WO2017106937A1 - Assemblages supramoléculaires à activité enzymatique - Google Patents

Assemblages supramoléculaires à activité enzymatique Download PDF

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Publication number
WO2017106937A1
WO2017106937A1 PCT/AU2016/051293 AU2016051293W WO2017106937A1 WO 2017106937 A1 WO2017106937 A1 WO 2017106937A1 AU 2016051293 W AU2016051293 W AU 2016051293W WO 2017106937 A1 WO2017106937 A1 WO 2017106937A1
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Prior art keywords
assembly
self
enzyme
assembling
bca
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PCT/AU2016/051293
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English (en)
Inventor
Lizhong He
Bhuvana Kamath SHANBHAG
Victoria Haritos
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Monash University
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Priority claimed from AU2015905394A external-priority patent/AU2015905394A0/en
Application filed by Monash University filed Critical Monash University
Publication of WO2017106937A1 publication Critical patent/WO2017106937A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/008—Supramolecular polymers
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18—Multi-enzyme systems
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/01001—Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals

Definitions

  • the present invention generally relates to enzymatically active supramolecular assemblies and methods of production thereof.
  • the present invention relates to an enzymatically active supramolecular assembly comprising a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme.
  • Enzymes are catalysts bearing some excellent properties (high activity, selectivity and specificity) that permit performing complex chemical processes.
  • proteins such as enzymes become unstable after purification, undergoing irreversible conformational changes, denaturing, and loss of biochemical activity.
  • enzyme properties have to be usually improved before their implementation at industrial scale (for example, where many cycles of high yield processes are desired).
  • soluble enzymes have to be immobilized to be reused for long times in industrial reactors.
  • immobilisation technique also depends on the conditions required for the process and optimal enzyme activity. For example, immobilisation of enzymes can be used for preventing enzyme residue contamination in the final enzymatically treated product, rather than for cost reduction.
  • enzymes immobilised onto surfaces by non-specific covalent bonding can exist in a large number of possible orientations, for example, with some enzymes oriented such that their binding or active sites are exposed whereas others may be oriented such that their active sites are not exposed, and thus not able to undergo selective catalytic reactions with the substrate of interest.
  • protein density may also be poorly controlled. Proteins are also subject to time-dependent denaturing, denaturing during immobilization, and leaching of the entrapped protein subsequent to immobilization. Furthermore, immobilisation may limit contact between an enzyme and the substrate of interest.
  • the present invention provides in a first aspect an enzymatically active supramolecular assembly comprising a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme.
  • each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme.
  • the present inventors have demonstrated that a self-assembly polypeptide bound to an enzyme allows the formation of a supramolecular assembly where the catalytic activity of the enzyme in the supramolecular assembly is not reduced relative to the free enzyme.
  • the assembly, components and methods avoid problems associated with conventional enzyme immobilisation systems.
  • the plurality of self-assembling enzymatic components are covalently linked.
  • the self-assembly polypeptide and the enzyme are covalently linked.
  • the present inventors have demonstrated that enzymatically active nanoparticles can be formed in the absence of a carrier, a solid support, the need to covalently link the enzyme to a solid support, or to immobilise the enzyme.
  • the present invention provides a supramolecular assembly which can be formed without the need for immobilisation of the enzyme on a solid support.
  • the supramolecular assembly can be formed without the need to covalently link the enzyme to a solid support.
  • the self-assembly polypeptide is a pH responsive self- assembly polypeptide.
  • the self-assembling enzymatic components are responsive to the concentration of Mg 2+ .
  • the assembly is selected from the group consisting of a particle, fibre, sheet or a combination thereof.
  • the particle is spherical.
  • the particle has a diameter of from about 20 nm to about 200 nm, and more preferably a diameter of from about 50 to about 100 nm.
  • the particle has a diameter of about 20 nm, 30 nm, 40 nm, 100 nm, 200 nm, 400 600 nm, 1000 nm or about 1500 nm.
  • the assembly is a hydrogel or an aggregate.
  • the present inventors have demonstrated that a supramolecular assembly provided herein can convert carbon dioxide to bicarbonate. Accordingly, in one aspect the present invention provides an assembly as described herein, wherein the enzyme converts carbon dioxide to bicarbonate.
  • the enzyme is selected from the group consisting of a formate dehydrogenase, a carbonic anhydrase, a RuBisCO and combinations thereof. [033] In another embodiment, the enzyme is selected from the group consisting of a cutinase, a tyrosinase and an aminotransferase.
  • the present inventors have demonstrated that enzymatically active nanoparticles can be formed in the absence of a carrier or other components. Accordingly, in one aspect the invention provides a supramolecular assembly wherein the assembly self assembles in the absence of a carrier. In another embodiment, the present invention provides an assembly wherein the assembly consists of the plurality of self-assembling enzymatic components.
  • the present inventors have also demonstrated that an assembly as described herein is stable across a range of temperatures for a period of months. Accordingly, in one aspect the present invention provides an assembly as described herein wherein the assembly is stable between 25 to 50°C. In another aspect, the assembly is stable for at least 2 months.
  • the catalytic activity of an enzyme of the assembly is at least 80% of the catalytic activity of the free enzyme.
  • the catalytic activity of the enzyme is at least 98% of the catalytic activity of the free enzyme.
  • the assembly is switchable between the supramolecular assembly state and an unassembled state.
  • the assembly is pH responsive.
  • the self-assembly polypeptide is a P family polypeptide. In another embodiment, the self-assembly polypeptide is Pn-4.
  • a linker binds the self-assembly polypeptide to the enzyme of the self-assembling enzymatic component.
  • the linker covalently binds the self-assembly polypeptide to the enzyme.
  • the linker comprises a polypeptide.
  • at least 90% of the amino acids in the linked polypeptide are glycine or serine or a combination thereof.
  • the linker is a glycine- serine (GS) linker, and in another embodiment the GS linker comprises SEQ ID NO: 2.
  • the present invention provides a process for sequestering carbon dioxide from a carbon dioxide containing fluid or gas, wherein the method comprises the steps of: providing an enzymatically active supramolecular assembly described herein, wherein the enzyme is carbonic anhydrase; contacting the assembly with the carbon dioxide containing fluid or gas; and converting carbon dioxide in the carbon dioxide containing fluid or gas to bicarbonate.
  • the enzymatically active supramolecular assembly is immobilised.
  • the assembly is immobilised on a film or membrane.
  • the fluid or gas is a fluid or gas stream.
  • the present invention provides an enzymatically active supramolecular assembly comprising a first self-assembling enzymatic component and a second self-assembling enzymatic component, wherein the first self-assembling enzymatic component comprises a self-assembly polypeptide bound to a first enzyme, and wherein the second self-assembling enzymatic component comprises a self-assembly polypeptide bound to a second enzyme.
  • the present invention provides a self-assembling enzymatic component wherein the self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, and wherein the self-assembling enzymatic component is capable of forming an enzymatically active supramolecular assembly in the presence of at least one further self- assembling enzymatic component comprising a self-assembly polypeptide bound to an enzyme.
  • a supramolecular assemblies can be formed using tyrosinase, a cutinase and a carbonic anhydrase, and can produce reaction products of these enzymes.
  • the present invention provides a process for producing a tyrosinase reaction product from a solution comprising a substrate of tryosinase, wherein the process comprises the steps of:
  • the present invention provides a process for producing a tyrosinase reaction product from a solution comprising a substrate of tryosinase, wherein the process comprises the steps of:
  • a solution comprising a substrate selected from the group consisting of tyrosine, DOPA, Tyrosol, Phenols and Catechols, Sericin, Phenolic species, Monophenols, diphenols, and a-lactalbumin
  • the present invention provides a process for producing an aminotransferase reaction product from a solution comprising a substrate of aminotrasferase, wherein the process comprises the steps of:
  • - contacting the assembly with solution comprising at least one substrate selected from the group consisting of amine enantiomers, pyruvate, alanine, ketones, ⁇ -keto acid ester, acetophenone, 1 - phenylethylamine, ketone and isopropylamine; and
  • the present invention a process for producing chiral amines from a solution comprising amino acids, wherein the process comprises the steps of:
  • aminotransferase is ⁇ - aminotransferase.
  • the present invention provides a cutinase reaction product from a solution comprising a substrate of cutinase, wherein the process comprises the steps of:
  • the present invention provides a process for producing a cutinase reaction product from a solution comprising a substrate of cutinase, wherein the process comprises the steps of:
  • the present invention provides a method of forming an enzymatically active supramolecular assembly, the method comprising the steps of (i) forming a solution containing a plurality of self- assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, and (ii) contacting the solution containing a plurality of self-assembling enzymatic components with a buffer to form an enzymatically active supramolecular assembly.
  • the assembly is a particle.
  • the particle is spherical.
  • the particle has a diameter of about 100 nm, and wherein the buffer comprises about 50mM Tris-HCI at about pH 6.8. In another embodiment, the particle has a diameter of about 400 nm, and wherein the buffer comprises about 50mM Tris-HCI at about pH 6.5. In another embodiment, the particle has a diameter of about 1500 nm, and wherein the buffer comprises about 50mM Tris-HCI at about pH 5.6. In another embodiment, wherein the particle has a diameter of about 1500 nm, and wherein the buffer comprises about 10 mM MgCI 2 at about pH 6.1 .
  • the particle has a diameter of about 1000 nm, and wherein the buffer comprises about 25 mM MgCI 2 in about 10mM Tris at about pH 8.0. In another embodiment, wherein the particle has a diameter of about 200 nm, and wherein the buffer comprises about 5 mM MgCI 2 in about 10mM Tris at about pH 8.0. In another embodiment, the particle has a diameter of about 400 nm, and wherein the buffer comprises about 50 mM MgCI 2 in about 10mM Tris at about pH 8.0. In another embodiment, the particle has a diameter of about 30 to about 40 nm or about 120 to about 200 nm, and wherein the buffer comprises about 50 mM NaN0 3 in about 10 mM Tris at about pH 6.8.
  • the particle has a diameter of about 30 to about 200 nm, and wherein the plurality of self-assembling enzymatic components are present at a concentration of about 1 .0 mg/ml in the solution containing the plurality of self-assembling enzymatic components. In another embodiment, the particle has a diameter of about 600 nm, and wherein the plurality of self-assembling enzymatic components are present at a concentration of about 3 mg/ml in the solution containing the plurality of self-assembling enzymatic components.
  • the particle has a diameter of about 200 nm, and wherein the buffer comprises about 6 mM MgC ⁇ at about pH 7.5 and wherein the plurality of self-assembling enzymatic components are present at a concentration of about 0.5 mg/ml in the solution containing the plurality of self-assembling enzymatic components.
  • the particle has a diameter of about 633 nm, and wherein the buffer comprises about 8 mM MgCI 2 at about pH 7.0 and wherein the plurality of self-assembling enzymatic components are present at a concentration of about 1 mg/ml in the solution containing the plurality of self-assembling enzymatic components.
  • the present inventors have demonstrated the ability to switch an enzymatically active supramolecular assembly between a supramolecular assembly state and an unassembled state.
  • the present invention provides a method of switching an enzymatically active supramolecular assembly between a supramolecular assembly state and an unassembled state, said method comprising contacting a solution comprising a supramolecular assembly as described herein, wherein the self-assembling enzymatic component comprises a tandem repeat of self-assembly polypeptides, with a buffer of increased pH to unassemble the supramolecular assembly.
  • the buffer of increased pH is a buffer is at about pH 8.0.
  • the present invention also provides a method of switching an enzymatically active supramolecular assembly between a supramolecular assembly state and an unassembled state, said method comprising contacting a solution comprising an unassembled supramolecular assembly of a supramolecular assembly as described herein, wherein the self-assembling enzymatic component comprises a tandem repeat of self-assembly polypeptides, with a buffer of decreased pH to assemble the supramolecular assembly.
  • the buffer of decreased pH is a buffer is at about pH 6.0.
  • the self-assembly polypeptide is a P family polypeptide. In another embodiment, the self-assembly polypeptide is Pn-4.
  • the method further comprises the step of recovering the supramolecular assembly from solution.
  • the present invention provides a method of forming an enzymatically active supramolecular assembly having a diameter 'd', the method comprising the steps of (i) forming a solution containing a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, and (ii) contacting the solution containing a plurality of self-assembling enzymatic components with a buffer to form a solution having a plurality of self-assembling enzymatic components at a concentration 'c' wherein the solution formed has a pH 'a' and comprises MgCI 2 at a concentration 'd' to form an enzymatically active supramolecular assembly, wherein:
  • the present invention provides a method of modulating the size of an enzymatically active supramolecular assembly, said method comprising the steps of contacting a solution comprising a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, with a buffer to form an enzymatically active supramolecular assembly, wherein the buffer comprises a component to modulate the size of the enzymatically active supramolecular assembly formed.
  • the component to modulate the size of the enzymatically active supramolecular assembly formed is Mg 2+ and/or a pH modulating compound.
  • the enzyme is a bovine carbonic anhydrase.
  • the self-assembly polypeptide is a P family polypeptide. In another embodiment, the self-assembly polypeptide is Pn-4.
  • a linker binds the self-assembly polypeptide to the enzyme of the self-assembling enzymatic component.
  • the linker covalently binds the self-assembly polypeptide to the enzyme.
  • the linker comprises a polypeptide.
  • at least 90% of the amino acids in the linked polypeptide are glycine or serine or a combination thereof.
  • the linker is a glycine- serine (GS) linker, and in another embodiment the GS linker comprises SEQ ID NO: 2.
  • Figure 1 shows the self-assembling enzymatic component BCA-P i 4, which comprises an enzyme, bovine carbonic anhydrase, fused to a self- assembly polypeptide, BCA-P i 4.
  • Figure 2 shows self-assembling enzymatic components can form a supramolecular assembly. TEM Image of BCA-P i 4 nanoparticles at two different lower magnifications.
  • Figure 3 shows self-assembling enzymatic components can form a supramolecular assembly.
  • Figure 4 shows enzymatic activity of a supramolecular assembly comprising self-assembling enzymatic components.
  • a hydrase activity test shows colour change of BTB dye from black to grey indicating the conversion of CO 2 to bicarbonate ion.
  • Figure 5 shows the stability and enzymatic activity of a supramolecular assembly comprising self-assembling enzymatic components. Comparison of particle size and relative esterase activity of BCA-P i 4 at different temperatures. The % relative activity at different temperatures was calculated relative to the esterase activity of BCA-Pn-4 at 25°C.
  • FIG. 6 shows free enzyme does not form supramolecular assemblies.
  • Figure 7 shows a supramolecular assembly comprising self-assembling enzymatic components has the same unfolding curve/three dimensional structure as the free enzyme. Derivative curves of DSF for BCA-P 4 (light grey) and WT- BCA (dark grey) in Tris-chloride and 200mM NaCI pH 8.0 buffer.
  • Figure 8 shows the size distribution of a supramolecular assembly. Size distribution of nanoparticles measured using Dynamic Light Scattering technique.
  • Figure 9 shows the nanoparticle size of BCA-P i 4 over time using Dynamic Light Scattering technique. Pdl; polydispersity index.
  • Figure 10 shows a schematic of A; a self-assembling enzymatic component, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme via a linker, and B; a self- assembling enzymatic component, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme via a tandemly repeated linker.
  • Figure 1 1 shows the formation of BCA-P i 4 nanoparticles.
