GB2628106A - Method - Google Patents

Abstract

This invention relates to methods of predicting whether an enzyme can be immobilised to a solid support while retaining catalytic activity over time under specified reaction conditions, comprising the steps of (a) identifying two or more enzymes with different polypeptide sequences that catalyse a chemical reaction of interest; (b) identifying one or more solid supports; and (c) determining whether at least one of the enzymes can be immobilised to one or more of the solid supports in an active state for the desired period of time under the specified conditions. The determination is made by comparing a characteristic of two or more of the enzymes to a characteristic of one or more solid supports, and selecting the enzyme and solid support to provide active enzyme when immobilised to the selected solid support.

Description

METHOD

Field of the Invention

This invention relates to enzyme immobilisation. In particular, the invention relates to methods of predicting whether an enzyme can be immobilised to a solid support while retaining catalytic activity over time under specified reaction conditions.

Background of the Invention

Enzymes can be immobilised on a wide variety of natural and synthetic carriers, which allows for the enzymes to be re-used. Sheldon et all (Chemical Society Reviews, 30 Mar 2021, 50, 5850-5862) note that immobilising enzymes is advantageous as it suppresses unfolding of an enzyme's tertiary structure, affording a more stable biocatalyst which can be used under a wider range of reaction conditions. However, they also note a limitation of immobilisation is that it generally leads to some loss of enzyme activity. Sheldon et all review the state of the art regarding enzyme immobilisation, comparing various immobilisation methods, and consider the integration of protein engineering and enzyme immobilisation protocols.

Padrosa et all (Bioinformatics, 18 January 2021, Volume 37, Issue 17, Pages 2761-2762) recognises that protein immobilisation, while widespread to unlock enzyme potential in biocatalysis, remains tied to a trial-and-error approach. The authors describe a computer-implemented application that can be used to help select papers that may be useful to perform immobilisation and produce visualisations that could help advise in making some immobilisation suggestions.

However, immobilising an enzyme of interest still relies upon trial and error to identify a solid support onto which to immobilise the enzyme of interest. There is a need to improve the development of immobilised enzymes.

Summary of the Invention

The present disclosure relates in general to the selection of one or more enzyme sequences to pair with a solid support such as a bead, wherein the selection is based on the expected stability of the immobilisation of the enzyme to the support. The disclosed method therefore pre-selects an enzyme and a solid support on which the enzyme is expected to retain activity over a period of time, before the immobilisation is physically carried out.

In a first aspect, the invention provides a method of identifying an enzyme and a solid support to which the enzyme can be immobilised in an active state for a desired period of time under specified conditions, comprising the steps of: identifying two or more enzymes with different polypeptide sequences that catalyse a chemical reaction of interest; identifying one or more solid supports; and determining whether at least one of the enzymes can be immobilised to one or more of the solid supports in an active state for the desired period of time under the specified conditions, wherein the determination is made by comparing a characteristic of two or more of the enzymes to a characteristic of one or more solid supports, and selecting at least one enzyme and at least one solid support, wherein the selected enzyme is predicted to provide active immobilised enzyme when combined with the selected solid support under the specified conditions and over the desired period of time when the enzyme is immobilised to its paired support under the specified conditions.

The specified conditions are typically reaction conditions under which the immobilised enzyme is required to catalyse the reaction of a substrate into a product. Typically, this is an industrial biocatalysis reaction.

In certain embodiments, the specified conditions comprise one or more of a specified temperature, pH, salt concentration, substrate or cofactor to which the immobilised enzyme will be subjected to perform a catalytic process.

When the specified condition is a temperature, the temperature may be a temperature range. In some embodiments, the temperature range is 0°C to 100°C, or 10°C to 90°C, or 20°C to 80°C. In some embodiments, the temperature range is 30°C to 60°C, or 35°C to 55°C, or 40°C to 50°C. In some embodiments, the temperature range is 60°C to 80°C, or 65°C to 75°C.

Typically, when the temperature range is outside of normal (mesophilic) biological ranges, the two or more enzymes that are included in the method of the invention includes an enzyme homologue from an extremophile adapted to very high or very low temperatures. For example, when the specified reaction condition is above 40°C more typically above 50°C or above 60°C, an enzyme from a thermophile is typically included in the panel of enzymes to assess. When the specified reaction condition is below 15°C more typically below 10°C or below 4°C, an enzyme from a cryophile is typically included in the panel of enzyme to assess.

When the specified condition is a pH, the pH may be a pH range. In some embodiments, the temperature range is 1 to 13, or 2 to 12, or 3 to 11. In some embodiments, the pH is basic, for example pH8 or pH9. In some embodiments, the pH is acidic, for example pH5 or pH6.

Typically, when the pH is outside of normal (mesophilic) biological ranges, the two or more enzymes that are included in the method of the invention includes an enzyme homologue from an extremophile adapted to high or low pH. For example, when the specified reaction condition is acidic, an enzyme from an acidophile is typically included in the panel of enzymes to be assessed.

When the specified condition is a salt concentration, for example greater than 1% NaCI, greater than 3% NaCI or greater than 10% NaCI, the two or more enzymes that are assessed in the method of the invention may include an enzyme homologue from an extremophile adapted to high salt concentration, namely a halophile. In some embodiments, the salt (e.g. NaCI) concentration range is 1% to 30%, or 2% to 30%, or 3% to 20%.

The invention provides, in some embodiments, the assessment of a panel of enzymes. In some embodiments, the two or more enzymes comprise 10 or more, 50 or more, 100 or more or 500 or more different polypeptide sequences that catalyse a chemical reaction of interest. In some embodiments, 1000 or more enzyme sequences are assessed, for example 2,00 or more, 3,000 or more, 4,000 or more, 5,000 or more, 6,000 or more, 7,000 or more, 8,000 or more, or 10,000 or more.

In some embodiments, the two or more enzymes comprise homologues of one enzyme from different species. The Examples below exemplify the assessment of 8961 nonredundant sequences for a single enzyme activity, identified by a BLAST search of known sequences. In some embodiments, the two or more enzymes comprise at least one enzyme from an extremophile species. As noted above, exemplary extremophile species form which an enzyme can be identified include a thermophile, an acidophile or a halophile. In some embodiments, homologues from multiple extremophiles are included in the panel to be assessed. In some embodiments, homologues from at least one extremophile species and at least one mesophile species are included in the panel to be assessed.

The two or more enzymes may comprise at least one enzyme having a mutation compared to the wild-type sequence. Typically, the mutation is compared to a wild type sequence from one species. In some embodiments, the mutation is purposely engineered into the enzyme by a recombinant technique such as site directed mutagenesis. The mutation may provide a functional benefit, for example to increase or decrease the stability of the enzyme, or to increase or decrease the activity of the enzyme, or to increase or decrease the stability and the activity of the enzyme.

