Immobilised enzymes and methods thereof

Abstract

This disclosure concerns a bioprocess comprising loading a lysate comprising a His- tagged enzyme, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His-tagged enzyme immobilized on a charged resin, washing the His-tagged enzyme immobilized on a charged resin, and contacting the His-tagged enzyme immobilized on a charged resin with a precursor in order to convert the precursor into a product; wherein the enzyme is selected from isomerase and oxidoreductase; wherein the enzyme is His-tagged.

Description

[0001] Immobilised Enzymes and Methods Thereof

[0002] Technical Field

[0003] The present invention relates, in general terms, to immobilised enzymes and their methods of use and fabrication thereof.

[0004] Background

[0005] The global biocatalyst market is experiencing robust growth, driven by increasing demand across pharmaceuticals, food and beverage, biofuels, and environmental applications. For example, the market size in 2025 is estimated to be about USD 12.4 billion, and forecast to grow to USD 30.3 billion in 2034, with a CAGR of 10.4% from 2025 to 2034.

[0006] For example, hydrolases such as proteases, lipases and amylases, which accounts for 50-55% of the total biocatalyst market, are widely used in food processing, detergents, biofuels, and pharmaceuticals. Oxidoreductases such as alcohol dehydrogenases, glucose oxidases and laccases, are used in pharmaceutical synthesis, biosensors, environmental remediation, and wastewater treatment. Transferases such as aminotransferases, methyltransferases and acyltransferases are important in antibiotic production, glycosylation of APIs, and agriculture. Other biocatalysts such as lyases, isomerases, ligases, are used in synthetic biology and advanced bioprocessing.

[0007] Biocatalysts for industrial scale production of bulk chemicals are often associated with significant costs arising from production (reactor cost) and purification / processing. Furthermore, there are requirements for maintaining their stability and activity.

[0008] For example, D-Allulose, also known as D-psicose, is a rare sugar low-calorie sweetener with many health benefits. It can prevent diabetes and be consumed by diabetic patients. D-Allulose has been approved for use as a sugar additive in USA, Japan, South Korea, Mexico, Singapore and several other countries. This rare sugar sweetener has high commercial potentials. The global allulose market size was valued at $95.4 million in 2021, and is projected to reach $308.2 million by 2032, growing at a CAGR of 11.7% from 2022 to 2032. The production of allulose is carried out by enzymatic epimerization of fructose but the process is hampered by poor stability and short lifetime of the free enzyme. It would be desirable to overcome or ameliorate at least one of the above-described problems.

[0009] Summary

[0010] The present disclosure concerns a bioprocess comprising : a) loading a lysate comprising a His-tagged enzyme, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His-tagged enzyme immobilized on a charged resin; b) washing the His-tagged enzyme immobilized on a charged resin; and c) contacting the His-tagged enzyme immobilized on a charged resin of step b) with a precursor in order to convert the precursor into a product; wherein the enzyme is selected from isomerase and oxidoreductase; wherein the enzyme is His-tagged.

[0011] In some embodiments, the enzyme is His-tagged at its N-terminus, C-terminus and / or in an internal polypeptide sequence of the enzyme.

[0012] In some embodiments, the enzyme is selected from D-psicose-3-epimerase, ketose 3- epimerases, D-tagatose 3-epimerase (DTEase), cholesterol oxidase, glucose oxidase (GOX) and glucose-methanol-choline oxidoreductase.

[0013] In some embodiments, the charged resin comprises Ni2+, Co2+or a combination thereof.

[0014] In some embodiments, the loading step comprises circulating the lysate through the charged resin for at least two cycles.

[0015] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by an impurity selected from a protein, a peptide, a carbohydrate, lipid, nucleic acid and / or metabolite.

[0016] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by an impurity loading of less than about 5%.

[0017] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterised by an enzyme to resin loading of about 0.01 mg / g to about 50 mg / g.

[0018] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterised by an average resin particle size of about 1 pm to about 300 pm.

[0019] In some embodiments, the method is performed at a temperature of about 40 °C to about 80 °C.

[0020] In some embodiments, the immobilised enzyme is characterised by an activity of more than 60% for at least 15 days.

[0021] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by a storage stability of at least 80 days at less than 10 °C.

[0022] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterised by a thermal stability of more than 5 days at 5 °C relative to a free enzyme.

[0023] The present disclosure concerns a method of converting fructose to Allulose, comprises: a) loading a lysate comprising a His-tagged D-psicose-3-epimerase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged D-psicose-3-epimerase immobilized on a charged resin; b) washing the His-tagged D-psicose-3-epimerase immobilized on a charged resin; and c) contacting the His-tagged D-psicose-3-epimerase immobilized on a charged resin with fructose in order to convert the fructose into Allulose; wherein D-psicose-3-epimerase is His-tagged.

[0024] The present disclosure also concerns a method of converting cholesterol to cholest-4- en-3-one, comprises: a) loading a lysate comprising a His-tagged cholesterol oxidase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged cholesterol oxidase immobilized on a charged resin; b) washing the His-tagged cholesterol oxidase immobilized on a charged resin; and c) contacting the His-tagged cholesterol oxidase immobilized on a charged resin with cholesterol in order to convert cholesterol to cholest-4-en-3-one; wherein cholesterol oxidase is His-tagged.

[0025] Brief description of the drawings

[0026] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0027] FIG. 1A shows a schematic representation for immobilisation of His-tagged DPEase on metal containing resins.

[0028] FIG. IB shows comparison of activities of immobilised and free enzymes (isomerase 1).

[0029] FIG. 2 shows gel analysis of concurrent purification and immobilisation of lysate from isomerase 2.

[0030] FIG. 3A shows a temperature profile of immobilised DPEase (isomerase 1).

[0031] FIG. 3B shows a temperature profile of free DPEase (isomerase 1).

[0032] FIG. 4A shows longevity of immobilised DPEase (isomerase 2) from lysate and purified enzyme.

