WO2017085217A1 - Moyen amélioré pour la purification d'eau - Google Patents

Moyen amélioré pour la purification d'eau Download PDF

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Publication number
WO2017085217A1
WO2017085217A1 PCT/EP2016/078065 EP2016078065W WO2017085217A1 WO 2017085217 A1 WO2017085217 A1 WO 2017085217A1 EP 2016078065 W EP2016078065 W EP 2016078065W WO 2017085217 A1 WO2017085217 A1 WO 2017085217A1
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Prior art keywords
bacterial cell
seq
tyrl
cell
tyrosinase
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PCT/EP2016/078065
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English (en)
Inventor
Gen Larsson
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Ragn-Sells Ab
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Priority claimed from SE1530180A external-priority patent/SE540415C2/en
Application filed by Ragn-Sells Ab filed Critical Ragn-Sells Ab
Priority to ES16802001T priority Critical patent/ES2866075T3/es
Priority to DK16802001.4T priority patent/DK3377619T3/da
Priority to EP16802001.4A priority patent/EP3377619B1/fr
Publication of WO2017085217A1 publication Critical patent/WO2017085217A1/fr
Priority to HRP20210696TT priority patent/HRP20210696T1/hr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/20—Heavy metals or heavy metal compounds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/34—Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32
    • C02F2103/343—Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32 from the pharmaceutical industry, e.g. containing antibiotics
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/02—Aerobic processes
    • C02F3/08—Aerobic processes using moving contact bodies
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/02—Aerobic processes
    • C02F3/12—Activated sludge processes
    • C02F3/1236—Particular type of activated sludge installations
    • C02F3/1268—Membrane bioreactor systems
    • C02F3/1273—Submerged membrane bioreactors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W10/00—Technologies for wastewater treatment
    • Y02W10/10—Biological treatment of water, waste water, or sewage

Definitions

  • the present invention relates to the field of water purification and to methods for performing water purification. More particularly, the invention relates to the field of purifying water from substances that are harmful to the environment, such as substances originating from pharmaceuticals, by the usage of purification means of biological origin capable of adsorbing such harmful substances.
  • a major source for contamination is release of pharmaceutical substances through household wastewater, and efficient removal at wastewater treatment plants could therefore go a long way towards reducing the amount of pharmaceutical substances released.
  • oxidation ozonation, UV treatment, use of peroxides
  • membrane filtration reverse osmosis
  • adsorbents active carbon
  • MBR Membrane bioreactor
  • MBBR moving bed biofilm reactor
  • MBBR moving bed biofilm reactor
  • both systems provide means for purifying waste water from pharmaceutical substances that are harmful to the environment albeit using different technology.
  • the present disclosure now solves or at least mitigates some of the problems previously associated with water purification.
  • the problems associated with purification of water from substances that are harmful to the environment, such as pharmaceutical substances are solved or at least mitigated.
  • an improved re-useable method for water purification in that the cells are both able to adsorb, but also release, adsorbed substances from the surface of the bacterial cells.
  • one or more bacterial cell(s) comprising a chemical-entity adsorbing composition on at least a part of the surface of said one or more bacterial cell(s).
  • Said bacterial cells comprising a chemical-entity adsorbing composition on at least a part of the surface thereof have been produced by a method as disclosed herein.
  • Said method comprises the introduction of an expression vector into said bacterial cells, wherein said expression vector comprises an expression cassette encoding a novel recombinantly produced tyrosinase (Tyrl) as a passenger in a Type V secretion ⁇ - autotransporter system.
  • the tyrosinase (Tyrl) in the Type V secretion ⁇ -autotransporter system is expressed as a recombinant protein in the bacterial cell.
  • the tyrosinase (Tyrl) is transported to the outside of the bacterial membrane where it is exposed externally on the cell surface towards the medium.
  • the tyrosinase Tyrl was shown to be catalytically active.
  • the novel synthetic tyrosinase, Tyrl comprises the amino acid sequence of SEQ ID NO : 1.
  • Such a tyrosinase (Tyr 1 ) was herein proven to possess excellent characteristics in a method for producing one or more bacterial cell(s) at least partially coated with a chemical-entity absorbing composition as further described herein.
  • a fragment or variant of Tyrl for positioning in a Type V secretion ⁇ -autotransporter system said fragment or variant of Tyrl having at least 95% identity therewith, such as at least 96, 97, 98 or 99% identity therewith and comprising amino acids 1-4 of SEQ ID NO: l in positions 1-4.
  • tyrosine or a functional equivalent thereof
  • the bacterial cells at least partially comprising said chemical-entity adsorbing composition on their surface are capable of adsorbing harmful substances present in water, when said water is allowed to encounter said cells.
  • an expression vector for expressing a tyrosinase Tyrl to be presented on the surface of a bacterial cell comprising an expression cassette encoding a tyrosinase (Tyrl) as a passenger in a Type V secretion ⁇ -autotransporter system, wherein said tyrosinase when expressed comprises an amino acid sequence of SEQ ID NO: 1 , or a fragment or variant as disclosed herein. Examples of such expression vectors are mentioned herein.
  • a method for producing one or more bacterial cell(s) at least partially coated with a chemical entity-absorbing composition comprising the steps of: a) selecting and culturing one or more bacterial cell(s) comprising an expression vector for expressing a tyrosinase (Tyrl), or a host cell comprising said expression vector as defined herein b) inducing the expression of tyrosinase (Tyrl) from said expression vector to allow presentation of said tyrosinase (Tyrl) on the surface of said one or more bacterial cell(s), c) adding tyrosine, or a functional equivalent thereof, to said one or more bacterial cell(s) to allow the formation of said chemical entity-adsorbing composition at least partially on the surface of said one or more bacterial cell(s), and thereafter d) retrieving the cells from step c).
  • bacterial cell(s) at least partially coated with a chemical entity- adsorbing composition obtainable by the method as disclosed herein.
  • one or more bacterial cell(s) as defined herein, for the removal of one or more chemical entities, from water, such as waste water.
  • Said one or more bacterial cell(s) may be suspended in a solution or be present in a biofilm.
  • the biofilm may be present on a solid support, such as a stationary or moving solid support.
  • the chemical entity may be a pharmaceutical substance or a heavy metal compound.
  • a device such as a MBR (Membrane Bioreactor), comprising one or more bacterial cell(s) obtained herein suspended in a solution.
  • MBR Membrane Bioreactor
  • a device such as a MBBR (Moving bed biofilm reactor) comprising one or more bacterial cell(s) obtained herein present in a biofilm on a solid support.