  • Figure 12 shows the effect of temperature on (a) enzyme activity of BCA- Pii-4 nanoparticles and WT-BCA measured by pNPA assay. The relative activity (%) at different temperatures was calculated using the esterase activity of respective enzymes at 25°C as standard (b) Particle size distribution of BCA-P i 4 nanoparticles measured at various temperatures via dynamic light scattering.
  • Figure 13 shows the evaluation of BCA-Pn-4 under C0 2 capture conditions at two temperatures (a) formation of bicarbonate and hydrogen ion measured by pH reduction curves of the reaction at ambient temperature (22 ⁇ 2°C) compared with WT-BCA; (b) TEM image of BCA-Pn-4 at end of reaction under ambient condition (22 ⁇ 2°C); (c) pH reduction curves of the reaction at 50°C; (d) TEM image of BCA-Pn-4 at end of reaction at 50°C.
  • Figure 14 shows the influence of pH, ionic strength and added salts on BCA-P 4 assembly size formation
  • a low ionic strength buffer of 10 mM Tris
  • b increased ionic strength buffer of 50 mM Tris
  • c 50 mM NaCI in 10 mM Tris
  • d 50 mM NaN0 3 in 10 mM Tris. Protein concentration 0.5 mg/mL; pH 8.0; pH 7.5; pH 6.8; pH 6.5 and pH 5.6.
  • Figure 15 shows the forces affecting BCA-P 4 nanoparticle formation
  • Figure 1 6 shows dynamic light scattering results showing effect of protein concentration BCA-Pn4 in 50 mM Tris buffer at pH 6.8.
  • Figure 1 7 shows dynamic light scattering results showing effect of temperature on 0.5mg/ml_ of BCA-P 4 in 50mM Tris buffer at pH 8.0
  • Figure 1 8 shows BCA-P 4 nanoparticle assembly facilitated by MgCI 2 in 10mM Tris pH 8.0 buffer
  • FIG. 19 shows the proposed mechanism of BCA-P 4 self-assembly in the presence of Mg 2+
  • Mg 2+ Mg 2+ with large Debye sphere (shaded circle) coordinated to four glutamic acid (E) residues (light grey dashed arrow bonds) and two water molecules (dark gray filled arrow bonds).
  • Self-assembly is further stabilized by electrostatic interaction (dashed line) between arginine (R) and glutamic acid (E) residues of adjacent strands,
  • (b) Under high ionic strength, smaller Debye sphere of Mg 2+ crowded by CI " ions preventing coordination with glutamic acids (E) that are surround by Na + . Crowding of CI " and Na + ions around arginine (R) and glutamic acid (E) residues prevent electrostatic interaction disrupting self-assembly.
  • Figure 20 shows two-level full factorial model describing factors that control BCA-P 4 nanoparticle formation, (a) Half-Normal Plot indicating factors with significant effect - A: pH, C: Protein concentration, D: MgCI 2 concentration, AD: pH and MgCI 2 concentration interaction (un-labelled symbols show positive effect, labelled symbols show negative effect and the triangles are error estimates); (b) 3-D Contour plot showing interaction effect between MgCI 2 and pH; (c) Comparison of predicted and actual particle size measured by dynamic light scattering.
  • Condition 1 0.5 mg/mL BCA-Pn4, pH 7.5, 6 mM MgCI 2 (Actual size - solid bold curve, predicted value-vertical dashed line).
  • Condition 2 1 .0 mg/mL BCA-P 4, pH 7.0, 8 mM MgCI 2 (Actual size -dotted curve, predicted value-vertical dashed line).
  • Figure 21 shows esterase activity of BCA-P 4 nanoparticles of various sizes measured as a percentage of the individual BCA-P 4 fusion protein. Shaded areas on the graph indicate various particle sizes. Average activity of all the sizes (solid bold line) along with standard deviations (black dashed lines) are shown.
  • Figure 22 shows purification of BCA-(P 4) 3 by Immobilised Metal-ion Affinity Chromatography a) Chromatography profile b) SDS-PAGE showing various chromatography fractions; lane 1 -Protein load, lane 2-flow through, lane 3-wash, lane 4-Eluate E1 fraction, lane 5-Eluate E2 fraction, lane 6- Regeneration, M-Molecular weight marker.
  • Figure 23 shows SDS-PAGE gel showing recombinant expression of BCA- (P 4)3 enzyme-peptide fusion systems a) using different induction method (S- soluble; IN-insoluble) b) comparison of expression levels of WT-BCA, BCA-P 4 and BCA-(Pii4)3 induced at 1 mM IPTG 20°C for 16 h.
  • Lanes 1 -5 WT-BCA- uninduced soluble, uninduced insoluble, soluble 12 h, soluble 16 h, insoluble 16 h.
  • Lanes 10-14 BCA-P 4(3) - uninduced soluble, uninduced insoluble, soluble 12 h, soluble 16 h, insoluble 16 h (All samples were diluted to OD600 -4.0 prior to gel loading). Arrows indicate BCA-(P 4) 3 protein band.
  • Figure 24 shows polishing of BCA-(P 4) 3 by AIEx a) Chromatography profile b) and c) SDS-PAGE showing various chromatography fractions; b) lane 1 - Protein load, lane 2-flow through, lane 3-wash, lane 4-Eluate E1 fraction, lane 5- Eluate E2 fraction, lane 6- Regeneration, M-Molecular weight marker, c) lane 1 - E8+E9 fraction, Iane2-E10+E1 1 fraction, Iane3-E12 to E16 fraction, E17 fraction and Iane5-E18 fraction. Arrows indicate bands analysed for Intact mass.
  • Figure 25 shows intact mass data of protein bands from AIEx SDS-PAGE ( Figure 24c) using MALDI-TOF
  • Figure 26 shows a) pH induced precipitation of BCA-(Pn4) 3 and b) SDS- PAGE comparing supernatant and pellet sample of BCA-(P 4) 3 following pH induced precipitation; lane-1 BCA-Pn4, lane-2 BCA-(Pn4) 3 supernatant showing cleaved protein bands, lane-3 BCA-(P 4) 3 pellet and M-molecular weight marker.
  • Figure 27 shows Dynamic Light scattering data comparing particle states of BCA-(P 4) 3 initial at pH8.0 (solid line), as a precipitate at pH 6.0 (dashed line) and after re-solubilisation (dotted line).
  • Figure 28 shows SDS-PAGE showing comparing WT-BCA and BCA-P 4 nanoparticle separation using " l OOkDa MWCO Centrisart® device.
  • Lane-1 , 2, 3 BCA-P 4 load, retentate and permeate respectively.
  • Lanes-4, 5, 6 WT-BCA load, retentate and permeate respectively.
  • Figure 29 shows BCA-Pn4 nanoparticle separation using 100 kDa MWCO Centrisart a) Dynamic light scattering data comparing particle sizes of Load (solid line), permeate (dot-dash line) and retentate (dashed line) samples, b) TEM image showing BCA-P 4 nanoparticle present in retentate sample.
  • Figure 30 shows SDS-PAGE showing comparing WT-BCA and BCA- (Pii4) 3 particle separation using 300 kDa MWCO Centrisart® device.
  • Lane-1 , 2, 3 WT-BCA load, permeate and retentate respectively.
  • Lanes-4, 5, 6 BCA-(P 4) 3 load, permeate and retentate respectively. Note fractions were diluted prior to SDS-PAGE analysis.
  • Figure 31 shows SDS-PAGE showing BCA-(P 4) 3 particle separation by centrifugation following three biocatalytic cycles.
  • M Molecular weight marker
  • lane 1 - BCA-(P 4) 3 reaction 1 sample lane 2-supernatant 1
  • lane 3- supernatant 2 lane 4- final pellet after 3 reactions.
  • Figure 32 shows protein expression (a) WT-TmCA: Lane 1 - Uninduced soluble, lane 2-Uniduced insoluble, lane 3-lnduced soluble, lane 4-lnduced insoluble (b) TmCA-P 4: Lane 1 - Uninduced soluble, lane 2-Uniduced insoluble, lane 3-Auto-induction soluble (1 L), lane 4-Auto-induction insoluble (1 L) lane 5- Auto-induction soluble (0.5L), lane 6-Auto-induction insoluble (0.5L), lane 7-IPTG induced soluble (1 L), lane 8-IPTG induced insoluble (1 L) lane 9-IPTG induced soluble (0.5L), lane 10-IPTG induced insoluble (0.5L).
  • Figure 33 shows SDS-PAGE analysis of Ni-IMAC purification fractions
  • WT-TmCA Lane 1 -load, lane 2-Flow thorough, lane 3-Wash 1 , lane 4- Wash 2, lane 5-Eluate E1 fraction, lane 6-Eluate E2+E3 fraction
  • TmCA-P ⁇ Lane 1 - load, lane 2-Flow thorough, lane 3-Wash 1 +2, Eluate E1 fraction, lane 5-Eluate E2 fraction, M-molecular weight marker.
  • Figure 34 shows particle sizes measured by Dynamic Light Scattering technique (a) WT-TmCA (b) TmCA-Pn4
  • Figure 35 shows TmCA-P 4 nanoparticles formed at 50°C (a) TEM image (b) Corresponding histogram for (a) TEM image.
  • Figure 36 shows temperature stability measured by differential Scanning Fluorimetry (a) Derivative of melting curve for WT-TmCA (b) Melting temperature in various buffers for WT-TmCA (c) Derivative of melting curve for TmCA-P 4 (d) Melting temperature in various buffers for TmCA-P
  • FIG 37 shows protein expression (a) WT-Tyrosinase (b) Tyrosinase- Pii4. S- Soluble, IN-lnsoluble. Arrows indicate protein of interest.
  • FIG 38 shows Ni-IMAC purification (a) Chromatography profile for WT- Tyrosinase (b) Chromatography profile for Tyrosinase ⁇ i4 (c) SDS-PAGE analysis of Ni-IMAC fractions. Lanes 1 -7 WT-Tyr: Load, Flow thorough, Wash, Eluate E1 fraction, Eluate E2 fraction, Eluate E3 fraction and Eluate E4 fraction. Lane 8-14 Tyr-P 4: Load, lane, Flow thorough, Wash, Eluate E1 fraction, Eluate E2 fraction, Eluate E3 fraction and Eluate E4 fraction M-molecular weight marker. [0108] Figure 39 shows particle sizes measured by Dynamic Light Scattering technique (a) WT-Tyrosinase (b) Tyrosinase ⁇ i4.
  • Figure 40 shows Tyrosinase-Pn4 nanoparticles formed with 10mM MgC (a) Particle size range 40 - 200nm (b) Large particles of size 100 -500nm.
  • Figure 41 shows protein expression (a) WT-Cutinase (b) Cutinase-Pn4. Al- Auto-lnduction, IP- IPTG Induction, S- Soluble, IN-lnsoluble. Arrows indicate protein of interest in soluble form.
  • Figure 42 shows protein expression of Cutinase-Pn4 at various IPTG concentrations. S- Soluble, IN-lnsoluble.
  • Figure 43 shows Ni-IMAC purification of Cut-Pn4 (a) Chromatography profile (b) SDS-PAGE analysis of Ni-IMAC fractions. Lanes 1 -6: Load, Flow thorough, Wash, Eluate E1 fraction, Eluate E2 fraction and regeneration. Box indicates Cutinase-Pn4 band.
  • Figure 44 shows nanoparticle formation of Cut-Pn4 (a) Particle size formed with and without 10 mM MgC added (b) TEM image showing large enzyme particles.
  • Figure 45 shows enzyme Activity of Cut-Pn4 measured at different concentration of substrate (pNPB).
  • Figure 46 shows protein expression (a) WT-ATA (b) ATA-P 4. Al- Auto- Induction, IP- IPTG Induction, S- Soluble, IN-lnsoluble. Arrows indicate protein of interest in soluble form.
  • Figure 47 shows Ni-IMAC purification (a) Chromatography profile for WT- ATA (b) Chromatography profile for ATA-Pn4 (c) SDS-PAGE analysis of Ni-IMAC fractions. Lanes 1 -5 WT-ATA: Load, Flow thorough, Wash 1 +2, Eluate E1 fraction, Eluate E2 fraction. Lane 6-1 1 ATA-P 4: Load, Flow thorough, Wash 1 , Wash 2, Eluate E1 fraction, Eluate E2 fraction, M-molecular weight marker.
  • an enzymatically active supramolecular assembly can be formed using an enzyme bound to a self- assembly polypeptide. Accordingly, the present invention provides an enzymatically active supramolecular assembly comprising a plurality of self- assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme.
  • the supramolecular assembly is a nanomaterial, such as a nanoparticle, a nanofibre, a sheet, or a combination thereof.
  • the supramolecular assembly macrostructure such as a hydrogel or an aggregate.
  • Nanomaterials including nanostructures offer certain advantages over conventional supports which include a high surface are to volume ratio, increased enzyme loading, enhanced mobility in flow systems and improved mass-transfer.
  • the small size of nanomaterials provides a large surface area for enzymes to be immobilised. This large surface area increases interfacial area allowing faster reaction rates and conversion efficiency by reducing diffusional limitations which is encountered in conventional immobilisation systems.
  • Nanostructures can have a stabilising effect on enzymes. Also the mobility, nanospatial confinement and solution behaviour of nanoscale systems are very different from conventional enzyme immobilisation systems and can confer unique and desirable properties to enzymes.
  • nanomaterials are challenged by problems of reduced enzyme activity, because techniques used to immobilise enzymes on nanomaterials are the same as those used for conventional supports. Furthermore, the enzyme may be denatured as a result of nanoparticle aggregation which occurs when attempting to separate them from bulk solution.
  • a self-assembly polypeptide bound to an enzyme allows the formation of a supramolecular assembly where the catalytic activity of the enzyme in the supramolecular assembly is not reduced relative to the free enzyme.
  • the supramolecular assembly is selected from the group consisting of a particle, fibre, sheet or a combination thereof.
  • immobilised enzymes For example, the most robust form of immobilised enzymes is those using the covalent immobilisation technique.
  • immobilising enzymes using the covalent method onto solid supports often leads to reduced enzyme activity compared to their free soluble forms. The reasons for this reduced activity is attributed to one or more of the following induced changes in structural conformation, enzyme rigidification or deactivation due to agents used in the immobilisation process.
  • Particular limitations associated with immobilised enzymes include:
  • the present invention is based in part on the surprising demonstration that enzymatically active nanoparticles can be formed in the absence of absence of a carrier, a solid support, the need to covalently link the enzyme to a solid support, or to immobilise the enzyme.
  • the present invention provides a supramolecular assembly which can be formed without the need for immobilisation of the enzyme on a solid support.
  • the supramolecular assembly can be formed without the need to covalently link the enzyme to a solid support.
  • the supramolecular assemblies of the present invention avoid problems of the immobilized enzymes of the prior art such as those discussed above.
  • the term “comprising” is intended to mean that the assemblies, components, compositions and methods include the recited elements, but not excluding others.
  • the term “enzymatically active” as used herein includes the ability of an enzymatic component, a supramolecular assembly, or an enzyme as described herein to catalyze the conversion of a substrate into a product.
  • a substrate for the enzyme comprises the natural substrate of the enzyme, but can also comprise analogues of the natural substrate, which can also be converted, by the enzyme into a product or into an analogue of a product.
  • the activity of the enzyme can be measured, for example, by determining the amount of product in the reaction after a certain period of time, or by determining the amount of substrate remaining in the reaction mixture after a certain period of time.
