We design to improve the activity and stability of bromelain by directed evolution. We used engineered bacteria to heterogeneously express bromelain, so as to ameliorate the traditional production methods. Bromelain has been recombinant expressed in E. coli in the current literature, so we plan to use E. coli to express it to verify the success of the directed evolution. In the practical application scenario of feed processing, B. subtilis was designed as engineered bacteria to heterogeneously express bromelain. We also designed the complete characterization experiment and conceived how the project could be practically applied in the future.
In order to get better bromelain with higher enzyme activity, we carried out directional evolution on the selected target fragments. The screening tool for point mutations was Hotspot Wizard, the 3D structure of the protease was demonstrated by Pymol, and the protease stability after mutation was calculated by RELAX section in R2, an online platform.
After Hotspot Wizard is used to screen out the possible mutation sites, pymol is used to mutate them to the amino acids suggested by Hotspot Wizard, and then the mutation with the lowest energy is selected as the target variant. Second mutation is carried out on this variant to obtain our optimal enzyme mutant.
First we performed a proof-of-concept in E. coli. To obtain a large number of heterologous proteins, we selected the E. coli BL21 Star (DE3) strain from TSINGKE and purchased TSINGKE TSC-E01 BL21(DE3) Chemically Competent Cell directly. For the proof-of-concept experiments, the strains were cultured in Luria-Bertani (LB) medium containing 0.5 mM ampicillin at 37°C and 220 rpm. 2% agar was added to the liquid medium for the preparation of the solid medium.
We used a lactose manipulator system for heterologous protein expression and therefore chose isopropylthiogalactoside (IPTG) as the inducer. In order to determine the most appropriate timing for the addition of iptg for induction, we designed a controlled assay to determine the best timing for the addition of iptg. Monoclonal strains were picked into a small volume of liquid medium for expansion and iptg was added at four times at its OD600=0.6, 0.8, 1.0 and 1.2 with a final concentration of 0.5 mM. Protein was collected by lysing the bacteria after incubation at 37 degrees and 220 rpm for 3-4 h. The amount of protein produced was later verified by sds-page.
To avoid the effect of endogenous proteases of Bacillus subtilis on protein expression, the bacterial strain used in this study was B. subtilis WB800N, which was obtained from the BeNa Culture Collection (BNCC). All of the strains used in this study were cultivated in Luria–Bertani (LB) medium at 37°C and 200 rpm. When required, 100 μg ml–1 of ampicillin, 0.03%MgCl2, were supplemented into the culture medium; 2% agar was also added to the liquid medium in order to prepare solid medium.
LB medium was used, with different temperatures, pH, inoculum concentrations and incubation times for each group of the experiment.The bacteria were incubated at 37±0.5°C and 200 r-min- 1 with other components fixed. Afterwards, the biomass of the bacteria was measured, compared and the most suitable culture conditions were selected.
The test was then arranged using an orthogonal table also below, with 3 factors at 3 levels, to screen for the optimum temperature, pH, inoculum concentration and incubation time by comparing peak biomass and fermentative bacteria. Each treatment was replicated.
The concentration of the bacterial solution is determined by the turbidimetric method, in which Bacillus subtilis is inoculated in LB medium and the optical density of the bacterial suspension is measured every hour using a spectrophotometer. The growth curve of the bacterium under certain conditions can then be plotted by graphing the measured OD600 against its corresponding incubation time.
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.( Part: BBa_K4228017 )
In order to complete the heterogenous expression of mutant bromelain in Bacillus subtilis, we cloned the sequence as BamHI-XhoI inserts in the pBER-2 expression vector as well. pBER-2 is a P43 promoter based shuttle vector which can propagate in Escherichia coli and Bacillus subtilis. The desired polypeptide can be expressed as a fusion protein with His6 tag at the C-terminus and Alkaliophilic Bacillus alkaline protease signal peptide and propeptide at the N-terminus to induce protein secretion.
Since our bromelain product is a recombinant product, in order to obtain a relatively pure protease that can provide biochemical analysis, we must eliminate all other impurity as much as possible, and the best way is to highlight the ‘specificity’ of the recombinant protein. A more classical and reliable method is to introduce a "tag" on the recombinant protein and take the tag as the focus of separation and purification, so that targeted separation can be carried out.
Therefore, we chose to construct a his-tag on the recombinant protease, and purified it by immobilized metal affinity chromatography (IMAC) using a chromatography column containing Ni-NTA gel after physical fragmentation of the engineered bacteria.
