Part:BBa_K863006

ecol laccase from E. coli

E.coli laccase ORF

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 225
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

In the last few years a lot of attention has been drawn to laccases due to their ability to oxidize both phenolic and nonphenolic lignin related compounds as well as highly recalcitrant environmental pollutants. This makes them very useful for applications concerning several biotechnological processes. This includes the detoxification of industrial effluents, for example from the paper and pulp, textile and petrochemical industries. Laccases are also valuable as a tool as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Furthermore these enzymes are also used as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics^[1]. Their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. In our project laccases are used as cleaning agents for a water purification system. Laccases are copper-containing polyphenol oxidase enzymes (EC 1.10.3.2) that can be found in many plants, insects, microorganisms and mainly in fungi. These enzymes fulfill several functions in different metabolic pathways. Laccases are able to oxidize a broad range of substrates due to the contained copper-cluster, by reducing oxygen to water. The active site of the enzyme includes a four-copper-ion-cluster, which can be distinguished by spectroscopic analyses. This cluster consists of one blue copper-ion (type 1), one type 2 and two type 3 copper-ions. Because of the blue copper-ion, the laccases belong to the big family of the blue copper proteins. This specific blue copper ion is essential for the enzyme mediated radical oxidation of the phenolic groups. In this reaction the electron from the oxidation is transferred to the other three copper ions. These ions form a trinuclearic cluster, which transfers electrons to the terminal electron acceptor oxygen. By receiving four electrons the molecular oxygen is finally reduced to water.
[1] Susana Rodríguez Couto & José Luis Toca Herrera;Industrial and biotechnological applications of laccases: A review; 2006; Biotechnology Advances 24 500–513

Cultivation, Purification and SDS-PAGE

Shaking Flask Cultivations

The first trials to produce ECOL were produced in shaking flask with various designs (from 100 mL^-1 to 1 L flasks, with and without baffles) and under different conditions. The parameters tested during our screening experiments were temperature (27 °C,30 °C and 37 °C), concentrations of chloramphenicol (20-170 µg mL^-1), various induction strategies ([http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction] and manual induction) and cultivation time (6 - 24 h). Furthermore it was cultivated with and without 0.25 mM CuCl₂ to provide a sufficient amount of copper, which is needed for the active center of the laccase. Based on the screening experiments we identified the best conditions under which ECOL was expressed. The addition of CuCl₂ did not increase the activity, so it was omitted.

• flask design: shaking flask without baffles
• medium: [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium]
• antibiotics: 60 µg mL^-1 chloramphenicol
• temperature: 37 °C
• cultivation time: 12 h

The reproducibility of the measured data and results were investigated for the shaking flask and bioreactor cultivation.

3 L Fermentation E. coli KRX with BBa_K863005

Figure 1: Fermentation of E. coli KRXwith BBa_K863005 (ECOL) in an Infors Labfors Bioreactor, scale: 3 L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold pO₂ at 50 %, OD₆₀₀ measured every 30 minutes.

After the positive measurement of activity of ECOL we made a scale-up and fermented E. coli KRX with BBa_K863005 in an Infors Labfors fermenter with a total volume of 3 L. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 1. The exponential phase started after 1.5 hours of cultivation. The cell growth caused a decrease in pO₂. After 2 hours of cultivation the agitation speed increased up to 629 rmp (5.9 hours) to hold the minimal pO₂ level of 50 %. Then, after 4 hours there was a break in cell growth due to induction of protein expression. The maximal OD₆₀₀ of 2.78 was reached after 5 hours. In comparison to E. coli KRX (OD_600,max =4.86 after 8.5 hours) and to E. coli KRX with BBa_K863000 (OD_600,max =3.53 after 10 hours, time shift due to long lag phase) the OD_{600 max} is lower. In the following hours, the OD₆₀₀ and the agitation speed decreased and the pO₂ increased, which indicates the death phase of the cells. This is caused by the cell toxicity of ECOL (reference: [http://www.dbu.de/OPAC/ab/DBU-Abschlussbericht-AZ-13191.pdf DBU final report]). Hence, cells were harvested after 12 hours.

Purification of ECOL

The harvested cells were resuspended in [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA- equilibration buffer], mechanically disrupted by [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation homogenization] and cell debris were removed by centrifugation. The supernatant of the cell lysate was loaded on the Ni-NTA column (15 mL Ni-NTA resin) with a flow rate of 1 mL min^-1 cm^-2. Then the column was washed with 10 column volumes (CV) [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA equilibration buffer]. The bound proteins were eluted by an increasing [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA elution buffer] step elution from 5 % (equates to 25 mM imidazol) with a length of 50 mL, to 50 % (equates to 250 mM imidazol) with a length of 60 mL, to 80 % (equates to 400 mM imidazol) with a length of 40 mL and finally to 100 % (equates to 500 mM imidazol) with a length of 80 mL. This strategy was chosen to improve the purification caused by a step by step increasing Ni-NTA-elution buffer concentration. The elution was collected in 10 mL fractions. Due to the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak only the UV-detection signal of the wash step and the elution are shown. A typical chromatogram of purified laccases is illustrated here. The chromatogram of the ECOL elution is shown in Figure 2:

Figure 2: Chromatogram of wash and elution fractions from FLPC Ni-NTA His tag Purification of ECOL produced by 3 L fermentation of E. coli KRX with BBa_K863005. ECOL was eluted by a concentration of 50 % (equates to 250 mM imidazol) with a maximal UV-detection signal of 292 mAU.

