Latest revision as of 09:00, 12 October 2022

Brief introduction

Considering that the molecular genetic technology is more mature in Escherichia coli, we chose the basic pET-32a vector to perform preliminary validation at first. We cloned the sequence as BamHI-XhoI inserts in the pET-32a expression vector. pET-32a is a T7 promoter vector which can propagate in Escherichia coli after appropriate induction by IPTG.The desired polypeptide can be expressed as a fusion protein with 6xHis tag at the C-terminus for simplified purification.

Characterization

Expression of Bromelain

Bromelain wild type, bromelain variant 1 and bromelain variant 2 were synthesised at 37°C, pH=7 and induced by iptg (50mg/mL, Sangon Biotech). After 3-4 hours all sluices containing the three proteins were collected and sonicated to lyse the bacteria. Correctly transformed and induced E. coli expressed bromelain wild type, bromelain variant 1 and bromelain variant 2 (Figure 1). We can see the bromelain (variant) bands around 25-35 kDa. By isolating and purifying pineapple protease variant 1 and variant 2 by Ni columns, we obtained a protein solution containing all three of these proteins.

Figure 1: Electrophoresis of proteins before and after transformation

Enzyme activity assay

In the experiment of formulating the working curve, we configured several standard L-tyrosine with gradient concentrations of 0, 10, 20, 30 and 40 respectively μg/ml; After a thorough reaction with Folin reagent for 20min, microplate reader was applied to measure the absorbance of the system after reaction at the wavelenth680nm. The A values obtained are 0.001, 0.436, 0.801, 1.096 and 1.293 respectively. From this series of data, the following working curve can be obtained:

The formula we applied to is: A=0.03246c+0.076, and the reaction is carried out at 40℃ then the absorbance of the system is measured:

Taking the wild stem bromelain for an example. The average value of the data is 0.552, and the concentration of L-tyrosine can be calculated as 14.66μg/ml according to the function. The amount of substance consumed in the reaction time is 0.08091μmol per minute. According to the formula provided in GB/T23527-2009:

Where X is the enzyme activity of the sample, A₁ is the enzyme activity of the reaction diluent obtained from the working curve (0.08091μmol here), V₁ is the volume of reaction vessel (15ml here), V₀ is the volume of the reaction system (10ml here), n is the dilution multiple of the sample (2 here), m₀ is the mass of the sample taken (0.5g here), and T is the time consumed (10min here). It can be calculated that the activity of our p.(S16G), p.(S16G,W67L) and wild type are respectively 6900U, 7344U, 4854U per gram of coarse cell extracts in the buffer.

Enzyme stability assay

——Only p.(S16G,W67L)

The experimental process is basically consistent with the contents above. The reaction temperature is raised to 50, 55 and 60℃, and three parallel groups are still set at each temperature. After reacting with Folin reagent, the absorbance of the system is measured at 680nm:

The calculation process will not be repeated. Therefore, the average absorbance values at three temperatures are 0.510, 0.266 and 0.087, and the enzyme activities at these three temperatures are 4412U, 1915U and 1541U per gram of coarse cell extracts in the buffer respectively. The enzyme activity-temperature diagram can be plotted from the above data:

Overall, our enzyme activity data confirms the success of the initial mutant as well as the model. We produced a bromelain variant with a 1.5-fold increase in activity in our final design.

Functional Parameters