Team:Stuttgart/Results
Cloning
Cloning of the bacterial laccase S.cyaneus
We were able to successfully clone the laccase sequence of S.cyaneus (synthesized by IDT), modified with a N-terminal 6xHis-Tag for purification, into the pET28a expression vector. For cloning, the NEBuilder® HiFi DNA Assembly Cloning kit was used. With this method, an overhang on both termini of the laccase sequence was generated by primers, which creates an overlap of about 20 to 40 bp to the vector backbone pET28a. The amplification was verified on a 1% agarose gel (Fig.1A) and cut at the desired length (laccase S.cyaneus: 1890 bp; pET28a: 5.273bp). Furthermore, the cloned vector was confirmed by restriction digest and sequencing (Figure 1B).
Figure 1: (A) PCR products of the amplified laccase S.cyaneus (LSC) and linearized backbone pET28a run on a 1 % agarose gel . M = molecular- weight size marker (NEB, 1 kb DNA Ladder), BB = backbone. The desired PCR products are labeled with a red asterisk. (B) restriction digest of the cloned plasmid containing the laccase gene of S.cyaneus with XbaI and NotI. The positive clone was labeled with an arrow. (1-5 = digested vectors of colony 1-5).
Unfortunately, due to a problem in the design of the laccase, there was a stop codon introduced in front of the N-terminal 6xHis-tag. Therefore, we had to order a new sequence at IDT, whereby we also modified sequences to introduce a 3x poly-Lysine tag and a 6x poly-Lysine tag at the C-terminus across the active side. This new sequence should be cloned into the new expression vector pET22b since cloning by restriction digest was a lot easier than with pET28a. We therefore introduced the restriction sites NotI and NdeI, to clone the laccase genes into the expression vector via ligation. The laccase genes and the vector backbone were digested and run on a 1% agarose gel. The desired fragments were cut from the gel and the DNA isolated (Fig. 2A). However, we had issues with the cloning of the new vector due to the low activity of the restriction enzyme NdeI. A test digest showed, that the NdeI enzyme did not digest all of the vector backbone, leading to the possible relegation of vectors cut with only NotI (Fig. 2B). Because of time restraints and COVID-19, we were not able to finish the cloning of the new vector.
Figure 2: (A) Digested laccase S.cyaneus (LSC) and linearized backbone pET22b with NotI and NdeI run on a 1 % agarose gel. M = molecular-weight size marker (NEB, 1 kb DNA Ladder), BB = backbone. The desired PCR products are labeled with a red or white asterisk. (B) Test digest of the expression vector pET22b. c = control with undigested plasmid pET22b; “1” = pet22b digested with NotI, “2”= pet22b digested with NdeI.
Expression and purification of the laccase S.cyaneus
The cloned plasmid containing the expression vector pET28a and the laccase of S.cyaneus was transformed into E. coli BL21 (DE3) and selected on agar- plates containing 50 µg/ml kanamycin (Kan50). The transformed BL21 (DE3) were used to express the enzyme for 4 hours by cultivation in LB+Kan50 medium with addition of 400 µl IPTG for induction. For the purification of the recombinant expressed laccase S.cyaneus, we used the Thermo Scientific HisPur Ni-NTA Resin, which enables effective immobilized metal affinity chromatography (IMAC) purification of poly-histidine-tagged proteins from a soluble protein extract. Since both the modified laccase S.cyaneus and T.versicolor should contain a 6x His-Tag, we would be able to purify the expressed proteins. Unfortunately, due to the stop codon in the sequence in front of the N- terminal 6xHis-tag, the His- Tag was not expressed. This led to the problem, that we were not able to purify the expressed laccase S.cyaneus. Nevertheless, we used the obtained supernatant to analyze the laccase by measuring ABTS oxidation. Using a control of the supernatant of non-induced BL21 allowed us to determine that the laccase was clearly expressed. This is because the induced sample showed a strong color change in contrast to the control which remained in its initial state (Fig. 3A). To further analyze the laccase, we performed the ABTS assay on the supernatant measuring every 20 seconds for 20 minutes (Fig. 3B).
Figure 3: (A) ABTS cuvettes containing 10 µl culture supernatant “1” of induced or “2” of uninduced BL21 cells, 100 µl 5 mM ABTS and 890 µl citric acid- phosphate buffer pH 4.0. (B) Measured absorbance at 420 nm with 0.5 mM ABTS for 20 minutes every 20 seconds at room temperature. Lac 1,2,3= measured samples.