  • MBBR Microving bed biofilm reactor
  • a method for the removal of one or more chemical entities from water comprising the steps of: a) providing one or more bacterial cell(s) as disclosed herein or a biofilm comprising said cells, b) allowing water to pass through the one or more bacterial cell(s) or the biofilm comprising said cells thereby adsorbing any chemical entities present in the water thereto, c) removing any adsorbed chemical entities from the one or more bacterial cell(s) or the biofilm comprising said cells by rinsing with an aqueous composition having an acidic pH, such as a pH ⁇ 7, to thereafter allow for reuse of the one or more bacterial cell(s) or the biofilm comprising said cells, and thereafter d) repeating steps b) and c).
  • a novel expression vector pAIDAl-Tyrl (SEQ ID NO:4). Also encompassed by the present disclosure is the recombinant protein that is expressed from said vector which comprises the amino acid sequence of SEQ ID NO:3, a fragment or a variant thereof, as well as a protein comprising the amino acid sequence of SEQ ID NO:l, or a fragment thereof, and an nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5.
  • FIGURES Figure 1A-E Display of tyrosinase (Tyrl) on the surface of E. coli; A) Schematic representation of the imagined position on the cell surface of the enzyme and the flanking peptide tags; His6 (six histidine peptide) and Myc (peptide from the c-myc gene) both specific antibody binding. B) The surface expression cassette; C) Agar plate showing E. coli expression of the Tyrl -AIDA 0 fusion protein as compared to a reference. Tyrosinase (Tyrl) is produced in an active form since melanin production is indicated by the black colour.
  • the Myc-tag was absent from both the soluble and inner membrane protein fractions (lanes 2,3/5,6 respectively), while a band was visible in the outer membrane protein fraction at the expected molecular weight for Tyrl -AIDA 0 (lanes 4 and 7). Partial proteolytic degradation was evident from bands at the approximate size of the AID Ac ⁇ -barrel.
  • FIG. 2A-E Production of melanin by surface displayed tyrosinase Tyr 1;
  • FTIR Fourier-transform infrared
  • FIG. 3 E. coli expressing Tyrl -AIDA 0 deposits melanin on the surface and are able to adsorb a drug substance; E. coli were probed with fluorescent anti-melanin antibodies and analysed by fluorescence microscopy. Cells expressing Tyrl -AIDA 0 were clearly visible (A, B), while reference cells without tyrosinase remained unstained (C, D). White bars in micrographs correspond to 20 ⁇ . Light and fluorescence microscope images to the left and right, respectively. (E) Adsorption efficiency of the antimalaria drug chloroquine to melanin coated cells.
  • Adsorbance efficiency is pH- dependent. At low drug concentrations, relevant in wastewater treatment plants, there is an approx. 90% reduction in the binding between pH 8 and 3.
  • FIG 4 Flow cytometric analysis of cells after external treatment with proteases to reveal enzyme (Tyrl) present on surface. Untreated cells and trypsinized cells are shown.
  • Figure 5 Flow cytometric analysis of cells expressing enzymes Tyrl or Tyr2 after exposure to proteolytic cleavage.
  • Figure 6 Flow cytometric analysis of cells comprising the vector pAIDA-Tyrl and reference cells indicating presence or non-presence of melanin on the surface of the cells.
  • “Expression” is used to mean the production of a protein from a gene and refer herein to the genes of and comprises the steps of "the central dogma” i.e. the successive action of transcription, translation and protein folding to reach the active state of the protein all as described in standard biochemistry textbooks. All these steps and elements are controlled in the bacterial cell by multiple means.
  • an "expression vector” as defined herein which may also be referred to as an expression construct, comprises gene sequences to achieve protein expression in cells.
  • the expression vector herein is used to introduce a specific gene into a cell, to thereafter direct the cell machinery for protein synthesis to produce the protein encoded by the gene.
  • a bacterial plasmid is an example of an expression vector encompassed by the present disclosure.
  • Plasmid is a small circular extra-chromosomal DNA molecule that can replicate independently of the cell and are found in bacteria. Plasmids are often used as vectors for molecular cloning i.e. to transfer and introduce selected DNA to a host cell. Plasmids are built-up from specific and necessary elements and may contain genes that can be homo- or heterologous to the bacterial host cell. Plasmids contain e.g. always an origin of replication and most often a gene for specific antibiotics resistance.
  • a " ⁇ -autotransporter system” as referred to herein, comprises the family of ⁇ -autotransporters (Leyton et al, 2012), including the ⁇ -autotransporter AIDA (Adhesin Involved in Diffuse Adherence) herein called AIDA-I (Benz and Schmidt, 1992), which provides a specific mechanism to present a protein on the surface of a bacterial cell.
  • the ⁇ -autotransporter system is a part of the Type V secretion system, and is therefore also referred to herein as the Type V secretion ⁇ -autotransporter system. It may also be referred to as the ⁇ -autotransporter system only.
  • Said protein is called a "passenger” in said Type V secretion ⁇ -autotransporter system and the passenger herein is tyrosinase (Tyrl).
  • an "expression cassette” is herein meant the specific part of the sequence of an expression vector that comprises the genes for expression of a protein, said expression cassette herein comprising a ⁇ - autotransporter system including the passenger tyrosinase Tyr 1 and appropriate regulatory sequences.
  • An example of such an expression cassette is shown in figure IB.
  • novel tyrosinase protein presented herein was successfully expressed and proven active on the cell surface when a ⁇ - autotransporter system was used, herein exemplified by AIDA-I and the plasmid pAIDAI-Tyrl .
  • a “recombinant” protein as mentioned herein, is meant a synthetic protein manufactured by DNA manipulation, which are techniques well-known to the person skilled in the art.
  • amino acid sequence having a particular "identity" to a nucleic or amino acid sequence it is intended herein that such a sequence may include up to 5% “modifications" of the original amino acid reference sequence, or up to 20% “modifications", of the original nucleic acid reference sequence.
  • Such a modification may be in the form of addition of one or more nucleic acid(s) or amino acid(s) to the reference sequence. These one or more amino acids or nucleic acids may have been intentionally added to one or both ends of the reference sequence or they may have been added within the sequence, all resulting in a longer nucleic acid or amino acid sequence than the reference sequence. Furthermore, a modification of a particular sequence may also arise where nucleic acids or amino acids have been removed/deleted/exchanged within the sequence but wherein the full sequence of the modified sequence still comprises at least 95% identity with the original sequence.
  • SEQ ID NO: l up to 15 amino acids may have been modified in SEQ ID NO: l (by addition, and/or deletion/removal/exchange), such as 1 , 2, 3, 4, 5, 6, 7, 8, 10, 1 1 , 12, 13, 14, or 15 amino acids.