In the process of protein purification by IMAC, we used high concentration imidazole solution to elute the protease with his-tag. Now the whole system theoretically contains imidazole, which is not suitable for long-term protein storage. Therefore, after a short period of low-temperature storage, we need to replace the high concentration imidazole with a buffer suitable for long-term storage of proteins, so we use gel chromatography to complete this step.
The protease can hydrolyze the casein substrate at a certain temperature and pH to produce amino acids containing phenolic groups (tyrosine, tryptophan, etc.). Under alkaline conditions, Folin reagent is reduced to produce molybdenum blue and tungsten blue, and spectrophotometer is used to measure the absorbance of the solution at the wavelength of 680nm. The enzyme activity is proportional to the absorbance, from which the enzyme activity of the product can be calculated.
We refer to the Chinese national standard GB/T23527-2009 for the specific operation. The activity determination mainly consists of the drawing of the standard curve and the enzyme activity reaction. 1g L-tyrosine is added to the phosphate buffer solution (pH=7.5), and then treated with NaOH and HCl to adjust the pH. After the pH value is calibrated, it becomes the standard tyrosine solution; Then, standard solutions with a gradient concentration is prepared by different proportions of solution and water, mixed with 1ml 0.5M Folin reagent and 5ml 0.25M Na2CO3, reacted at 40℃ for 20 min, and finally measured the absorbance of standard solution with different concentrations at 680nm to draw a standard curve.
For the determination of enzyme activity, dilute 0.5ml of enzyme solution with a small amount of buffer, and add 1.00ml 100 μg/ml tyrosine solution to the system for a 20-minute reaction after preheating at 40 ℃, and finally TCA was used to terminate the reaction; The unreacted casein in the reaction system is filtered out, and then sodium carbonate solution and Folin reagent are added to develop color again at 40 ℃ for 20min. After that, the absorbance is measured at 680nm, and the corresponding tyrosine concentration can be found on the standard curve, so as to obtain the enzyme activity.
The stability of enzyme is also a very important physicochemical property of enzyme. We used the method of changing the temperature (heating up) to carry out the enzyme activity reaction to determine the stability of the enzyme reaction. In other words, the activity of the enzyme at different temperatures directly reflects the stability of the enzyme. We selected the temperatures of 45℃, 50℃ and 60℃ as the reaction conditions. The basic process of the experiment is almost consistent with the operation of enzyme activity determination, except that the reaction temperature is different. In view of the fact that higher temperature may cause the situation that the buffer capacity of the buffer is exceeded (excessive pH change), we did not choose higher temperature for the reaction.
After obtaining higher temperature data, we can compare it with the data at 40℃ to obtain a relative activity of recombinant bromelain. In the subsequent experimental design, we also anticipated more control variable conditions, such as pH. However, due to the replacement of buffer solution and the need to remeasure the standard curve and other data, we will put such a huge workload into our future experimental.
We carried out molecular docking of bromelain and P. (S16G, W67L) with BAEE, and thus conducting molecular simulation using GROMACS.2020, and obtained RMSD, RMSF and other relevant values. You can see further information in the Model page.
Based on surveys in Human Practices, we selected soybean meal as a proof-of-concept sample.
If the subsequent time is sufficient, we will carry out the following detailed experiments. We have also designed a Kjeldahl method that is more friendly to application scenarios, and hope that it can be used for product detection in the future. In addition, we hope to detect the hydrolysis ability of bromelain on allergenic proteins in feed in the future by means of detailed design of control experiments.
- First use a Kjeldahl method to measure the protein content in the feed before the reaction;
- Preparation of bromelain-treated feed;
- Precipitate the above-treated feed with trichloroacetic acid, and perform a Kjeldahl determination on the supernatant.
- Degree of protein hydrolysis:
Soybean meal powder (4 g) was added with 50 mL (0.5%) bromelain and incubated for 3 h in a shaker water bath at 50 °C. After protein hydrolysis, 20% (w/v) TCA (trichloroacetic acid) was added to the protein hydrolyzate at a volume ratio of 1:1 to obtain a 10% TCA soluble material. The mixture was allowed to stand for 20 min to allow precipitation, and then diluted to a volume of 100 mL. Samples (35 mL) were placed in conical tubes and centrifuged for 20 min to analyze the protein content of the supernatant (1.5 mL).
Soybean meal powder (4 g) was added with 50 mL of distilled water and incubated for 3 h in a shaker water bath at 50 °C.
W5 solution is not practical because soybean meal has destroyed the cell structure.
A 1.5 mL suspension sample was taken before analysis.
Nitrogen is one of the five main elements found in organic materials such as proteins.
This fact was endorsed by Danish chemist Johan Kjeldahl, who used it as a way to determine the amount of protein in samples taken from various organisms. In 1883, Kjeldahl proposed to the Danish Chemical Society a method (which has been extensively revised since his time) for determining the nitrogen content in mixtures of substances containing ammonium salts, nitrates or organic nitrogen compounds.
