Part:BBa_K4695210

pnbA-docscaB device

This device is a fully functional gene capable of expressing PnbA-DocScaB fusion protein (BBa_K4695110).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1795
  Illegal XbaI site found at 96
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1795
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1795
  Illegal BglII site found at 30
  Illegal BamHI site found at 726
  Illegal BamHI site found at 1029
  Illegal BamHI site found at 1979
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1795
  Illegal XbaI site found at 96
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1795
  Illegal XbaI site found at 96
  Illegal NgoMIV site found at 795
  
• 1000
  COMPATIBLE WITH RFC[1000]

Figure 1 | Model of the PnbA-DocScaB fuision protein predicted by AlphaFold2 software. Enzymatic domain PnbA shown in gold. Linker containg TEV site shown in light blue. Dockerin domain DocScaB shown in green. HisTag at the C-terminal end shown in grey.
Catalytic triad is showcased in the center of the enzymatic domain: Ser189 in green, Glu310 in red and His399 in blue.

Introduction

In response to the problem of phthalic acid esters (PAEs) pollution we came up with an idea of a protein complex capable of efficient degradation of those pollutants. Based on literature and iGEM 2023 DNA Distribution Kit we designed enzymes fused to dockerin domains that enable immobilization to scaffoldin. PnbA-DocScaB is one of those fusion proteins. This construct is capable of hydrolyzing ester bonds in PAEs and can form complexes with the scaffoldin, stabilizing the protein and improving degradation efficiency. It has a modular build - two domains are connected with a TEV linker, allowing to separate them if needed. This design approach and usage of RFC1000 system is unified across all our fusion proteins and should allow other teams to use our parts with ease.

PnbA esterase BBa_K4695010

Origin

The enzymatic domain of our fusion protein PnbA-DocScaB has the sequence obtained from the genome of Bacillus subtilis BJQ0005 by Xu et al. 2021 . Genome annotation conducted and described in that article unraveled 33 potential ester bond hydrolysis-capable enzymes, and one of them - p-nitrobenzyl esterase (PnbA) (locus tag: GTW28_17760) - showed a di-ester bond hydrolysis activity, which is the reason we grew an interest in this particular protein. It is important to mention, that an enzyme with identical sequence was discovered before - Streit et al. in their paper describe a carboxylesterase with 100% similarity to PnbA .

Biology

Enzymes capable of performing a hydrolysis reaction - hydrolases - can be divided into many classes (e.g., lipases, cutinases, esterases). Some of those enzymes are capable of catalyzing an ester bond hydrolysis. PnbA belongs to a group of classic Ser-His-Glu triad esterases from the 3.1.1 EC family of proteins. Those enzymes follow a distinct pattern of chemical reactions between the substrate and active triad to achieve a hydrolysis of an ester bond.

Some ester bond hydrolases are capable of degrading phthalates. According to their properties, they can be classified into one of 3 groups:

• Group I hydrolases - are capable of performing a hydrolysis of only one ester bond of phthalic acid esters.
• Group II hydrolases - are capable of performing a hydrolysis only of the ester bond of mono-ester phthalates.
• Group III hydrolases - are capable of performing a hydrolysis of both ester bonds of phthalic acid esters.

Group III is the most versatile, because, in theory, enzymes from this group are able to completely degrade PAE’s to pthtalic acid. PnbA is the only group III enzyme identified in the above mentioned paper. Unfortunately, the carboxylic group remaining after first ester bond hydrolysis possibly inhibits the catalytic efficiency of the enzyme on second ester bond. For this reason, our project relies on usage of additional enzymes (see parts: BBa_K4695020, BBa_K4695021, BBa_K4695022, BBa_K4695023). Bacterial supernatant containing PnbA shows promising phthalate esters degrading properties (1).

Sequence and structure

PnbA is a 489 amino acid long protein, with molecular weight of 55,2 kDa. It contains a catalytic triad, present in native form, consisting of Ser189, Glu310 and His399 (1).

DocScaB dockerin BBa_K4695041

Origin

Type I Dockerin ScaB used in our fusion protein originates from a cellulolytic enzyme form bacterium Acetivibrio cellulolyticus .