  • Enzymes suitable for the enzymatically active supramolecular assembly will depend on the desired application for the supramolecular assembly.
  • enzyme refers generally to polypeptides that catalyze biochemical reactions.
  • a compound for which a particular enzyme catalyzes a reaction is typically referred to as a "substrate" of the enzyme.
  • six classes or types of enzymes are recognized. Enzymes catalyzing reduction/oxidation or redox reactions are referred to generally as EC 1 (Enzyme Class 1 ) Oxidoreductases. Enzymes catalyzing the transfer of specific radicals or groups are referred to generally as EC 2 Transferases. Enzymes catalyzing hydrolysis are referred to generally as EC 3 hydrolases.
  • Enzymes catalyzing removal from or addition to a substrate of specific chemical groups are referred to generally as EC 4 Lyases. Enzymes catalyzing isomeration are referred to generally as EC 5 Isomerases. Enzymes catalyzing combination or binding together of substrate units are referred to generally as EC 6 Ligases.
  • the enzyme is selected from the group consisting of enzymes from EC 4.2.1 .1 (Lyase), 4.2.1 .1 (Lyase), 1 .14.18.1 (Oxidoreductase) 3.1 .1 .74 (Hydrolase), and EC 2.6.1 .18 (Transferase).
  • Exemplary enzymes used in the present invention include enzymes selected from the group consisting of Bovine carbonic anhydrase (BCA), Microbial carbonic anhydrase (TmCA), Tyrosinase (Tyr), Cutinase (Cut) and Aminotransaminase (ATA).
  • Exemplary enzymes used in the present invention include carbon dioxide capture/carbon dioxide converting enzymes selected from the group consisting of formate dehydrogenase, carbonic anhydrase, RuBisCO and combinations thereof.
  • the carbon dioxide capture/carbon dioxide converting enzyme is carbonic anhydrase.
  • the present invention provides enzymatically active supramolecular assemblies wherein the assembly comprises self-assembling enzymatic components comprising different enzymes. Accordingly, the assembly will have at least two enzymatic activities.
  • the present invention comprises an enzymatically active supramolecular assembly comprising a first self-assembling enzymatic component and a second self-assembling enzymatic component, wherein the first self-assembling enzymatic component comprises a self-assembly polypeptide bound to a first enzyme, and wherein the second self-assembling enzymatic component comprises a self-assembly polypeptide bound to a second enzyme.
  • the enzymatically active supramolecular assembly comprises at least three, at least four or at least five different self-assembling enzymatic components.
  • the term "supramolecular assembly” as used herein includes polymers, homo- and/or co- polymers, capable of associating with one another by means of covalent bonds, non-covalent interactions including ionic interactions and Van der Waals forces, including hydrogen bonding and dispersion forces, or a combination thereof.
  • the term includes polymers, homo- and/or co- polymers of self-assembling enzymatic components as described herein.
  • the supramolecular assembly is a nanostructure or a macrostructure.
  • One exemplary nanostructure of the present invention is a nanoparticle.
  • the choice of nanostructure or macrostructure will depend on the desired application of the supramolecular assembly, and can be determined in part by the characteristics of the self-assembly polypeptide to be used in the self- assembling enzymatic component.
  • self-assembly includes the process of a self- assembling polypeptide or a component comprising the self-assembling polypeptide to form higher order structures or aggregates, including regularly ordered higher order structures or aggregates.
  • the self-assembly may occur in response to conditions in the environment, such as when added to an aqueous medium or in response to a particular environmental condition such as a pH of an aqueous medium.
  • self-assembly polypeptide as used herein includes a peptide comprising a self-assembling motif. Self-assembly polypeptides that are capable of self-assembly or self-assembly of components comprising the self-assembly polypeptide into higher order structures.
  • polypeptide includes amino acid polymers of any length.
  • the protein may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labelling component.
  • proteins containing one or more analogs of an amino acid including, for example, unnatural or non-natural amino acids, etc.
  • Proteins can occur as single chains or associated chains. Associated chains may be joined by non-covalent or covalent interactions.
  • Polypeptides of the invention can be prepared by various means (e.g. isolation and purification from source, recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g. native, fusions, non-glycosylated, lipidated, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from host cell proteins). Typically, the polypeptide is substantially pure when it is at least 60%, by weight, of total protein present.
  • the preparation is at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, more preferably at least 90%, by weight, of total protein present.
  • Self-assembling enzymatic components comprising a self-assembly polypeptide bound to an enzyme can be prepared by various means (e.g. isolation and purification from source, recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g. native, fusions, non-glycosylated, lipidated, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from host cell proteins). Typically, the self- assembling enzymatic component is substantially pure when it is at least 60%, by weight, of total protein present.
  • the preparation is at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, more preferably at least 90%, by weight, of total protein present.
  • the self-assembling enzymatic component is purified from cell culture.
  • the supramolecular assembly comprising self- assembling enzymatic components is substantially free of other components.
  • a supramolecular assembly that contains at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% self-assembling enzymatic component, by weight, of total protein present.
  • the present inventors have generated polynucleotides encoding the self-assembling enzymatic components described herein. Accordingly, the present invention provides polynucleotides encoding the self-assembling enzymatic components described herein.
  • polynucleotide includes DNA and RNA, and also their analogues, such as those containing modified backbones (e.g. phosphorothioates, etc.), and also peptide nucleic acids (PNA), etc.
  • the invention includes nucleic acid comprising sequences complementary to those described above (e.g. for antisense or probing purposes).
  • the skilled person understands that strict compliance with the polynucleotide and protein sequences defined herein is not necessary, and functional equivalents are included in the scope of the invention.
  • Various strains and species of organisms may have differences at various amino acid and/or nucleotide residues without substantially affecting enzyme activity or structure of the protein.
  • nucleic acid which can hybridise to these nucleic acid molecules, preferably under "stringent” conditions (e.g. 65°C in a 0.2 x SSC).
  • Nucleic acid according to the invention can be prepared in many ways (e.g. by chemical synthesis, from genomic or cDNA libraries, from the organism itself, etc.) and can take various forms (e.g. single stranded, double stranded, vectors, probes, etc.). They are preferably prepared in substantially pure form (i.e. substantially free from other host cell nucleic acids).
  • the skilled person understands that strict compliance with any amino acid sequence disclosed herein is not necessarily required, and he or she could decide by a matter of routine whether any further mutation is deleterious or preferred.
  • the polypeptides of the present invention include sequences having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to any protein disclosed herein.
  • the polypeptides also include variants (e.g. allelic variants, homologs, orthologs, paralogs, mutants, etc.).
  • the molecules may lack one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or more amino acids (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N- terminus.
  • sequence identity includes, in the context of two or more nucleic acids or polypeptide sequences, to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection. Sequence identity, homology and the like may be determined using standard methods known the skilled person, for example, using any computer program and associated parameters, such as BLAST or FASTA.
  • nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of the molecule, e.g., an exemplary nucleic acid of the invention. For example, they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400 or more residues in length. Nucleic acids shorter than full length are also included.
  • nucleic acids are useful as, e.g., hybridization probes, labelling probes, PCR oligonucleotide probes, antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites, binding domains, regulatory domains and the like.
  • nucleic acids of the invention are defined by their ability to hybridize under high stringency comprises conditions of about 50% formamide at about 37°C to 42°C. In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 30°C to 35°C. Alternatively, nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 42°C in 50% formamide, 5X SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 ug/ml sheared and denatured salmon sperm DNA). In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35°C.
  • the filter may be washed with 6X SSC, 0.5% SDS at 50°C. These conditions are considered to be “moderate” conditions above 25% formamide and “low” conditions below 25% formamide.
  • a specific example of “moderate” hybridization conditions is when the above hybridization is conducted at 30% formamide.
  • a specific example of “low stringency” hybridization conditions is when the above hybridization is conducted at 10% formamide.
  • the temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly.
  • Nucleic acids of the invention are also defined by their ability to hybridize under high, medium, and low stringency conditions as set forth in Ausubel and Sambrook. Variations on the above ranges and conditions can be used to practice the invention and are well known in the art.
  • native promoter includes a promoter that is endogenous to the organism or virus and is unmodified with respect to its nucleotide sequence and its position in the viral genome as compared to a wild- type organism or virus.
  • heterologous promoter includes a promoter that is not normally found in the wild-type organism or that is at a different locus as compared to a wild type organism.
  • a heterologous promoter is often not endogenous to a cell or virus into which it is introduced, but has been obtained from another cell or virus or prepared synthetically.
  • a heterologous promoter can refer to a promoter from another cell in the same organism or another organism, including the same species or another species.
  • a heterologous promoter can be endogenous, but is a promoter that is altered in its sequence or occurs at a different locus (e.g., at a different location in the genome or on a plasmid).
  • a heterologous promoter includes a promoter not present in the exact orientation or position as the counterpart promoter is found in a genome.
  • a synthetic promoter is a heterologous promoter that has a nucleotide sequence that is not found in nature.
  • a synthetic promoter can be a nucleic acid molecule that has a synthetic sequence or a sequence derived from a native promoter or portion thereof.
  • a synthetic promoter can also be a hybrid promoter composed of different elements derived from different native promoters.
  • a heterologous nucleic acid (also referred to as exogenous nucleic acid or foreign nucleic acid) includes a nucleic acid that is not normally produced in vivo by an organism from which it is expressed or that is produced by an organism but is at a different locus, expressed differently, or that mediates or encodes mediators that alter expression of endogenous nucleic acid, such as DNA, by affecting transcription, translation, or other regulatable biochemical processes.
  • Heterologous nucleic acid is often not endogenous to a cell or virus into which it is introduced, but has been obtained from another cell or prepared synthetically.
  • Heterologous nucleic acid can refer to a nucleic acid molecule from another cell in the same organism or another organism, including the same species or another species.
  • Heterologous nucleic acid can be endogenous, but is nucleic acid that is expressed from a different locus or altered in its expression or sequence (e.g., a plasmid).
  • heterologous nucleic acid includes a nucleic acid molecule not present in the exact orientation or position as the counterpart nucleic acid molecule, such as DNA, is found in a genome.
  • nucleic acid encodes RNA and proteins that are not normally produced by the cell or virus or in the same way in the cell in which it is expressed.
  • Any nucleic acid, such as DNA that one of skill in the art recognizes or considers as heterologous, exogenous or foreign to the cell in which the nucleic acid is expressed is herein encompassed by heterologous nucleic acid.
  • the invention provides nucleic acid (e.g., DNA) sequences of the invention operatively linked to an expression regulatory sequence (including transcriptional regulatory sequence or translational regulatory sequence) e.g., promoters or enhancers, to direct or modulate RNA synthesis/expression.
  • the expression control sequence can be in an expression vector.
  • Exemplary bacterial promoters include lacl, lacZ, T3, T7, gpt, lambda PR, PL and trp.
  • Exemplary eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein I.
  • the promoter is trc.
  • the expression control sequence is inducible.
  • portion is understood to refer to a portion of a polypeptide of the invention which maintains a defined characteristic or activity of the full- length polypeptide. For example, having the ability to self-assemble.
  • a portion or a biologically active fragment of a polypeptide of the invention may be capable of forming higher order structures or aggregates, including regularly ordered higher order structures or aggregates.
  • Exemplary self-assembly polypeptides include short peptides of 2-20 amino acid residues which self-assemble through molecular recognition to form structures of various orders. Factors that drive molecular recognition and self- assembly include weak non-covalent interactions like hydrogen bonds, ionic interactions, Van der Waals forces, dispersion forces and hydrophobic interactions. Without wishing to be bound by theory, though these forces are weak, the present inventors propose they can collectively interact among individual units to yield stable and robust peptide structures. At a critical polypeptide concentration, self-assembly can be triggered by external stimuli.
  • the external stimulus is selected from the group consisting of salt concentration, pH, temperature, ions, light or an enzyme.
  • the self-assembly polypeptide is an external stimuli responsive self-assembly polypeptide.
  • the self-assembly polypeptide is selected from the group consisting of a pH responsive self-assembly polypeptide, a temperature responsive self-assembly polypeptide; a metal ion responsive self-assembly polypeptide, an enzyme responsive self-assembly polypeptide, and a light responsive self-assembly polypeptide.
  • the self-assembly polypeptide can be designed or chosen depending on the desired characteristics of the self-assembly and/or the supramolecular assembly.
  • Table 1 includes exemplary self-assembly polypeptides used in the present invention.
  • Self-assembly polypeptides can be classified based on the secondary structure of the monomeric units, namely, a-helix peptides, ⁇ -sheet peptides and peptide amphiphiles.
  • Peptide amphiphiles (PA) are divided into peptide-only amphiphiles and lipidated amphiphiles. Alternate classification types can be based on the self-assembled structure formed and/or based on the stimuli responsive property of the self-assembly peptide.
  • Self-assembly polypeptides can form several types of higher order structures, including nanostructures and macrostructures.
  • nanostructures and “nanomaterials” as used herein includes nanofibres, nanoparticles, nanospheres, vesicles and sheets.
  • nanoparticle refers to particles sized between about 0.5 to about 1500 nanometers in at least one dimension, although the nanoparticles need not be spherical in shape.
  • the particles are sized between about 30 to about 1000 nanometers in at least one dimension, more preferably about 40 to about 1000 nanometers in at least one dimension, more preferably about 100 to about 1000 nanometers in at least one dimension, more preferably about 200 to about 633 nanometers in at least one dimension.
  • the nanoparticles have a diameter of from about 30 nm to about 40 nm, from about 120 nm to about 200 nm. In another embodiment, the average diameter is from about 50 nm to about 100 nm.
  • the type of nanostructure formed by self-assembly polypeptide depends on two factors: a) sequence of the self- assembly peptide monomer which decides the type of functional side chains and b) the kind of interactions between the side chains of these self-assembly peptide monomers.
  • sequence of the self- assembly peptide monomer which decides the type of functional side chains
  • kind of interactions between the side chains of these self-assembly peptide monomers are similar to those of protein secondary and tertiary structures.
  • proline is a rigid amino acid well-known as a " ⁇ -sheet breaker", and this feature can be used to control the nanostructures formed.
  • a ⁇ -strand peptide Fl, H-PKFKIIEFEP-OH forms nanofibers instead of nanosheets; the proline residues on either ends of the peptide prevented the hydrogen bonding required for sheet formation and favoured elongation.
  • a nano-web or beads-on-a-thread structure can be formed by using the peptide Ac-PSFCFKFEP-NH2 which has both a ⁇ -turn and sheet in its structure; and this favours formation of filaments and globular structures to yield the nano- web or beads-on-a-thread structure.
  • Fmoc-dipeptides with combinations of phenylalanine (F) and glycine (G) show different forms at the nanoscale.
  • Fmoc-FF can be used to form fibre bundles
  • Fmoc-GG can be used to form thin fibres with entangled network morphology
  • the combination Fmoc-FG can be used to form twisted ribbons.
  • the self-assembly polypeptides used in the present invention can form different nanostructures depending on the individual peptide sequence despite having the same secondary structure.
  • the interaction between self-assembly peptide monomers used in the present invention can also be used to influence nanostructure morphology.
  • the interaction among hydrophobic tails is the major driving force to self-assemble and the hydrophilic head group involve in hydrogen bonding or electrostatic interactions resulting in nanofibers formation.
  • a series of peptide amphiphiles A6D1 , V6D1 , V6D2 and L6D2 with tails of increasing hydrophobicity can be used to form both nanofibres as well as nano-vesicles.