The method assesses at least two enzymes, sometimes referred to as a panel of enzymes. These enzymes may be identified by a search of known enzymes that catalyse the chemical reaction of interest. The search is typically a bioinformatic search, such searching for homologues of a reference enzyme. The bioinformatic search may comprises a BLAST search of a reference enzyme sequence, and may be a search based on a reference nucleic acid sequence or a reference protein sequence.

The method of the invention identifies an enzyme and a solid support to which the enzyme can be immobilised in an active state for a desired period of time under specified conditions.

Typically, the period of time is the length of time taken to complete an industrial biocatalysis under the specified conditions. In typical embodiments, the period of time is at least 3 days, at least 1 week, at least 2 weeks, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months or at least 2 years, optionally wherein the period of time is at least one month or at least one year.

In industrial biocatalysis, having an enzyme that is stable in a reaction chamber for weeks, months or even years allows for great economies of scale to be achieved, for example because the reaction can proceed without interruption for a long period of time, or because multiple batches of reactions can be carried out without having to replace the enzyme.

The method assesses one or more characteristics of an enzyme and one or more characteristics of a solid support, typically a bead. These characteristics may be chemical characteristics such as surface charge or physical characteristics such as shape or porosity.

This assessment of the characteristics of what is typically a panel of beads and a panel of enzymes allows for a prediction to be made of which enzyme sequence will remain active when immobilised to which bead. In some embodiments, the panel of enzymes comprises enzyme homologues from multiple species and the panel of beads comprises at least two different types of beads, for example any two or more of controlled pore glass, polystyrene, sepharose, alginate, epoxy-resin, ion-exchange resin, amine-resin or adsorbent resin beads.

In some embodiments, the panel of enzymes comprises at least 10 enzyme homologues from multiple species and the panel of beads comprises at least 3 different types of beads, at least 4 different types of beads, at least 5 different types of beads, at least 10 different types of beads or at least 20 different types of beads.

In some embodiments, the panel of enzymes comprises at least 50 enzyme homologues from multiple species and the panel of beads comprises at least 3 different types of beads, at least 4 different types of beads, at least 5 different types of beads, at least 10 different types of beads or at least 20 different types of beads.

In some embodiments, the panel of enzymes comprises at least 100 enzyme homologues from multiple species and the panel of beads comprises at least 3 different types of beads, at least 4 different types of beads, at least 5 different types of beads, at least 10 different types of beads or at least 20 different types of beads.

In some embodiments, the panel of enzymes comprises at least 1000 enzyme homologues from multiple species and the panel of beads comprises at least 3 different types of beads, at least 4 different types of beads, at least 5 different types of beads, at least 10 different types of beads or at least 20 different types of beads.

In some embodiments, the panel of enzymes comprises at least 1000 enzyme homologues from multiple species and the panel of beads comprises at least 5 different types of beads.

In some embodiments, the panel of enzymes comprises at least 5000 enzyme homologues from multiple species and the panel of beads comprises at least 10 different types of beads.

In some embodiments, the panel of enzymes comprises at least 5000 enzyme homologues from multiple species and the panel of beads comprises at least 20 different types of beads.

The assessment of which enzyme sequence is likely to immobilised with stable activity onto which bead is made taking into account the specified reaction conditions for which the immobilised enzyme is being designed, for example a specified biocatalysis reaction. The comparison may include assessing the enzyme and bead against known results from the immobilization and experimental testing of an enzyme and a bead, or of multiple enzymes and beads. The comparison may also include scoring the expected activity, stability, and combined activity and stability, and comparing that to scores obtained experimentally for other enzymes and beads.

In some embodiments, the characteristic of the enzyme comprises the enzyme sequence. In some embodiments, the characteristic of the enzyme comprises the proportion of charged residues. In some embodiments, the characteristic of the enzyme comprises the proportion of surface-exposed charged residues. In some embodiments, the characteristic of the enzyme comprises the secondary structure. In some embodiments, the characteristic of the enzyme comprises the tertiary structure. In some embodiments, the characteristic of the enzyme comprises the isoelectric point (pi) of the enzyme. These characteristics are assessed under the reaction conditions in which the enzyme is to be used in the catalytic reaction process.

In some embodiments, the characteristic of the solid support comprises the charge of the support. In some embodiments, the characteristic of the solid support comprises the chemistry of the conjugation site. In some embodiments, the characteristic of the solid support comprises the porosity of the support. These characteristics are assessed under the reaction conditions in which the solid support is to be used in the catalytic reaction process.

As described elsewhere herein, the solid support is typically a bead.

The immobilised enzyme is typically assessed for covalent attachment to the solid support.

The solid support is typically a bead. Other immobilisation means such as physical adsorption are also within the scope of the present invention.

In some embodiments the bead may be an epoxy-functionalised bead and the immobilisation is between one or more chemical groups of the amino acid backbone or amino acid side chains on the enzyme that are covalently bound to one or more epoxy groups, optionally wherein the residue bound to the epoxy group is lysine or cysteine. The bead may alternatively be an amine functionalised bead and one or more chemical groups of the amino acid backbone or amino acid side chains on the bead-immobilised enzyme are covalently bound to one or more amine groups. The bead may, in some embodiments, be a methacrylate bead. In some embodiments, the bead is an epoxy-functionalised methacrylate bead.

The method may identify more than one active enzyme-solid support pair that is expected to be stable and active over a period of time. The multiple identified enzyme-solid support pairs can be ranked in terms of their expected activity over time under the specified conditions. The activity over time, for example the activity after 4 weeks, or 3 months, or more, is particularly important to the efficiency of industrial biocatalysis.

In certain embodiments, the activity over time is scored as the mathematical product of the activity and the time over which the activity is predicted to be provided under the specified conditions. Accordingly, a high initial activity that degrades over time will typically have a lower score (and so be less preferred) than an immobilised enzyme with a lower initial activity but that retains activity over weeks or months. An immobilised enzyme that retains at least 50% of its initial activity (e.g. the activity within an hour of immobilisation) after four weeks is therefore one useful output of the method. In another embodiment, the method identifies an immobilised enzyme that retains at least 60% of its initial activity (e.g. the activity within an hour of immobilisation) after three months.

The method may involve computational analysis. In some embodiments, at least steps (a) and (c) are carried out by a computer. In some embodiments, essentially all or all of the steps are carried out by a computer.

The method typically provides a prediction of which enzymes and supports can be combined to provide an enzyme that is expected to be active over a specified period of time under specified reaction conditions. In some embodiments, the method further comprises the step of immobilizing the, any or all of the identified enzymes to its identified support. The activity of the at least one of those immobilised enzymes can then be tested under the specified conditions, for example to verify the selected combination. The results of that activity test can then be used to inform subsequent predictions.

In some embodiments where the activity of the at least one immobilised enzyme under the specified conditions is tested, the activity is tested at a first time point (t1) and a second later time point (t2). The time between t1 and t2 may be the desired period of time, typically at least 3 days, at least 1 week, at least 2 weeks, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months or at least 2 years.