Biology

Some cellulolytic organisms use big, extracellular protein complexes to achieve effective cellulose degradation. Those complexes are called cellulosomes, and the essence of their functionality lies in the strong interaction between long, structural scaffolding proteins, containing cohesin domains and enzymes, that contain a high cohesin-affinity domains - dockerins.

Sequence and structure

ScaB dockerin is a relatively small domain (9,4 kDa) consisting of 74 amino acids. Its secondary structure consists mainly of alpha-helixes, of which two interact with the correspondent cohesin domain. This particular dockerin shows almost perfect internal symmetry, which allows it to achieve dual binding mode with the cohesin domain.

TEV site

TEV site is a specific amino acid sequence (ENLYFQ\S - the “\” shows exact cleavage site), that is recognised by the TEV protease. TEV protease (EC 3.4.22.44, Tobacco Etch Virus nuclear-inclusion-a endopeptidase) is a ~27 kDa highly sequence-specific cysteine protease, frequently used for controlled cleavage of fusion proteins . To add a TEV site-containing linker to our fusion protein we decided to use a part from E. coli Protein Expression Toolkit available with the iGEM 2023 distribution kit - DT4_TEV (BBa_J435395).

HisTag

PnbA-DocScaB has a 6x HisTag at the C-terminal end of the polypeptid chain. HisTag is often used as purification tag consisting of 6 or more histidine residues fused at the C-terminal or N-terminal end of the protein . Because histidines are strongly inivolved in coordination complexes with metal ions, this tag allows proteins to be easily purified using metal ion affinity chromatography (IMAC). To add a 6x HisTag to our fusion protein we decided to use a part from E. coli Protein Expression Toolkit available with the iGEM 2023 distribution kit - T5E_6H (BBa_J435369).

Design

This composite part consists of:

• AB_T7_lacO promoter (BBa_J435350)
• BC_BCD23 RBS (BBa_J435385)
• PnbA coding sequence (BBa_K4695010)
• DT4_TEV linker (BBa_J435395)
• DocScaB coding sequence (BBa_K4695041)
• C-terminal T5E_6H HisTag linker (BBa_J435369)
• EF_T7 terminator (BBa_J435361)

Figure 2 | SnapGene map of pnbA-docscaB device with part names of individual components. Different open reading frames (ORFs) shown in yellow and green. The longer, yellow ORF represents the sequence coding the desired protein.

All non-coding parts and linkers were chosen from the E. coli Protein Expression Toolkit collection.

Choosing the promoter, RBS and terminator

When choosing promoter, RBS and terminator we immediately decided to use parts from iGEM Distribution Kit. We wanted to work in the spirit of iGEM competition and promote the basic principles of synthetic biology: standardization and universality. We believed that the selected regulatory components were adequately tested, and thus we did not need to look for those elements outside of iGEM competition. In our opinion that is what this competition is all about - contribution by designing new parts but also using ones that are already available.

We needed a promoter that could be induced by IPTG and we chose the T7 promoter with LacO regulation regions (BBa_J435350). To follow up we have also chosen a T7 terminator (BBa_J435361). When choosing RBS, our only requirement was that there were no tags. We wanted the prefixes and suffixes to match the promoter and coding parts so we chose one of the available ones (BBa_J435385).

Figure 3 | Table showcasing the modular nature of fusion proteins designed by our team.

Modular fusion protein

Our project relies on using enzymes fused with dockerin domains, which allows binding to scaffoldin. We have come up with a design that allows user to swap between enzymatic and dockerin domains and achieve different fusion combinations (fig. 3). This maximizes the chances of finding an optimal phthalate degrading conditions. Because of the specificity of the dockerin domains to the scaffoldin cohesin domains, this also ensures precision while creating the protein complex of enzymes and scaffoldin.

This design also allows to separate enzymatic and dockerin domains using TEV protease.

Figure 4 | Table enlisting 5' and 3' flanks added to the parts used to compose our devices.