[0033] FIG. 4B shows the longevity study of immobilised isomerase 3.

[0034] FIG. 5 shows storage stability of immobilised DPEase (isomerase 1) at 4°C.

[0035] FIG. 6 shows activity comparison of immobilised cholesterol oxidase from Streptomyces Lavendulae at 35°C, pH 7.5. SDS-PAGE analyses of purified cholesterol oxidase. Purified free enzyme is unstable and does not retain its activity. Initial oxidase activity was taken as 100%. H2O2 released over a fixed time period was used to compare activity rates.

[0036] FIG. 7 shows a calibration curve of absorbance to amount of cholesterol present.

[0037] FIG. 8A shows 2 mg sieved LcPET (<150 pm) reaction with immobilised or free LCC- ICCG. 1.67 pM purified protein is used, either as free or as immobilized enzymes onto magnetic beads (HisPur Ni-NTA Magnetic Beads, ThermoFisher) or Ni-Resin. Reaction was conducted at 70°C, 650 rpm and 2h, 100 mM potassium phosphate (pH 8). N.D. Not detected.

[0038] FIG. 8B shows SDS Page analysis of protein eluted from magnetic Ni-NTA beads (lane 1) and Ni Resin (lane 2).

[0039] FIG. 8C shows Pymol structure of LCC-ICCG (PDB: 8JMP).

[0040] Detailed description

[0041] The present disclosure concerns the immobilisation of certain enzymes onto resin. It was found that certain classes of enzymes may be advantageously immobilised without loss of stability and / or activity. Enzyme engineering is only as fast as the proteins that can be screened and tested. At 1 mL protein expression scale, due to constraints of the reaction being assayed or tested, there are often time inhibitions or interference may occur to the assays by salt or impurities in the lysate. By adapting a one-step immobilisation and purification protocol, it was found that the cost and effort of enzyme screening may be reduced at a small and / or large scale. For example, a generalized cost-effective method for immobilisation and co-purification for His-tagged enzymes may be used for large scale production and / or lab scale screening. The method may comprise a strong binding of charged resin to a His-tag peptide on the enzyme. The enzyme may be D-psicose-3-epimerases and cholesterol oxidases. Immobilised D-psicose-3-epimerase demonstrated much improved stability than the free enzyme, retaining >60% >20 days under conditions for continuous production of D-allulose. In addition, the immobilised DPEase showed remarkable storage stability of >100 days at 4°C without decreased activity. The immobilisation can be carried out by direct loading of lysate instead of purified enzyme thereby greatly reduced the enzyme production cost at industrial scale. Through immobilisation, cholesterol oxidases also maintain stability over varied temperatures and also stability over 7 days at room temperature.

[0042] Furthermore, this protocol can be used at scales of 1 mL cultures (>5 pL resin) to enable accelerated high throughput lab scale screening of purified enzyme variants, including for enzyme families beyond D-psicose-3-epimerase, with a single step protocol without a need for columns or desalting.

[0043] Accordingly, the present disclosure concerns a bioprocess comprising: contacting a His-tagged enzyme or a variant, homolog or synthetic analogue thereof immobilized on a charged resin with a precursor in order to convert the precursor into a product; wherein the enzyme is selected from isomerase and oxidoreductase.

[0044] Immobilization is a process in which enzymes are fixed to or within solid supports, creating a heterogeneous immobilized enzyme system. As compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. In addition, heterogeneous immobilized enzymes systems allow the easy recovery of both enzymes and products, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs.

[0045] For example, enzyme immobilization may occur via (1) non-covalent adsorption and deposition, (2) physical entrapment, (3) covalent attachment, and (4) bio-conjugation. Support binding can be physical or chemical, involving weak or covalent bonds. The support can be a synthetic resin, an inorganic polymer such as zeolite or silica, or a biopolymer. Entrapment involves inclusion of an enzyme in a polymer network (gel lattice) such as an organic polymer or a silica sol-gel, or a membrane device such as a hollow fiber or a microcapsule. Entrapment requires the synthesis of the polymeric network in the presence of the enzyme. The last category involves cross-linking of enzyme aggregates or crystals, using a bifunctional reagent, to prepare carrier-free macroparticles.

[0046] Non-covalent methods of protein immobilization involve either passive adsorption onto hydrophobic surfaces or electrostatic interactions with charged surfaces. Nitrocellulose membranes or polylysine-coated slides for electrostatic binding may be used. The major advantage of this kind of immobilization is that neither additional coupling reagents nor modification of the protein of interest is required.

[0047] Enzyme immobilization may be by absorption on mesoporous silicates with pore sizes in the range of 20-300 A. Mesoporous silicates provide a sheltered protected environment in which reactions with selected substrates could proceed. Mesoporous structures have been synthesized using cationic, neutral, and block copolymer surfactants. These copolymers contain organic functional groups and metals located within their framework or grafted onto their surface and have been used as a scaffold to develop mesoporous carbon materials. It is possible to chemically modify their surfaces with various functional groups, enabling electrostatic attraction or repulsion between the mesoporous silicate support and the biological molecule of interest. As a result of their silicate inorganic framework, mesoporous silicates are chemically and mechanically stable and are resistant to microbial attack.

[0048] Enzyme immobilization may be on polyketone polymer by hydrogen bonds. Polyketone polymer -[-CO-CH2CH2-]n- immobilization procedure may be carried out in diluted aqueous buffer, gently mixing the proteins with the polymer. No bi-functional agents or spacer arms are required for the immobilization, which occurs exclusively via a large number of hydrogen bonds between the carbonyl groups of the polymer and the -NH groups of the polypeptidic chain.