The activity of the at least one immobilsied enzyme under the specified conditions can be tested over multiple cycles of a batch reaction. In certain embodiments, the enzyme remains active for at least 2, 3, 4, 5 or 10 batch cycles. In other embodiments, the activity of the at least one immobilsied enzyme under the specified conditions is tested in a flow reaction.

A second aspect of the invention provides an enzyme that is immobilsied to a solid support that is identified by the method of the first aspect.

A third aspect of the invention provides a method of identifying a solid support to which an enzyme can be immobilised in an active state for a desired period of time in a specified reaction process, comprising the steps of: identifying one or more solid supports; and determining whether at least one of the solid supports can be used as a support for an enzyme in a specified reaction process for a desired period of time, wherein the determination is made by comparing characteristics of one or more solid supports to the conditions of the specified reaction process and selecting a solid support that can support an active enzyme in the specified reaction process.

All of the typical embodiments of the first aspect apply as appropriate, to the third aspect.

Further aspects and embodiments will be apparent to the skilled person from the description provided below.

Brief Description of the Drawings

Figure 1 shows the identification of the best set of hits along the x-axis that will work best under specified process conditions.

Figure 2 shows reaction hits at different time points, with the best performers taken forward and scaled using the same expression protocol at 50mL for screening on immobilization supports: FN667, FN1048, FN1098, FN1101, FN1045, FN1099, FN1113, FN1043, FN1047, FN1046, FN1109, FN1108.

Figure 3 shows 50mL scale-up and 1mL parallel free enzyme specific yields for the homologue hits.

Figure 4.1 shows reuse cycle data for FN667.

Figure 4.2 shows reuse cycle data for FN1041.

Figure 4.3 shows reuse cycle data for FN1043. Figure 4.4 shows reuse cycle data for FN1044.

Figure 4.5 shows reuse cycle data for FN1045. Figure 4.6 shows reuse cycle data for FN 1046. Figure 4.7 shows reuse cycle data for FN 1096. Figure 4.8 shows reuse cycle data for FN1107.

Figure 5 shows flow results at 0.5 m L per min. Figure 6 shows results of K-modes analysis performed as a route to predict the compatibility of 39 immobilization supports, with the 39 immobilization supports grouped into 3 clusters based on material types and immobilization chemistries.

Detailed Description of the Invention

The invention is based on the development of new methods to select enzymes and solid supports for enzyme immobilisation to perform biocatalysis under specified reaction process conditions.

The invention relates in part to accelerating the rate at which the successful immobilization of an enzyme can be achieved in the dimension within which it should matter i.e. stabilisation of enzymes under known process conditions. Without wishing to be bound by theory, the method described herein can explicitly pair the bead type with the enzyme that will most benefit from the type of immobilization.

The invention therefore relates to a systematic approach that preselects the enzyme design space of interest, as required by the known biocatalysis process, and provides accuracy and precision to an otherwise imprecise and poorly-formed process.

The invention is based in part on a method of identifying an enzyme and a solid support to which the enzyme can be immobilised in an active state under known reaction conditions. The invention is also based on a method of identifying a solid support to which an enzyme can be immobilised in an active state, under known reaction conditions. The invention is particularly based on the development of new methods to select an enzyme in the context of the support and process conditions, based on the expected stability of the immobilised enzyme under the process conditions.

The invention improves the precision, accuracy and speed at which stability can be engineered into an enzyme by immobilising it under specified process conditions.

In one aspect, the method allows the user to assess the correct part of the design space and provide the required precision in pairing an enzyme with a bead. This is in contrast to the conventional trial-and-error approach of immobilising an enzyme to a bead and testing whether it is active, which is reliant on luck to identify a functional immobilisation. The benefit of the accuracy of a repeatable design-led process is clear, where knowledge of the physical and/or characteristics of the enzyme and bead directs the user right path at the outset and therefore provides the fastest route to a stable immobilised enzyme in a given reaction process. The alternative of protein engineering and using free enzymes would be a poor choice because the user is then less likely to take advantage of cell-free systems, reuse of catalysts will be more difficult, and continuous processing in the form of flow will not be an option.

In one aspect, the invention utilises one or more, optionally all, of the following features: a means to generate homologue libraries; computational means of preselecting the homologue library space based on knowledge of the immobilization support and industrial process conditions; computational means to preselect beads for immobilization or by conventional routes or by using a library; means to evaluate the library of homologues prior to immobilization; and means to screen for stability under process conditions post-immobilization.

Where the process conditions are known, the supports of choice are limited (to some extent) followed by selection of enzymes that match process criteria limited by bead optionality and their respective surface chemistry.

Typically beads are used that are process compatible and preselecting for enzyme that appears to show process compatibility i.e. if the reaction process needs to run at 70°C then there can be an emphasis on thermophiles, and the analysis can focus in the dimension of lysine rich surfaces for covalent chemistry over adsorptions.

In certain aspects, the invention relates to the preselection of enzyme homologues and bead pairing using computational methods for enzyme immobilization and maximal process stability.

The invention typically uses a computationally generated panel of enzyme homologues, wherein the homologues perform the same reaction chemistry, generated using conventional bioinformatic methods.

The computationally generated enzymes can then be preselected based on their likelihood of being immobilized onto an immobilization support under specific process conditions to preselect for the ideal pairing which maximises for stability of the enzyme.

Alternatively, the immobilization supports are computationally pre-selected (or rejected as unsuitable) when immobilization support requirements are unknown. In either instance of preselection or rejection of the immobilization support, the assessment of the immobilization supports is based on characteristics that impart stability to the appropriate enzyme.

A clustering analysis can be used in some embodiments, to assist in selecting the enzyme and/or the solid support (e.g. bead). Clustering algorithms are known in the art, for example the K-modes clustering algorithm used in the Examples and depicted in Figure 6.

Typically, once the pre-selected enzymes and/or beads are selected, responses across two dimensions are searched to identify preferred enzyme-support pairing: 1) enzyme stability under the required process conditions and 2) enzyme activity. Without wishing to be bound by theory, assessing activity first to ensure that the enzyme is functional and then potentially compromising on activity by focussing the search on maximised stability, is expected to provide advantages in identifying enzymes that retain activity over a useful time period for industrial biocatalysis. The invention focuses on the stability of the immobilsied enzyme under specified process conditions, but does still require that the free enzyme is active under the process conditions before moving to assessment of stability.

Accordingly, and again without wishing to be bound by theory, certain aspects of the invention provide methods that maximise the stability of enzymes under process conditions, by searching for the best enzyme-immobilization support pairing using computation to accelerate the selection of enzymes based on bead and process requirements which can also be applied to select beads in instances where bead requirements unknown.

Accordingly, it is possible to can take a diverse enzyme space and use computational methods to select for the best immobilisation conditions.