The chromatogram shows two distinguished peaks. The first peak was detected at a [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibration buffer] concentration of 5 % (equates to 25 mM imidazol) and resulted from the elution of weakly bound proteins. After increasing the Ni-NTA elution buffer concentration to 50 % (equates to 250 mM imidazol), an UV-detection signal peak of 292 mAU was measured. The area of this peak indicates that a high amount of protein was eluted. The corresponding fractions were analyzed by SDS-PAGE to detect ECOL. There were no further peaks detectable. The following increasing UV detection signal results from the rising imidazol concentration of the Ni-NTA elution buffer. The corresponding SDS-PAGES are shown in Figure 3.

SDS-PAGE of ECOL purification

Figure 3: SDS-Pages of purified E. coli KRX containing BBa_K863005 lysate (fermented in 3 L an Infors Labfors fermenter). The flow-through and elution fraction 2-9 are shown. The arrow marks the ECOL band with a molecular weight of 53.4 kDa.

In Figure 3 the SDS-PAGE of the Ni-NTA His tag purification of the lysed culture (E. coli KRX containing BBa_K863005) is shown including the flow-through and the fractions 2 to 9. The red arrow indicates the band of ECOL with a molecular weight of 53.4 kDa, which appears in all fractions. The strongest bands appear in fractions 6 and 7. These were the first two fractions (each 10 mL) eluted with 50 % Ni-NTA elution buffer (equates to 250 mM imidazol), in which the distinguished peak appeared. These bands were analyzed by [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#MALDI MALDI-TOF] and identified as CueO (ECOL). In contrast, the second, faint band with a lower molecular weight could not be identified.

6 L Fermentation of E. coli KRX with BBa_K863005

Figure 4: Fermentation of E. coli KRX with BBa_K863005 (ECOL) in a Bioengineering NFL22 fermenter, scale: 6 L, [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium] + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation increased when pO₂ was below 30 %, OD₆₀₀ taken every hour.

Another scale-up of the fermentation of E. coli KRX with BBa_K863005 was made up to a final working volume of 6 L in a Bioengineering NFL 22 fermenter. Agitation speed, pO₂ and OD₆₀₀ were determined and illustrated in Figure 3. There was no noticeable lag phase and the cells immediately began to grow. The cells were in an exponential phase between 2 and 4 hours of cultivation, which results in a decrease of pO₂ value and therefore in an increase of agitation speed. After 4 hours of cultivation the maximal OD₆₀₀ of 2.76 was reached, which is comparable to the 3 L fermentation of E. coli KRX with BBa_K863005. Due to induction of protein expression there is a break in cell growth. The death phase started, which is indicated by an increasing pO₂ and a decreasing OD₆₀₀. This demonstrates the cytotoxicity of the laccase for E. coli, which was reported by the [http://www.dbu.de/OPAC/ab/DBU-Abschlussbericht-AZ-13191.pdf DBU]. In comparison to the fermentation of E. coli KRX with BBa_K863000 under the same conditions (OD_600,max= 3.53), the OD_600,max was lower. Cells were harvested after 12 hours.

Purification of ECOL

The harvested cells were resuspended in [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-equilibration buffer], mechanically disrupted by [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Mechanical_lysis_of_the_.28bio-reactor.29_cultivation homogenization] and cell debris were removed by centrifugation. The supernatant of the cell lysate was loaded on the Ni-NTA column (15 mL Ni-NTA resin) with a flow rate of 1 mL min^-1 cm^-2. The column was washed by 10 column volumes (CV) [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA- equilibration buffer]. The bound proteins were eluted by an increasing [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA- elution buffer] gradient from 0 % to 100 % with a length of 200 mL and the elution was collected in 10 mL fractions. Due to the high UV-detection signal of the loaded samples and to simplify the illustration of the detected product peak only the UV-detection signal of the wash step and the eluate are shown. A typical chromatogram of purified laccases is shown here. The chromatogram of the ECOL elution is shown in Figure 5:

Figure 5: Chromatogram of wash and elution from FLPC Ni-NTA His tag purification of ECOL produced by 3 L fermentation of E. coli KRX with BBa_K863005. ECOL was eluted between a process volume 670 mL to 750 mL with a maximal UV-detection signal of 189 mAU.

After washing the column with 10 CV [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography Ni-NTA-elution buffer] the elution process was started. At a process volume of 670 mL to 750 mL the chromatogram shows a remarkable widespread peak (UV-detection signal 189 mAU) caused by the elution of a high amount of proteins. The run of the curve show a fronting. This can be explained by the elution of weakly bound proteins, which elutes at low imidazol concentrations. A better result could be achieved with a step elution strategy ([http://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#Purification_of_ECOL see purification of the 3 L Fermentation above]). To detect ECOL the corresponding fractions were analyzed by SDS-PAGE.

SDS-PAGES of ECOL purification

Figure 6: SDS-Pages of lysed E. coli KRX culture containing BBa_K863005 (fermented in a 6 L Bioengineering NFL22) after purification. The flow-through, wash and the elution fraction 1 to 15 are shown (except from fraction 11/12). The arrow marks the ECOL band with a molecular weight of 53.4 kDa.

In Figure 6 the SDS-PAGE of the Ni-NTA His tag purification of the lysed culture E. coli KRX containing BBa_K863005 (6 L fermentation) including the flow-through, wash and the fractions 1 to 15 (except from fraction 11/12) is shown. The red arrow indicates the band of ECOL with a molecular weight of 53.4 kDa, which appears in all fractions. The strongest bands appear from fractions 3 and 8 with a decreasing amount of other non-specific bands. In summary, the scale up was successful, improving protein production and purification once again.