[0049] It was found that using His-tag as the binding motif to metal (Ni, Co) containing resin may effectively immobilise the enzyme. This allows for concurrent purification and immobilisation of an enzyme from a lysate, which simplifies the process and reduces the cost of production. The results show an increased activity and thermostability compared to the free enzyme, and a high enzyme loading and activity. The immobilised enzyme may be used for high throughput screening, enzyme discovery and long term storage or transport of enzymes. The method allows for concurrent immobilisation and purification of an enzyme from a lysate, while keeping its activity and stability. The method may be adapted to a scale of more than 1 mL to 1 L to allow for one step salt free / buffer flexible purification and screening of enzymes. The method is thus flexible and scalable, enabling reduction of cost of biocatalysts (by reducing purification and stages for bioprocessing) whilst enabling recyclability.

[0050] A His-tag, also known as a polyhistidine-tag, is a sequence of histidine amino acids (typically six or more) attached to a protein, usually at its N- or C-terminus. The His- tag may also be attached to the internal polypeptide sequence of a protein. This approach may be used when N- or C-terminal tags impair protein function or structure. This tag allows for the efficient purification of the tagged protein using immobilized metal affinity chromatography (IMAC). The histidine residues bind to metal ions like nickel or cobalt immobilized on a resin, enabling the separation of the His-tagged protein from other cellular components.

[0051] In some embodiments, the enzyme is His-tagged at its N-terminus. In some embodiments, the enzyme is His-tagged at its C-terminus. In some embodiments, the enzyme is His-tagged in an internal polypeptide sequence of the enzyme. In some embodiments, the enzyme is His-tagged at both its N-terminus and C-terminus. In general, there are 6 histidine residues in the H-tag.

[0052] It was found that certain classes of enzyme are particularly advantageous for use in this bioprocess. Enzymes are classified into major classes based on the type of reaction they catalyze, as defined by the International Union of Biochemistry and Molecular Biology (IUBMB). These classes are: oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases and translocases. Each class is further subdivided into subclasses and sub-subclasses, based on the specific reaction and substrate. A unique EC number (e.g., EC 1.2.3.4) is assigned to each enzyme, providing a detailed classification based on the reaction it catalyzes. For example, it was found that when the enzyme is isomerase (in particular sub-class epimerase) or oxidoreductase, the stability and activity of the immobilised enzyme is at least maintained, if not improved. This was determined based on extensive screening of the enzyme classes and sub-class thereof. In contrast, hydrolases are found to have a reduced stability and / or activity when immobilised using this method. It is believed that the binding of these enzyme class to the resin causes some extend of denaturation. In some embodiments, the enzyme is selected from epimerase and oxidoreductase. In some embodiments, the enzyme is selected from D-psicose-3-epimerase and cholesterol oxidase. Examples of other suitable epimerases are ketose 3-epimerases, D-tagatose 3-epimerase (DTEase). Examples of other suitable oxidoreductases are glucose oxidase (GOX) and glucose-methanol-choline oxidoreductase. The enzyme may be a wild-type protein or a variant, homolog or synthetic analogue thereof. For example, the enzyme may be a mutant or variant with a single amino acid residue being modified. For example, the enzyme may be a variant with 2, 3 or 4 amino acids residues being modified. As the His-tag is at the N-, C terminus and / or in an internal polypeptide sequence of the enzyme, variations within the protein sequence does not adversely affect its binding to the resin.

[0053] The enzyme may be obtained from a suitable microorganism, or genetically modified strain thereof. For example, the enzyme (in particular D-psicose 3-epimerase) may be obtained from Clostridium cellulolyticum H10, Corynebacterium glutamicum strain FIS002, Escherichia coli strain K-12 W3110 (pWKLP), Microbacterium foliorum strain SYG27B, locasia fonsfrigidae strain SP3-1, Clostridium scindens, Agrobacterium tumefaciens, Bacillus sp. KCTC 13219, Clostridium bolteae, Dorea sp. CAG317, and / or Bacillus subtilis. For example, the enzyme (in particular oxidoreductase) may be obtained from a bacteria such as Nocardia species (such as Norcardia erythropolis, which is now considered the same species as Rhodococcus erythropolis), Streptomyces sp. (such as S. fradiae, S. violascens, and S. parvus), Mycobacterium species, Arthrobacter species, Corynebacterium species, Rhodococcus species, Brevibacterium species, Schizophyllum species, Rhodococcus species (Rhodococcus erythropolis and Rhodococcus equi) and / or a fungi.

[0054] The terms "enzyme", "polypeptide", "proteinaceous molecule", "peptide", "protein" and "biocatalyst" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non- naturally-occurring amino acid, such as a chemical analogue of a corresponding naturally-occurring amino acid, as well as to naturally-occurring amino acid polymers. These terms do not exclude modifications, for example, glycosylations, acetylations, phosphorylations and the like. Soluble forms of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids or polypeptides with substituted linkages. The polypeptide may comprise an amino acid sequence having at least 70% sequence identity, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to an amino acid sequence of a wild-type polypeptide. Sequence variations herein include amino acid substitutions, insertions, deletions and sequence inversions, and may be natural or engineered. It is to be understood that the sequence variations herein (such as the up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25% or 30% sequence variation) are variations which do not substantially affect any biological activity of the polypeptide. For example, the variations may be conservative amino acid substitutions and / or are located in segments of the polypeptide which do not contain the following : an active site, an allosteric site, a chelating site, a site for protein modification (e.g., a phosphorylation, acetylation, glycosylation or cleavage site), a site for intramolecular interaction (e.g., a site of a disulphide or other covalent or non-covalent bond), a binding site for a receptor, ligand, antigen, nucleic acid, protein, lipid, ion or metabolite, a site for an intermolecular covalent or non-covalent interaction, or a multimerisation site (including a dimerisation site). A skilled person can identify appropriate segments and sites which minimally affect a biological activity of a polypeptide using, for example, structural or homology data for the polypeptide.