The core basis of the process is the oxidation of organic compounds using strong sulfuric acid. When organic material is oxidized, the carbon it contains is converted to carbon dioxide and hydrogen to water.
Nitrogens from amine groups found in peptide bonds of polypeptide chains are converted to ammonium ions, which dissolve in an oxidizing solution and can then be converted to ammonia gas.
The Kjeldahl method of analysis is the global standard for calculating protein content in a variety of materials, including human and animal food, fertilizers, wastewater and fossils.
After the instrument is washed, take a 100ml conical flask, add 5ml of boric acid solution, and insert the glass tube at the lower end of the condenser into the boric acid solution. Remove the 11 rod-shaped glass stopper, accurately add 2ml of the digested sample solution to the reaction chamber with a 2ml pipette, then put the glass stopper back, add 10ml of 30% NaOH solution to the 7-glass cup, rotate the rod-shaped glass stopper, put the hydrogen The sodium oxide solution was slowly put into the reaction chamber, and a small amount of liquid was left as a water seal. Wait until the boric acid solution in the conical flask changes from purple to bright green and start timing, continue to distill for 3 minutes, then move the conical flask so that the liquid level is about 1 cm away from the mouth of the condenser, and continue to distill for 1 min. Wash the periphery of the condensation port with a small amount of distilled water, and remove the conical flask. Immediately.
Titrate with standard hydrochloric acid solution and wash the instrument as described above for the next distillation. Repeat the distillation and titration three times. Change 2ml of digested sample solution to 2ml of digested blank control solution, other operations are the same as above, and three groups are measured. In the three groups of blank measurements, if the boric acid solution in the conical flask does not change color, titration is not required.
The soluble nitrogen content in the protein hydrolyzate was determined using the following formula.
Calculate the degree of hydrolysis (DH) using the following formula.
Soluble protein composition and distribution in feed after bromelain treatment, using Tricine SDS-PAGE.
SDS-PAGE analysis is mainly used to separate peptides with molecular weights above 14 kDa. In order to clearly determine the distribution of protein sub-fractions with molecular weights below 30 kDa (to extract the hydrolysis ability of bromelain, to analyze the hydrolysis ability of bromelain to sensitive proteins in soybean meal), Tricine-SDS-PAGE analysis was used in this study[2].
Soybean meal powder (4 g) was added with 50 mL (0.5%) bromelain and incubated for 3 h in a shaker water bath at 50 °C. Another soybean meal powder (4 g) was added with 50 mL of distilled water and incubated for 3 h in a shaker water bath at 50 °C. The treated soybean meal is dried at 55-60°C to a moisture content of approximately 10%.
Two groups were treated separately. 100 mg of sample was mixed thoroughly with 400 μl of protein extract for 10 minutes. Centrifuge the slurry at 7,000 × g for 30 min. The supernatant was collected and the protein concentration was measured by Coomassie brilliant blue method.
protein extract
50 mM Na2CO3, 100 mM NaCl, 0.05% Triton X-100, 0.05% Tween-20, 1 mM PMSF, 1% β- mecaptoethanol.
The soluble protein profile of SBM contains polypeptides and subunits, including β-conglycinin of α, α' and β, and acidic and basic glycinin, with estimated molecular weights of 83, 72, 48.4, 38.9 and 18 ( kDa).
After identifying the major peaks in the PAGE, the ratio of each band was determined by densitometric analysis. The protein subfractions were divided into three fractions based on molecular weight. Protein bands of 55 kDa and above are large-sized fractions, 16-55 kDa are medium-sized fractions, and those less than 16 kDa are small-sized fractions[1].
The proportion of small protein fractions was increased in soybean meal after bromelain treatment, and the increment was calculated according to the ratio. The proportion of large-sized fractions (especially the allergenic protein β-conglycinin component) decreased; the reduction was calculated according to the ratio.
We propose some methods to optimize the current production process of bromelain. We use a simple control panel in fermenter, and a computer to control the whole process. In addition, we also processed the resulting bromelain to make it more stable. You can see further details on the Implementation Page.
[1] C. C. Chen, Y. C. Shih, P. W. S. Chiou and B. Yu (May 2010). “Evaluating Nutritional Quality of Single Stage- and Two Stage-fermented Soybean Meal”. Asian Australasian Journal of Animal Sciences. 23(5): 598 - 606.doi:10.5713/ajas.2010.90341
[2] Schaegger, H., and von Jagow, G., "Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa". Anal. Biochem. 166(2), 368-379.