  • the type of amino acid used as head group in a peptide amphiphiles influences the morphology of the nanostructure. Long, helical fibres are formed with aliphatic amino acids whereas short, straight fibres are formed with aromatic amino acids. Electrostatic forces are also employed to initiate fibril formation which uses two different peptides monomers having complementary charges. Examples of this kind are peptide pairs Pn-13 and Pn-14 (negative glutamic acid and positive ornithine residues respectively) and the self-complementary EAK16 peptides.
  • Model peptides like EAK16/RADA16 and certain peptide amphiphiles can be used for nanotube and nano-vesicle formation respectively.
  • the invention provides macrostructures and macromaterials.
  • the formed nanostructures can lead to the formation of macrostructures.
  • microstructures as used herein includes hydrogels and aggregates.
  • hydrogels and aggregates [0190] Several self-assembly polypeptides self-assemble and progress to form macroscopic hydrogels or aggregates.
  • hydrogel includes a three dimensional network structure that itself is insoluble in water but which is capable of retaining large quantities of water to form a stable, often soft and pliable structure via surface tension.
  • a hydrogel may contain over 90% water, or preferably over 99% water.
  • hydrogel formation involves the initial stage of fibre/fibril formation which grows into network structures resulting in hydrogel.
  • the formation of hydrogels can be initiated by one or a combination of stimuli, the most common stimuli being temperature, pH and salt ions.
  • the self-assembly polypeptides of the present invention include polypeptides that are responsive to pH.
  • pH responsive self-assembly polypeptide refers to a self-assembly polypeptide that drives assembly or disassembly of a supramolecular assembly in response to a particular pH or a pH change.
  • the present inventors have shown herein that when a pH responsive self- assembly polypeptide, Pn-4, is bound an enzyme, the enzyme-self-assembly polypeptide component assembles into an enzymatically active supramolecular structure.
  • the self-assembly polypeptide is the Pn- 4, which is a member of the P family of polypeptides.
  • Pii-4" as used herein relates to a self-assembling polypeptide comprising the sequence Gln-Gln-Arg-Phe-Glu-Trp-Glu-Phe-Glu-Gln-Gln (SEQ ID NO: 1 ).
  • the self-assembly polypeptide is a P family polypeptide.
  • the P family of polypeptides are ⁇ -sheet forming 1 1 residue polypeptides with anti-parallel arrangement.
  • the P family of polypeptides self-assemble to form nanostructures like tapes and fibrils, and beyond a certain critical peptide concentration (CPC) they form reversible hydrogels. This transition is triggered by pH stimuli and can be fine-tuned by varying salt concentration.
  • the peptides in this family can be used to respond to change in pH to switch between a stable fluid and stable gel states.
  • the peptide gelation occurs either at acidic pH (Pn-4) or at basic pH (Pn-5).
  • Chiral tri-peptides using both D-and L-amino acids can be used to create pH responsive hydrogels.
  • the D-amino acid at the N-terminus is essential for molecular packing and fibre elongation.
  • the tri-peptides Val-Phe-Phe, Phe- Phe- Val and Leu-Phe-Phe can be used to form hydrogels at pH 7.4 in sodium phosphate buffer.
  • the optimal pH of the enzyme of the present invention can be paired with a self-assembly polypeptide with desired characteristics at that pH.
  • bovine carbonic anhydrase has its optimal activity is at pH -8.0 and therefore the P11-4 peptide is an ideal choice as it exists in soluble form at pH > 7.0 and gels at pH ⁇ 3.
  • the self-assembly polypeptides of the present invention include polypeptides that are responsive to salt.
  • the EAK16 peptide can be used to spontaneously form membranes when Na + ions are added. This is attributed to the complementary ionic interactions of alternating amino acids in its sequence. Chiral forms of the same peptide (d-EAK16 and I-EAK16) form gels in the presence of Na7K + ions at concentrations of 1 mg/mL. Using rheological tests, the d-form is more responsive to the ions than its l-form.
  • Peptide (TIGYG) based on potassium ion channel epitope can be used to gel in the presence of K + ions but not with Na + ions.
  • the peptide gels at various concentrations of K+ ions but Na + ions have no effect. Without wishing to be bound by theory, this is attributed to the ability of K + ions to crosslink between nanofibers and act as glue to promote gelation.
  • Ca 2+ ions can be also used to induce gelation.
  • short peptides of sequence n-FGLDD, n-FFCGLD, n-FFCGLDD and longer peptide amphiphile can be used to form gels with Ca ⁇ and this can be reversed by addition of EDTA solution.
  • Peptide amphiphiles can also be used to respond to divalent cations (Ca 2+ and Mg 2+ ) but not to monovalent cations (Na + ), to form gels. Without wishing to be bound by theory, this is attributed it to the ability of divalent ions to form effective salt bridges.
  • Dual-responsive peptides that respond to pH and Ca 2+ ions can also be used in the present invention.
  • the ability of ions to be involved in electrostatic interactions depends on the charged state of the ions as well as peptide. Since charge depends on pH, their effects are often cumulative and result in peptides that can respond to both pH and ions.
  • Peptide amphiphiles (PA) all having four distinct regions: lipophilic tail (palmityl), ⁇ -sheet segment, spacer segment and biological epitope can be used to form gels at pH 7.4 or in 50mM CaCI 2 solution.
  • the rate of gelation can be varied by varying the amino acid residues used in the ⁇ -sheet and spacer segments.
  • Acidic ⁇ -sheet forming peptides of sequence Pro-Y-(Z-Y) 5 -Pro (where Y-Leu/Phe and Z-Asp/Glu) were investigated for dual response.
  • Peptide concentration of -1 .6 %w/v formed gels at pH range 6-8 and the exact gelling pH depended on the residues.
  • the addition of ⁇ 20mM CaCI 2 solution yields gels that are stable and formed instantly even at lower peptide concentration ⁇ 1 .34 % w/v.
  • the self-assembly polypeptides of the present invention include polypeptides which respond to temperature and enzyme action.
  • Tri-peptides with hydrophobic residues (Val, Leu and Phe) dissolved under basic conditions (pH 1 1 .5-13.5) with application of heat and on cooling to room temperature can be used to form self-supporting gels.
  • Synthetic peptides modified to have phosphate groups e.g. Nap-GFFYp-OMe
  • the dephosphorylation of the peptides triggers the formation of hydrogel at peptide concentration as low as -0.01 wt. %.
  • Another macroscopic form of the supramolecular assembly of the present invention is an aggregate.
  • aggregate refers to a bulk material composed of a plurality of hydrogel particles held together by inter-particle and particle-liquid forces, such as, without limitation, hydrogen bonds.
  • a salt/ion responsive peptide used in the present invention is the bolaamphiphile bis (N-R-amido- glycylglycine)-1 ,7-heptane dicarboxylate. At low Ni 2+ concentration these peptides can be used to form nanofibres and at moderate Ni 2+ concentration the nanofibres bundle together to form macroscopic aggregates. Addition of EDTA can be used to revert back the bundle form to nanofibres. Similar effects can be achieved using with Cu 2+ ions but not Na + , emphasizing the role of divalent cations to stabilise salt bridge interactions.
  • Elastin-like polypeptides which have repetitive penta-peptides (Val- Pro-Gly-Xaa-Gly; Xaa-any amino acid except proline) derived from the tropoelastin protein found in muscles can be used for temperature responsive aggregation. Beyond a certain critical solution temperature, ELP converts from soluble to insoluble aggregates. The critical solution temperature of the peptide can be tuned by changing the amino acid sequence.
  • the self-assembling enzymatic component comprises at least two self-assembly polypeptides bound to an enzyme. In one embodiment, the self-assembling enzymatic component comprises at least 3 self-assembly polypeptides bound to an enzyme. In another embodiment, the self-assembling enzymatic component comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 self-assembly polypeptides bound to an enzyme. [0219] In one aspect, the plurality of self-assembling enzymatic components are treated to fix the supramolecular assembly in the assembled state.
  • the supramolecular assembly may be treated such that the self-assembling enzymatic components are covalently linked.
  • the self-assembling enzymatic components are covalently linked via disulphide bonds or amines such that the supramolecular assembly is not responsive to external stimuli.
  • the assembly does not switch between the assembly state and the soluble component state in response to a stimulus.
  • the non-responsive assembly can be reverted to its soluble component state by disrupting the covalent bonds linking the plurality of self- assembling enzymatic components.
  • bound includes both covalent and non-covalent interactions bridging one polypeptide chain to another.
  • an enzyme polypeptide may be covalently or non-covalently bound to a self-assembly polypeptide.
  • Non-covalent interactions include ionic interactions, Van der Waals forces, hydrogen bonding and dispersion forces, or a combination thereof.
  • bonds may form between backbone atoms, side chain atoms or both.
  • the term "plurality” refers to more than one.
  • at least two self-assembling enzymatic components at least two self-assembling enzymatic components.
  • the supramolecular assembly comprises at least two self-assembling enzymatic components comprising a first enzyme.
  • the supramolecular assembly comprises at least two self-assembling enzymatic components comprising a first enzyme, and at least two self- assembling enzymatic components comprising a second enzyme.
  • the self-assembly polypeptide and the enzyme of the self-assembling enzymatic components are not covalently linked.
  • a self-assembly polypeptide can be expressed as a fusion protein fused to an enzyme of the invention, to form a component that can self-assemble into a supramolecular structure.
  • the self-assembling polypeptide and the enzyme of the self-assembling enzymatic components are covalently linked.
  • the self-assembling enzymatic component comprises a tandem repeat of self-assembly polypeptides.
  • the self-assembling enzymatic component comprises a tandem repeat of self-assembly polypeptides.
  • the repeats of the self-assembly polypeptide can be separated by one or more amino acid residues.
  • a self- assembly polypeptide can be expressed as a fusion protein fused to an enzyme of the invention, to form a component that can self-assemble into a supramolecular structure, wherein a linker binds the self-assembly polypeptide to the enzyme.
  • the self-assembling enzymatic component comprises at least 2 tandemly repeated self-assembly polypeptides bound to an enzyme.
  • the self-assembling enzymatic component comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 tandemly repeated self-assembly polypeptides bound to an enzyme.
  • a linker as used herein includes one or more amino acids which function to allow a space between the enzyme and the self-assembly polypeptide.
  • linkers with different amino acid sequences are suitable to function to allow a space between the enzyme and the self-assembly polypeptide.
  • Glycine which does not have any functional group attached to the primary carbon atom allowing free rotational movement about the carbon-carbon bond axis, can be used as a linker in the components described herein.
  • the Gly-Gly linker can be used to link short coils or domains to allow flexibility and space for interaction.
  • Glycines can be interspersed with serine molecules to form a GS-linker. Due to individual characteristics of glycine and serine, the resultant GS-linker is flexible and a random coil with no defined secondary structure.
  • a link with stable secondary structure is desired to improve stability of the fusion protein.
  • Short sequences that form a-helical structures can be used to link fusion proteins. If a more rigid linker is needed then proline residues can be used in the linker sequence.
  • Poly-proline sequences can be used to form rigid rod structures and as molecular rulers.
  • Protease cleavage sites can be incorporated into the linker region. The linker allows for sufficient space for proteases to bind to the specific site to initiate cleavage.
  • linker length depends on the specific application of the supramolecular assembly. Linker length can be chosen to ensure there is sufficient space for enzyme-substrate interaction to take place.
  • a self-assembly polypeptide can be expressed as a fusion protein fused to an enzyme of the invention, to form a component that can self-assemble into a supramolecular structure, wherein a GS linker binds the self-assembly polypeptide to the enzyme.
  • the linker is a GS linker, wherein at least 90% of the amino acids in the linked polypeptide are glycine or serine or a combination thereof.
  • An exemplary GS linker is GGGGSGGGGS (SEQ ID NO: 2).
  • the amino acid sequence of the self-assembling enzymatic component is:
  • the amino acid sequence of the self-assembling enzymatic component is:
  • the amino acid sequence of the self- assembling enzymatic component is:
  • the amino acid sequence of the self- assembling enzymatic component is:
  • the amino acid sequence of the self-assembling enzymatic component is:
  • the present invention provides a self-assembling enzymatic component as described herein, wherein the self-assembling enzymatic component is capable of switching between a soluble and insoluble (e.g. assembled into a supramolecular assembly) state.
  • a self-assembling enzymatic component comprising a self-assembly polypeptide bound to an enzyme can self-assemble into a supramolecular assembly in response to an external stimulus.
  • a supramolecular assembly as described herein can reverse back to the soluble component state of the self-assembling enzymatic components, in response to an external stimulus.
  • the Pup-sheet peptide family can be used to switch between soluble-gel states in response to pH at peptide concentration of 6-7mM.
  • the mechanism controlling this behaviour is due to three main interactions between peptide monomers which are charged interactions between arginine and glutamic acid residues, hydrophobic interactions of glutamate and 7 ⁇ -7 ⁇ interaction between aromatic residues of tryptophan and phenylalanine residues.
  • a peptide amphiphile (C12-GAGAGAGY) based on the silk fibroin protein can be used to form reversible hydrogels. At 0.2 wt. % and at pH 1 1 , the peptide is soluble; at pH 8 it shows aggregation before forming a gel at pH 4. When the tyrosine residue was replaced by serine, the gelling property is lost but reversible aggregates are formed at similar pH values.
  • An a-helical peptide AFD19 can be used for pH responsive reversible gelation at 0.1 wt. %. Importantly, this peptide it has two gelation and solution states unlike the other systems which have only one. At the extremes of pH 3.0 and 1 1 .5 the peptide remains soluble but at pH 6 and 10.5 it forms a stable gel and at pH 7.5 it forms a precipitate.
  • Peptide amphiphiles SA2 and SA7 can be used to form switchable aggregates; the peptides self-assemble into nano-vesicles of diameter ⁇ 60 nm at pH 7.0 and at pH 5.0 the vesicles clump into visible aggregates. However, once vesicles are formed they cannot revert back to the monomeric state, but the aggregation of vesicles is reversible.
  • the ELPs can be used as a reversible stimuli-responsive system. These peptides can be modified in different ways to alter their switchable behaviour.
  • a grafted 20 repeat sequence of ELP using methacryloyl polymer forms cylindrical polymeric brushes. These brushes show dual response to high level of salt and temperature to form aggregates which were reversible at lower levels. Incorporation of cysteine residues in the ELP promotes cross-linking hydrogels. At 2.5 wt. % the peptides exhibit dual response to both temperature and oxidative conditions resulting in reversible gels.
  • An advantage of such supramolecular assemblies is that the enzymatic components can be selectively separated from the reaction mixture on demand by switching between their soluble and insoluble states. For example; enzymes which respond to external stimuli could be temporarily immobilised in an enzymatically active supramolecular assembly and then switched back to their soluble component state to be reused. Such a property will be beneficial for enzymes used in the pharmaceutical and fine chemical industries where free or soluble enzymes are preferred over immobilised forms.
  • the present inventors have also demonstrated that a plurality of self- assembling enzymatic components as described herein are able to form an enzymatically active supramolecular assembly without the need for additional self- assembling peptides to drive the formation the assembly.
  • the enzymatically active supramolecular assembly consists of a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme.
  • the enzymatically active supramolecular assembly consists essentially of a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme.
  • compositions and methods refers to excluding other elements of any essential significance to the composition and methods.