[0055] As used herein "sequence identity" refers to the number (or fraction expressed as a percentage %) of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by sequence alignment of nucleic acid or protein sequences to identify regions of similarity or identity. For purposes herein, sequence identity is generally determined by alignment to identify identical residues. Alignment can be local or global, but for purposes herein alignment is generally a global alignment where the full-length of each sequence is compared. Matches, mismatches and gaps can be identified between compared sequences. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there can be internal and terminal gaps. Sequence identity can be determined by taking into account gaps as the number of identical residues / length of the shortest sequencexlOO. When using gap penalties, sequence identity can be determined with no penalty for end gaps (e.g., terminal gaps are not penalised). Alternatively, sequence identity can be determined without taking into account gaps as the number of identical positions / length of the total aligned sequencex lOO. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known to those skilled in the art, for instance, using publicly available computer software available on internet web sites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0056] As used herein, the phrase "consisting essentially of" in the context of a recited subunit sequence (e.g., amino acid sequence) indicates that the sequence may comprise at least one additional upstream subunit (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more upstream subunits; e.g., amino acids) and / or at least one additional downstream subunit (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more upstream subunits; e.g., amino acids), wherein the number of upstream subunits and the number of downstream subunits are independently selectable.

[0057] The terms "wild-type protein" or "parent protein" are used interchangeably herein and refer to the non-mutated version of a polypeptide as it appears naturally. The terms "mutant", "variant", "engineered polypeptide" and "engineered protein" are used interchangeably herein to refer to a polypeptide derived from a wild-type protein and comprising one or more amino acid modifications, e.g., an amino acid substitution, insertion and / or deletion. The variants may be obtained by various techniques well known in the art, e.g., site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.

[0058] The term "modification" or "alteration" as used herein in relation to a position in a polypeptide sequence or an amino acid means that the amino acid in the particular position has been modified compared to the amino acid of the wild-type protein.

[0059] A "substitution" means that an amino acid residue is replaced by another amino acid residue. An amino acid residue may be replaced by another selected from the naturally- occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g. hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine, N- ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non-naturally occurring amino acid residue, often made synthetically, (e.g. cyclohexyl-alanine). Preferably, the term "substitution" refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T). The sign "+" indicates a combination of substitutions. The following terminology is used herein to designate a substitution: N202C denotes that the amino acid residue at position 202 (asparagine, N) of the parent sequence is changed to a cysteine (C). F207V / I denotes that the amino acid residue at position 207 (phenylalanine, F) of the parent sequence is substituted with either a valine (V) or an isoleucine (I).

[0060] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows: a) Amino acid sub-classification

[0061] Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying its activity. Conservative substitutions are shown in the table below under the heading of exemplary substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. b) Exemplary Amino Acid Substitutions

[0062] The term "deletion", used in relation to an amino acid, means that the amino acid has been removed or is absent. The term "insertion" means that one or more amino acids have been added.

[0063] In some embodiments, the charged resin is a positively charged (cationic) resin. In some embodiments, the charged resin comprises multivalent metal ions. In some embodiments, the charged resin comprises a chelate. In some embodiments, the charged resin comprises Ni2+, Co2+or a combination thereof. In some embodiments, the charged resin comprises Ni2+. In some embodiments, the charged resin is selected from nickel-nitrilotriacetic acid (Ni-NTA) resin and / or Ni2+loaded iminodiacetic acid (IDA) resin. Here, a support bearing a chelating moiety such as nitrilotriacetic acid or iminodiacetic acid is treated with a solution of the relevant metal salt to produce a support presenting the metal ions.

[0064] It was found that Ni2+resin is particularly advantageous over Co2+resin. The nickel ions in the resin form a stable complex with the histidine residues of the His-tag, allowing for efficient binding and elution of the target protein. The complexation is robust; it can withstand a range of buffer conditions and is compatible with a variety of detergents, reducing agents, and other additives. Ni2+resins may sometimes result in higher levels of non-specific binding, leading to co-purification of contaminant proteins. This is due to the strong metal-protein interactions that may also attract non-His-tagged proteins with histidine-rich sequences. As a result, additional steps may be required in the purification process, such as increased wash stringencies or secondary purification methods. On the other hand, Co2+resins form a slightly weaker complex with the His- tag compared to nickel ions, but this reduced binding strength translates into higher specificity for His-tagged proteins. Consequently, Co2+resins exhibit lower levels of nonspecific binding.

[0065] In order to overcome or at least reduce the non-specific binding, imidazole may be added to facilitate competitive binding. A low imidazole concentration (e.g., 10-25 mM) in the binding and wash buffers may help to minimize the binding of non-specifically interacting proteins. While some proteins may be lost during this purification and polishing step, it was found that it improves the overall efficiency of the bioprocess.

[0066] The His-tagged enzyme or a variant, homolog or synthetic analogue thereof may be a purified enzyme. A purified enzyme is an enzyme that has been isolated from its natural source and separated from other cellular components, resulting in a high degree of purity.

[0067] In some embodiments, the bioprocess comprises: contacting a purified His-tagged enzyme or a variant, homolog or synthetic analogue thereof immobilized on a charged resin with a precursor in order to convert the precursor into a product; wherein the enzyme is selected from isomerase and oxidoreductase. In some embodiments, the method further comprises a step before the contacting step of loading a lysate comprising the His-tagged enzyme onto the charged resin.

[0068] In some embodiments, the bioprocess comprises a) loading a lysate comprising a His-tagged enzyme onto a charged resin in order to form a His-tagged enzyme immobilized on a charged resin; b) contacting the His-tagged enzyme immobilized on a charged resin with a precursor in order to convert the precursor into a product; wherein the enzyme is selected from isomerase and oxidoreductase; wherein the enzyme is His-tagged.

[0069] The lysate comprises the His-tagged enzyme. The lysate may further comprise lysed cells, components and / or organelles thereof. The lysate may be obtained by lysing a cell culture. In this regard, the His-tagged enzyme may not be secreted extracellularly. For example, the host cells transfected with the appropriate polynucleotide sequence, and may be cultivated by methods well known in art, e.g., by shake flask cultivation or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and / or isolated.

[0070] Where the enzyme is secreted extracellularly into the culture medium, the enzyme in the culture supernatant may be separated from the cells and loaded onto the charged resin.