To solve the problem of the unknown protein concentration in the supernatant, we determined the total protein concentration via Bradford assay. Therefore, we generated a standard curve using BSA solutions (bovine serum albumin) with defined concentrations (Fig. 4).
Figure 4: standard curve of BSA for Bradford Assay.
By inserting Abs595 = 0.0965 for x in y [mg/ml] = 1.56x [absorption at 595 nm], a total protein concentration of c(protein, total) = 15 mg/ml was determined. Using the information of this measurement and the information of the previously performed ABTS assay, we could calculate the concentration of the laccase S.cyaneus in the supernatant.
The specific activity is defined as
Formula 1:
From the literature, a specific activity of laccase from S.cyaneus for ABTS was found to be Uspez= 6.3 U/mg [1].
Formula 2:
Since the total volume measured was 1 ml a laccase S.cyaneus concentration of 1,06 mgLSc/ml crude extract was determined.
Cloning of the fungal laccase T. versicolor
In addition to the bacterial laccase, we were able to successfully clone the laccase sequence of T. versicolor (synthesized by IDT) based on the already existing BioBrick of Stockholm 2018 (BBa_K2835003) which contained a N-terminal 6xHis Tag for purification, into the pPICZa A expression vector. For cloning, the NEBuilder® HiFi DNA Assembly Cloning kit was used. With this method, an overhang on both termini of the laccase sequence was generated by primers, which creates an overlap of about 20 to 40 bp to the vector backbone pPICZa A. The amplification was verified on a 1% agarose gel (Fig.5A and Fig.5B) and cut at the desired length (LTV: 1528bp; pPICZa A: 3543 bp) . Furthermore, the cloned vector was confirmed by sequencing.
Figure 5: PCR products of the amplified laccase T.versicolor (LTV) and linearized backbone pPICZa A run on a 1 % agarose gel . M= molecular- weight size marker (NEB, 1 kb DNA Ladder), BB = backbone. The desired PCR products are labeled with a red asterisk.
Transformation and Expression of the laccase T.versicolor
The cloned plasmid containing the expression vector pPICZa A and the laccase of T. versicolor was transformed into P. pastoris X-33 and selected on YPD agar-plates containing 100 µg/ml zeocin for 2 to 3 days. Subsequently, we selected the grown colonies for Mut+ phenotypes on MD and MM agar plates. The P. pastoris genome contains the Alcohol Oxidases AOX1 and AOX2, which if both genes are present and functional, are Mut+. Transformation of X-33 with linearized constructs favor single crossover recombination at the AXO1 locos. Therefore, there is a chance that recombination will occur in the AOX1 regions, creating MutS transformants. These MutS transformants show a significantly slower growth rate, about 5% or 10% of the activity compared to the wildtype, because its reliant only on the AOX2 gene for methanol metabolism [2]. After selection, the transformed X-33 were used to express the enzyme for 5 days by cultivation in BMGY & BMMY with daily addition of 0.5% methanol and 0.2 mM copper sulfate for induction.
The resulting supernatant was then subjected to an ABTS assay to examine the laccase obtained and determine activity. Unfortunately, neither the supernatant nor the cell lysate showed any activity or colour change. We measured the induced samples as well as an uninduced sample for 20 minutes every 2 minutes, shown in Fig. 6. Even with repetitions and changes in expression, we could not express laccase. Since we used the laccase sequence of T.versicolor based on the already existing BioBrick of Stockholm 2018 (BBa_K2835003), we suspected that the expression could be influenced negatively due to codon optimization or bad genome integration. As time was limited due to laboratory restrictions, we therefore concentrated on bacterial laccase S.cyaneus for further experiments.
Figure 6: Measured absorbance at 410 nm with 0.5 mM ABTS for 20 minutes every two minutes at room temperature.
Analysis
LSC Kinetics
Laccase activity of cell lysate was measured at 9 different substrate concentrations. The substrate-dependent activity of the laccase was evaluated with a Hanes-Woolf plot and a Lineweaver-Burk plot. Both the Lineweaver-Burk plot and the Hanes-Woolf-Plot provide a graphical method for analysis of the Michaelis-Menten equation. Since the Hanes-Woolf-Plot is thought to be more accurate for the determination of kinetic parameters, we determined Vmax and Km according to said plot (Fig.7).
Figure 7: Enzymatic activity in different substrate concentrations at pH 4 at room temperature. (A) Lineweaver-Burk-Plot (B) Hanes-Woolf-Plot.