  • an assembly consisting essentially of the a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme would not exclude other elements that do not materially affect the of the invention.
  • the catalytic activity of the enzyme in the supramolecular assembly is at least 70% of the catalytic activity of the free enzyme, such as at least 70%, 74%, 80%, 85%, 90%, 95%, or 98% of the catalytic activity of the free enzyme.
  • the present inventors have shown that the self-assembling enzymatic components comprising a self-assembly polypeptide bound to an enzyme interact primarily between the self-assembly polypeptides of the plurality of components, and this is expected to minimise any distortion to the enzyme active site allowing greater retention of activity in the nanoparticle form compared to conventional immobilisation strategies.
  • the present inventors have also demonstrated the supramolecular assembly is stable between 25 to 50°C, such as between 25 to 40°C.
  • the catalytic activity of the enzyme in the supramolecular assembly is at least 70% of the catalytic activity of the free enzyme, such as at least 70%, 74%, 80%, 85%, 90%, 95%, or 98% of the catalytic activity of the free enzyme at a range of temperatures.
  • the present inventors have also demonstrated the supramolecular assembly is at least as catalytically active as the free enzyme between 25 to 50°C.
  • the present inventors have also demonstrated the supramolecular assembly is at least as catalytically active as the free enzyme at 4°C.
  • the present inventors have also demonstrated the supramolecular assembly is stable over a storage period of at least 2 months. [0270] The present inventors have produced a self-assembling enzymatic component comprising a first enzyme, and a self-assembling enzymatic component comprising a second enzyme.
  • the present invention provides a process for forming an enzymatically active supramolecular assembly as described herein, wherein the process comprises the step of contacting a first self-assembling enzymatic component with at least a second self-assembling enzymatic component, wherein each self-assembling enzymatic components each comprise a self-assembly polypeptide bound to an enzyme.
  • a supramolecular assembly comprising a first self-assembling enzymatic component comprising a first enzyme and a second self-assembling enzymatic component comprising a second enzyme brings the first and second enzymes into close proximity, thereby facilitating the catalytic activity of the two enzymes and of the supramolecular assembly, and/or increases the catalytic activity such that reversible reactions can be driven to completion more frequently compared to free enzymes in solution and/or compared to enzymes immobilised using conventional immobilisation systems, and/or decreases the reaction time compared to free enzymes in solution and/or compared to enzymes immobilised using conventional immobilisation systems, and/or allows the enzymes to act co-operatively.
  • co-operatively refers to two or more enzymes functioning in a concerted manner.
  • the two or more enzymes form an enzyme complex, or alternatively react with the same substrate simultaneously, to carry out their catalytic activities.
  • the product of the first enzyme is a substrate for the second enzyme.
  • the first enzyme is carbonic anhydrase and the second enzyme formate dehydrogenase
  • the bicarbonate produced by the self-assembling enzymatic component comprising carbonic anhydrase is used as a substrate for the self-assembling enzymatic component comprising formate dehydrogenase.
  • the first and second enzymes form part of a cascade reaction.
  • cascade reaction includes a series of enzymatic reactions where the product of one reaction is a substrate for a subsequent reaction.
  • a first enzyme reacts with the substrate first, producing a product, which in turn, is a substrate that reacts with a second enzyme.
  • the enzymatic reactions of the cascade reaction can be part of the same metabolic pathway.
  • metabolic pathway refers to a series of chemical reactions occurring within a cell. In each pathway, a principal chemical is modified by a series of chemical reactions. Lists of metabolic pathways are available publicly from, e.g., the KEGG Pathway Database (available at www.genome.jp/kegg/pathway.html).
  • the present invention provides a self-assembling enzymatic component capable of forming an enzymatically supramolecular assembly, wherein the self-assembling enzymatic component comprises a self- assembly polypeptide bound to an enzyme.
  • the present inventors have demonstrated enzymatic activity of the supramolecular assemblies. [0282] Accordingly, the assemblies provided herein can be used for a number of industrial applications.
  • the assemblies described herein can be used in the conversion of carbon dioxide to bicarbonate as part of carbon capture and sequestration processes.
  • CCS carbon capture and sequestration
  • the capture of C0 2 is the bottle neck of the three-step process of carbon capture and sequestration (CCS) process including its transport and storage, and contributes to up to 82% of capital cost.
  • CCS carbon capture and sequestration
  • capture methods are the area of focus for improvement in the CCS process to improve overall efficiency and reduce cost.
  • Chemical based capture technologies like electrochemical pumps, chemical looping and selective membranes focus mainly on concentrating C0 2 content in flue gas rather than conversion. Chemical methods also use toxic, corrosive substances and demand additional energy costs for catalyst and adsorbent regeneration.
  • CA enzymes have been integrated with C0 2 capture methods in both their free form such as the Carbozyme permeator or immobilised to support materials for industrial scrubbers.
  • the form of CA used strongly influences the overall efficiency of the capture process. Increased hydration of C0 2 at gas-liquid interface is achieved by free CA that cannot be reused.
  • ultrafiltration and nanofiltration membranes are used but are limited by low flow rates, high transmembrane pressure drop and membrane fouling.
  • the present inventors have demonstrated that a carbon dioxide capture/ carbon dioxide converting enzyme can be formed into an enzymatically active supramolecular assembly.
  • the carbon dioxide converting/carbon dioxide converting enzyme is selected from the group consisting of formate dehydrogenase, carbonic anhydrase, RuBisCO and combinations thereof.
  • the present invention comprises an enzymatically active supramolecular assembly comprising a first self-assembling enzymatic component and a second self-assembling enzymatic component, wherein the first self-assembling enzymatic component comprises a self-assembly polypeptide bound to a first enzyme, and wherein the second self-assembling enzymatic component comprises a self-assembly polypeptide bound to a second enzyme.
  • the first enzyme is carbonic anhydrase and the second enzyme is formate dehydrogenase.
  • the invention provides an enzymatically active supramolecular assembly comprising a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to carbonic anhydrase.
  • the enzyme bovine carbonic anhydrase (BCA) was fused with the self-assembly polypeptide Pn-4 connected by a GS-linker ( Figure 1 ) to form a self-assembling enzymatic component comprising a self- assembly polypeptide bound to an enzyme via a linker.
  • the self-assembling peptide Pn-4 has a ⁇ -strand structure and spontaneously self-assembles to form nanofibers under suitable conditions.
  • the Pn-4 peptide is relatively small in size compared to the enzyme BCA itself and sufficient space is provided for peptide interaction to promote self-assembly of two or more fusion proteins.
  • BCA and Pn-4 were connected by the GS-linker to provide flexibility between protein domains.
  • BCA-P i 4 was purified and the formation of nanoparticles was demonstrated with TEM images ( Figure 2).
  • BCA- Pn-4 samples show the presence of uniform distribution of particles with spherical morphology. This data demonstrates that a plurality of self-assembling enzymatic components comprising a self-assembly polypeptide bound to an enzyme are able to produce a supramolecular assembly, wherein the assembly is a nanoparticle.
  • the present invention provides an enzymatically active supramolecular assembly comprising a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to carbonic anhydrase, wherein the assembly is a nanoparticle.
  • Figure 3 demonstrates the particles observed had an average size in the range of 50 - 100 nm with some as large as 180 nm. In contrast, there was no formation of spherical particles observed in the TEM images of free enzyme WT- BCA ( Figure 6), confirming that the self-assembly polypeptide plays an important role in driving the self-assembly of individual monomers to form nanoparticles of defined size and shape.
  • the present invention provides an assembly wherein the catalytic activity of the enzyme is at least 98% of the catalytic activity of the free enzyme.
  • BCA-P i 4 nanoparticles In addition to high activity, BCA-P i 4 nanoparticles also have attractive high stability at increased temperatures, an important feature for C0 2 capture a high temperature. The influence of high temperature on the nanoparticle stability and enzyme activity was tested. Results from dynamic light scattering technique showed that an average particle size range of 100 - 120nm was stable between 25°C to 50°C ( Figure 4).
  • the present invention provides an assembly wherein the assembly is stable between 25 to 50°C.
  • the present invention provides an assembly wherein the assembly is catalytically active between 25 to 50°C.
  • the structural stability of the BCA-P i 4 nanoparticles is essential for its integration with existing methods for C0 2 capture process.
  • the relatively large size of BCA-P i 4 nanoparticles allows its integration with large pore membrane as opposed to the small pore membranes used in existing methods to retain free carbonic anhydrase.
  • the preservation of nanoparticle structure of BCA-P i 4 over time would ensure long-term containment within membrane minimizing the replenishing of fresh enzyme.
  • the use of BCA-P -4 within large pore membrane will allow operations at high flow rates and easy membrane maintenance, thereby improving the overall process efficiency.
  • the present invention provides a process for sequestering carbon dioxide from a carbon dioxide containing fluid or gas, wherein the method comprises the steps of:
  • the present invention provides a self-assembling enzymatic component wherein the self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, and wherein the self-assembling enzymatic component is capable of forming an enzymatically active supramolecular assembly in the presence of at least one further self- assembling enzymatic component comprising a self-assembly polypeptide bound to an enzyme.
  • the supramolecular assembly is immobilized.
  • the present invention provides a process for sequestering carbon dioxide from a carbon dioxide containing fluid or gas, wherein the method comprises the steps of:
  • the present invention provides a process for sequestering carbon dioxide from a carbon dioxide containing fluid or gas, wherein the method comprises the steps of:
  • carbon dioxide containing fluid or gas is a fluid or gas stream.
  • an assembly as described herein is formulated as an agent designed to ameliorate or treat a condition such as a pathological condition.
  • the agent may be administered via a conventional route normally used to administer a medicament including, but not limited to, oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal (including nasal), transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) routes.
  • Intravenous delivery may take place via a bolus injection or via infusion; infusion may be done over a period ranging from less than a minute to several hours to continuously.
  • a course of treatment will involve administration by a combination of routes.
  • Example 1 1 As described in the Examples (e.g. Example 1 1 ), the present inventors have demonstrated the controlled formation of BCA-P 4 nanoparticles using pH and metal-ion Mg 2+ as two independent key parameters, and using difference concentrations of self-assembling enzymatic components. It is also demonstrated that salt type and ionic strength of the solution influence nanoparticle size and degree of self-assembly. A two-level full factorial model was used to determine that pH and MgCI 2 concentration can be varied alone or combined to control nanoparticle size.
  • the present invention provides a method of forming an enzymatically active supramolecular assembly, the method comprising the steps of (i) forming a solution containing a plurality of self- assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, and (ii) contacting the solution containing a plurality of self-assembling enzymatic components with a buffer to form an enzymatically active supramolecular assembly.
  • buffer includes those agents which maintain a solution pH in an acceptable range.
  • a buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugate acid. Its pH changes very little when a small amount of strong acid or base is added to it and thus it is used to prevent any change in the pH of a solution. Buffer solutions are used in protein formulations as a means of keeping proteins within a narrow pH range to optimize shelf life.
  • buffer refers to a solution that resists changes in pH by the action of its acid-base conjugate components.
  • Various buffers which can be employed depending, for example, on the desired pH of the buffer.
  • An example of a suitable buffer is a buffer comprising 50mM Tris-HCI.
  • the buffer comprises 50 mM NaN0 3 in 10 mM Tris.
  • the buffer comprises 50 mM NaN0 3 in 10 mM Tris.
  • the present invention provides a method of forming an enzymatically active supramolecular assembly, the method comprising the steps of (i) forming a solution containing a plurality of self- assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, and (ii) contacting the solution containing a plurality of self-assembling enzymatic components with a buffer to form an enzymatically active supramolecular assembly, wherein the buffer comprises 50mM Tris-HCI.
  • the size of the supramolecular assembly formed increased with reducing pH.
  • the solution formed has a pH in the range from about 5.6 to about 6.8.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 1 00 nm, and wherein the buffer comprises 50mM Tris-HCI at pH 6.8.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 400 nm, and wherein the buffer comprises 50mM Tris-HCI at pH 6.5.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 1500 nm, and wherein the buffer comprises 50mM Tris-HCI at pH 5.6.
  • Example 1 1 The present inventors have also demonstrated in Example 1 1 that supramolecular assembly size is influenced by the concentration of the self- assembling enzymatic component. Importantly, formation of enzyme nanoparticles requires a relatively low concentration of self-assembling enzymatic components (e.g. 0.025 mg/mL P ⁇ ⁇ 4 in 0.5 mg/mL BCA-P 4) compared to the concentration of self-assembling polypeptide alone (e.g. peptide P ⁇ ⁇ 4 alone requires > 10 mg/mL for self-assembly).
  • concentration of self-assembling enzymatic components e.g. 0.025 mg/mL P ⁇ ⁇ 4 in 0.5 mg/mL BCA-P 4
  • self-assembling polypeptide alone e.g. peptide P ⁇ ⁇ 4 alone requires > 10 mg/mL for self-assembly.
  • the solution formed has a concentration of self- assembling enzymatic components in the range from about 0.5 mg/mL to about 3mg /mL.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 30 to about 200 nm, and wherein the plurality of self-assembling enzymatic components are present at a concentration of about 1 .0 mg/ml in the solution containing the plurality of self-assembling enzymatic components.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 600 nm, and wherein the plurality of self-assembling enzymatic components are present at a concentration of about 3 mg/ml in the solution containing the plurality of self- assembling enzymatic components.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 200 nm, and wherein the buffer comprises 6 mM MgC at pH 7.5 and wherein the plurality of self-assembling enzymatic components are present at a concentration of about 0.5 mg/ml in the solution containing the plurality of self-assembling enzymatic components.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 633 nm, and wherein the buffer comprises 8 mM MgC at pH 7.0 and wherein the plurality of self-assembling enzymatic components are present at a concentration of about 1 mg/ml in the solution containing the plurality of self-assembling enzymatic components.
  • the present inventors have shown that in addition to ionic strength, the type of salts affect supramolecular assembly.
  • the type of salt affects BCA-P 4 nanoparticle formation.
  • the solution formed comprises an ionic strength of less than 200 mM.
  • the solution formed comprises NaN0 3 at 50mM, NH 4 CI at 50 mM or NH 4 CI at 100 mM.
  • the solution formed does not comprise NaCI.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 30 to about 40 nm or about 120 to about 200 nm, and wherein the buffer comprises 50 mM NaN0 3 in 10 mM Tris at pH 6.8.
  • Example 1 25 mM Mg 2+ promoted BCA-P 4 self-assembly.
  • the divalent cation is Mg 2+ .
  • the Mg 2+ is provided by MgCI 2 present at a concentration of about 5 mM MgCI 2 , about 10 mM MgCI 2 , or about 25 mM MgCI 2 .
  • the present invention provides a method as described herein wherein the particle has a diameter of about 1500 nm, and wherein the buffer comprises 10 mM MgCI 2 at pH 6.1 .
  • the present invention provides a method as described herein wherein the buffer comprises 25 mM MgCI 2 in 10mM Tris at pH 8.0.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 200 nm, and wherein the buffer comprises 5 mM MgCI 2 in 10mM Tris at pH 8.0.
  • the present invention provides a method as described herein, wherein the particle has a diameter of about 400 nm, and wherein the buffer comprises 50 mM MgC ⁇ in 10mM Tris at pH 8.0.
  • Example 1 1 demonstrates that protein concentration, MgCI 2 and pH allow prediction of BCA-P1 14 particle size under various conditions.