[0071] The lysate may be partially purified by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Alternatively, the lysate may be totally purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS- PAGE, or extraction to obtain substantially pure polypeptides.

[0072] It was found that compared to loading purified enzymes on the charged resin, loading a lysate (with impurities such as lysed cells, cell debris, organelles and components such as proteins, carbohydrates and lipids) on the charged resin also allows for the chelation of His-tagged enzymes. While some small amount of impurities may also attach to the charged resin, it was found that this does not reduce the stability and / or activity of the enzyme. It is believed that the presence of some impurities act as "spacers" to reduce inter-enzyme interactions, as well as form a corona around the charged resin, which may help slow down or minimize degradation of the enzyme. This is evidenced when purified protein is released from the charge resin; in most cases, the purified protein tends to aggregate and / or denature. Purified proteins may thus require additional purification step (this could be based on affinity, surface charge, size) and buffer exchange step before it can be loaded onto the resin.

[0073] Further, the number of steps and cost of production in the process is reduced.

[0074] In some embodiments, the loading step comprises circulating the lysate through the charged resin for at least two cycles. In some embodiments, the loading step comprises continuously circulating the lysate through the charged resin. This may be for at least 3, 4, 5, or 10 cycles.

[0075] In some embodiments, the method further comprises a step after the loading step of washing the His-tagged enzyme immobilized on a charged resin. The washing step may be performed using a buffer.

[0076] In some embodiments, the washing step removes some of the impurities associated with the charged resin. In some embodiments, most of the impurities is removed.

[0077] In some embodiments, the bioprocess comprises a) loading a lysate comprising a His-tagged enzyme onto a charged resin in order to form a His-tagged enzyme immobilized on a charged resin; b) washing the His-tagged enzyme immobilized on a charged resin; c) contacting the His-tagged enzyme immobilized on a charged resin of step b) with a precursor in order to convert the precursor into a product; wherein the enzyme is selected from isomerase and oxidoreductase; wherein the enzyme is His-tagged.

[0078] For example, the washing may be performed using 20 mM imidazole phosphate buffer (pH7.0). The washing may be performed using an aqueous medium. The term 'aqueous medium' used herein refers to a water based solvent or solvent system, and which comprises of mainly water. Such solvents can be either polar or non-polar, and / or either protic or aprotic. Solvent systems refer to combinations of solvents which resulting in a final single phase. Both 'solvents' and 'solvent systems' can include, and is not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethylether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol or water. Water based solvent or solvent systems can also include dissolved ions, salts and molecules such as amino acids, proteins, sugars and phospholipids. Such salts may be, but not limited to, sodium chloride, potassium chloride, ammonium acetate, magnesium acetate, magnesium chloride, magnesium sulfate, potassium acetate, potassium chloride, sodium acetate, sodium citrate, zinc chloride, HEPES sodium, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate and sodium phosphate. As such, biological fluids, physiological solutions and culture medium also falls within this definition.

[0079] As mentioned, strong metal-protein binding between the charged resin and proteinaceous material may also cause non-specific binding of other proteins, carbohydrates, and / or lipids, which are impurities.

[0080] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by an impurity. The impurity may be a protein or peptide (which is not the His-tagged enzyme), a carbohydrate, lipid, nucleic acid (DNA, RNA) and / or metabolite.

[0081] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by an impurity loading of less than about 5%. In other embodiments, the impurity loading is less than about 4.5%, about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.8%, about 0.7%, about 0.6%, or about 0.5%.

[0082] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by a protein or peptide loading of less than about 5%. In other embodiments, the protein or peptide loading is less than about 4.5%, about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.8%, about 0.7%, about 0.6%, or about 0.5%.

[0083] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by a carbohydrate loading of less than about 5%. In other embodiments, the carbohydrate loading is less than about 4.5%, about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.8%, about 0.7%, about 0.6%, or about 0.5%. In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by a lipid loading of less than about 5%. In other embodiments, the lipid loading is less than about 4.5%, about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.8%, about 0.7%, about 0.6%, or about 0.5%.

[0084] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by a nucleic acid loading of less than about 5%. In other embodiments, the nucleic acid loading is less than about 4.5%, about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.8%, about 0.7%, about 0.6%, or about 0.5%.

[0085] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by a metabolite loading of less than about 5%. In other embodiments, the metabolite loading is less than about 4.5%, about 4%, about 3.5%, about 3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.8%, about 0.7%, about 0.6%, or about 0.5%.

[0086] Depending on the enzyme, the precursor may vary. For example, when the enzyme is D-psicose-3-epimerase, the precursor may be D-fructose. D-fructose is converted to D- psicose by D-psicose-3-epimerase. For example, when the enzyme is cholesterol oxidase, the flavoenzyme precursor may be cholesterol. Oxygen may additionally be present. Cholesterol is oxidised to cholest-4-en-3-one with the reduction of oxygen to hydrogen peroxide.

[0087] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterised by an enzyme to resin loading of about 0.01 mg / g to about 50 mg / g. In some embodiments, the enzyme to resin loading is about 0.1 mg / g to about 50 mg / g, about 0.5 mg / g to about 50 mg / g, about 1 mg / g to about 50 mg / g, about 12 mg / g to about 50 mg / g, about 14 mg / g to about 50 mg / g, about 16 mg / g to about 50 mg / g, about 18 mg / g to about 50 mg / g, about 20 mg / g to about 50 mg / g, about 22 mg / g to about 50 mg / g, or about 24 mg / g to about 50 mg / g. In some embodiments, the enzyme to resin loading is about 0.01 mg / g to about 45 mg / g, about 0.01 mg / g to about 40 mg / g, about 0.01 mg / g to about 35 mg / g, or about 0.01 mg / g to about 30 mg / g.

[0088] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterised by an average resin particle size of about 1 pm to about 300 pm. It is found that this size range allows for higher enzyme activity compared to a size range of more than 300 pm. In other embodiments, the average resin particle size is about 10 pm to about 300 pm, about 50 pm to about 300 pm, about 100 pm to about 300 pm, about 150 pm to about 300 pm, or about 200 pm to about 300 pm.