Prefixes and suffixes

Individual elements of this composite part were flanked as shown in fig. 4. As a result, the whole part is flanked by prefix GGAG and suffix CGCT. It allowed us to ligate this part in silico and in vitro into pJUMP23-1A, which is an AF vector.

Sequence optimization

Source coding sequences were optimized and checked for BsaI and SapI restriction sites using: http://genomes.urv.es/OPTIMIZER. Before ordering, the coding sequences were optimised once more due to synthesis limitations. No promoter, RBS, TEV site, HisTag or terminator was altered.

Cloning

Golden Gate Assembly

We had come to conclusion that pJUMP23-1A (BBa_J428347) would be the most promising available vector for expression of our devices, because of the pBBR1 origin, which translates to medium plasmid copy number. Therefore, we cloned the pnbA-docscaB device into this vector using Golden Gate Assembly. Mixture was prepared according to New England Biolabs’ protocol for using NEBridge® Golden Gate Assembly Kit (BsaI-HF®v2) (fig. 5) and the reaction was incubated following conditions for library preparation (37 °C for 1 hour followed by 60 °C for 5 minutes). In the next step, E. coli strain BL21 (DE3) was transformed with the product (LA with 30 ug/ml of kanamycin). Plasmid isolation from overnight culture was performed using A&A Plasmid Mini AX kit.

Figure 5 | Table showing amounts and proportions of components used for the Golden Gate Assembly (NEBridge® Golden Gate Assembly Kit (BsaI-HF®v2))

Restriction analysis

We treated the pJUMP23-1A-pnbA_docscaB and the unmodified pJUMP23-1A with XbaI alone and with combination of XbaI+BamHI. XbaI alone linearises the unmodified pJUMP23-1A and cuts out short 113 bp parts from the same expression vectors containing our device, which gives very similar band height to linearisation. XbaI and BamHI combination should have the same effect as sole XbaI on unmodified pJUMP23-1A, but cut out parts of specific length from correctly cloned expression vectors:

• for pnbA-docscaB – 950 bp, 630 bp and 303 bp

Gel simulation (fig. 6a) and the results (fig. 6b) are shown. We simultaneously performed similar analysis for constructs pJUMP23-1A-dcx1-docxylY and pJUMP23-1A-estJ6-docscaB. Results for pJUMP23-1A-pnbA-docscaB are very similar to the simulation results, indicating proper assembly: the construct cut with XbaI only has around 5.7 kbp and parts cut of from the construct by the combination of XbaI+BamHI give bands at around 1050 kbp and 750 kbp, with the 3rd fragment not seen probably because of small size. Those results are not giving any information about sequence, but based on them we estimated, that the cloning of pnbA-docscaB into pJUMP23-1A was succesfull.

Figure 6 | a) Verification of proper pJUMP23-1A vector constructs assembly with restriction analysis (fragments)(SnapGene simulation). b) Verification of proper pJUMP23-1A vector constructs assembly with restriction analysis (fragments).

PCR

In order to confirm the assembly more precisely, we ordered primers complementary to every part and linker used to design this device, as well as for the pJUMP23-1A vector (fig. 7). We performed 16 PCR reactions with following primers pairs:

• fJUMP_1_F and pJUMP_1_R
• fJUMP_1_F and RBS_348_R
• fJUMP_1_F and Est_1_R
• fJUMP_1_F and DT4_357_R
• fJUMP_1_F and Ock_2_R
• fJUMP_1_F and T5E_332_R
• fJUMP_1_F and Term_324_R
• Prom_296_F and Est_1_R

Every pair was used in two reactions: one with template DNA and one without template DNA as a negative control. Each one of the 16 PCR tubes was filled with 1.25 ul of each primer, 10 ul of the template DNA (pJUMP23-1A-pnbA-docscaB diluted 1:9 - template:NF water)(10 ul of NF water for control instead) and 12.5 ul of the Q5® High-Fidelity 2X Master Mix (NEB). Reaction was performed in the thermocycler with conditions shown in fig. 8. Results shown in fig. 9b compared to SnapGene simulation shown in fig 9a indicate that the construct is assembled correctly, as all the control reactions were negative and all the bands are on the correct height on agarose gel.

