Enzyme Activity Experiment Report (DEX and FruA)
The main purpose of this experiment is to measure the activity of target enzymes, Dextranase in a TOP 10 expression system. The sequences of Dextranase is extracted from Escherichia coli K-12. Testing the efficiency and the effects of these enzymes’ expression is a fundamental portion of the whole project. We hypothesized that the enzyme activity (defined by active unit divided by bacteria population) is positively related to a given period of time. We obtained factors from bacteria density in cul-ture to the degradation amount of substrate via visible spectrophotometer.
Considered all the properties of E. coli expression system, there are several things that can influence the expression of a particular enzyme. To understand and catego-rize the factors, the expression process of enzyme (protein, specifically) need to be broke down into three stages, which are target plasmid replication, transcription and translation (include further process to structure protein). Among these stages, tran-scription and translation affects the expression efficiency mainly.
In this experiment, the influence to Dextranase expression in TOP10 expression sys-tem is mainly measured in a factor, which is enzyme activity. To be specific, enzyme activity is the ability or effectiveness of catalyzing reactions.
Considered that the condition in natural environment will be much more complicate and unpredictable, designing appropriate experiments and dividing factors correctly are especially vital.
II Background and Experiment Design
It has been mentioned in the literature that this enzyme can decompose and detach the dextran plaque adhering to the wire. For the product design, we want to express the protein with lactic acid bacteria and make a yoghurt-based project.
Due to the limitation of experimental equipment, we were unable to obtain the suc-cessfully transformed lactic acid bacteria, so E. coli was used as the expression vec-tor, and the experiment of enzyme activity measurement was designed with the con-dition variable near the actual range.
1. Selection of experimental variable pH: The data shows that the pH of the yogurt is about 6 and the pH of the saliva is about 6.6-7.1. Therefore, our pH in experimental group selected is 7.
2. Selection of experimental variable temperature: The lower limit of the tempera-ture variable we choose is 25 degrees Celsius, because the body temperature is 37 degrees Celsius, and the chilled yogurt will cause the temperature of the mouth to decrease; on the other hand, the upper limit of the temperature variable is 50 degrees Celsius. Thus, we could explore the stability of enzyme activity in the higher tem-perature state and understand the enzyme activity in the production and storage pro-cess.
Therefore, we can get a reasonable range of dextranase enzyme activity curve to predict the effect of product enzymes to some extent.
According to the background above, we predict that the enzyme activity is positively related to time within a period.
IV Experiment Protocol
1.Centrifuge for 5 min in a 2 ml centrifuge tube at 5000 r/min.
2.Add 2ml of 0.1mol/L Tris-HCl buffer solution(400W) start the ultrasonic wave for 3s and stop for 8s. 90 cycles in total. last for 990s (about 16min)
3.Use the Ultrasonic Cell Crusher to centrifuge the cells at 12,000 g for 20 minutes at 24 ° C, and after remove the supernate, the rest is the crude enzyme solution. It is expected to mention 2 ml of crude enzyme solution.
Material Prep: Substrate solution: confect 2% dextran solution (add0.1 g dextran to 1 ml, 1 mol/L Tris-HCl buffer, then add 4 ml dd water)
4.Reaction substrate solution: Sulfuric acid was added dropwise to the substrate solution prepared in the previous step to prepare reaction substrates having pH of 8, 7.5, 7, 6.5, 6, 5.5 separately.
5.Use 1.5ml centrifuge tubes as the reaction vessels; add 10ul of the crude enzyme solution and 90 ul of the substrate solution. (divided into thirty-six experimental groups, one control group). The control group was crude enzyme solution which is inactivated at 100 ° C for 5 min reacts with the substrate at 37 ° C for 10 min.
6.After the reaction, add 150 ul DNS developer and boil at 100 ° C for 5 min, then add 2 ml dd water.
7.Measure the absorbance of each group at a wavelength of 540 nm, and adjust the control group to a zero point.