  • the present invention provides a method of forming an enzymatically active supramolecular assembly having a diameter 'd', the method comprising the steps of (i) forming a solution containing a plurality of self-assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, and (ii) contacting the solution containing a plurality of self-assembling enzymatic components with a buffer to form a solution having a plurality of self- assembling enzymatic components at a concentration 'c' wherein the solution formed has a pH 'a' and comprises MgCI2 at a concentration 'd' to form an enzymatically active supramolecular assembly, wherein:
  • the present invention provides a method of modulating the size of an enzymatically active supramolecular assembly, said method comprising the steps of contacting a solution comprising a plurality of self- assembling enzymatic components, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme, with a buffer to form an enzymatically active supramolecular assembly, wherein the buffer comprises a component to modulate the size of the enzymatically active supramolecular assembly formed.
  • the component to modulate the size of the enzymatically active supramolecular assembly formed is Mg 2+ and/or a pH modulating compound.
  • the enzyme is a bovine carbonic anhydrase.
  • the self-assembly polypeptide is a P family polypeptide. In another embodiment, the self-assembly polypeptide is Pn-4.
  • a linker binds the self-assembly polypeptide to the enzyme of the self-assembling enzymatic component.
  • the linker covalently binds the self-assembly polypeptide to the enzyme.
  • the linker comprises a polypeptide.
  • at least 90% of the amino acids in the linked polypeptide are glycine or serine or a combination thereof.
  • the linker is a glycine- serine (GS) linker, and in another embodiment the GS linker comprises SEQ ID NO: 2.
  • membrane filtration can be used as an alternate processing route because it offers benefits of continuous operation mode, improved product recovery and reduced energy consumption.
  • Cross-flow filtration composed of a series of membranes is being used as an efficient purification method for the recovery of proteins and enzymes from complex solutions.
  • membrane based processes are useful for enzymatic reactions since they provide high-surface area for enzyme loading, easy separation of products and reusability of the enzymes when immobilised on the membrane surface.
  • the selection of membrane is often limited.
  • Supramolecular assemblies and particles formed by self-assembly do not require additional chemical agents. Also they can be efficiently retained within available membrane systems and also eliminate the need to immobilise them on the membrane surface, consequently offering additional benefits of reduced process time and cost associated with membrane preparation and maintenance.
  • the supramolecular assemblies such as the precipitates formed from the enzyme fused with 3 peptides offer another mode to recover enzymes without the need for membranes. Hence reusability of enzyme particles can be achieved in two complementary modes: with and without membranes.
  • the self-assembled, enzymatically active supramolecular assemblies of the present invention present a carrier-free method to immobilise enzymes with retained activities and reusability for advanced biocatalysis.
  • a supramolecular assembly with self-assembling enzymatic component comprises a tandem repeat of self- assembly polypeptides forms an about 1000-1 100 nm precipitate at pH 6.0, but this precipitate can be solubilized at pH 8.0.
  • the present invention provides a method of switching an enzymatically active supramolecular assembly between an insoluble state and a soluble state, said method comprising contacting a solution comprising an insoluble supramolecular assembly as described herein wherein the self- assembling enzymatic component comprises a tandem repeat of self-assembly polypeptides, with a buffer of increased pH to solubilize the supramolecular assembly.
  • the buffer of increased pH is at pH 8.0.
  • the present invention provides a method of switching an enzymatically active supramolecular assembly between a supramolecular assembly state and an unassembled state, said method comprising contacting a solution comprising a supramolecular assembly as described herein wherein the self-assembling enzymatic component comprises a tandem repeat of self- assembly polypeptides, with a buffer of increased pH to unassemble the supramolecular assembly.
  • the buffer of increased pH is at pH 8.0.
  • the present invention provides a method of switching an enzymatically active supramolecular assembly between an insoluble state and a soluble state, said method comprising contacting a solution comprising a plurality of self-assembling enzymatic components as described herein wherein the self- assembling enzymatic component comprises a tandem repeat of self-assembly polypeptides, with a buffer of increased pH to solubilize the supramolecular assembly.
  • the buffer of decreased pH is at pH 6.0.
  • the present invention also provides a method of switching an enzymatically active supramolecular assembly between a supramolecular assembly state and an unassembled state, said method comprising contacting a solution comprising a plurality of self-assembling enzymatic components as described herein, wherein the self-assembling enzymatic component comprises a tandem repeat of self-assembly polypeptides, with a buffer of decreased pH to assemble the supramolecular assembly.
  • the buffer of decreased pH is at pH 6.0.
  • the present invention provides a method as described herein, wherein the method further comprises the step of recovering the supramolecular assembly.
  • the filtering of the supramolecular assemblies can be performed using a back-pulse or back- flush filter array, a cross flow membrane, a flow-through membrane, a sintered metal filter, a single media filter, a multimedia filter, or a combination thereof.
  • the filtering of the supramolecular assemblies can further comprise a back flush of a filter with liquid medium to produce the recovered supramolecular assemblies.
  • filter configurations for solid/liquid filtration include (1 ) outside-in filtration where a traditional solid/liquid barrier separation occurs on the outer perimeter of a tubular filter element, (2) inside-out filtration where a solid/liquid barrier separation occurs on the inside of a tubular filter element, (3) inside-out (multimode) filtration where a solid/liquid (barrier or crossflow) separation that occurs on the inside of open- ended tubular filter element and filtration is with multi-option top or bottom feed inlet.
  • the supramolecular assemblies can be separated from liquid medium using a gravity-assisted separation technique.
  • the supramolecular assemblies can be separated from liquid medium using centrifugation.
  • the supramolecular assemblies can be separated from liquid medium using precipitation.
  • the present inventors have demonstrated that by increasing the number of self-assembly polypeptides in the self-assembling enzymatic component, the self- assembly behavior of these components can be controlled. For example, the present inventors have demonstrated that increasing the number of self-assembly polypeptides in the self-assembling enzymatic components form large aggregates, rather than nanoparticles, that were easily precipitated. Therefore altering the peptide length and number of repeats in the fusion protein design provides an option to specifically design enzyme-peptide fusion systems with a controllable assembly feature.
  • the present inventors have generated self-assembling enzymatic components wherein the self-assembling enzymatic component comprises a self- assembly polypeptide bound to a number of enzymes.
  • the self-assembling enzymatic component comprises a self- assembly polypeptide bound to a number of enzymes.
  • the inventors have fused a self-assembly polypeptide to thermostable carbonic anhydrase from Thiomicrospira crunogena (TmCA), Tyrosinase (Tyr), Cutinase (Cut) and ⁇ -Aminotransf erase (ATA). All four enzymes fused a self- assembly polypeptide were successfully expressed and purified, and their chromatogram profiles were similar to those of their wild-type counterparts.
  • TmCA-P 4 self-assembly can be controlled using temperature. That temperature can be used as a trigger for certain types of enzymes without comprising their structure and function is useful for forming nanoparticles of thermostable enzymes which have a wide range of industrial applications.
  • the enzymes characterized in the Examples are of different classes, source, structure and function, demonstrating supramolecular assemblies can be formed using the methods of the invention for a wide array of enzymes. All enzyme-fusion systems investigated showed similar activity and stability in comparison to their wild-type counterparts.
  • TmCA is one promising candidate of the a number of carbonic anhydrases being explored for C0 2 removal in flue gas scrubbing systems, and carbonic anhydrases have already be tested in immobilised form on solid supports to allow reuse and continuous biocatalytic processes.
  • 'BCA' refers to bovine carbonic anhydrase.
  • carbonic anhydrase gene includes a cDNA molecule, genomic gene or nucleotide sequence which is capable of encoding a CA enzyme or a polypeptide fragment thereof or alternatively, a nucleotide sequence which is complementary to said cDNA molecule, genomic gene or nucleotide sequence.
  • Carbonic anhydrase gene encoding a carbonic anhydrase are well known in the art.
  • Tyrosinase is used in the manufacture of 3,4-dihydroxyphenylalanine (L- DOPA), a potent drug used in treatment of several neural diseases (Zaidi et al., 2014). Attempts to reuse this enzyme for L-DOPA production by immobilising on DEAE-Granocel (cellulose) support showed that this method was not feasible due to enzyme inactivation and use of native enzyme would be more productive.
  • L- DOPA 3,4-dihydroxyphenylalanine
  • tyrosinase is an enzyme that is also referred to as monophenol monooxygenase; phenolase; monophenol oxidase; cresolase; monophenolase; tyrosine-dopa oxidase; monophenol monooxidase; monophenol dihydroxyphenylalanine:oxygen oxidoreductase; N-acetyl-6-hydroxytryptophan oxidase; monophenol, dihydroxy-L-phenylalanine oxygen oxidoreductase; o- diphenol:02 oxidoreductase; phenol oxidase.
  • Tyrosinase catalyzes an oxidation reaction of phenols.
  • Tyrosinase (EC 1 .14.18.1 ) is a type III copper protein found in a broad variety of bacteria, fungi, plants, insects, crustaceans, and mammals, which is involved in the synthesis of betalains and melanin.
  • the enzyme which is activated upon binding molecular oxygen, can catalyse both a monophenolase reaction cycle (reaction 1 , above) or a diphenolase reaction cycle (reaction 2, above).
  • one of the bound oxygen atoms is transferred to a monophenol (such as L-tyrosine), generating an o-diphoenol intermediate, which is subsequently oxidized to an o-quinone and released, along with a water molecule.
  • the enzyme remains in an inactive deoxy state, and is restored to the active oxy state by the binding of a new oxygen molecule.
  • the enzyme binds an external diphenol molecule (such as L-dopa) and oxidizes it to an o-quinone that is released along with a water molecule, leaving the enzyme in the intermediate met state.
  • the enzyme then binds a second diphenol molecule and repeats the process, ending in a deoxy state.
  • the second reaction is identical to that catalysed by the related enzyme catechol oxidase (EC 1 .10.3.1 ). However, the latter cannot catalyse the hydroxylation or monooxygenation of monophenols.
  • tyrosinase gene includes a cDNA molecule, genomic gene or nucleotide sequence which is capable of encoding a tyrosinase enzyme or a polypeptide fragment thereof or alternatively, a nucleotide sequence which is complementary to said cDNA molecule, genomic gene or nucleotide sequence.
  • Tyrosinase genes encoding a tyrosinase are well known in the art.
  • Tyrosinase substrates products are shown below:
  • DOPA 2,5-di-S- cysteinyl-DOPA, 6- S-cysteinyl-DOPA, 5-S-cysteinyl-3, 4- DOPA, and di- DOPA cross-links in
  • Pork and chicken Improves gel Meat Processing Food industry meat formation ability of
  • Cutinase has wide industrial applications ranging from oil industry, production of flavour and phenolic compounds to polymer synthesis and enantioselective esterification reactions.
  • Cutinase (EC 3.1 .1 .74) acts on cutin a polymeric structural component of plant cuticles, which is a polymer of hydroxy fatty acids that are usually C16 or C18 and contain up to three hydroxy groups.
  • the enzyme from several fungal sources also hydrolyses the p-nitrophenyl esters of hexadecanoic acid. It is however inactive towards several esters that are substrates for non-specific esterases.
  • cutinase gene includes a cDNA molecule, genomic gene or nucleotide sequence which is capable of encoding a cutinase enzyme or a polypeptide fragment thereof or alternatively, a nucleotide sequence which is complementary to said cDNA molecule, genomic gene or nucleotide sequence. Cutinase genes encoding a cutinase are well known in the art.
  • Cutinase substrates, products, and commercial applications are shown below:
  • NH2 amine
  • the amino acid becomes a keto acid
  • the keto acid becomes an amino acid.
  • ⁇ -amino transferase is an enzyme that catalyzes the transfer of an amino group from a primary amino donor to a carbonyl acceptor with pyridoxal 5'-phosphate (PLP) as cofactor.
  • the reaction can be divided into two half reactions, where the amino group is first transferred to PLP to form PMP (pyridoxamine phosphate) and then from PMP onto the carbonyl group, ⁇ -amino transferase (ATA) which is a highly valuable enzyme for the R-selective transamination reactions used to produce pharmaceutically- important drug intermediates (Hohne and Bornscheuer, 2012).
  • (S)-selective ⁇ -transaminases are well known, and (R)-amines have mainly been prepared by kinetic resolution of racemic amines by (S)- transaminases. However, with this method (R)-amines are obtained only with a maximum yield of 50%. Recently, several (R)-selective enzymes have been described. [0388] There is no particular restriction to a method for measuring aminotransferase activity, and it can be measured using the methods described herein.
  • aminotransferase gene includes a cDNA molecule, genomic gene or nucleotide sequence which is capable of encoding a cutinase enzyme or a polypeptide fragment thereof or alternatively, a nucleotide sequence which is complementary to said cDNA molecule, genomic gene or nucleotide sequence.
  • Aminotransferase genes encoding aminotransferases are well known in the art.
  • sitagliptin [0391 ] The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only, and the invention is not limited to these examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.
  • DNA encoding bovine carbonic anhydrase (BCA) with linker and Pn-4 peptide were codon optimized, synthesized and ligated into pET28a expression plasmid between Ncol and Xhol restriction sites by GenScript (Piscataway, NJ, USA).
  • GenScript GenScript (Piscataway, NJ, USA).
  • the recombinant plasmid was transformed using NEB High efficiency transformation protocol into BL21 (DE3) competent E.coli cells (New England Biolabs Inc.). Cells were grown using terrific broth (TB) medium and kanamycin (50 ⁇ g/mL) for 4 hours at 37°C.
  • Protein production was induced by adding 1 mM isopropyl-p-D-thiogalactopyranoside to the medium after 4 hours and incubated at 20°C for 16 hours.
  • Harvested cells were suspended in lysis buffer (50mM Tris-HCI pH8.0, 50mM NaCI, 1 mM EDTA, 0.5% Triton-X) and incubated for 20 minutes at room temperature.
  • Cells were lysed using a homogeniser (Avestin Emulsiflex C5, Ottawa, Canada) for 2 passes at 15,000 psi. Cell supernatant solution was collected by centrifugation at 14,000 rpm 4°C for 20 minutes.
  • BCA-P 4 was purified by immobilized metal-ion affinity chromatography on a Profinity IMAC (BioRad laboratories) column (1 .5 ⁇ 8 cm) charged with nickel ion with BioRad BioLogic DuoflowTM Chromatography system. Fusion protein was eluted step-wise from 24 to 200 mM imidazole in 50 mM Tris-HCI buffer (pH 8.0) containing 0.5M NaCI. Eluate containing the fusion protein was desalted using size exclusion chromatography on a G25 Sephadex (GE Healthcare) column (1 .5 ⁇ 17 cm) with 20mM Tris-HCI buffer (pH 8.0). Purified BCA-P 4 was stored at 4°C in desalting buffer for subsequent analysis. Wild-type BCA (Sigma Aldrich) dissolved in desalting buffer at 0.5-1 .0 mg/mL concentration was used for comparative studies.
  • Protein samples of 10 ⁇ _ volumes having concentration 0.5 -1 .0 mg/mL were applied on individual freshly glow-discharged carbon coated 400 mesh copper grids and left on grids for 5 minutes. Grids were washed with 5 ⁇ _ of water and blotted with filter paper and stained using 2%v/v uranyl acetate for 30 seconds.
  • Samples were viewed using transmission electron microscope at 80 kV (Hitachi, HT7710 120kV FEG) and at 200 kV (FEI, Tecnai G2 T20 TWIN LaB6) and electron micrographs were recorded using CCD camera and Gatan "Digital Micrograph" software.