Figure 7 | SnapGene map of the pJUMP23-1A-pnbA-docscaB vector with complementary primers designed to each component for the PCR verification of the correct assembly.

Figure 8 | Conditions for PCR reaction (verification of the correct pJUMP23-1A-pnbA-docscaB assembly).

Figure 9 | a) 1% agarose gel with the results of PCR analysis of pJUMP23-1A-pnbA-docscaB vector assembly (SnapGene simulation). b) 1% agarose gel with the results of PCR analysis of pJUMP23-1A-pnbA-docscaB vector assembly.

Overexpression and overproduction

Small scale

In previous step we obtained E. coli BL21 (DE3) clone with vector pJUMP23-1A-pnbA-docscaB. First, we overproduced the desired PnbA-DocScaB fusion protein on small scale. 50 ml of sterile LB medium containing 30 ug/ml of kanamycin was inoculated with 2 ml of overnight culture of above mentioned strain with the same concentration of kanamycin. Culture was incubated in 37 °C with 160 RPM shaking. At OD600=0.8 the IPTG was added to the final concentration of 1 mM, the temperature was set to 16 °C and culture was incubated overnight with 160 RPM. Samples before and after induction were taken. Figure 10a shows the result of the induction. A thick band apperas on the second path, on the right height corresponding to the molecular weight of the desired protein PnbA-DocScaB (64.6 kDa).

Large scale

After succesful small scale overproduction we set to produce PnbA-DocScaB on a bigger scale. We inoculated 3 liters of sterile LB medium containing 30 ug/ml kanamycin with E. coli strain BL21 (DE3) containing vector pJUMP23-1A-pnbA-docscaB. This time, after incubating in 37 °C with 160 RPM shaking, IPTG induction (final concentration of 1mM) was done when OD600 reached 2.5. Ideally it should be performed at values ranging from 0.5-0.8. Unfortunately, the scale-up of the process changed the dynamic of bacterial growth and we missed the optimal spot. Culture was incubated at 16 °C overnight with shaking 160 RPM. Samples before and after induction were taken. After that, cells were harvested by centrifugation at 5000 RCF and the product – bacterial paste containing desired protein PnbA-DocScaB – was frozen. Results of the big scale overproduction are shown in fig. 10b - similarly to a small scale overproduction, a thick band at the same height appears on the poliacrylamide gel after induction. This time overproduction seems to be less abundant, possibly due to the late induction.

Figure 10 | a) SDS-PAGE gel (fragments) showing PnbA-DocScaB overexpression (1mM of IPTG, 16 °C overnight) on a small scale (50 ml). Samples pre- and post-induction are shown. b) SDS-PAGE gel (fragments) showing PnbA-DocScaB overexpression (1mM of IPTG, 16 °C overnight) on a large scale scale (3 l). Samples pre- and post-induction are shown.

Purification

Bacterial lysis

Bacterial paste with PnbA-DocScaB was resuspended in lysis buffer (10 mM Phosphate buffer pH=7.4, 137 mM NaCl, 2.7 mM KCl, 10% v/v glycerol), homogenized and lysed by sonication (Q-sonica q700 sonicator, 4420 microtip used, amplitude 13, pulse-on time - 15 s, pulse-off time - 45 s, total pulse time - 5 min). Next the lysate was centrifuged at 75 000 RCF to separate soluble and insoluble fractions. All the samples were checked for the presence of the proteins of interest on SDS-PAGE (fig. 11). Most od the protein was in the supernatant fraction, so this fraction was chosen for further purification steps.