Advantageously, the invention provides robust methods to provide immobilised enzymes that do not require living cells to catalyse the reaction or reactions of interest. The reactions do not typically take place within a cell, for example in a living bacterial cell. The systems and methods described herein can therefore be described as relating to cell-free, or alternatively as artificial, industrial, abiotic or synthetic systems and methods. In certain embodiments, the invention utilises one or more enzymes immobilised to a support such as a bead, which does not exist inside a living cell.

Without wishing to be bound by theory, the invention solves problems faced by existing biocatalysis immobilisation methods, Immobilisation of enzymes imparts benefits of binding directly to the enzyme and therefore maximising the surface area:volume ratio issues faced by whole cells. Cells are more likely to lyse under harsher industrial conditions and therefore release unwanted materials in the milieu making downstream processing more difficult.

Immobilisation of an enzyme onto a bead enables processes based on flow and other immobilized catalysts, which are otherwise not afforded by whole-cell biocatalysis and cell-free enzymes alone.

In some embodiments, immobilisation uses a chemical linker to connect surface amino acid residues, typically one or more lysines or cysteines, of the enzyme to the support. Such chemical linkers can inactivate the enzyme because the protein structure is disrupted by bonding surface residues on the enzyme to the functionalised support or chemical linker. The inactivating effect of chemical immobilisation is also unpredictable for any given enzyme, because each enzyme sequence contains different numbers and locations of reactive surface residues. Accordingly, one particular advantage of the present invention is the maintenance of enzyme activity when immobilised on the bead, by selecting for appropriate enzyme sequences and beads. The maintenance of enzyme activity is particularly useful in the context of industrial biocatalysis, where the large scale of reactions that are catalysed means that maintaining activity can lead to very large increases in the efficiency of the overall process.

The solid support is typically a bead, which can impart stability benefits onto the enzyme which are not provided when whole cells are immobilised. Examples of suitable beads include a resin bead such as an epoxy-functionalised resin bead or amine-functionalised resin bead. In some embodiments, the bead is porous. In porous beads, binding sites can also be internal, leading to higher loading of enzyme. In some embodiments a cofactor is immobilised onto the support in addition to an enzyme immobilised onto the support. In some embodiments, a cofactor is immobilised as part of the same molecular complex as the enzyme, for example forms one continuous covalently linked molecule with the enzyme and support.

Chemical reaction of interest The chemical reaction of interest is catalysed by an enzyme. The chemical reaction of interest is typically a biocatalysis reaction to produce an industrial chemical product. The chemical reaction of interest typically converts a substrate to a product.

In some embodiments, the chemical reaction of interest is catalysed an oxidoreductase, transferase, hydrolase, lyase, ligase, isomerase, or translocase enzyme.

In some embodiments, the chemical reaction comprises any of oxidation, reduction, transfer of a group of atoms, hydrolysis, breakdown of chemical bonds through methods other than hydrolysis or oxidation, ligation of two molecules, conversion of one isomer to another, or movement of ions or molecules across membranes or their separation within membranes.

The reaction conditions for the chemical reaction of interest are applied to the determination of which enzyme sequence and which solid support will provide functional, active and stable immobilised enzyme to catalyse that reaction efficiently.

Enzymes The term enzyme is to be given its usual meaning in the art, i.e. a protein that accelerates or catalyses chemical reactions. An enzyme may have one or more active sites that bind to a substrate or selection of substrates. An enzyme may be naturally occurring or it may be of synthetic origin.

An enzyme is capable of catalysing a given reaction, and is sometimes thereof rereferred to as "an activity" or an "enzyme activity". For example, a protein capable of catalysing the conversion of glycerol to 3-H PA is a glycerol dehydratase activity.

Suitable enzymes can include, for example: enzymes comprising natural and unnatural amino acids, chemical modifications or post-translational modifications; natural enzymes; wild-type enzymes; recombinant enzymes; enzymes produced by directed evolution, de novo design or the genetic fusion of peptide or protein domains; peptide catalysts; nucleic acid enzymes (e.g. ribozymes or DNA enzymes); hybrid catalysts; monomeric, dimeric or multimeric enzymes; enzymes produced in vivo or in vitro for example by solid-phase synthesis.

In an active enzyme system, the enzymes are provided under conditions suitable for enzyme activity, for example in the necessary physical and chemical conditions for enzyme activity. For the reaction to proceed, reagents will be needed, along with any necessary cofactor such as NAD/H or coenzyme such as B12. Metal ions may also be included in the active enzyme system.

Enzyme activity and stability An enzyme in an active state, i.e. an active enzyme, is capable of catalysing a chemical reaction.

In one embodiment, an enzyme is stable if it remains in an active state for a period of time. In some embodiments, the period of time is at least a day, at least three days, at least 1 week, at least 2 weeks, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months or at least 2 years, optionally wherein the period of time is at least one month or at least one year.

In some embodiments, an enzyme is stable if it remains in an active state over one or more-re-use cycles. In some embodiments, each cycle can be about 1 minute or more, about 5 minutes or more, about 10 minutes or more, about 20 minutes or more, about 30 minutes or more, about 60 minutes or more, about 120 minutes or more, about 180 minutes or more, about a day, or about a week.

In some embodiments, the enzyme is active over two or more, three or more, four or more, five or more, ten or more, fifteen or more, twenty or more, thirty or more, forty or more, fifty or more, sixty or more, seventy or more, eighty or more, ninety or more, or one hundred or more re-use cycles.

In some embodiments, the activity of an enzyme can be measured by the yield of the product produced by the enzymatic reaction, or by measuring conversion, depletion or consumption of the substrate. In some embodiments, the enzyme is active if the yield of product is about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% of the yield from the initial use cycle, when assessed under the same reaction conditions.

In some embodiments, the enzyme is stable over a given time period if, over that time period, the yield of product is about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% of the activity within one hour of immobilisation, when assessed under the same reaction conditions.

In some embodiments, the enzyme is active after 4 weeks or three months. In some embodiments, the enzyme is active over that time period if the yield of product is about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100% of the activity within one hour of immobilisation, when assessed under the same reaction conditions.

In one aspect of the invention, a homologue of a reference enzyme has higher stability than the reference enzyme under the specified reaction process conditions. For example, a reference enzyme from a first species may catalyse a stated reaction when immobilised, but a homologue of that enzyme catalyses the same stated reaction with higher activity when immobilised to the same solid support. Typically, the homologue enzyme will have higher activity after a period of time, for example after weeks or months, and the long-term stable activity can be more valuable in terms of the efficiency of large scale biocatalysis than the initial activity shortly after immobilisation. Therefore, identifying homologues that are active and stable for a period of weeks, months or longer, is particularly advantageous.

Generating homologue libraries The method of the present invention accelerates the identification and selection of an enzyme and a solid support to which the enzyme can be immobilised. One way that this can be achieved, is by identifying multiple homologues of a single enzyme activity and interrogating which homologue (or homologues) is optimised for immobilisation under the required reaction process conditions.