[0089] In some embodiments, the method is performed at a temperature of about 40 °C to about 80 °C. In other embodiments, the temperature is about 40 °C to about 75 °C, about 40 °C to about 70 °C, about 40 °C to about 65°C, about 40 °C to about 60 °C, about 40 °C to about 55°C, about 40 °C to about 50 °C, about 40 °C to about 45 °C, about 45 °C to about 80 °C, about 45 °C to about 75 °C, about 45 °C to about 70 °C, about 45 °C to about 65 °C, about 45 °C to about 60 °C, about 45 °C to about 55 °C, about 45 °C to about 50 °C, about 50 °C to about 80 °C, about 50 °C to about 75 °C, about 50 °C to about 70 °C, about 50 °C to about 65 °C, about 50 °C to about 60 °C, about 50 °C to about 55 °C, about 55 °C to about 80 °C, about 55 °C to about 75 °C, about 55 °C to about 70 °C, about 55 °C to about 65 °C, about 55 °C to about 60 °C, about 60 °C to about 80 °C, about 60 °C to about 75 °C, about 60 °C to about 70 °C, about 60 °C to about 65 °C, about 65 °C to about 80 °C, about 65 °C to about 75 °C, about 65 °C to about 70 °C, about 70 °C to about 80 °C, or about 70 °C to about 75 °C.

[0090] In some embodiments, the method is performed at a pH of about 6 to about 8.5. In other embodiments, the method is performed at pH of about 6 to about 8, about 6 to about 7.5, about 6 to about 7, about 6 to about 6.5, about 6.5 to about 8.5, about 6.5 to about 8, about 6.5 to about 7.5, about 6.5 to about 7, about 7 to about 8.5, about 7 to about 8, about 7 to about 7.5, about 7.5 to about 8.5, about 7.5 to about 8, or about 8 to about 8.5.

[0091] In some embodiments, the immobilised enzyme is characterised by an activity of more than 60% for at least 15 days. In some embodiments, the immobilised enzyme is characterised by an activity of more than 60% for at least 16, 17, 18 or 20 days.

[0092] In some embodiments, the immobilised enzyme is characterised by an activity of more than 50% for at least 5 days. In some embodiments, the immobilised enzyme is characterised by an activity of more than 50% for at least 6, 7, 8 or 9 days.

[0093] In some embodiments, the immobilised enzyme is characterised by an improvement in activity relative to an un-immobilised enzyme. In some embodiments, the improvement in activity is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%. In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterized by a storage stability of at least 80 days at less than 10 °C. In other embodiments, the storage stability is at least 85 days, 90 days, 95 days, 100 days, 110 days, 120 days, or 130 days. In other embodiments, the temperature is less than 8 °C, 6 °C, 5 °C, or 4 °C. In some embodiments, the storage stability is at least 100 days at 4°C.

[0094] In some embodiments, the His-tagged enzyme immobilized on a charged resin is characterised by a thermal stability of more than 5 °C relative to a free enzyme. The thermal stability may be for more than 5 days, 6 days, 8 days, 10 days, 15 days, 20 days, 30 days, 40 days, or 50 days.

[0095] The present disclosure concerns a method of converting fructose to Allulose, comprising : contacting a His-tagged D-psicose-3-epimerase or a variant, homolog or synthetic analogue thereof immobilized on a charged resin with fructose in order to convert the fructose into Allulose; wherein D-psicose-3-epimerase is His-tagged.

[0096] The method thus catalyses the epimerisation of fructose to allulose.

[0097] In some embodiments, the method of converting fructose to Allulose, comprises: a) loading a lysate comprising a His-tagged D-psicose-3-epimerase, or a variant or synthetic analogue thereof onto a charged resin in order to form a His-tagged D-psicose- 3-epimerase immobilized on a charged resin; b) contacting the His-tagged D-psicose-3-epimerase immobilized on a charged resin with fructose in order to convert the fructose into Allulose; wherein D-psicose-3-epimerase is His-tagged.

[0098] In some embodiments, the method of converting fructose to Allulose, comprises: a) loading a lysate comprising a His-tagged D-psicose-3-epimerase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged D-psicose-3-epimerase immobilized on a charged resin; b) washing the His-tagged D-psicose-3-epimerase immobilized on a charged resin; and c) contacting the His-tagged D-psicose-3-epimerase immobilized on a charged resin with fructose in order to convert the fructose into Allulose; wherein D-psicose-3-epimerase is His-tagged. In some embodiments, the method of converting fructose to Allulose, comprises: a) loading a lysate comprising a His-tagged D-psicose-3-epimerase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged D-psicose-3-epimerase immobilized on a charged resin; b) washing the His-tagged D-psicose-3-epimerase immobilized on a charged resin; and c) contacting the His-tagged D-psicose-3-epimerase immobilized on a charged resin with fructose in order to convert the fructose into Allulose; wherein D-psicose-3-epimerase is His-tagged at its N-terminus.

[0099] The present disclosure concerns a method of converting cholesterol to cholest-4-en-3- one, comprising : contacting a His-tagged cholesterol oxidase or a variant, homolog or synthetic analogue thereof immobilized on a charged resin with cholesterol in order to convert cholesterol to cholest-4-en-3-one; wherein cholesterol oxidase is His-tagged.

[0100] In some embodiments, the method is performed in the presence of oxygen. In some embodiments, the method is performed in an aqueous medium.

[0101] In some embodiments, the method of converting cholesterol to cholest-4-en-3-one, comprises: a) loading a lysate comprising a His-tagged cholesterol oxidase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged cholesterol oxidase immobilized on a charged resin; b) contacting the His-tagged cholesterol oxidase immobilized on a charged resin with cholesterol in order to convert cholesterol to cholest-4-en-3-one; wherein cholesterol oxidase is His-tagged.