  • SEQ ID NO: l when being part of SEQ ID NO:3 (in an ⁇ - autotransporter system).
  • tyrosinase having at least 96, 97, 98 or 99%> identity with SEQ ID NO: l , or where 1 or 2 amino acids have been modified in said sequence.
  • such a modified version with a particular identity to Tyrl still comprises amino acids 1 -4 of SEQ ID NO: l in positions 1 -4 as further explained herein.
  • amino acids 1 -4 of SEQ ID NO: l in positions 1 -4 as further explained herein.
  • amino acids 1 -4 of SEQ ID NO: l in positions 1 -4 as further explained herein.
  • up to about 180 nucleic acids may have been modified in SEQ ID NO:2.
  • tyrosinase which is encoded by a sequence having at least 85, 90, 95, 96, 97, 98 or 99% identity with SEQ ID NO:2.
  • the tyrosinase (Tyrl) as disclosed herein may differ from SEQ ID NO: l , or SEQ ID NO:2 by one or more modifications, such as i.e. by deletion or addition of one or more amino acids or nucleic acids, such as by a point mutation. Still, such a modified version of Tyrl still comprises amino acids 1 -4 of SEQ ID NO: l in positions 1 -4.
  • the percentage of identity between two sequences may e.g. be determined by the sequence alignment program BLAST (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ)
  • a "fragment" of a protein as defined herein comprises a protein having an amino acid sequence which has been shortened with reference to the original protein, such as by a total of 1 -15 amino acids in one or both ends of the protein, and which still results in a protein having substantially the same biological function as the protein comprising the amino acid sequence of SEQ ID NO: l . Still, such a fragment of Tyrl still comprises amino acids 1 -4 of SEQ ID NO: l in positions 1 -4.
  • a modified protein when expressed, such as having a particular sequence identity as described herein and/or being a fragment of the original protein, this will result in a protein having substantially the same biological function as the protein comprising the amino acid sequence of SEQ ID NO: l or encoded by the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO: 15 or SEQ ID NO:5 i.e. being a functional equivalent thereof.
  • a tyrosinase which when expressed in a Type V secretion ⁇ -autotransporter system is capable of being transported to the surface of a bacterial cell where it thereafter can execute its function as a tyrosinase in the context of this disclosure i.e.
  • a “promoter” is a region of DNA which initiates transcription of a gene.
  • a "host cell” as referred to herein relates to a bacterial cell which has been transformed by an expression vector as disclosed herein.
  • a host cell herein is a bacterial cell. In the example given herein, the host cell is Escherichia coli, or E. coli.
  • At least a part of the surface of one or more bacterial cell(s) comprises chemical- entity adsorbing composition
  • this is intended to mean that a sufficient part of the surface of the cell comprises the chemical entity adsorbing composition to allow the adsorption of chemical entities, as mentioned herein, thereto.
  • An example of a successful adsorption is provided in the experimental section.
  • a “bio film” comprises an aggregate of microorganisms in which cells adhere to each other and/or to a surface. Cells in a biofilm may be embedded within a self-produced matrix of extracellular polymeric substance (EPS).
  • EPS extracellular polymeric substance
  • a novel and inventive approach to water purification including the use of a novel tyrosinase (Tyrl) for cell-surface expression and subsequent catalysis of tyrosine and formation of a chemical-entity adsorbing composition on said surface.
  • the method provided herein comprising the combination of a successful expression of a novel active tyrosinase (Tyrl) on the surface of a bacterial cell, and the subsequent formation of a chemical-entity adsorbing composition on at least a part of a surface of said bacterial cell was proven to result in an efficient material (a coated bacterial cell) for water-purification purposes.
  • the specific research challenge of the present disclosure was threefold: (1) the design of the expression system and the transport of tyrosinase (Tyrl) to the outer part of the outer membrane with a proven exposure to the environment and in an active configuration, (2) from this enzyme, provide the conditions necessary for catalysis of the polymerisation reaction and complete oxidation to the polymer and (3) the use of the inherent characteristics of melanin, such as its well-known adsorption of e.g. pharmaceuticals 4 to prove the benefits of the surface deposited product melanin.
  • This challenge was successfully for the first time completed by the present disclosure by using a tyrosinase, herein named Tyrl in a Type V secretion ⁇ -autotransporter system.
  • Tyrl a synthetic protein, was shown to be a particularly suitable tyrosinase for the purpose of the present disclosure, as illustrated by the experimental results presented herein.
  • two enzymes (Tyr 1 , Tyr 2) were introduced as passengers into the ⁇ -autotransporter system autotransporter AIDA (Adhesin Involved in Diffuse Adherence).
  • the vectors were subsequently introduced into an OmpT-negative strain to avoid the AIDA-inherent cleavage of the passenger from the surface 8 .
  • Expression of Tyrl and subsequent melanin formation was initially detected already on agar plates as blackish colonies (figure 1C). To understand the efficiency of melanin formation the degree of localization of the enzyme on the surface was investigated.
  • Tyr2 was found to be inefficiently expressed on the cell surface as verified both by flow cytometry and Western blot analysis and in particular, the His6-tag is proteolytically cleaved (figure 5). Without wishing to be bound by theory, this is partly likely to be due to its larger size and the presence of cysteine residues 7 ' 9 . To try to remove these obstacles, homology modelling based on smaller tyrosinases was used and revealed extensions 16 both in the C- and N-terminal and these were engineered to give a minimised R. etli domain. The modified Tyr2 (p-AIDAl -Tyr2_core) was more efficiently expressed, but did still not to the level of Tyrl . Hence, Tyrl was identified as a particularly suitable enzyme for the present purpose and was selected for further analysis.
  • tyrosinase (Tyrl) expression was verified the biocatalysis reaction was introduced by addition of tyrosine. Since melanin polymerisation from tyrosine is subjected to several constraints process conditions were screened. This was done in bioreactors since constant pH, temperature and oxidative conditions were imperative qualities needed for the polymerisation. Furthermore, tyrosine is not readily soluble at pH7 in the quantities needed for the processing and had eventually to be solubilised at pH 10.5 in a stock solution. After the final process was established cells were grown in batch until the carbon source was depleted where after tyrosine was added at dissolved oxygen concentrations above 30%. The biomass growth and melanin formation were followed by frequent sampling throughout the cultivation ⁇ (figure 2A).
  • the adsorption capacity with respect to a selected drug substance was thereafter investigated.