Identification of one or more homologues In one embodiment, an enzyme for immobilisation is identified by identifying two or more enzymes with different polypeptide sequences that catalyse a chemical reaction of interest. In some embodiments, the two or more enzymes comprises the enzyme of interest and one or more enzymes that are homologues of that enzyme of interest. In some embodiments, identifying the two or more enzymes is performed in a search, typically a search of one or more bioinformatic databases using one or mor bioinformatic search tools.

Bioinformatic databases and tools are well-known in the art. For example, a summary of useful bioinformatics tools can be found at www.ebi.ac.uk/tools and with sequence similarity searching tools collected at https://www.ebi.ac.uk/Tools/sss/ In some embodiments, identifying the two or more enzymes is performed in a search wherein an amino acid sequence is an input. In some embodiments, identifying two or more enzymes is performed in a search wherein a nucleic acid sequence is an input. The input sequence is typically a reference enzyme sequence from a first species. In some embodiments, the search output is one or more amino acid sequences. In some embodiments, the search output is one or more nucleic acid sequences. Typically, the search output includes an enzyme sequence from a different species from the reference sequence, that has a different sequence from the sequence of the reference enzyme, and that catalyses the same chemical reaction.

In some embodiments, the search comprises use of one or more sequence similarity searching methods. Optionally, the sequence similarity searching method is one or more of: BLAST (Basic Local Alignment Search Tool), optionally NCB! BLAST, PSI-BLAST, or PHI-BLAST; FASTA, optionally FASTA, SSEARCH, PSI-SEARCH, PSI-SEARCH2, GGSEARCH, GLSEARCH, or FASTM/S/F. Optionally, the sequence similarity searching method uses one or more of: heuristics, a scoring matrix, Smith-Waterman algorithm, modified Smither-Waterman algorithm, Needleman-Wunsch algorithm, or a modified Needleman-Wunsch algorithm. Typically, the sequence similarity searching method is BLAST. Typically, the sequence similarity searching method uses heuristics.

The sequences obtained from the sequence similarity searching method are typically aligned. In some embodiments, two or more sequences are aligned. In some embodiments, multiple sequences are aligned. In some embodiments, the sequences are aligned using a multiple sequence alignment tool. Optionally, the multiple sequence alignment tool is MAFFT (Multiple alignment program for amino acid or nucleotide sequences), Clustal Omega, (https://www.abi.acuktroolsimsaldustak") CLUSTALW (httos://www.genomejpitoolf.'-bintclustalw) COBALT (https://www.ncbi.nlm.nih.qov/tools/cobalt/cobaltcai?CMD=Web) Mutlin, (https://www.hsls.pitt. edu/obrc/index.php?paqe=UR L1051812386) Kalign (https://www.ebi.ac.uk/Tools/msa/kaliqn/) MUSCLE (Multiple Sequence Comparison by Log-Expectation), T-Coffee (https://www.ebi.ac.uk/Tools/msa/tcoffee/) or WebPRANK (https://www.ebi.ac.uk/qoldman-srv/webprank/). Typically, the multiple sequence alignment tool is MAFFT. Optionally, keyword searches can be used to ensure a wide range of extremophile host organisms, for example thermophiles, acidophiles, halophiles, etc. Alignment of sequences allows for redundant sequences (i.e. 100% match) sequences to be removed. Redundant sequences may also be removed by other methods, including by eye.

In some embodiments, the search comprises identifying an enzyme of interest and identifying sequences that are similar to the enzyme of interest using a database of protein families. Two enzyme sequences may be similar if they are, for example, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical or at least 90% identical. Optionally, the database of protein families is one or more of InterPro, CATHGene3D, CDD, HAMAP, PANTHER, Pfam, PIRSF, PRINTS, PROSITE profiles, PROSITE patterns, SFLD, SMART, SUPERFAMILY, TIGRFAMs. Typically, the database of protein families is InterPro (previously known as Pfam).

In some embodiments, the search comprises a classical literature search to identify additional mutations of the enzyme of interest.

In some embodiments, identifying the two or more enzymes comprises search which comprises one or more, optionally all, of use of one or more sequence similarity searching methods, alignment of multiple sequences, identifying an enzyme of interest and identifying sequences that are close to the enzyme of interest using a database of protein families, and identify additional mutations of the enzyme of interest. Optionally, two or more sequences are aligned again to remove redundancies.

Selection of one or more homologues The method of the present invention accelerates the identification and selection of an enzyme and a solid support to which the enzyme can be immobilised.

In one aspect of the invention, the enzyme is identified by a method that comprises a step comprising identifying two or more enzymes with different polypeptide sequences that catalyse a chemical reaction of interest. In some embodiments, the two or more enzymes comprises one enzyme which is the enzyme of interest and one or more enzymes which are homologues of the enzyme of interest.

In some embodiments, properties of the one or more homologues are compared to properties of the enzyme of interest to select for one or more homologues for immobilisation.

Immobilised enzymes Compared to free enzymes in solution, immobilised enzymes are typically more robust and more resistant to environmental changes.

Multiple copies of a single enzyme may be attached to a single support, typically a single bead.

These may be attached in an identical fashion, or different copies of a single functional molecule may be attached differently. This can provide, for example, improved activity for a single type of functional molecule when the structure is placed under different bulk environmental conditions.

When different enzymes are attached to the support, each type of enzyme may be attached identically, or may be attached differently as discussed above. Typically, each copy of the same enzyme (e.g. each Enzyme "A" in a given multi-step pathway, and each Enzyme "B" in that pathway) will be attached using the same attachment to create the same local environment for that molecule.

In one embodiment a single species of functional molecule is provided on each support (e.g. each individual bead). In other embodiments, 2, 3, 4, 5 or more species of enzyme are provided on each support (e.g. each indivisible bead). The or each species of functional molecule may be present only once on the support (i.e. a bead contains only a single functional molecule attached thereto). In other embodiments, each species of functional molecule may be present on each support (e.g. each individual bead) in multiple copies, for example 10 or more, 50 or more or 100 or more of the same species of functional molecule.

In some embodiments, two or more different enzymes are attached to each support, e.g. each bead. These may each be present once, or may each be present multiple times.

In some embodiments, a population of supports, typically a population of beads, comprises individual supports (e,g. beads) each with different enzymes immobilised thereon. For example, the population could contain a bead or beads having only a first enzyme (or first combination of enzymes) immobilised thereon and a second bead or beads having a different, second enzyme (or second combination of enzymes) immobilised thereon. Third, fourth or fifth, or more, different enzyme-beads could be included as appropriate. Accordingly, a reaction pathway of two or more enzymes, such as a multi-reaction enzyme reaction cascade can be provided by combining multiple beads in a population. The population of beads will be able to perform the functions of all of the attached enzymes. When the attached enzymes form a reaction pathway, the population will then be able to catalyse that pathway. Each reaction step of a sequence of reaction steps may be catalysed by an enzyme and optionally one or more cofactors.