  • the pH drop in the solution due to conversion of C0 2 to HCO 3" is visualized using the indicator bromothymol blue (BTB) which changes color from blue (pH >7.6) to green (pH 6.0 - 7.6) and yellow (pH ⁇ 6.0) accordingly.
  • Blank solution was prepared as mentioned above and volume of enzyme was replaced with water.
  • the time taken for the pH change from 8.0 to 7.0 was recorded in seconds and the hydrase activity was calculated in terms of Wilbur Anderson Units (WAU) using the formula (Tc - Te) / Te; where Tc is the time in seconds for change in pH to 7.0 for the blank solution and Te is the time in seconds for change in pH to 7.0 for the enzyme solution.
  • Nanoparticle sizes were determined using Zetasizer Nano (Malvern Instruments). Protein particle sample of volume 1 ml_ was taken in cuvette and measured for particle size using the zetasizer software. For temperature dependent DLS measurement, the sample chamber was set at specific temperature using in-built temperature control and protein was incubated at respective temperature for 5 minutes before taking measurements. Average particle sizes were determined from 3 runs each comprising 16 cycles
  • esterase activity was performed in a 96 well assay plate using reaction buffer (50mM sodium sulphate + 50mM HEPES pH 8.0) with a sample volume containing 3 ⁇ g of enzyme. To this p-nitrophenyl acetate was added as a substrate at a final concentration of 1 mM. Absorbance was measured at 405 nm every 15 seconds for 10 minutes using microplate reader (Infinite 200PRO, Tecan). Similar blank solution was prepared using water instead of enzyme solution. Following blank correction of absorbance, the slope in terms of absorbance per second was calculated to determine reaction rate and esterase activity. For temperature dependent assay, enzyme was incubated at specific temperature in a heating block for 5 minutes before adding to the reaction buffer.
  • reaction buffer 50mM sodium sulphate + 50mM HEPES pH 8.0
  • EXAMPLE 7 Formation of nanoparticles comprising bovine carbonic anhydrase bound to a self-assembly polypeptide.
  • BCA bovine carbonic anhydrase
  • the self-assembling peptide P i 4 was selected because of its ⁇ -strand structure and its ability to spontaneously self-assemble to form nanofibers under suitable conditions.
  • the Pn-4 peptide is relatively small in size compared to the enzyme BCA itself and sufficient space is required for peptide interaction to promote self-assembly of two or more fusion proteins.
  • BCA and Pn-4 were connected by the GS-linker known to provide flexibility between protein domains.
  • BCA- P i 4 was purified and the formation of nanoparticles was demonstrated with TEM images ( Figure 2).
  • BCA- P -4 samples show the presence of uniform distribution of particles with spherical morphology.
  • This data demonstrates that a plurality of self-assembling enzymatic components comprising a self-assembly polypeptide bound to an enzyme are able to produce a supramolecular assembly.
  • This data also demonstrates that a plurality of self-assembling enzymatic components comprising a self-assembly polypeptide bound to an enzyme are able to produce a supramolecular assembly wherein the assembly is a nanoparticle.
  • Figure 3 demonstrates the particles observed had an average size in the range of 50 - " l OOnm with some as large as 180nm. In contrast, there was no formation of spherical particles observed in the TEM images of free enzyme WT- BCA ( Figure 6), confirming that the self-assembly polypeptide plays an important role in driving the self-assembly of individual monomers to form nanoparticles of defined size and shape.
  • EXAMPLE 8 Enzymatically active supramolecular assemblies.
  • Figure 4 demonstrates a supramolecular assembly comprising BCA- P i 4 is enzymatically active. Furthermore, the enzymatically active nanoparticles retain full catalytic activity of the enzyme. As demonstrated in Figure 4, the hydrase activity of BCA- P i 4 nanoparticles is comparable to that of free enzyme (WT- BCA) using the Wilbur-Anderson method. After 25 seconds, both enzyme solutions changed color to green indicating enzymatic conversion of C0 2 .
  • the structural stability of the BCA-P i 4 nanoparticles is essential for its integration with existing methods for C0 2 capture process.
  • the relatively large size of BCA-P -4 nanoparticles allows its integration with large pore membrane as opposed to the small pore membranes used in existing methods to retain free CA.
  • the preservation of nanoparticle structure of BCA-P -4 over time would ensure long-term containment within membrane minimizing the replenishing of fresh enzyme.
  • the use of BCA-Pn-4 within large pore membrane will allow operations at high flow rates and easy membrane maintenance, thereby improving the overall process efficiency.
  • EXAMPLE 10 Enzymes bound to self-assembly polypeptides.
  • Table 4 sets out self-assembling enzymatic components produced, wherein each self-assembling enzymatic component comprises a self-assembly polypeptide bound to an enzyme.
  • Cutinase hydrolyses the colourless molecule p-nitrophenyl butyrate to the yellow molecule p-nitrophenol, which allows for colorimetric analysis of cutinase activity.
  • 0.25-1 .5 ⁇ cutinase is analysed by adding 1 ⁇ . of 0.1 M p-nitrophenyl butyrate in dimethyl sulfoxide and measured p-nitrophenol absorbance at 405 nm for 3 min using a spectrophotometric plate reader.
  • the initial velocity, vo, of p-nitrophenyl butyrate hydrolysis to p-nitrophenol is calculated from the linear portion of a plot of A405 nm versus time.
  • the catalytic activity of aminotransaminase from cell lysate can be determined by using a standard photometric assay at 25°C containing 0.25 mg/mL of total lysate protein, 0.1 mM PLP, 5mM (R)-a-methylbenzylamine and 5 mM pyruvate or 5 mM butanal in 50 mM potassium phosphate buffer pH 7.5. The increase of acetophenone was measured at 300 nm (extinction coefficients.28 mM "1 cm "1 ).
  • Microbial carbonic anhydrase activity is determined using the methods of Example 4 and Example 6.
  • Nanoparticle sizes are determined using the method of Example 5.
  • EXAMPLE 11 Modulation of enzymatically active supramolecular assembly formation
  • the fusion protein BCA-P 4 was expressed in E.coli cells and purified as described above. Briefly gene expressing enzyme bovine carbonic anhydrase linked to Pn4 peptide via a GS-linker was cloned as a single construct into pET28a between Ncol and Xhol (GenScript, Piscataway, NJ, USA). The plasmid pET28a-BCA-Pii4 was transformed into E.coli BL21 (DE3) and cells were grown in terrific broth. Expression of BCA-P1 14 was initiated by 1 mM IPTG and culturing continued for 16 h at 20°C.
  • Enzymatically active nanoparticle formation and size was determined using a Zetasizer Nano (Malvern Instruments). BCA-P 4 in solution (1 ml_) was measured under a range of conditions for particle size using the zetasizer software. Data were collected from 3 independent experiments with each comprising 12 cycles.
  • the sample chamber of Zetasizer Nano was adjusted using the in-built temperature control and BCA-P 4 solution, diluted to 0.5 mg/mL in 50 mM Tris-HCI pH 8.0 to final volume of 1 ml_, was incubated at the respective temperature in a heating block for 5 min before taking measurements.
  • Stock solutions of 0.5 M NH 4 CI, MgCI 2 , NaCI, NaN0 3 and Na 2 S0 4 were prepared in 10 mM Tris-HCI pH 8.0 and the required volume added to BCA-P 4 sample diluted to 0.5 mg/mL in 10 mM Tris-HCI pH 8.0, to achieve a final concentration of 50, 100 and 200 mM.
  • BCA-P 4 with added salts was prepared as above and adjusted to pH 6.5 ⁇ 0.1 using 1 M acetic acid.
  • Negative staining of enzymatically active supramolecular assemblies was performed on carbon coated 200 mesh copper grids (GSCu200CC, Proscitech, QLD Australia) by an established protocol (Booth et al., 201 1 ) using uranyl acetate stain. Samples were visualized at 200 kV using FEI, Tecnai G2 T20 TWIN LaB6 and electron micrographs were recorded using CCD camera and Gatan "Digital Micrograph" software.
  • a Factorial Design experiment using a two-level design was used to model the BCA-P 4 enzymatically active supramolecular assembly formation. This method examined 4 factors (pH, temperature, MgCI 2 concentration and protein concentration) at two levels (Table 5) to determine the major factors and interaction effects on BCA-P 4 supramolecular assembly formation.
  • 4 factors pH, temperature, MgCI 2 concentration and protein concentration
  • Table 5 A 2 4 full factorial design with 4 center points was performed in 20 experiments where supramolecular assembly sizes measured by dynamic light scattering were used as the response variable. The center point values were determined as the average of low and high level values. Response data were analyzed using statistical software Design- Expert 10 (Stat-Ease, Inc. Minneapolis, USA).
  • Table 5 shows Two-level Factorial Design:
  • Protein concentration (mg/mL) 0.5 1 .25 2.0
  • the present inventors propose two interactive forces that influence BCA-P 4 self-assembly: firstly, there is the interaction between peptide-peptide monomers (F p-P ) in anti-parallel orientation that is a combination of the attractive and repulsive forces as shown in Figure 15, where attractive forces exist between oppositely positioned arginine and glutamic acid residues and the ⁇ - ⁇ interaction of aromatic residues while glutamic acid residues at the 5 th and 6 th peptide position impart repulsive forces.
  • F e-P peptide-peptide monomers
  • Table 6 shows estimated charge of peptide Pn4, enzyme BCA and fusion protein BCA-P 4 from pH 4.0 to pH 8.0 calculated using PROTEIN CALCULATOR v3.4:
  • Nanoparticles were produced in 50 mM NaN0 3 at pH 6.5 (Figs 14c-d).
  • the effect of NaN0 3 on nanoparticle formation may be attributed to its specific interaction with wild-type BCA through the first hydration shell confirmed by molecular dynamic simulation studies (Warden et al., 2015).
  • the most effective salt for facilitating BCA-P 4 self-assembly in low ionic strength buffer was NH 4 CI.
  • Ammonium chloride promoted self-assembly at pH 6.5 and salt concentrations up to 100 mM. Without wishing to be bound by theory, this behavior might be attributed to the unique ability of the ammonium cation to form cation- ⁇ interactions (Dougherty, 1996) with aromatic amino acids present in the peptide.
  • Table 7 Influence of added salts and pH on self-assembly of BCA-P 4.
  • Mg 2+ ions exist in a stable coordination complex with 6 oxygen atoms from water molecules (Bock et al., 1994). However, in solution with proteins, Mg 2+ has preference to interact with oxygen atoms from carboxyl and hydroxyl groups in the side chain residues of amino acids (Zheng et al., 2008).
  • Figure 19a illustrates the interaction of Mg 2+ with the carboxyl oxygen of glutamic acid residues in P 4 monomers.
  • the model generated using the two-level full factorial design not only encompasses the aforementioned findings regarding the effects of individual factors in self-assembly but it also identified an interaction between pH and MgCI 2 concentration.
  • the model generates Formula I which describes the factors and interactions that can be used to predict BCA-P 4 particle size under various conditions:
  • BCA-P 4 particle were predicted to be formed with sizes of 253 and 633 nm under the following conditions - (1 ) 0.5 mg/mL BCA- Pii4, pH 7.5, 6 mM MgCI 2 , and (2) 1 .0 mg/mL BCA-Pn4, pH 7.0, 8 mM MgCI 2 , respectively.
  • Experimental results confirmed the predicted sizes whereby peak centers for conditions 1 and 2 measured by dynamic light scattering were 201 and 669 nm ( Figure 20c), respectively.
  • the empirical formula predicts particle size of BCA-P 4 under specified conditions and encompasses the parameters that allow robust control over particle size.
  • Esterase activities were determined for the various enzyme nanoparticles formed under the two-factorial design conditions and expressed relative to the activity of the monomeric protein-peptide unit. Average activities of the protein particles of various sizes were 108% of the relative activity of individual enzyme- peptide units as shown in Figure 21 . This demonstrates that the self-assembly process allows proper orientation and access to active sites of individual enzyme molecules thereby retaining complete functionality. This is a much desired feature when designing enzyme nanoparticles and other protein assemblies with functionality for specific applications.
  • BCA-P 4 The gene encoding BCA and a single peptide (BCA-P 4) was inserted into pET28a vector and expressed in BL21 (DE3) E.coli cells as described above.
  • the second fusion construct was designed to attach three P 4 peptides in series to the C-terminus of BCA via GGGGSGGGGS linker sequence and is designated as BCA-(P 4) 3 .
  • the gene sequence for BCA-P 4(3) was codon optimised, synthesized and ligated into pET28a expression plasmid between Ncol and Xhol restriction sites by GenScript (Piscataway, NJ, USA).
  • the plasmid pET28a-BCA- (Pi i4) 3 was transformed into BL21 (DE3) E.coli cells using NEB High Efficiency Transformation protocol.
  • Cells were grown in terrific broth (TB) medium and kanamycin (50 ⁇ g/mL) for 4 hours at 37 °C followed by induction with 1 mM isopropyl-p-D-thiogalactopyranoside at 20 °C for 16 hours.
  • 2% v/v lactose was added in the initial TB medium.
  • fusion protein was eluted stepwise from 10, 24, 50 and 200 mM imidazole in 10 mM Tris-HCI buffer (pH 8.5). Eluate containing the fusion protein was diluted with water to decrease the conductivity 4-6mS/cm before loading on 1 ml_ HiTrap OFF anion exchange chromatography column (GE Healthcare). Protein fractions was eluted with 0- 100% gradient over 15 column volume (CVs) using 10 mM Tris-HCI buffer (pH 8.5) containing 1 M NaCI.
  • BCA-Pii4 nanoparticles [0468] Purified BCA-Pn4 was diluted to a protein concentration of 0.8 mg/mL with 10 mM Tris-HCI buffer (pH 8.0). Using 5 M acetic acid solution, the pH of the enzyme solution was adjusted to 6.1 ⁇ 0.1 to which MgCI 2 stock solution prepared in 10 mM Tris-HCI buffer (pH 8.0) was added to a final concentration of 10 mM. Nanoparticles were formed instantaneously and their size measured by Dynamic Light scattering technique using Zetasizer Nano (Malvern Instruments).
  • BCA-(P 4) 3 was diluted to 0.5 mg/mL protein concentration with 10 mM Tris-HCI buffer (pH 8.0). Using 5 M acetic acid, the pH of solution was adjusted to 5.7 ⁇ 0.1 , the pi of BCA-(P 4) 3 , to induce instant formation of aggregates. BCA-(P 4) 3 solution was then incubated under this condition for 15 min at 50 rpm. The solution was centrifuged at 8000 rpm for 5 min (Sigma, John Morris Scientific). The supernatant was collected in a separate tube and BCA- (Pii4) 3 particles were collected as a precipitated pellet. To the pellet, 10 mM Tris- HCI buffer (pH 8.0) was added to its initial volume and gently shaken for 5 min to completely solubilise the pellet for subsequent activity analysis.
  • 10 mM Tris- HCI buffer pH 8.0
  • the kinetic parameters of the enzyme were determined by measuring the esterase activity of BCA using para-nitrophenol acetate (pNPA) as substrate.
  • the assay was performed in a 96 well assay plate using reaction buffer (50 mM sodium sulphate + 50 mM HEPES pH 8.0) containing 3 ⁇ g of enzyme in a final sample volume of 0.2mL.