  • Chloroquine an antimalarial drug that has a well-established affinity for melanin 22 was chosen for this purpose. It is shown herein that cells coated with melanin (catalysis by Tyrl) successfully adsorbed up to 88% of the chloroquine in particular at concentrations relevant of most pharmaceutical pollution in wastewater i.e. nanograms to a few milligrams per liter 20 , while only minor binding occurred in reference samples (figure 3F).
  • the present inventors have now surprisingly made it possible to produce one or more bacterial cell(s) at least partially coated with a chemical-entity adsorbing composition, wherein said bacterial cells have been shown to efficiently adsorb harmful chemical substances, i.e. substances that are harmful to the environment when present in the water, such as pharmaceutical substances.
  • harmful chemical substances i.e. substances that are harmful to the environment when present in the water, such as pharmaceutical substances.
  • These cells hence finds a use in purifying water, such as waste water, from these substances.
  • Tyrl novel tyrosinase which when expressed in an Type V ⁇ -autotransporter system, e.g AID A, could be presented as a biologically active tyrosinase on the surface of a bacterial cell, and which thereafter, as described herein, further made it possible to generate bacterial cells at least partially coated with a chemical-entity forming composition.
  • Tyrl in a Type V secretion ⁇ -autotransporter system was surprisingly shown to result in the transport of Tyrl over the inner membrane, through the periplasm including the cell wall and through the outer membrane, all without premature folding of the protein and resulting in a large portion of full length protein on the cell surface. Thereafter, Tyrl was exposed in such a way that it was facing the exterior and was further not embedded in the outer membrane. In addition, it was shown to be folded in such a way that it was active i.e. that it can use utilise its substrate tyrosine, or a functional equivalent thereof, in such a way that it performs its enzymatic reaction at high catalytic activity.
  • the method for producing the bacterial cell(s) presented herein was designed to be tuned in such a way that the production of the tyrosine enzyme is not too fast which will lead to premature folding and/or degradation.
  • the tuning involved using a suitable amount of IPTG, choosing a suitable promotor (medium strong) and using a low plasmid copy number (about 10-20 copies).
  • This in combination with Tyrl as a passenger protein in a Type V secretion ⁇ -autotransporter system made it possible to produce bacterial cells at least partially coated with a chemical-entity adsorbing material.
  • Escherichia coli which resulted in at least partially chemical-entity coated or melanin-coated bacterial cells (E. coli) cell upon the addition of tyrosine thereto. These cells are further shown to efficiently adsorb a model pharmaceutical substance, chloroquine.
  • an expression vector for expressing a tyrosinase (Tyrl) to be presented on the surface of a bacterial cell comprising an expression cassette encoding a tyrosinase (Tyrl) as a passenger in a Type V secretion ⁇ -autotransporter system, wherein said tyrosinase when expressed comprises a protein comprising an amino acid sequence of SEQ ID NO: 1.
  • a variant or a fragment thereof having at least 95% identity therewith and comprising amino acids 1-4 of SEQ ID NO: l in positions 1-4.
  • an "expression vector” is referred to herein what is intended is a vector from which it is possible to express, by firstly transcribing, the gene encoding the protein and thereafter translate the RNA from said transcription into a protein.
  • An example of an expression vector herein is a plasmid.
  • the expression vector herein is usually a DNA vector.
  • An example of a Type V ⁇ - autotransporter system used herein is AIDA-I, which is illustrated in figure IB together with a vector backbone.
  • the expression cassette encoding a tyrosinase, Tyrl, as a passenger in a Type V secretion ⁇ - autotransporter system is referred to herein this also means that the expression cassette in addition to the recombinant tyrosinase encodes additional proteins, such as signal peptides, linker peptides or other protein tags or the like that aids in the transport, positioning and/or detection of the tyrosinase in the cell membrane. These entities form part of the ⁇ -autotransporter system as shown in figure IB.
  • the expression cassette further comprises regulatory sequences as previously mentioned herein.
  • an expression vector wherein said expression cassette encodes or comprises one or more of the following components: the promotor pLacUV5, a signal peptide, a linker region, a His-tag site, a Myc-Tag site, a 3C site, and/or a TEV site, or as illustrated in figure IB.
  • a Type V secretion ⁇ -autotransporter system in the present context, refers to an abundant group of translocators that use a specific set of ingoing elements in a vector cassette which renders it capable of transporting a recombinant passenger protein (herein the tyrosinase Tyrl) through a specific translocator domain (herein exemplified by AIDA C ) inserted into the cell outer membrane and thereafter exposing/presenting the passenger protein on the surface of said cells or releasing it to the medium.
  • a Type V ⁇ -autotransporter is displayed by the AIDA-I system.
  • the present disclosure extends to the usage of other Type V ⁇ -autotransporter proteins (systems) other than AIDA-I, such as exemplified in www.pfam.sanger.ac.uk, accession PF03787 (Pallen MJ, Chaudhuri RR, Henderson IR. Genomic analysis of secretion systems. Curr Opin Microbiol. 2003;6: 519-527.) As of today, 8209 sequences are listed. A specific feature is the generality of some of the Type V ⁇ -autotransporter parts i.e. the translocator domain (AIDA C in the AIDA-I system) which in many cases is interchangeable with other ⁇ -autotransporters due to the same functionality.
  • a novel recombinant tyrosinase, Tyrl as presented herein, was proven to possess the accurate characteristics to successfully allocate to the surface of the bacterial cell by using the Type V ⁇ - autotransporter system. It was also proven to be active, i.e. possessing tyrosinase activity, once exposed on the exterior of the cell membrane. Such a tyrosinase, Tyrl , was particularly useful in the context of the present disclosure making it possible to provide bacterial cells at least partly covered with a chemical-entity adsorbing composition once tyrosine, or a functional equivalent, is added to the cell culture. Such bacterial cells were thereafter proven to efficiently absorb a pharmaceutical substance from water.
  • tyrosinase wherein the amino acid sequence as compared to SEQ ID NO: 1 or the nucleic acid sequence as illustrated in SEQ ID NO:2 has been modified in a manner as previously described herein.
  • a variant or a fragment thereof having at least 95% identity therewith and comprising amino acids 1 -4 of SEQ ID NO: l in positions 1 -4.
  • tyrosinase consists of an amino acid sequence of SEQ ID NO: 1.
  • tyrosinase may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:2, or a sequence having at least 80% identity, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 97 % or 98% identity with SEQ ID NO:2 still encoding a functional equivalent of the protein. Accordingly, this also includes e.g. codon-optimized versions of the nucleic acid sequences.
  • said tyrosinase (Tyrl) is encoded by a nucleic acid sequence consisting of the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO: 15.