Cofactors may be co-located on the same bead as the requisite enzyme, or on different beads, or may be provided elsewhere in the reaction environment such as immobilised onto an oligonucleotide that is within the same reaction system. In some embodiments, an oligonucleotide-tethered cofactor is attached to a nucleotide-binding domain (e.g. a HUH domain) that is linked to the SpyCatcher polypeptide, for example as SpyCatcher-HUH fusion protein, as described below. The cofactor may be, for example, NADH or NAD+.

Solid Supports The invention typically identifies an enzyme that can be usefully immobilsied to a solid support to carry out a stated reaction. The solid support may comprise controlled pore glass, polystyrene, sepharose or alginate. In some embodiments, the solid support is particulate. The solid support may comprise beads, for example controlled pore glass beads, polystyrene beads or buoyant beads. In some embodiments, the solid support is a resin. In some embodiments, the solid support is magnetic, for example magnetic beads. In one embodiment, the solid support comprises Poly(styrene-divinylbenzene).

The solid support is typically a resin bead. Resin beads are well-known in the art. Resin beads are typically essentially spherical.

In some embodiments, the bead is porous. In porous beads, binding sites can also be internal, leading to higher loading of enzyme.

The beads may be from 10 micron to 1500 micron diameter, typically 100 micron to 1200 micron, for example 150 micron to 300 micron diameter.

In some embodiments, the beads are methacrylate beads, for example epoxy methacrylate beads, epoxy/butyl methacrylate beads, amino C2 methacrylate beads or Amino C6 methacrylate beads.

The inventors have successfully immobilised enzymes using a variety of beads including amine-functionalised beads and epoxy-functionalised beads.

Epoxy resins Beads that are functionalised with epoxides are known as "epoxy" functionalised resins, or epoxy-resins. The IUPAC name for an epoxide group is an oxirane. Epoxides react with nucleophiles and in the context of protein immobilisation, the reactive residues will typically be Lys and Cys (and sometimes His). Surface epoxy groups on beads therefore allow direct covalent binding of proteins or peptides, which can be achieved by simple incubation of the protein or peptide with the epoxy-bead, for example overnight.

Epoxy-functionalised beads can be used in the invention because they form very stable covalent linkages.

In some embodiments, the bead is an epoxy-methacrylate bead. In some embodiments, the bead is an epoxy/butyl methacrylate bead.

In some embodiments, the pore diameter of the epoxy beads is 300 to 1800 A. Optionally, the pore diameter is 300 to 600 A, 450 to 600 A, or 1200 to 1800 A. In some embodiments, the total moisture of the beads is 50 to 80%.

Optionally, the total moisture of the beads is 50 to 65%, or 70 to 80%.

In some embodiments, a minimum of 85% of the beads are perfectly spherical. Optionally a minimum of 95% of the beads are perfectly spherical.

In one embodiment, the epoxy beads are epoxy/butyl methacrylate beads, wherein the particle size range is 250 to 1000 micron, the pore diameter is 450 to 650 A, and the total moisture is 70 to 80%. The functional group is epoxy. The spherical beads are stable at pH 5 to pH 9, and are suitable for storage at 2 to 8°C. Optionally, a minimum of 85% of the beads are perfectly spherical.

In one embodiment, the epoxy beads are epoxy methacrylate beads, wherein the particle size range is 150 to 300 micron, the pore diameter is 300 to 600 A, and the total moisture is 5065%. The functional group is epoxy. The spherical beads are stable at pH 5 to pH 9, and are suitable for storage from 2 to 8°C. Optionally, a minimum of 95% of the beads are perfectly spherical.

In one embodiment, the epoxy beads are epoxy methacrylate, wherein the particle size range is 150 to 300 micron, the pore diameter is 1200 to 1800 A, and the total moisture is 70 to 80%. The functional group is epoxy. The spherical beads are stable at pH 5 to pH 9, and are suitable for storage at 2 to 8°C. Optionally, a minimum of 95% of the beads are perfectly spherical.

Amine-functionalised resins Beads that are functionalised with amines are known as amine-functionalised beads or aminefunctionalised resins. Amine functional groups form covalent amide bonds with proteins or polypeptides. Amine-functionalised beads are generally pre-activated with glutaraldehyde, prior to ionic polypeptide immobilisation. pH adjustment during incubation results in the formation of an amide bond between the bead and the protein or polypeptide.

In some embodiments, the bead is an amino C2 methacrylate bead. In some embodiments, the bead is an amino C6 methacrylate bead.

In some embodiments, the particle size range of the amine-functionalised beads is 150 to 300 micron.

In some embodiments, the pore diameter is 600 to 1800 A. Optionally, the pore diameter is 600 to 1200 A, or 1200 to 1800 A. In some embodiments, the total moisture of the beads is 62 to 80%. Optionally, the total moisture is 62 to 72%, or 70 to 80%.

In some embodiments, a minimum of 95% of the beads are perfectly spherical.

In one embodiment, the amine-functionalised beads are amino C2 methacrylate beads, wherein the particle size range is 150 to 300 micron, the pore diameter is 600 to 1200 A, and the total moisture is 62 to 72%. The functional group is amino (short spacer). The beads are typically stable at pH 3 to pH 10, and suitable for storage at 2 to 20°C. Optionally, a minimum of 95% of the beads are perfectly spherical.

In one embodiment, the amine-functionalised beads are amino C2 methacrylate beads, wherein the particle size range is 150 to 300 micron, the pore diameter is 1200 to 1800 A, and the total moisture is 70 to 80%. The functional group is amino (short spacer). The beads are typically stable at pH 3 to pH 10, and are suitable for storage at 2 to 20°C. Optionally, a minimum of 95% of the beads are perfectly spherical.

In one embodiment, the amine-functionalised beads are amino C6 methacrylate beads, wherein the particle size range is 150 to 300 micron, the pore diameter is 1200 to 1800 A, and the total moisture is 70 to 80%. The functional group is amino (short spacer). The spherical beads are stable at pH 3 to pH 10, and are suitable for storage at 2 to 20 oC. Optionally, a minimum of 95% of the beads are perfectly spherical.

Adsorbent resins In some embodiments, the bead is an adsorbent resin bead. Adsorbent beads immobilise the protein or peptide by adsorption. Proteins or polypeptides with hydrophobic properties can be efficiently immobilised onto adsorbent beads. Adsorbent resins are water-insoluble carriers that allow physical adsorption of an enzyme onto its surface.

In some embodiments, the bead is a polystyrenic, non-functionalised resin bead. In some embodiments, the bead is an octadecyl methacrylate bead. In some embodiments, the bead is a polymethacrylic/divinylbenzene bead. In some embodiments, the bead is a polymethacrylic/divinylbenzene macroporous bead. In some embodiments, the polymer structure is polymethacrylic crosslinked with divinylbenzene.

Adsorbent beads can be used with the invention because they immobilise proteins or polypeptides with high mechanical stability.