The obtained kinetic parameters vmax and Km by the Hanes-Woolf-Plot are:
vmax [µM/s] = 0.36764706
Km [µM] = 46.4814815
As already mentioned in chapter “Expression and purification of the laccase S.cyaneus”, we were able to successfully determine the total concentration of the supernatant c(protein, total) = 15 mg/ml and the concentration of the laccase with the concentration of 1,06 mgLSc/ml.
pH stability Assay of laccase S.cyaneus
We could then determine the activity U of the enzyme after three days for each pH value, shown in table 1. Based on the activity, we can see that pH 6 and pH 7 have a high initial activity, but then decrease strongly. Already after 24 hours we can see a clear decrease in activity, which shows a strong instability of the enzyme at these pH values. In contrast, pH 4 showed the lowest decrease in activity.
Figure 8: Measured activity using ABTS assay plotted over time. (A) pH stability assay at pH 4 (B) pH stability assay at pH 5 (C) pH stability assay at pH 6 (D) pH stability assay at pH 7.
In order to determine the pH stability of our recombinant expressed laccase S.cyaneus, we performed a pH stability assay at different pH values. Over 4 days, samples were drawn at certain time points and an ABTS assay was performed to determine laccase activity. Each measurement was done in triplicates. The BL21(DE3) lysate containing laccase S.cyaneus was diluted 1:10 in citrate-Na2HPo4 buffer (total volume 1 ml) with pH 4, 5, 6 or 7 and put into a heat bock at 25 °C. 10 µL of reaction mixtures were drawn at the time points 0h, 24h, 48h, 72h and 96h. Activity was measured at each time point for 20 minutes in 20 second intervals. The slope of the linear range was used to calculate the specific activity of the laccase (Fig.8).
Table 1: Calculated activities U for each pH value at the time points 0d, 1d, 2d for the laccase S.cyaneus.
This observation was also confirmed by us when considering the percentage decrease of activity after 4 days at each pH value. The percentage decrease can be calculated using the following formula (3). The calculated percentages are shown in table 1.
Formula 3:
On the basis of this calculation we could determine that pH 4 showed the lowest decrease in activity with only 56.8 % after four days. On the contrary, the measurements with pH 6 and pH 7 showed a decrease higher than 90%. With this assay, we could successfully demonstrate and confirm, that the laccase S.cyaneus showed the highest and best stability in the environment of pH 4. The gained information could then be used for further research of our laccase S.cyaneus.
Table 2: Calculated percentage decrease of activity after 4 days for the laccase S.cyaneus.
Long-term stability of laccase T.versicolor
To determine long-time activity of our laccase, a sample was stored at room temperature for several days and laccase activity of the sample was measured sporadically. Here, the commercial laccase of T. versicolor (Sigma Aldrich) was used. Each measurement was done in triplicates. 10 μl of laccase solution (1 mg/ml) with 100 μl of ABTS solution (0.5 mM) in a total volume of 1 ml citrate-Na2HPO4 buffer. Activity was measured for 20 minutes in 20 second intervals and plotted over time. The slope of the straight line was used to calculate the specific activity of the laccase (Fig.9).
Figure 9: Laccase activity of T.versicolor at pH 5, 2.5 hours.
The specific activity was plotted over the time and fitted with an exponential curve to represent the degradation of the laccase and therefore, the loss in enzyme activity (Fig.10).
Figure 10: Activity loss of the laccase T.versicolor at 25 °C over time.
We were able to determine an activity loss of the laccase S.cyaneus of 80 % after 9 days.
Diclofenac Assay – HPLC Method
To determine the degradation of DCF under the ideal pH conditions, which were taken from literature [3] and confirmed via ABTS assay a 3-day assay was performed. Comparatively the degradation was tested at pH 5. The assay was performed as described in the methods section. The mean values of the triplicates as well as the standard derivation are shown in table 3.
Table 3: values of the measured triplicates as well as the standard derivation at pH 4 and pH 5 over time.
All measurements were performed using liquid chromatography. The 100 µM starting concentration of DCF in the assay was defined as 100 % and all the other measurements were set relative to the respective starting concentration. The stability of DCF was already demonstrated under the assay conditions over a period of 3 days in previous experiments as well as in literature [3].
Figure 11: Degradation of DCF by the laccase of S. cyaneus compared at pH 4 and pH 5. All measurements are set relative to the 100 µM starting concentration in the assay which was defined as 100 %. The error bars indicate the standard derivation of the triplicates.