Immobilised Metal Affinity Chromatography (IMAC)

Because PnbA-DocScaB has a HisTag at a C-terminal end, we decided to use Ni-NTA resin (His60 Ni Superflow Resin, Takara) and apply an imidazole gradient for efficient, one-step purification. NiNTA resin was transferred to low-pressure, BioRad econo-column (1.5x15 cm) and the column was connected to AKTA Start system. Resin was equilibrated with an A buffer (10 mM Phosphate buffer pH=7.4, 500 mM NaCl, 2.7 mM KCl, 50 mM imidazole, 10% v/v glycerol). PnbA-DocScaB supernatant was loaded on the Ni-NTA resin and washed with A buffer. Then the gradient of imidazole was applied (0-100% B buffer: 10 mM Phosphate buffer pH=7.4, 500 mM NaCl, 2.7 mM KCl, 500 mM imidazole, 10% v/v glycerol). SDS-PAGE gel was performed with fractions collected from different stages of purification. Samples were chosen based on the UV absorbance signal acquired during purification (fig. 12).

Results from this gel are shown in figure 13. It can be observed, that the PnbA-DocScaB band (64,6 kDa) visible in the load sample is much less abundant in the flow-through fraction, which indicates binding of the PnbA-DocScaB to the resin. Wash (50 mM imidazole) eluted most of the non-specifically bound proteins. Imidazole gradient (50-500 mM) proved to be effective in eluting the desired protein. At a concentration of around 150 mM of imidazole, a thick band with height corresponding to the overproduction band in the first path started to appear on the gel. The purity and the concentration of eluted samples are decent. After visualisation on gel, fractions 15-24 from the PnbA-DocScaB gradient were collected.

10 fractions (15-24) containing purified PnbA-DocScaB were mixed (total volume: 20 ml) and dialyzed to the final buffer (10 mM Phosphate buffer pH=7.4, 137 mM NaCl, 2.7 mM KCl, 10% v/v glycerol) overnight (1:100 sample:buffer ratio). On the next day purified protein solution was portioned and frozen. Densitometry was performed with a BSA calibration curve, and the protein concentration (22,3 uM) was established using ImageLab software.

Figure 11 | SDS-PAGE gel (fragments) containing samples of lysate, supernatant and pellet of the E. coli strain BL21 (DE3) harboring overproduced protein PnbA-DocScaB.

Figure 12 | a) Chromatogram of PnbA-DocScaB purification on Ni-NTA resin (load and wash steps). The UV signal shown in blue. b) Chromatogram of PnbA-DocScaB purification on Ni-NTA resin (imidazole gradient). The UV signal shown in thick black.

Figure 13 | SDS-PAGE gel showing fractions collected during the PnbA-DocScaB purification on Ni-NTA resin (load, wash and imidazole gradient). Visible bands of height corresponding to PnbA-DocScaB molecular weight (64,6 kDa) start to appear at around 12th fraction, indicating elution of the protein of interest from the resin.

TEV protease digestion

Since we incorporated TEV protease recognition site between the enzymatic domain (PnbA) and dockerin domain (DocScaB), we set to obtain the native form of PnbA. TEV protease was borrowed from the resources of the Laboratory of Protein Biochemistry from our faculty. It was not a commercially available protein, and we used ratios according to the Laboratory’s protocols. 3 ml of the purified protein solution was mixed with 200 µl of TEV protease solution. This reaction mixture was left overnight with 400 RPM shaking in 16 °C. After the TEV protease cleavage we tried to separate the native enzyme from cleaved dockerin domain on the Ni-NTA resin, taking advantage of the fact that the His-tag remained linked to the dockerin. Overnight mixture of PnbA-DocScaB and TEV protease was loaded onto a small amount of Ni-NTA resin in batch in order to separate PnbA and DocScaB domains. Elution was done with the same buffer, as during the protein purification (500 mM imidazole). The flow-through and elution fractions were collected. Samples of load, flow-through and elution were collected along the way to visualize the process on a SDS-PAGE gel.