In some embodiments, the particle size range is 300 to 1200 micron. Optionally, the particle size range is 300 to 710 micron, or 350 to 1200 micron.

In some embodiments, the pore diameter is 220 to 1200 A. Optionally, the pore diameter is 900 to 1100 A, 400 to 650 A, 220 to 340 A, or 350 to 1200 A. In some embodiments, the total moisture is 57 to 78%. Optionally, the total moisture is 67 to 78%, 58 to 63%, 57 to 68%, or 60-66%.

In some embodiments, the typical pore diameter by nitrogen adsorption is 300 A. In some embodiments, the typical pore volume by nitrogen adsorption is 1.2 mUg. In some embodiments, the typical surface area by nitrogen adsorption is 490 m2/g.

In some embodiments, the specific gravity is 1.1.

In some embodiments, a minimum of 95% of the beads are perfectly spherical.

In some embodiments, the adsorbent bead has a functional group. Optionally the adsorbent bead has an amino functional group. Optionally, the functional group is non-ionic.

In one embodiment, the adsorbent bead is a polystyrenic, non-functionalised resin bead, wherein the particle size range is 300 to 710 micron, the pore diameter is 900 to 1100 A, and the total moisture is 67 to 78%. The spherical beads are stable at pH 1 to pH 14, and are suitable for storage at 2 to 20°C. Optionally, a minimum of 95% of the beads are perfectly spherical.

In one embodiment, the adsorbent bead is an octadecyl methacrylate bead, wherein the particle size range is 300 to 710 micron, the pore diameter is 400 to 650 A, and the total moisture is 58 to 63%. The functional group is amino (short spacer). The spherical beads are stable at pH 2 to pH 10, and are suitable for storage at 2 to 20°C Optionally, a minimum of 95% of the beads are perfectly spherical.

In one embodiment, the adsorbent bead is a polymethacrylic/divinylbenzene, nonfunctionalised resin bead, wherein the particle size range is 300 to 710 micron, the pore diameter is 220 to 340 A, and the total moisture is 57 to 68%. The beads are typically stable at pH 1 to pH 14, and are suitable for storage at 2 to 20°C. Optionally, a minimum of 95% of the beads are perfectly spherical.

In one embodiment, the adsorbent bead is a polymethacrylic/divinylbenzene macroporous, adsorbent resin bead in a non-ionic form, wherein the particle size range is 350 to 1200 micron, the typical pore diameter by nitrogen adsorption is 300 A, the typical pore volume by nitrogen adsorption is 1.2 mUg, the typical surface area by nitrogen adsorption is 490 m2/g, the specific gravity is 1.1, and the moisture retention is 60-66%. The functional group is non-ionic and the polymer structure is polymethacrylic crosslinked with divinylbenzene. The spherical beads are stable at pH 0 to pH 14, and the temperature limit is 100°C.

Ion exchange resins In some embodiments, the bead is an ion-exchange resin. In some embodiments, the ion-exchange resin is a cation exchange resin or an anion exchange resin.

In some embodiments, the ion-exchange resin comprises polystyrene crosslinked with divinylbenzene.

Examples of epoxy, amine and adsorbent beads that are commercially available from Purolite Ltd. (Llantrisant, Wales, UK) are provided below along with their catalogue numbers.

Epoxy resins: ECR8204F * Epoxy-functionalised resin * Purolite "perfect bead": min 95%.

* Epoxy methacrylate beads, wherein the particle size range is 150 to 300 pm, the pore diameter is 300 to 600 A, and the total moisture is 50-65%. The functional group is epoxy. The spherical beads are stable at pH 5 to pH 9, and are suitable for storage from 2 to 8°C.

ECR8285 * Epoxy-functionalised resin * Purolite "perfect bead": min. 85%.

* Epoxy/butyl methacrylate beads, wherein the particle size range is 250 to 1000 pm, the pore diameter is 450 to 650 A, and the total moisture is 70 to 80%. The functional group is epoxy. The spherical beads are stable at pH 5 to pH 9, and are suitable for storage at 2 to 8°C.

ECR8215F * Epoxy-functionalised resin * Purolite "perfect bead": min. 95%.

* Epoxy methacrylate beads, wherein the particle size range is 150 to 300 pm, the pore diameter is 1200 to 1800 A, and the total moisture is 70 to 80%. The functional group is epoxy. The spherical beads are stable at pH 5 to pH 9, and are suitable for storage at 2 to 8°C.

Adsorbent resins: ECR1090M * Adsorbent resin * Purolite "perfect bead": min. 95%.

* Functional group: none.

* Polystyrenic, non-functionalised resin beads, wherein the particle size range is 300 to 710 pm, the pore diameter is 900 to 1100 A, and the total moisture is 67 to 78%. The spherical beads are stable at pH 1 to pH 14, and are suitable for storage at 2 to 20°C. 15 ECR8806M * Adsorbent resin * Purolite "perfect bead": min. 95%.

* Octadecyl methacrylate beads, wherein the particle size range is 300 to 710 pm, the pore diameter is 400 to 650 A, and the total moisture is 58 to 63%. The functional group is amino (short spacer). The spherical beads are stable at pH 2 to pH 10, and are suitable for storage at 2 to 20°C.

ECR1030M * Adsorbent resin * Purolite "perfect bead": min. 95%.

* Functional group: none.

* Polymethacrylic/divinylbenzene, non-functionalised resin beads, wherein the particle size range is 300 to 710 pm, the pore diameter is 220 to 340 A, and the total moisture is 57 to 68%. The spherical beads are stable at pH 1 to pH 14, and are suitable for storage at 2 to 20°C.

PAD610 * Adsorbent resin * Polymethacrylic/divinylbenzene macroporous, adsorbent resin beads in a non-ionic form, wherein the particle size range is 350 to 1200 pm, the typical pore diameter by nitrogen adsorption is 300 A, the typical pore volume by nitrogen adsorption is 1.2 mL/g, the typical surface area by nitrogen adsorption is 490 m2/g, the specific gravity is 1.1, and the moisture retention is 60-66%. The functional group is non-ionic and the polymer structure is polymethacrylic crosslinked with divinylbenzene. The spherical beads are stable at pH 0 to pH 14, and the temperature limit is 100°C.

Amine resins: ECR8309F * Amine-functionalised resin * Purolite "perfect bead": min. 95%.

* Amino C2 methacrylate beads, wherein the particle size range is 150 to 300 pm, the pore diameter is 600 to 1200 A, and the total moisture is 62 to 72%. The functional group is amino (short spacer). The spherical beads are stable at pH 3 to pH 10, and suitable for storage at 2 to 20°C.

ECR8315F * Amine-functionalised resin * Purolite "perfect bead": min. 95%.

* Amino C2 methacrylate beads, wherein the particle size range is 150 to 300 pm, the pore diameter is 1200 to 1800 A, and the total moisture is 70 to 80%. The functional group is amino (short spacer). The spherical beads are stable at pH 3 to pH 10, and are suitable for storage at 2 to 20°C.