  • ⁇ -autotransporter system is AIDA-I (Adhesin Involved in Diffuse Adherence).
  • Tyrl part of such a protein comprises amino acids 1 -4 of SEQ ID NO: l in positions 1 -4.
  • an expression vector wherein the recombinant tyrosinase Tyr 1 in the Type V secretion ⁇ -autotransporter system AIDA-I is encoded by a nucleic acid sequence comprising SEQ ID NO:5, or a sequence having at least 80% identity with SEQ ID NO:5, such as at least at least 85%, 90%, 91%, 92%, 93%, 94%, 97 % or 98% identity with SEQ ID NO:5.
  • an expression vector which is pAIDAl -Tyr (SEQ ID NO:4), or expression vector having at least 80% sequence identity with the sequence of SEQ ID NO:4, such as a vector having at least 85%, 90%, 91%, 92%, 93%, 94%, 97 % or 98% identity with SEQ ID NO:4.
  • an expression vector which is pAIDAl -Tyr (SEQ ID NO: 4) and which comprises or consists of the nucleic acid sequence of SEQ ID NO:4.
  • pAIDAl -Tyr SEQ ID NO:4 where the vector backbone (excluding the expression cassette comprising the Type V secretion ⁇ -autotransporter system) has been modified in the manner that some structural parts thereof have been exchanged for equally suitable structural parts in a functional context. For example, an origin of replication or the antibiotics resistance may be changed to another one but still provide the same function. Furthermore, it is also encompassed the modification of any non-coding sequences, such as one or more of the regulatory part(s) of the vectors, such as the translation initiation site of the vector etc. These are modifications to the vector backbone which the skilled person can do without undue experimentation. Such expression vectors are also within the scope of this disclosure.
  • the expression vector provided herein may be a plasmid, such as a low-copy and/or a plasmid comprising an inducible promoter or a promotor for constitutive expression.
  • a low copy plasmid is a plasmid generating about 10-50 copies, such as 10-20 copies, of the plasmid in the cell and is being particularly suitable in the present context to generate a sufficient amount of the tyrosinase Tyrl .
  • An plasmid comprising an inducible promoter or promotor for constitutive expression is also particularly suitable in the present context, as the transcription and subsequent expression (translation) of the protein may thereby be controlled allowing for controlled expression of the protein when the expression vector has been introduced into the bacterial cells. It may be so that it is preferable to await a particular stage of cultivation of the bacterial cells before the expression (transcription) of the surface expression cassette is induced. This may be made possible due to the usage of such a controlled system. Furthermore, the synthesis of the protein will also be slower when the expression can be controlled in this manner, which is particularly preferred due to that otherwise one may compromise with the structure and function, particularly proteolysis and folding of the tyrosinase.
  • IPTG Isopropyl ⁇ -D-l - thiogalactopyranoside
  • lactose lactose
  • the bacterial cell culture medium used in the present context may be a defined minimal salts medium. Glucose is then added as the carbon source to reach a certain amount of cell mass where after the induction takes place.
  • the expression vector disclosed herein comprises a promotor which is not dependent upon the sugar concentration in the cell medium used for cultivating the bacterial cells.
  • a promotor is the promotor pLacUV5, but other promoters are also envisaged.
  • a host cell comprising an expression vector as disclosed herein.
  • a host cell which is a bacterial cell.
  • a host cell such as an E.coli cell, suitably lacks OmpT (an aspartyl protease found on the outer membrane of Escherichia coli) to avoid the presence of a protease that may cleave AIDA in the outer membrane of the cell.
  • the host cell may be an E.coli cell, such as an E.coli K12 strain, such as E.coli K12 0:17.
  • the host cell may also be the strain E.coli O ⁇ 7K ⁇ 2AompT 23 .
  • the gene for OmpT has been mutated by general means generating a bacterial cell that lacks this protease.
  • a method for producing one or more bacterial cell(s) at least partially coated with a chemical entity-adsorbing composition comprising the steps of: a) selecting and culturing one or more bacterial cell(s) comprising an expression vector as disclosed herein, or an host cell comprising an expression vector as disclosed herein, b) inducing the expression of tyrosinase (Tyr 1) from said expression vector to allow presentation of said tyrosinase (Tyrl) on the surface of said one or more bacterial cell(s), c) adding a suitable amount of tyrosine, or a functional equivalent thereof, to said one or more bacterial cell(s) to allow the formation of said chemical entity-adsorbing composition at least partially on the surface of said one or more bacterial cell(s), and thereafter d) retrieving the cells from step c).
  • a functional equivalent of tyrosine is referred to herein as a composition which will have substantially the same effect as tyrosine when added to the tyrosinase on the surface of the one or more bacterial cell(s). This means that it will be catalyzed by the tyrosinase Tyrl in the same manner as tyrosine generating one or more bacterial cell(s) which are at least partially covered with a chemical-entity adsorbing composition, such as melanin or a structurally related variant thereof.
  • a chemical-entity adsorbing composition herein is referred to due to that even though it is known that tyrosinase catalyzes tyrosine to melanin, it may be so that other structural components are formed during the catalysis of the tyrosine at the cell surface generating an adsorbing composition which does not entirely consist of melanin, even though a part of the composition may comprise melanin.
  • the culturing of the cells may be performed at a pH of about 7 to 8, such as at about pH 7.5. Maintaining the pH in the cell culture within such ranges was shown to improve the activity of the tyrosinase when positioned in the cell membrane and exposed on the exterior of the cell surface. Furthermore, adjusting the pH in the cell culture was shown to improve the formation of the chemical-entity absorbing composition on the surface of the bacterial cells.
  • the administration mode in the form of timing of and/or amount of induction agent, such as IPTG, added to the cell culture had an effect on the formation of the chemical entity- absorbing composition on at least a part of the surface on at least one or more bacterial cell(s).
  • the activation in step b) of the method may be performed by adding IPTG or lactose to the cell culture, such as in an amount of about 200 ⁇ per activation, and further by adding copper (Cu2+), such as in the form of CuSO i.
  • CuSO i may be in a concentration of about ⁇ , as exemplified herein.
  • the tyrosinase once expressed is also activated by the addition of Copper (in the form of CUSC ).
  • tyrosinase Tyrl is allowed to continue during a minimum of two cell generations after the induction in step b), such as 2, 3, 4, 5 or 6 generations. This is particularly useful when attempting to generate increased amounts of chemical- entity adsorbing material on the cell surface.
  • tyrosine is in the form of a powder composition.