[0102] In some embodiments, the method of converting cholesterol to cholest-4-en-3-one, comprises: a) loading a lysate comprising a His-tagged cholesterol oxidase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged cholesterol oxidase immobilized on a charged resin; b) washing the His-tagged cholesterol oxidase immobilized on a charged resin; and c) contacting the His-tagged cholesterol oxidase immobilized on a charged resin with cholesterol in order to convert cholesterol to cholest-4-en-3-one; wherein cholesterol oxidase is His-tagged. In some embodiments, the method of converting cholesterol to cholest-4-en-3-one, comprises: a) loading a lysate comprising a His-tagged cholesterol oxidase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged cholesterol oxidase immobilized on a charged resin; b) washing the His-tagged cholesterol oxidase immobilized on a charged resin; and c) contacting the His-tagged cholesterol oxidase immobilized on a charged resin with cholesterol in order to convert cholesterol to cholest-4-en-3-one; wherein cholesterol oxidase is His-tagged at its N-terminus.

[0103] The present disclosure also concerns an immobilised enzyme, comprising : a) a His-tagged enzyme; and b) a charged resin, the His-tagged enzyme chelated or conjugated to the charged resin via the His-tag; wherein the enzyme is selected from isomerase and oxidoreductase; wherein the enzyme is His-tagged.

[0104] In some embodiments, the charged resin comprises a protein, peptide, carbohydrate and / or lipid.

[0105] Examples

[0106] As an initial demonstration, D-psicose-3-epimerase (named "DPEase" hereafter) was selected as the biocatalyst to be immobilised. We have also extended this technology to other enzyme families, such as oxidases. Additionally, we have applied this technology for high throughput screening of enzymes, allowing for the purification of the protein and eliminating the need for specific buffer systems (used in purification) in just one step.

[0107] Table 1. Enzymes

[0108] Isomerase 1 is a wild type enzyme, the sequences of which may be obtained from, for example, www.uniDrot.org based on the Genbank ID.

[0109] Oxidoreductase 2 is obtained following Yamada, Keith, et al. "Characterization and overproduction of cell-associated cholesterol oxidase ChoD from Streptomyces lavendulae YAKB-15." Scientific reports 9.1 (2019): 11850, the reference of which is herein incorporated in its entirety.

[0110] Identification of a suitable resin for immobilisation of DPEase

[0111] Since the DPEase contains a His-tag sequence (isomerase 1), metal (Ni2+and Co2+) loaded iminodiacetic acid (IDA) resins were investigated as potential supports for immobilisation of the enzyme (FIG. 1A). Comparison of activities for allulose conversion showed that Ni2+immobilised enzyme variants (isomerase 2 and 3) had much higher conversion than Co2+immobilised and the respective free enzymes (FIG. IB). Based on these results, Ni2+IDA resin was chosen as a suitable support for immobilisation of DPEase.

[0112] Concurrent purification and immobilisation of lysate

[0113] Initial immobilisation of DPEase was carried out with purified enzyme. The purification was achieved by immobilised metal ion affinity chromatography (IMAC), which selectively captured the His-tagged DPEase from many other unwanted proteins. The captured enzyme was then eluted from the column by washing with imidazole buffer solution. Further desalting provided the purified enzyme. However, the purified DPEase is rather unstable, precipitated out from the buffer solution right after desalting. This resulted in loss of enzyme and activity. In addition, the process is very costly due to the consumables (IMAC and desalting columns) used. In fact, purified DPEase cost nearly 70 times more than the processed lysate. As such, it would be most effective and cost- efficient if purification and immobilisation could be achieved in a single process from the fermentation lysate. Taking advantage of metal affinity of the His-tag sequence expressed in the DPEase, the lysate from isomerase 2 (FIG. 2, lane (L) 1) was loaded onto the Ni2+IDA resin by continuous circulation (at 4 °C for at least 1 hour). Up to about 24 mg / mL of enzymes may be loaded per 1g of resin. The protein loaded resin was washed with 20 mM imidazole phosphate buffer (pH7.0), which removed most of the unbound proteins ((FIG. 2, L2). Selective binding of the His-tagged DPEase was confirmed by gel analysis of the eluted protein (FIG. 2, L4 vs LI) with 1.0 M imidazole phosphate buffer (pH7.0). This simple process provided a cost-effective method for concurrent immobilisation and purification of DPEase.

[0114] Table 2. SDS-PAGE gel analyses

[0115] Maintenance or enhancement of activity and stability by immobilisation protocol Increased temperature stability of immobilised DPEase

[0116] Thermostability is an important factor for DPEases as the reaction is carried out at higher temperatures (~60 °C). Comparison of allulose conversion showed that immobilised DPEase (isomerase 1) had maximal activity at ~80 °C (FIG. 3A) whereas the free enzyme was <75 °C (FIG. 3B). The results showed the immobilised DPEase is more stable than the free enzyme.

[0117] Comparison of lysate and purified protein loading: Longevity studies of immobilised DPEase

[0118] Longevity of immobilised enzyme is crucial for its productivity and cost-effectiveness. The immobilised DPEase were continuously operated under flow conditions until their activities dropped below 60% of their initial activities (based on % allulose conversion). The results of both immobilised lysate and purified DPEase (isomerase 2) are shown in FIG. 4A, indicated purified enzyme has slightly longer lifetime than the lysate (22 vs 18 days). However, there is a trade off in that purified enzyme costs ~70 times higher than the lysate. FIG. 4B shows the longevity study of immobilised isomerase 3. Crosslinked enzymes are intermolecularly chemically attached using bifunctional crosslinkers. The immobilised enzyme demonstrates more than 54 days of stability at process conditions. The results also show that mutants or variants of the wild type isomerases can perform as well as the wild type. The effect is thus demonstrated across the enzyme class.

[0119] Storage stability of immobilised DPEase

[0120] Storage stability of immobilised DPEases is crucially important from operational point of view as it allows batch preparation and storage of the immobilised enzyme. The immobilised DPEase (isomerase 1) showed excellent storage stability when incubated with fructose substrate, maintaining its initial activity after storing nearly 100 days at 4 °C (FIG. 5). On the contrary, the free enzyme quickly precipitates within hours.