As visible in Fig. 11 at pH 4, 42 ± 7 % of the starting DCF concentration was still detectable in comparison to 63 ± 8% at pH 5. As expected, the degradation is more efficient under more acidic conditions at the laccases pH optimum as already seen in the ABTS assay. After 24 and 48hrs no real difference in degradation can be determined due to the high standard derivations in the assay. To achieve even greater degradation rates the assays temperature could be optimized as well as the reaction time. Furthermore, an enzyme engineering could be performed to optimize the laccases activity under more basic conditions as can be found in sewage treatment plants.
MCF
Synthesis and Glutaraldehyde Immobilization of the MCF
In order to use our laccase to degrade pollutants in wastewater, we immobilized the enzymes onto a mesostructural cellular silica foam (MCF) via glutaraldehyde. This could lead to improved stability and extended half-life. We were successfully able to synthesize the mesostructural cellular silica foam (Fig.13A) and to immobilize the commercial laccase of T.versicolor onto the foam. The foam was synthetized and immobilized according to the respective protocols (hier verlinken: Glutaraldehyde Immobilization). In order to characterize the Mesocellular Silica Foam (MCF) we performed Scanning Electron Microscopy (SEM) to gain insight into the surface and pore size of our MCF. Therefore a small sample of our Foam was spin-coated on a mini slide and analyzed by a Scanning Electron Microscope (SEM; Hitachi S-4800) at 2,0kV. Fig.12 shows a representative SEM image.
Figure 12: Mesostructural cellular silica foam (MCF) under a scanning electron microscope.
For the first immobilization mix, 10 ml of 5mg/ml laccase T.versicolor (commercially available) stock solution were added to 1 g of glutaraldehyde activated MCF. 10 ml crude extract with the concentration of 7.5 mg/ml total protein concentration with the laccase S.cyaneus were added to 1 g of glutaraldehyde activated MCF. Both tubes were incubated overnight at 4 °C and under shaking at 165 rpm for 24 h. In order to analyze if the laccases were immobilized and if an activity was detectable, wei first measured the amount of protein left in the supernatant. After 24 h of incubation, both 15 ml tubes containing the MCF and the protein solutions were centrifuges for 5 minutes with 4600 rpm and at 4 °C. The supernatant was transferred and tested for their protein content using a Bradford assay. Since the initial protein concentration was known, we could determine the amount of immobilized protein. The initial protein concentration and the concentration after immobilization are shown in table 4. No protein was immobilized in our crude extract sample, since the protein concentration in the supernatant was the same as the start concentration. This could be due to the other components in the crude extract, which clogged the pores of the MCF, so no protein was able to immobilize. Future procedure here would be the purification of the protein for another attempt at immobilization. Luckily, almost 99% of the commercially available laccase T.versicolor was immobilized, since the remaining protein concentration was about 1% of the starting concentration auf 5 mg/ml.
Table 4: Initial protein concentration and after immobilization [mg/ml] measured by Bradford Assay for the laccases S.cyaneus and T.versicolor.
As an initial test, we performed an ABTS assay using the full amount of immobilized MCF with the laccase concentration of 5 mg/ml in a total volume of 15 ml with 0.5 mM ABTS of 20 minutes (Fig. 12C) and a control with non-immobilized MCF. Fig. 12B shows the centrifuged supernatant with the oxidized ABTS next to the control.
Figure 13: (A) Synthetized MCF (B) Supernatant of the ABTS assay with the MCF after 20 minutes. Right: negative control MCF without immobilization. Left: with immobilized laccases (C) 15 ml falcon containing the immobilized MCF with 0.5 mM ABTS after 20 minutes.
Further testing for activity of the immobilized laccase T.versicolor was done by ABTS assay using diluted samples. 10 samples each of foam with a protein concentration of 1 mg/ml and ABTS concentration 0.5mM were measured in a final volume of 1ml. Since the foam requires centrifugation of the samples and only the supernatant can be measured, each sample was centrifuged and measured at a different time point (Fig.14).
Figure 14: Measured absorbance at 420 nm with 0.5 mM ABTS for 20 minutes every 2 minutes at room temperature of the supernatant of the immobilized MCF.
The slope of the straight line was used to calculate the activity [U] = 0.0075 of the immobilized laccase T.versicolor
References
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- Moser, J.W., Prielhofer, R., Gerner, S.M. et al. Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris . Microb Cell Fact 16, 49 (2017). https://doi.org/10.1186/s12934-017-0661-5
- Margot J, Bennati-Granier C, Maillard J, Blánquez P, Barry DA, Holliger C. Bacterial versus fungal laccase: potential for micropollutant degradation. AMB Express. 2013 Oct 24;3(1):63. doi: 10.1186/2191-0855-3-63. PMID: 24152339; PMCID: PMC3819643.