  • adding the tyrosine in a powder format could improve the formation of the chemical entity-adsorbing composition on at least a part of the surface on at least one or more bacterial cell(s).
  • the tyrosine, or a functional equivalent thereof may be contained in an aqueous basic composition or solution having a pH of about 9-12, such as about pH 10, or about pH 10.5
  • the basic pH of this composition was shown to increase the solubility of tyrosine which in turn resulted in increased access to tyrosine providing a higher binding to melanin.
  • the method may be performed in an oxidative environment, wherein the DOT (Dissolved Oxygen Tension) is at least about > 30%.
  • DOT Dissolved Oxygen Tension
  • the chemical entity-adsorbing composition may comprise melanin, an intermediate thereof, a functional equivalent or a structurally related variant thereof.
  • the chemical entity-adsorbing composition may hence comprise melanin in combination with other compounds aiding in the adsorption of chemical entities to the bacterial cells.
  • the chemical entities in a method as disclosed herein comprise one or more pharmaceutical substances or compound(s), such as small molecules, or one or more degradation products thereof.
  • a chemical- entity adsorbing composition may also be a pharmaceutical substance-adsorbing composition.
  • An example of a pharmaceutical substance that binds to a bacterial cell at least partially coated with a chemical- entity adsorbing composition is chloroquine, but the present disclosure is not limited thereto.
  • heterogeneity of melanin affinity is rather large.
  • chemical entities in the present context are organic amines and metal ions.
  • Further examples comprise toxins (beta-N- methylamino-L-alanine, BMAA, alfatoxin Bl), various drugs (e.g. phenothiazines, tricyclic antidepressants, aminoglycosides, and chlorpromazine), other compounds (herbicides, illicit drugs (amphetamine), alkaloids and metals. 4
  • a chemical-entity binding to melanin or a melanin- like composition is a heavy metal compound, such as divalent cations (Larsson et al, Pigment Cell Research, 1993;
  • one or more bacterial cell(s) at least partially coated with a chemical entity-adsorbing composition obtainable by the method as disclosed herein.
  • use of said one or more bacterial cell(s) at least partially coated with a chemical entity-adsorbing composition as disclosed herein for purifying waste water, e.g. by removing one or more chemical entities from said waste water as exemplified herein.
  • Such one or more bacterial cell(s) comprising said bacterial cells may be present in a biofilm present on a solid support, or they may be suspended in a solution (suspended growth).
  • the solid support may be in the form of a stationary solid support or a moving solid support.
  • MBR Membrane Bioreactor
  • MBBR Muscle Biofilm Reactor
  • a device for water purification such a reactor, comprising said one or more at least partially coated bacterial cell(s).
  • MBR multi-reactive protein mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated adsorption-mediated stystylene glycol, g., adionitride, adionitride, adionitride, adionitride, adionitride, adionitride, adionitride-mediated adionitride-mediated adionitride-mediated adionitride-mediated adionitride-mediated adionitride-sulfate-mediated adion-s-
  • MBBR Moving Bed biofilm reactor
  • cells are growing as a biofilm on a moving solid support (e.g. on particles).
  • the moving media is maintained in the reactor by the same principle as for MBR.
  • An example of a moving solid support is a floating plastic substrate.
  • An example of a stationary solid support (or bed) is a trickling filter or a biotower. Stationary particles e.g. in the form of rock or plastic containing biofilm growth bacteria are packed in a bed or a column, respectively. Water is introduced in the top, trickles through the bed and clean water thereafter leaves in the bottom of the bed.
  • biofilm may be present on a solid support, such as a stationary solid support or a moving solid support.
  • the chemical entity adsorbed by a bacterial cell at least partially coated with a chemical- entity adsorbing composition may be pharmaceutical substance, or a heavy metal compound.
  • a device such as a MBR (Membrane Bioreactor), comprising one or more bacterial cell(s) as disclosed herein suspended in a solution.
  • MBR Membrane Bioreactor
  • a device such as a MBBR (Moving bed biofilm reactor) comprising one or more bacterial cell(s) as disclosed herein present in a biofilm on a solid support.
  • a method for the removal of one or more chemical entities from water, such as wastewater comprising the steps of: a) Providing one or more bacterial cell(s) or a biofilm comprising said cells as disclosed herein, b) allowing water to pass through the one or more bacterial cell(s) or the biofilm comprising said cells as disclosed herein thereby adsorbing any chemical entities present in the water thereto, c) removing any adsorbed chemical entities from the one or more bacterial cell(s) or the biofilm comprising said cells as disclosed herein by rinsing with an aqueous composition at an acidic pH, to thereafter allow for reuse of the one or more bacterial cell(s) or biofilm comprising said cells, and thereafter d) repeating steps b) and c).
  • the one or more chemical entities may be pharmaceutical substances, or heavy metal compounds, or degradation products thereof.
  • the one or more chemical entities comprise substances that have affinity to the chemical entity-adsorbing composition e.g. substances that are harmful to the environment.
  • a recombinant protein comprising the amino acid sequence of SEQ ID NO: l, a fragment thereof, or a protein having at least 95% identity with SEQ ID NO: l, such as 96%, 97%), 98% or 99% identity, as the identity has been defined herein, i.e. wherein position 1-6 comprises amino acids 1-6 of SEQ ID NO: l .
  • an nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:2, a fragment thereof, or a functional equivalent thereof encoded by a nucleic acid sequence having 80% identity therewith, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 97 % or 98% identity therewith.
  • a protein comprising the amino acid sequence of SEQ ID NO:3, a fragment thereof, or a functional equivalent thereof, having at least 95% identity with SEQ ID NO:3, such as 96%), 97%, 98% or 99% identity, as explained herein.
  • an expression vector comprising the nucleic acid sequence of SEQ ID NO:4 or comprising a nucleic acid sequence having 80%) identity therewith, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 97 % or 98% identity therewith and/or a vector wherein parts therein have been exchanged for functionally equivalent parts as further described herein.
  • nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:5, or a functional equivalent thereof encoded by a nucleic acid sequence having 80% identity therewith, such as at least 85%, 90%, 91%, 92%, 93%, 94%, 97 % or 98% identity therewith.
  • Tyrl is a synthetic protein.