  • Substrate was added at the following concentrations 0.1 , 0.25, 0.5, 0.75 and 1 mM and absorbance measured at 405 nm every 30 sec for 15 min using a microplate reader (Infinite 200PRO, Tecan).
  • a blank solution was prepared with the same components except that water was added instead of enzyme solution.
  • a volume of 1 ml_ BCA-(Pn4) 3 at 0.8 mg/mL in 10 mM Tris-HCI buffer (pH 8.0) buffer solution was taken into a microcentrifuge tube.
  • the enzyme activity was determined for a small subsample of this solution by removing 3.5 ⁇ _ (targeting 3 ⁇ g) and testing CO 2 hydration using the Wilbur Anderson method modified to 0.2 ml_ total volume in a microtiter plate set-up.
  • the initial enzyme activity was designated Reaction 1 .
  • the pH of BCA-(P 4) 3 was adjusted to pH 5.7 ⁇ 0.1 with 5 M acetic acid and incubated for 15 min at 50 rpm.
  • the solution was centrifuged at 8000 rpm for 5 min. The supernatant was collected in a separate tube and the enzyme precipitate was solubilized with 10 mM Tris-HCI buffer (pH 8.0). This was repeated for two more cycles and enzyme performance was measured after every precipitation and solubilisation step. The supernatant and final resuspended pellet were analyzed for protein concentration using the Bradford method and SDS-PAGE analysis in 4-12% Bis-Tris Bolt gels.
  • the BCA-(P 4) 3 fusion system was designed to have 3 repeats of peptide in contrast to the single peptide in BCA-P 4 fusion system as illustrated in Figure 10. All other design parameters such as linker length and C-terminal fusion position were maintained.
  • BCA- (Pii4) 3 was significantly reduced in comparison to WT-BCA and BCA-P 4 as shown in Figure 22b.
  • the reduction in protein yield for BCA-(P 4) 3 illustrates the direct consequence of the additional peptide units in this design, possibly due to its repetitive nature.
  • the E.coli expression system is known to perform poorly when expressing peptides or proteins rich in repetitive sequences. For example, a 60% reduction in peptide yield was reported when 6-repeats of Pn4 peptide fused to KSI was expressed using E.coli system.
  • low yield of BCA-(P 4) 3 may indicate the need for increased protein folding time due its longer peptide length during the translational phase. Without wishing to be bound by theory, fusion to C- terminus may further slow-down protein folding due to the presence of a C- terminal knot structure in BCA which is inherently difficult to refold.
  • Figure 25a shows that the pre-peak fraction corresponds to a mass of 31 .75 kDa while Figure 25b shows that the peak-tail fraction to have a mass of 34.41 kDa which corresponds to the size of BCA- (Pi i4) 3 .
  • the probable sequence of 31 .75 kDa protein mass was determined using Protparam computational tool and identified as a partially cleaved sequence of BCA-(P 4) 3 , with cleavage of the last 19 amino acids of the C-terminal sequence. Due to the close sequence similarity and similar peptide fragment length, this difference was unidentifiable through mass spectrometry of trypsin digested samples, but is revealed by exact mass measurement.
  • the data provides evidence that repeat sequences coupled with complex knot formation at the C-terminal of the enzyme were truncated by the E.coli expression system.
  • the present inventors propose the reduced yield of BCA-(P 4) 3 may relate to the C-terminal knot structure of BCA which is specific only to certain enzyme families.
  • V max and increased K m values of BCA-(P 4) 3 is proposed to be an effect of the length of the 3 peptide repeats causing steric effect on accessibility of the substrate to the enzyme as well as aforementioned difficulty in folding at the C-terminal knot region.
  • the presence of knot structures in proteins is known to provide a stabilization effect protecting it from unfolding under denaturing conditions.
  • the tightening of the C- terminal knot in BCA has been associated with formation of the active site and thereby has a direct influence on the enzyme activity.
  • Table 8 Kinetic parameters of WT-BCA, BCA-Pn4 and BCA-(Pn4) 3
  • This data also demonstrates the assembly of self-assembling enzymatic components comprising a tandem repeat of a self-assembly polypeptide bound to an enzyme form an enzymatically active supramolecular assembly.
  • This data demonstrates the assembly of self-assembling enzymatic components comprising a tandemly repeated self-assembly polypeptide bound to an enzyme into a supramolecular assembly can be unassembled into the self- assembling enzymatic components using pH. This allows recovery of the self- assembling enzymatic components for re-use.
  • Table 9 Impact of pH switching on the enzyme activity of WT-BCA, BCA- Pii4 and BCA-(Pn4) 3 by Wilbur-Anderson method.
  • One objective of engineering enzymes with self-assembly feature is to allow recovery and reuse of enzymes.
  • Table 10 Separation efficiency of BCA-P 4 nanoparticle using 1 00kDa MWCO Centrisart device
  • the permeate fraction shows particles size ⁇ 5 nm which corresponds to the monomeric form of BCA- Pii4.
  • Table 1 1 Separation efficiency of BCA-(Pn4) 3 particle using 300 kDa MWCO Centrisart device
  • BCA-(P 4)3 was shown to be robust to the pH switch and particles formed were large enough to be precipitated out of solution. Therefore the reusability of BCA-(P 4) 3 system was investigated in a coupled process that included biocatalysis in an industrial scenario. For this purpose, pH induced precipitation of the enzyme-peptide for recovery was performed for three consecutive cycles with activity tested after each cycle.
  • Table 12 Separation efficiency and biocatalytic performance of BCA- (Pi i4) 3 particle following three biocatalytic cycles.
  • EXAMPLE 14 Production of enzymatically active supramolecular assemblies using different enzymes.
  • Each of the gene sequences were codon optimised, synthesized and ligated into pET28a expression plasmid between Ndel and Xhol restriction sites by GenScript (Piscataway, NJ, USA) thereby incorporating an N-terminal 6X-Histag with thrombin cleavage site sequence pre-existing in the pET28a plasmid.
  • the plasmids were transformed into BL21 (DE3) E.coli competent cells using NEB High Efficiency Transformation protocol only with the exception of plasmids for WT-Cutinase and Cutinase-Pn4 that were transformed into SHuffle T7 express cells to facilitate correctly formation of disulphide bond for cutinase (4 cysteines and 2 disulphide bonds).
  • TmCA tyrosinase and cutinase-expressing cells
  • growth in terrific broth (TB) medium and kanamycin (50 ⁇ g/mL) was initiated with 2% v/v inoculum for 4 h at 37°C for the first growth phase.
  • Protein expression was examined by adding isopropyl-p-D-thiogalactopyranoside to a final concentration of 1 mM in the medium at 37°C for 3 h and at 20 °C for 16 h.
  • 2% v/v lactose was added in the initial TB medium as an inducer and incubated at 37°C and 20°C for 16 h.
  • a second washing step was used to remove contaminant proteins.
  • Fusion protein was eluted step-wise with appropriate ratio of buffers A and B to target specific imidazole concentration for at least 3 column volumes. Fractions were pooled based on purity determined by SDS-PAGE 4-1 2% Bis-Tris Bolt gels and bulk eluate sample was desalted using size exclusion chromatography on a G25 Sephadex (GE Healthcare) column (1 .5 ⁇ 17 cm) with specific desalting buffer. Purified samples were stored at 4°C in desalting buffer for subsequent analysis. The specific buffers used for each enzyme are tabulated below (Table 14). Identical purification protocols were performed for WT-enzyme and their corresponding fusion system.
  • Tris-HCI pH 50 mM Tris-HCI Protein NaCI, 1 mM
  • Tris-HCI 50 mM Tris-HCI 50 mM Tris-HCI 20 mM Tris-HCI
  • Tris-HCI 50 mM Tris-HCI 50 mM Tris-HCI 20 mM Tris-HCI 50 mM Tris-HCI
  • IMAC buffer B buffer pH 8.0+0.5 buffer pH 7.5+0.5 buffer pH 8.0+0.5 buffer+0.3M NaCI
  • Cut- P ⁇ 4 nanoparticles [0525] Purified Cut-Pn4 at 0.5 mg/mL protein concentration in 20 mM Tris-HCI buffer pH 8.0 was incubated with addition of MgC (final concentration 10mM) for 2 min at 25°C and nanoparticle formation was monitored by Dynamic Light scattering technique using Zetasizer Nano (Malvern Instruments). Particle sizes were determined as triplicate measurements each consisting of 10 cycles and compared with particle size determined without addition of MgCI 2 .
  • the esterase activity using para-nitrophenyl acetate (pNPA) as substrate was used to determine the kinetic parameters of both WT-TmCA and TmCA-P 4.
  • the assay was performed in a 96 well assay plate using reaction buffer (1 M sodium sulphate + 50 mM HEPES pH 8.0) in final assay volume of 0.2 ml_ containing 3 ⁇ g of enzyme and pNPA at the following concentrations 0.1 , 0.25, 0.5, 0.75 and 1 mM.
  • Absorbance was measured at 405 nm every 30 sec for 15 min using a microplate reader (Infinite 200PRO, Tecan).
  • a blank sample was prepared using assay buffer and substrate with water replacing the enzyme solution.
  • the esterase activity of Cut-P 4 was determined using para-nitrophenyl butyrate (pNPB) as substrate at concentrations 0.1 and 0.5 mM.
  • the assay was performed in a 96 well assay plate using reaction buffer (50 mM Tris-HCI pH 8.0 +10 mM NaCI) in final assay volume of 0.2 ml_ containing 0.35 ⁇ g enzyme. Absorbance was measured at 405 nm every 15 sec for 2 min using a microplate reader (Infinite 200PRO, Tecan). A blank solution was prepared using water instead of enzyme solution. Following blank correction of absorbance, the slope in terms of absorbance per second was converted to reaction rate determined by calculating the concentration of p-nitrophenol from a standard curve.
  • the native TmCA enzyme is originally obtained from the deep-sea chemolithoautotroph microorganism Thiomicrospira crunogena (Dobrinski et al., 2010), and the enzyme's structure is more stable in buffers containing high salt rather than low salt. For this reason, the TmCA desalting buffer contained 200 mM NaCI which prevents the enzyme from precipitating out of the solution. Conditions were explored to promote self-assembly. As demonstrated above, BCA-P 4 nanoparticles at pH 6.8 showed increase in particle size at higher temperatures. Based on this observation, temperature was used as a controlling factor to induce particle self-assembly in TmCA-P 4.
  • TmCA-Pii4 nanoparticles of size ranging from 200-500 nm Figure 34b
  • WT-TmCA showed no self-assembly at 50 °C ( Figure 34a). This confirms that the self-assembly was driven by the fused Pn4 peptide.
  • the formation of TmCA-P 4 nanoparticles was confirmed by transmission electron microscopy which showed particles of average 200 nm in size which correlated with value shown by dynamic light scattering ( Figure 34b).
  • TmCA-Pri4 Temperature and pH stability of TmCA-Pri4
  • the self-assembly of TmCA-P 4 is triggered by a higher temperature of 50 °C and this condition may unintentionally change enzyme properties, it was essential to confirm that the self-assembly did not alter the enzyme's structural stability.
  • the unfolding characteristics of TmCA-P 4 were investigated by differential scanning fluorimetry. The melting curves shown in Figure 36a and c illustrate that both WT-TmCA and TmCA-P 4 unfold at -60 °C. This is similar to the unfolding temperature of another dimeric CA isolated from the same microorganism Thiomicrospira crunogena (Diaz-Torres et al., 2015).
  • This data demonstrates that a plurality of self-assembling enzymatic components comprising a self-assembly polypeptide bound to an enzyme are able to produce an supramolecular assembly wherein the structure of the enzyme is preserved in the supramolecular assembly.
  • Table 15 Kinetic parameters of WT-TmCA and TmCA-Pn4.
  • Cut-Pn4 alone formed large particles of size ⁇ 800 nm without added MgCI 2 ( Figure 44a) and the addition of MgCI 2 showed no significant change in particle size. Cutinase being closely related to the lipase family has several hydrophobic loops in its structure that are accessible to the solvent (Carvalho et al., 1998) and capable of movement upon interfacial binding (Martinez et al., 1992). It is possible that the cutinase is prone to self- aggregation. Under transmission electron microscopy, large aggregate like particles were observed whose size was ⁇ 800-900nm ( Figure 44b) which correlated with the dynamic light scattering results.
  • Cut-Pn4 displayed significant enzyme activity.
  • Substrate p-nitrophenyl butyrate at low concentration (0.1 mM) resulted in a slower reaction rate when compared to 0.5 mM which reached enzyme saturation within 45 sec of reaction initiation ( Figure 45). This may be attributed to the low solubility of pNPB in aqueous buffers and its subsequent availability to the enzyme for conversion.
  • the increased specific activity of enzyme with high pNPB concentration (Table 18) indicates that Cut-P 4 displays high activity and conversion if sufficient substrate can be provided to it.
  • YUZBASHEVA E., YUZBASHEV, T., LAPTEV, I., KONSTANTINOVA, T. & SINEOKY, S. 201 1 .
  • HARTMANN B. M., KAAR, W., YOO, I. K., LUA, L. H., FALCONER, R. J. & MIDDELBERG, A. P. 2009.

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Abstract

La présente invention concerne de manière générale des assemblages supramoléculaires à activité enzymatique et des procédés de production de ces assemblages. La présente invention concerne en particulier un assemblage supramoléculaire à activité enzymatique comprenant une pluralité de composants enzymatiques à autoassemblage, chaque composant enzymatique à autoassemblage comprenant un polypeptide à autoassemblage lié à une enzyme.
PCT/AU2016/051293 2015-12-24 2016-12-23 Assemblages supramoléculaires à activité enzymatique WO2017106937A1 (fr)

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CN108611267A (zh) * 2018-04-02 2018-10-02 中山大学 一种实时动态光散射定量pcr仪
CN112391376A (zh) * 2020-11-03 2021-02-23 天津科技大学 一种固定化脂肪酶杂化纳米花及其制备方法与用途
EP3789427A1 (fr) * 2019-09-09 2021-03-10 Université de Strasbourg Gel supramoléculaire supporté sur une mousse de polymère à cellules ouvertes
US11058725B2 (en) 2019-09-10 2021-07-13 Obsidian Therapeutics, Inc. CA2 compositions and methods for tunable regulation

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108611267A (zh) * 2018-04-02 2018-10-02 中山大学 一种实时动态光散射定量pcr仪
CN108611267B (zh) * 2018-04-02 2022-02-18 中山大学 一种实时动态光散射定量pcr仪
EP3789427A1 (fr) * 2019-09-09 2021-03-10 Université de Strasbourg Gel supramoléculaire supporté sur une mousse de polymère à cellules ouvertes
WO2021048199A1 (fr) * 2019-09-09 2021-03-18 Université De Strasbourg Gel supramoléculaire supporté sur mousse polymère à cellules ouvertes
US11879038B2 (en) 2019-09-09 2024-01-23 Universite De Strasbourg Supramolecular gel supported on open-cell polymer foam
US11058725B2 (en) 2019-09-10 2021-07-13 Obsidian Therapeutics, Inc. CA2 compositions and methods for tunable regulation
CN112391376A (zh) * 2020-11-03 2021-02-23 天津科技大学 一种固定化脂肪酶杂化纳米花及其制备方法与用途
CN112391376B (zh) * 2020-11-03 2022-11-01 天津科技大学 一种固定化脂肪酶杂化纳米花及其制备方法与用途

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