group 5

5

4

4

4

Group 6

5

4

5

3

SD

0.41833001

0.66458007

0.83666003

0.61237244

2 minutes                                    Mean ± SD

4.75

4.08333333

3.5

3.75

Table 1.3 data after 4min

Group 1

4.5

4.5

3.5

3

Group 2

4

4

3

3

Group 3

4

3

2

4

Group 4

3

4

2.5

3

Group 5

5

4

4

4

Group 6

5

4

4

3

SD

0.75828754

0.49159604

0.81649658

0.51639778

4 minutes                                  Mean ±

4.25

3.91666667

3.16666667

3.33333333

Table 1.4 data after 8min

Group 1

4.5

4

3.5

3.5

Group 2

5

4

2

2

Group 3

5

3

2

3

Group 4

3.5

3.5

2.5

4

Group 5

5

4

4

4

Group 6

5

5

4

2

SD

0.60553007

0.66458007

0.9486833

0.91742393

8 minutes                                 Mean

4.66666667

3.91666667

3

3.08333333

Table 1.5 data after 10min.

Group 1

5

3.5

3.5

4

Group 2

5

4

1

2

Group 3

5

3

2

4

Group 4

3.5

2.5

2.5

3

Group 5

5

4

4

5

Group 6

5

5

4

2

SD

0.61237244

0.87559504

1.21106014

1.21106014

10 minutes                                   Mean ± 

4.75

3.66666667

2.83333333

3.33333333

Table 2. shows the combined analysis of bacterial amylase per stipulated time

Bacterial Amylase data analysis

Temp (°C)

0

25

55

85

Time (min)

Group 1

4.5

5

5

5

Group 2

5

5

5

5

Group 3

5

4

4

4

Group 4

4.5

4.5

4.5

5

Group 5

4

5

5

5

Group 6

4

5

5

5

± SD

0.447213595

0.418330013

0.418330013

0.40824829

0 minutes                                    Mean

4.5

4.75

4.75

4.833333333

Group 1

5

5

3

4.5

Group 2

5

4

4

5

Group 3

5

3

3

3

Group 4

3

3

1.5

2

Group 5

4

5

1

4

Group 6

3

5

1

5

± SD

0.98319208

0.98319208

1.25499004

1.200694244

2 minutes                                   Mean

4.166666667

4.166666667

2.25

3.916666667

Group 1

5

5

3

4

Group 2

5

4

3

4

Group 3

5

3

3.5

2

Group 4

3

3

1.5

2

Group 5

4

5

1

4

Group 6

3

5

1

5

± SD

0.98319208

0.98319208

1.125462868

1.224744871

4 minutes                                  Mean

4.166666667

4.166666667

2.166666667

3.5

Group 1

5

5

2.5

3.5

Group 2

4

3

2

4

Group 3

4

3

3.5

2

Group 4

3

3

1.5

2

Group 5

4

5

2

4

Group 6

3

5

1

5

± SD

0.752772653

1.095445115

0.861200712

1.200694244

6 minutes                                Mean

3.833333333

4

2.083333333

3.416666667

Group 1

5

5

2

4

Group 2

5

4

3

4

Group 3

4

3

3.5

2

Group 4

3

3

1.5

2

Group 5

4

5

2

4

Group 6

3

5

1

5

± SD

0.894427191

0.98319208

0.930949336

1.224744871

8 minutes                                  Mean

4

4.166666667

2.166666667

3.5

Group 1

5

5

2

4.5

Group 2

Group 3

4

3

3.5

2

Group 4

3

3

1.5

2

Group 5

4

5

2

4

Group 6

3

5

1

5

± SD

0.836660027

1.095445115

0.935414347

1.414213562

10 mintues                                   Mean

3.8

4.2

2

3.5

Observation of spots

Figure 3: Group number spot plate during bacterial amylase experiment showing the amylase reaction during each temperature

Discussions

               After obtaining the effects of temperature analyzed in table 1 and 2, it can be deduced that the outcome of the experiment offers sufficient grounds to prove the theoretical understanding or even the anticipation in biological learning field that enzymes are cut off or denatured when optimum temperature is not achieved. The effect indicated that high or low temperatures have an impact for enzyme activity as well demonstrated by the graph 1 and 2. When temperature effects are compared with color coding design offered figure 1, the maximum temperatures for all amylases could be determined. The maximum enzyme temperature had a bright starch color, implying that the amylase was able to be digest starch in the solution. Upon no change in blue-black the enzyme could have been denatured indicating that it was unable to break down the starch figure 1 and 2.

               The parametric quantities which were more import taken into history to acquire the ancient effects included clip as well as temperature. Investigating the reaction color, between amylum and amylase, by utilization of iodine test, it could be postulated that for fungal amylase maximum temperature was attained at 25 degree Celsius and bacteria maximum temperature at 55 degrees Celsius. As per Harisha, (2006), some ancient effects could be as well obtained in the above graphs. Effects obtained by every group are about to be actual as compared, and the variation between the sample data collected by a singular group and norm of data is very low indicating that the effects of experiment are consistent for entire groups. Test outlooks happened with the effects, since old understanding of enzymes were provided in the lab manual, in addition, the maximum temperature were not acknowledged in prior since enzymes work best depending on the surrounding. For future studies, more variability should be in temperature scope and even adding negative values. In-fact greater understanding would have been available if enzymes beginnings had more fluctuations which could have led to better apprehension of maximum conditions as well as enzyme temperatures.

Literature cited/ Reference

Lab manual

Jose et al., General Biology Manual, second edition

Raven P., Johnson G. B., Mason K. A., Losos J. B., Singer S. S. (2008). Biology 8th edition. New York: The McGraw Hill Companies.

Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Jackson, R. B. (2011). Campbell biology(Vol. 9). Boston: Pearson.

Harisha S. (2006). Introduction to Practical Biotechnology. India: Laxmi Publications.

Willems, N., Lelimousin, M., Koldsø, H., & Sansom, M. S. (2016). Interfacial Enzymes and Their Interactions with Surfaces: Molecular Simulation Studies. Understanding Enzymes: Function, Design, Engineering, and Analysis, 297.

Whitehurst R. J., Van Oort M. (2009). Enzymes in Food Technology: Wiley-Blackwell; 2nd edition.