  • SEQ ID NO:2 Nucleic acid sequence of Tyrl resulting in protein of SEQ ID NO:l (Open reading frame) ggtaacaagtatagagttagaaaaacgtattacatcttaccgacacggaaaaagagattttgttcgtaccgtgctaatactaaaggaaaagggat atatgaccgctatatagcctggcatggtgcagcaggtaaatttcatactcctccgggcagcgatcgaaatgcagcacatatgagttctgctttttaccg tggcatcgtgaataccttttacgattcgaacgtgaccttcagtcaatcaatccagaagtaacccttcctttattgggaatgggaaaacctttcagtcaatcaatccagaagtaacccttcctttt
  • SEQ ID NO:3 Protein expressed from pAIDAl-Tyrl
  • SEQ ID NO:4 Nucleotide sequence expression vector pAIDAl-Tyrl
  • SEQ ID NO:5 Nucleic acid sequence encoding protein expressed from the vector pAIDAl-Tyrl (encoding protein of SEQ ID NO:3) ATGAATAAGGCCTACAGTATCATTTGGAGCCACTCCAGACAGGCCTGGATTGTGGCCTCA GAGTTAGCCAGAGGACATGGTTTTGTCCTTGCAAAAAATACACTGCTGGTATTGGCGGTT GTTTCCACAATCGGAAATGCATTTGCAGTCGACCACCATCACCATCACCATCTGGAAGCG CTGTTCCAGGGTCCGGGTACCGGTAACAAGTATAGAGTTAGAAAAAACGTATTACATCTT ACCGACACGGAAAAAAGATTTTGTTCGTACCGTGCTAATACTAAAGGAAAAAGGGAT ATATGACCGCTATATAGCCTGGCATGGTGCAGCAGGTAAATTTCATACTCCTCCGGGCAG CGATCGAAATGCAGCACATATGAGTTCTGCTTTTTTACCGTGGCATCGTGAATACCTTTTA CGATTCGAACGTGACCTTCAGTCAG
  • SEQ ID NO:6 Amino acid sequence protein AIDA C IGNTLTVSNYTGTPGSVISLGGVLEGDNSLTDRLWKGNTSGQSDIVYVNEDGSGGQTRDGIN IISVEGNSDAEFSLK RWAGAYDYTLQKGNESGTDNKGWYLTSHLPTSDTRQYRPENGSYA TNMALANSLFLMDLNERKQFRAMSDNTQPESASVWMKITGGISSGKLNDGQNKTTTNQFIN QLGGDIYKFHAEQLGDFTLGIMGGYANAKGKTINYTSNKAARNTLDGYSVGVYGTWYQNG ENATGLFAETWMQYNWFNASVKGDGLEEEKYNLNGLTASAGGGYNLNVHTWTSPEGITGE FWLQPHLQAVWMGVTPDTHQEDNGTWQGAGK IQTKAGIRASWKVKSTLDKDTGRRFR PYIEANWIHNTHEFGVKMSDDSQLLSGSRNQGEIKTGIEGVITQNLSV
  • SEQ ID NO: 10 Primer Tyr2 rev_SacI AGC GAG CTC GGC GGA CAC TAT GGC TAT TTC T
  • SEQ ID NO:ll Primer Core-tyr2 frw_KpnI AAA GGT ACC TCA AAT CCA AGG GAG AGT
  • SEQ ID NO:12 Primer Core-tyr2 _rev_SacI AAA GAG CTC GAC CTT GAA GTA GGT CCG
  • SEQ ID NO:13 Primer Tyr IC_frw_HindIII GAAAAGCTTCATATG GGTAACAAGTATAGAGTTAGAAAAAA
  • SEQ ID NO: 14 Primer Tyr IC_rev_SgsI
  • Supporting table 1 shows a summary of the plasmids and strains used in this study.
  • the plasmid pAIDAl 15 was used for surface expression of the tyrosinases.
  • This plasmid uses the AIDA-I surface expression system fused to a recombinant passenger protein flanked by an N-terminal His6-detection tag and a C-terminal Myc detection tag (figure 1A).
  • the strain E. coli 0:17AOmpT 23 previously used for AIDA 0 mediated surface expression 8 , was the host strain in all surface expression experiments.
  • the tyrosinases used in this study are fused to the autotransporter AIDA, generating Tyrl -AIDA 0 and Tyr2-AIDA°.
  • Tyrl (Supporting table 1) has some sequence similarity to the wild type B. megaterium tyrosinase while Tyr2 is identical to the R. etli wild type 6 . Both were PCR amplified from plasmids using the primers listed in supporting table 2 and ligated into the Kpnl and Sacl restriction sites in pAIDAl giving the plasmids. In addition, a truncated "core" version of Tyr2 was created using homology modelling and cloned into pAIDAl, generating Tyr2_core-AIDA c . Finally, Tyrl was also ligated into the Hindlll and SgsI(AscI) restriction sites of pAIDAl, creating an intracellular control (pTyr IC) for expressed Tyrl .
  • the E.coli K12 0:17 strain can e.g. be purchased from SSI Diagnostica (Statens Serum Institut), Denmark, http://www.ssi.dk/ssidiagnostica.
  • the ompT gene has been deleted by conventional means.
  • Core-tyr2 rev Sacl AAA GAG CTC GAC CTT GAA GTA GGT CCG (SEQ ID NO: 12)
  • Tyr lC rev S gsl AAA GGC GCG CCT TAT GAG GAA CGT TTT GAT TTT CTT A
  • Cultivation media and growth conditions All cell growth was performed using minimal salts medium as described previously 8 at pH 7.5 and 30°C in an orbital shaker incubator.
  • the culture medium was supplemented with and 100 ⁇ g/mL chloramphenicol for plasmid selection.
  • solid medium a 20X stock of minimal salts was prepared separately and added to 1.5% agar along with all of the above components.
  • Bioreactor cultures for design and enhancement of the surface reaction and melanin deposition were performed in a 10L bioreactor (Belach Bioteknik AB) with a working volume of 5L.
  • the bioreactor medium was supplemented with 15 g/L glucose.
  • Cells were grown at 30°C at a constant dissolved oxygen tension (DOT) of 40% air saturation and pH was kept at 7.5 through automatic titration with NFLtOH (25% w/v) and H3PO4 (10% w/v).
  • DOT dissolved oxygen tension
  • H3PO4 10% w/v
  • Melanin production was initiated by addition of sterile tyrosine solution to a concentration of 1 g/L as the culture entered the stationary phase due to glucose depletion under fully oxygenated conditions.
  • the access to tyrosine was significantly improved by a dissolving protocol where a stock solution was prepared in water and titrated by KOH to pH 10.5.
  • CDW cell dry weight
  • Samples of cells and medium were taken during cultivation and diluted to an OD600 of approximately 1, mixed 50/50 with sterile glycerol solution (50% v/v) and frozen at -80°C for preservation, as described previously8.