In the path with purified PnbA-DocScaB we can see a thick band of the protein with little contaminations. In the path with the post-reaction mixture (PnbA-DocScaB + TEV protease) we can see a thick band of PnbA and a band of cleaved DocScaB, both on the expected height, proving that protein was successfully cleaved. We can also see a distincs band at the same hight as uncut enzyme, meaning that the cleavage was not complete and in the mixture remained significant amounts of whole PnbA-DocScaB. In the path with Ni-NTA flow-through we can see all of the bands that we could observe in the post-reaction mixture, meaning that the purification was not very efficient. In the path with Ni-NTA elution we can once again see all 3 bands of PnbA-DocScaB, PnbA and DocScaB, but thinner, especially the PnbA. It means that even though the purification was not satisfactory some of the cleaved dockerin domain was removed. Surprisingly PnbA was also present in the elution, meaning that, even though His-tag should have been removed together with dockerin domain, native form of PnbA seems to still have some affinity to the Ni-NTA resin.

Figure 14 | SDS-PAGE gel with fractions obtained from enzymatic digestion of PnbA-DocScaB cut with TEV protease.

Activity testing

Tycho

Analysis of the purified protein on SDS-PAGE is conducted in denaturing conditions, so no information about protein folding and activity is obtained. After purification of PnbA-DocScaB, we wanted to characterise the acquired enzyme in terms of its stability and tertiary structure. We decided to use Tycho (NanoTemper). Usage of this system is very simple. According to the protocol, that guides you when using the system, in order to measure our sample we simply left it to reach the room temperature, and then transferred a fraction of the purified protein solution to the dedicated capillary and inserted the capillary into the device. The analysis was done, data were collected and analysed. Tycho allows to see how the rise of the temperature affects the tertiary structure of proteins in the sample, by measuring the intrinsic fluorescence of tryptophan and tyrosine residues. The higher the ratio, the more residues are emitting light and the more unfolded the protein is. Results of this particular experiment (fig. 15) show a big unfolding event occurring at around 54 °C and a smaller one at around 63 °C. Event at 54 °C might correspond to the unfolding of the enzymatic PnbA domain, and the 63 °C can be associated to the unfolding of the smaller dockerin domain. This shows, that the purified PnbA-DocScaB protein indeed has a functional tertiary structure and loses stability at >50 °C.

Figure 15 | Graph showing the relation of temperature with the ratio of intrinsic fluorescence at 350 nm and 330 nm, which directly translates to the folding state of measured protein.

PnbA-DocScaB activity testing using HPLC

In order to measure the concentrations of substrates and products of enzyme reactions in High-Performance Liquid Chromatography (HPLC) we plotted calibration curves of dibutyl phthalate (DBP), monobutyl phthalate (MBP), butyl benzoate (BB), phthalic acid (PA) and benzoic acid (BA). We worked on two different chromatographs, therefore we made the calibration curves twice. Fo PnbA-DocScaB activity we used the second calibration curve.

Figure 16 | Degradation pathway of dibuthyl phthalate to benzoic acid. Enzyme classes responsible for catalyzing each step are showcased.

After purifying PnbA-DocScaB we wanted to test its activity. The analyses were done in 20% ACN in 10 mM PBS (pH = 7.4) to increase substrate solubility. We used dibutyl phthalate (DBP) and butyl benzoate (BB) as substrates. In our proposed degradation pathway PnbA-DocScaB catalyzes the di-ester hydrolysis bond resulting in degradation of DBP to MBP and BB to BA (fig. 16) This experiment was carried out in triplicate for both DBP and BB. Enzymatic reaction was stopped by centrifugation on a membrane with 10 kDa weight cut off. This also separates proteins from the rest of the sample, preventing them from being loaded onto the HPLC column.