[0121] Application to stability of Cholesterol oxidases

[0122] The stability of cholesterol oxidases is crucial, particularly in diagnostic applications as biosensors for measuring cholesterol levels. With the His-tag immobilization, increased activity is observed compared to free enzymes (FIG. 6). Immobilized enzymes were also able to maintain >50% activity over 194 hours (FIG. 6). Of note, the purified free enzymes had minimal activity under the same conditions.

[0123] The assay consists of immobilizing lysate on Ni-NTA magnetic beads. Oxidases are incubated with cholesterol for reaction before H2O2 produced is measured by a fluorescence-based assay. Amount of H2O2 released from the samples is quantified indirectly with calibration curve using known H2O2 concentrations for determination (FIG. 7).

[0124] Adaption of protocol for high throughput screening of enzyme variants

[0125] Here, we also demonstrate the protocol adapted for small scale assays for activity of cholesterol oxidases. Assays performed with lysate or eluate have challenges due to background enzymes or high salt from lysis and eluate conditions. Here, the protocol allows for isolation and also exchange of buffer. Traditional methods; include His-tag pull down, eluate, buffer exchange or desalting via columns. These are multiple steps and also high cost (due to columns). In this co-purification and immobilization protocol, oxidases are isolated directly and specifically from the lysate to perform an activity assay. This allows us to reduce steps and cost while increasing the purity of enzyme screened. Table 3. Cholesterol Oxidase from microorganisms (Sigma Aldrich) was loaded onto Experiment 1 (with resin) and Experiment 5 (without resin). The fluorescence observed in both experiments was similar, indicating that there were no interactions between the assays and the resin. HisPur Ni-NTA Magnetic Beads was used as resin (ThermoFisher).

[0126] Comparative example: Immobilisation of PET degrading hydrolase

[0127] As shown in FIGS. 8A-8C, in these experiments involving His-tagged LCC-ICCG, equivalent immobilized proteins demonstrated less or no activity to free enzymes. Crystal structure of LCC-ICCG also does not predict possible bottlenecks with immobilization technology. The results show that immobilisation via his-tag does not work for all his-tagged proteins.

[0128] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

[0129] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0130] Throughout this specification and the claims which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other non-recited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims

Claims1. A bioprocess comprising : a) loading a lysate comprising a His-tagged enzyme, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His-tagged enzyme immobilized on a charged resin; b) washing the His-tagged enzyme immobilized on a charged resin; and c) contacting the His-tagged enzyme immobilized on a charged resin of step b) with a precursor in order to convert the precursor into a product; wherein the enzyme is selected from isomerase and oxidoreductase; wherein the enzyme is His-tagged.

2. The bioprocess according to claim 1, wherein the enzyme is further His-tagged at its N-terminus, C-terminus and / or in an internal polypeptide sequence of the enzyme.

3. The bioprocess according to claim 1 or 2, wherein the enzyme is selected from D-psicose-3-epimerase, ketose 3-epimerases, D-tagatose 3-epimerase (DTEase), cholesterol oxidase, glucose oxidase (GOX) and glucose-methanol-choline oxidoreductase.

4. The bioprocess according to any one of claims 1 to 3, wherein the charged resin comprises Ni2+, Co2+or a combination thereof.

5. The bioprocess according to any one of claims 1 to 4, wherein the loading step comprises circulating the lysate through the charged resin for at least two cycles.

6. The bioprocess according to any one of claims 1 to 5, wherein the His-tagged enzyme immobilized on a charged resin is characterized by an impurity selected from a protein, a peptide, a carbohydrate, lipid, nucleic acid and / or metabolite.

7. The bioprocess according to claim 6, wherein the His-tagged enzyme immobilized on a charged resin is characterized by an impurity loading of less than about 5%.

8. The bioprocess according to any one of claims 1 to 7, wherein the His-tagged enzyme immobilized on a charged resin is characterised by an enzyme to resin loading of about 0.01 mg / g to about 50 mg / g.

9. The bioprocess according to any one of claims 1 to 8, wherein the His-taggedenzyme immobilized on a charged resin is characterised by an average resin particle size of about 1 pm to about 300 pm.

10. The bioprocess according to any one of claims 1 to 9, wherein the method is performed at a temperature of about 40 °C to about 80 °C.

11. The bioprocess according to any one of claims 1 to 10, wherein the immobilised enzyme is characterised by an activity of more than 60% for at least 15 days.

12. The bioprocess according to any one of claims 1 to 11, wherein the His-tagged enzyme immobilized on a charged resin is characterized by a storage stability of at least 80 days at less than 10 °C.

13. The bioprocess according to any one of claims 1 to 12, wherein the His-tagged enzyme immobilized on a charged resin is characterised by a thermal stability of more than 5 days at 5 °C relative to a free enzyme.

14. A method of converting fructose to Allulose, comprises: a) loading a lysate comprising a His-tagged D-psicose-3-epimerase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged D-psicose-3-epimerase immobilized on a charged resin; b) washing the His-tagged D-psicose-3-epimerase immobilized on a charged resin; and c) contacting the His-tagged D-psicose-3-epimerase immobilized on a charged resin with fructose in order to convert the fructose into Allulose; wherein D-psicose-3-epimerase is His-tagged.

15. A method of converting cholesterol to cholest-4-en-3-one, comprises: a) loading a lysate comprising a His-tagged cholesterol oxidase, or a variant, homolog or synthetic analogue thereof onto a charged resin in order to form a His- tagged cholesterol oxidase immobilized on a charged resin; b) washing the His-tagged cholesterol oxidase immobilized on a charged resin; and c) contacting the His-tagged cholesterol oxidase immobilized on a charged resin with cholesterol in order to convert cholesterol to cholest-4-en-3-one; wherein cholesterol oxidase is His-tagged.

Images