  • 50 ⁇ of frozen samples were thawed and washed with 800 ⁇ PBS. After centrifugation (10 minutes, 4500 rpm, 4°C) the supernatant was discarded and the remaining cell pellets were resuspended in 100 ⁇ PBS containing antibody solution.
  • the excitation wavelength was 488 nm for the His-tag and Alexa488-tag analysis and emission was detected at 525/40 nm.
  • Soluble, inner membrane and outer membrane proteins were isolated as previously described24. Centrifugation (2500 rpm, 15 minutes, 4°C) (J-6B Centrifuge, Beckman) was used to harvest 500 ml cultures four generations after induction. The pellet was washed using 140 mL 50 mM Tris-HCl (pH 7.5) and centrifuged under the same conditions as previously. The obtained pellet was stored overnight in 4°C. The following day, 1.75 g of cell pellet was dissolved in 11 mL 50 mM Tris-HCl (pH 7.5) and disintegrated in a French press high-pressure homogenizer (SLM Aminco).
  • SLM Aminco French press high-pressure homogenizer
  • Non-disintegrated cells were removed by centrifugation (2000 g, 15 minutes, 4°C) (J-20 XP Centrifuge, Beckman). The supernatant was centrifuged (36000 g, 40 minutes, 4°C) to collect membrane bound proteins in the pellet. The resulting supernatant contains the soluble proteins and was collected and stored at -20°C until analysis. The remaining pellet was washed with 1.6 mL 50 mM Tris-HCl (pH 7.5) and centrifuged (36000 g, 40 minutes, 4°C).
  • the pellet was dissolved in 12 mL 50 mM Tris-HCl with 0.1 % (v/v) sarcosyl (N-laurylsarcosine sodium salt solution, Fluka) and incubated on a shaking table for 1 hour at 4°C.
  • the suspension was centrifuged (36000 g, 40 minutes, 4°C) and the supernatant containing the inner membrane proteins was collected and stored at -20°C until analysis.
  • the remaining pellet containing the outer membrane proteins was dissolved in 7 mL 50 mM Tris-HCl with 5 mM EDTA and 1 % (v/v) Triton-X-100 (SigmaUltra, Sigma- Aldrich) and stored at -20°C until analysis.
  • Soluble, inner membrane and outer membrane protein fractions were thawed and loaded on a 10 % SDS-PAGE gel (Bis-Tris, NuPAGE, Invitrogen) and run at 180 V. The gel was blotted to a nitrocellulose membrane, blocked overnight with PBS containing 5 % milk powder. The next day the membrane was incubated for 60 minutes in PBST containing 10 g/L Bovine serum albumin (BSA) and either 0.5 ⁇ g/mL antibody (THETM c-Myc [HRP], GenScript) or rabbit antiserum against AID Ac. After washing in PBST, the membranes were developed as described previously 8, 15.
  • BSA Bovine serum albumin
  • HRP 0.5 ⁇ g/mL antibody
  • GenScript rabbit antiserum against AID Ac.
  • melanin from 4 ml supernatant samples were precipitated by lowering the pH to 2 using 6M HC1 followed by centrifugation (4500 rpm, 5 min).
  • melanin from 4 ml supernatant samples were precipitated by lowering the pH to 2 using 6M HC1 followed by centrifugation (4500 rpm, 5 min).
  • cells were treated over night with 1M KOH at 60°C.
  • the alkaline solution was filtered (0.2 ⁇ sterile cellulose filters, VWR). The pH of the solution was adjusted to 2, HC1, and centrifuged for 5 min 4500 g. The precipitate were washed twice with deionized water and dried over night at 40°C.
  • FTIR Fourier transform infrared spectroscopy
  • API (Pharmaceutical substance) binding assay E. coli cells producing melanin on the surface where assayed for affinity to the antimalarial drug chloroquine, which has previously been reported to adsorb to melanin 17.
  • Cells and drugs dissolved in PBS buffer at pH 7.0 were used for all adsorption experiments.
  • Cell solution (5g/L) and chloroquine solution were mixed in equal parts to a total volume of 1 ml in a 1,5 ml microcentrifuge tubes.
  • Samples were incubated (25°C, 900 rpm) for 30 min in a microcentrifuge tube mixer (Thermomixer compact, Eppendorf) to avoid sedimentation. Cells were separated from the solution using a centrifuge (Heraus, Biofuge fresco) at 16060g for 10 min. 200 ⁇ were pipetted from the top of the solution and added to an HPLC vial containing 800 ⁇ PBS solution (pH 7.0). Chloroquine was varied in different concentrations in-between (35 ⁇ -2 ⁇ ). All adsorption experiments were performed in triplicates. Samples were analysed using high performance liquid chromatography (Alliance, Waters 2695).
  • a reversed-phase Novapak CI 8 column, 3.9x300mm, with 4 ⁇ particle size was used.
  • the mobile phase was prepared by mixing a solution of 15:85 acetonitrile:MilliQ water, afterwards 1% trimethylamine was added and pH was adjusted to 2.8 with 89% H3PO4 26 .
  • the mobile phase was degassed and set to flow at 1 ml/min.
  • the column effluent was monitored for 20 minutes at 343 nm using a Waters 2996 photodiode array detector.
  • the samples were treated with melanin specific antibodies as described above, redissolved in ⁇ PBS and viewed at 1000X in an Olympus BX51 microscopy using a fluorescein filter.

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Abstract

La présente invention concerne le domaine de la purification de l'eau et concerne un moyen amélioré correspondant comprenant des cellules bactériennes au moins partiellement revêtues d'une composition adsorbant des entités chimiques. Le moyen amélioré permet d'obtenir une manière efficace et réutilisable de purification de l'eau de substances pharmaceutiques qui sont nuisibles pour l'environnement. La présente divulgation concerne un procédé de production d'une ou plusieurs cellule(s) bactérienne(s) au moins partiellement revêtues d'une composition adsorbant des entités chimiques, ainsi qu'un nouveau vecteur d'expression codant pour une nouvelle tyrosinase synthétique (Tyrl) destinée à être présentée à la surface des cellules bactériennes.
PCT/EP2016/078065 2015-11-20 2016-11-17 Moyen amélioré pour la purification d'eau WO2017085217A1 (fr)

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WO2021255480A1 (fr) * 2020-06-19 2021-12-23 Prokarium Limited Système autotransporteur
CN113373077A (zh) * 2021-02-07 2021-09-10 南宁海关技术中心 一种氯霉素高效降解菌、高效降解菌剂及其应用
CN113373077B (zh) * 2021-02-07 2023-02-07 南宁海关技术中心 一种氯霉素高效降解菌、高效降解菌剂及其应用

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