We mixed 5ml of reaction mixture consisting of 20% ACN in PBS, 400 µM of dibutyl phthalate (DBP) or butyl benzoate (BB) and 223 nM (14.41 µg/ml) of purified PnbA-DocScaB. Reaction was set up in room temperature. Simultaneously the control sample was prepared with 50 µl of 10% glycerol in PBS instead of 50 µl of the enzyme. It is the same buffer in which the enzyme was dissolved. Enzymes were added last and right after that mixtures were vortexed and aliquots of 500 µl were sampled. Then the mixtures were kept for 30 minutes at 37 °C with shaking. After this time aliquots of 500 µl were taken from each sample again. Aliquots were directly applied to an Amicon® Ultra 0.5 ml and centrifuged at 10 000 RCF for 10 minutes. We transferred 50 µl of filtrate from each sample to an autosampler bottle. Then 30 µl were loaded on the Phenomenex Jupiter 5um C18 300A (250x4.6mm). 1 hour gradient of mobile phase (0% to 100% buffer B, buffer A – dH2O with 0,1% trifluoroacetic acid – TFA, buffer B – 80% ACN in dH2O with 0,1% TFA) at 40 °C was applied to separate the samples. We can see in figure 17a that the concentration of substrate in control sample is around ten times lower than 400 µM in DBP in time 0 and almost four times lower after 30 minutes. In the case of BB it even dropped below the calibration curve concentration range (fig. 17c). It indicates that this method requires redesigning. Amicons might be the potential cause of the concentration decrease. It requires investigation if these compounds interact with the filter. This experiments should be repeated with different method of reaction stopping and enzyme removal. We can see that in the control sample there is no MBP but in the samples with PnbA-DocScaB there has been a clear signal from MBP on the chromatogram (fig. 17b). Similarly in the experiment with BB as a substrate in the control sample there has been a minimal peak from BA, but there has been a significant signal from BA in the sample with the enzyme (fig. 17d). It should be kept in mind that we cannot say with confidence what was the concentration of the product in the sample, since substrate concentration measurements in control samples turned out to be unreliable. Nonetheless this data proves that PnbA-DocScaB catalyzes reactions of ester bond hydrolysis in both DBP and BB. Though there was no signal from PA, therefore we cannot say if this enzyme catalyzes hydrolysis of MBP in the experiment conditions.

We can see that with sampling time equal 0 minutes product was already created. It indicates that reaction stopping by centrifugation on Amicon was too slow to observe the change in reagents concentration in time. If we were to repeat this experiment we would try stopping the reaction with acid as it was dome by Xu et al. (1). Reaction mixture setting and sample preparation have been done in room temperature, therefore we can see that enzyme was functional in this temperature. Concentration of substrate significantly changes after 30 minutes in both control and research sample. Moreover it increases for DBP and decreases for BB. These unexpected changes in substrate concentration might be connected to the temperature change during incubation, since DBP and BB did not form homogenous mixture in 20% ACN in PBS, so temperature rise might have changed the distribution of the substrates between phases.

It is important to point out that after 30 minutes of incubation white flakes precipitated from the reaction mixture. It was probably denatured enzyme. It shows that PnbA-DocScaB has rather low stability in 20% ACN in PBS in these temperature conditions. Fast denaturation of the enzyme also gives plausible explanation for lack of product concentration rise after 30 minutes of incubation.

In conclusion, although this experiment failed to give data about reaction kinetics and enzyme activity it still confirms that PnbA-DocScaB catalyzes ester bond hydrolysis in MBP and BB in room temperature.

Figure 17 | Results of HPLC analysis of enzymatic activity of PnbA-DocScaB towards DBP and BB. Data about the area of peaks was collected, and translated to concentration (uM) using calibration curves. All experiments performed in triplicates. a) Mean DBP concentration in samples after centrifugation on Amicon, measured at 205 nm. b) Mean MBP concentration in samples after centrifugation on Amicon, measured at 205 nm. c) Mean BB concentration in samples after centrifugation on Amicon, measured at 205 nm. d)Mean BA concentration in samples after centrifugation on Amicon, measured at 205 nm.

Conclusions

Our result show, that a modular fusion protein PnbA-DocScaB designed by our team was successfully cloned to expression vector pJUMP23-1A, overproduced and purified using Immobilised Metal Affinity Chromatography. Then its activity was tested and we confirmed that this fusion enzyme is indeed capable of performing ester bond hydrolysis in dibuthyl phthalate (DBP) and butyl benzoate (BB). Those results are promising, but it requires more data for us to be able to properly establish this protein’s characteristics. In the future we plan on testing the activity of this protein in more detail, as well as its affinity to our scaffoldin fusion protein. Nevertheless, we are excited to present this part as our achievement and we hope it will insipre other teams to incorporate our ideas in their projects.

Sources and references