ECR8415F * Amine-functionalised resin * Purolite "perfect bead": min. 95%.

* Amino C6 methacrylate beads, wherein the particle size range is 150 to 300 pm, the pore diameter is 1200 to 1800 A, and the total moisture is 70 to 80%. The functional group is amino (short spacer). The spherical beads are stable at pH 3 to pH 10, and are suitable for storage at 2 to 20°C.

The invention is now described further with reference to the following illustrative examples. EXAMPLES Homologue generation A generic BLAST search of the non-redundant protein sequences (nr) database from NCBI was run to accumulate homologs for the enzyme. These were aligned using the online MAFFT server and the redundant (100% match) sequences removed. Keyword searches were used to ensure a wide range of extremophile host organisms, such as thermophiles, acidophiles, halophiles, etc. The Pfam database was used to find sequences that are close to the enzyme of interest. A final BLAST search looked at the metagenomic proteins (env_nr) database of NCBI.

Removing redundancies left a total of 8,961 sequences to analyse for the enzyme of interest. Using classical methods of literature search additional mutations were also identified and incorporated. The homologs were once again aligned and redundancies removed. 96 sequences were ordered of which FN1096 was preselected to work well on beads 12 and 13 with the outcome that these pairing would result in imparted process stabilisation.

Figure 1 shows how we were able to find the best set of hits along the x-axis that will work best under our process conditions. We were able to select a few (purple circles is FN1096) and the other hits cluster nearby and are shown in yellow.

Expression of homologues: 2.a -Homologue transformation and glycerol stocks Prepare 4 x 24-well kanamycin resistance agar plates. Dilute plasmid stocks to 10 ng/uL in the plates from TWIST.

Remove BL21*(DE3) tubes from the -70°C freezer, and place directly on ice for 2 minutes to thaw the competent cells Split each 50 uL BL21*(DE3) tube into 10 uL across 96 well Twin tech PCR plates.

In the sterile hood, add 1 pl of plasmid to each well containing 10 uL of cells using a multichannel pipette.

Incubate the plate on ice for 30 minutes Heat shock the cells at exactly 42°C for exactly 30 seconds by transferring the plate to a pre-warmed thermoblock with the 96-PCR attachment Place cells on ice for 5 minutes Pipette 90 pl of room temperature SOC into each well Place at 37°C for 60 minutes, shaking at 600 rpm in a thermoblock. In parallel, warm the 24-well LB-Agar plates to 37°C.

Dispense 10 pl from each well into a 24-well LB-Agar plate with kanamycin, rotate plate to try to spread the liquid across the well and allow to dry. Incubate overnight at 37°C Pick single colonies for each of the homologues and grow overnight in their respective kanamycin resistant growth medium overnight at 37°C and add 50% glycerol LB to create glycerol stocks. Free at -70°C for future use.

Expression Prepare 2 sterile 96 well deep well plates (DWP), one with 1 mL of LB AIM (auto-induction medium) and one with 1 mL of TB AIM, both supplemented with kanamycin (50ng/uL) in each well Inoculate each well of the DWP plates with 10 uL (1% v/v) of the required overnight culture Seal plate with a sterile breathable film Grow culture at 37°C and 1,000 rpm for 3 hours on a thermoblock, then grow at 30°C and 1,000 rpm overnight.

Resuspension & chemical lysis of cell pellets Centrifuge the DWP at 4000 rpm and 4 °C for 20 mins Discard the supernatant For the cultures grown in TB AIM, resuspend the pellets in 1 x PBS and centrifuge again at 4000 rpm and 4 °C for 20 mins.

Discard the supernatant Freeze pellets for at least 30 mins at -20 °C.

Supplement xTractor buffer with lysozyme and DNase I as per the manufacturers recommendations.

For 100 mL of xTractor buffer: Add 200 uL of 5 U/uL DNase I solution (Provided in kit) Add 500 uL of 100x Lysozyme solution (Provided in kit) Add 200 uL XTractor buffer to each pellet and resuspend by shaking on the thermoblock at 1000 rpm at 25 °C for 10 mins Incubate the resuspended pellets for 10 mins at 25 °C, shaking at 1000 rpm Centrifuge at 4300 x g for 2 hrs at 4 °C and discard the pellet (the supernatant is the soluble fraction).

Screening the homologue library To the lysed cells, add 600 uL 0.1M sodium phosphate buffer pH 7.0 to each well in the 96 deep well plate.

Centrifuge the plate for 30 min at 4'500 r.p.m.

In the meantime, prepare activity assay master mix.

Using the platemaster, take 50 uL of the activity assay master mix from above and add to every position 1 in a 384 well transparent plate.

Using the platemaster, transfer 5 uL of enzyme from the fermentation plate and add to positions 1 the 384 well transparent plate.

Add a film to prevent evaporation.

Scan the absorbance profiles at 340, 385, 500 nm using a plate reader. Make sure the path length is adjusted to 55 uL. Scanning every 1 min, with 2 seconds of shaking between runs for mins.

Leave the plate at RT for a further 1.5 hours.

Figure 2 shows reaction hits at different time points. The best performers were taken forward and scaled using the same expression protocol at 50mL for screening on immobilization supports: Best performers: FN667, FN1048, FN1098, FN1101, FN1045, FN1099, FN1113, FN1043, FN1047, FN1046, FN1109, FN1108 50mL expression of top homologue hits: Inoculate 1 mL of LB kanamycin resistance media with a glycerol scrape from the respective hit.

Grow overnight at 37 °C, shaking at 200 rpm.

Inoculate 50 mL of kanamycin resistant LB AIM media (in a sterile 500 mL flask) with 500 uL (1% v/v) of the primary culture.

Grow culture at 37°C for 3 hours, then move to 20 °C and 200 rpm and grow overnight.

To measure the final OD dilute 5 uL of overnight culture into 195 uL PBS in a 96 well U-bottom MWP and measure absorbance at 600 nm on the plate reader, blanking using 200 uL PBS Ensure that the final OD reading is <1 Do the same for any small scale 1mL parallels. Lysis of pellets 1. Transfer the contents from the shake flasks into 50 mL falcon tubes 2. Centrifuge the DWP and falcon tubes at 4000 rpm and 4 °C for 20 mins a. Discard the supernatant 3. Supplement xTractor buffer with lysozyme and DNase I as per the manufacturer's recommendations 4. Add 10 mL Xtractor buffer 5. Resuspend by pipetting and vortexing 6. Incubate the resuspended pellets for 10 mins at 25 °C 7. Centrifuge at 4300 x g for 1hr at 4 °C and discard the pellet (the supernatant is the soluble fraction).

QC of homolog hits prior to immobilization Create a plate from the top hits expressed at 50mL to enable plate based assaying.

Using the platemaster, take 50 uL of the activity assay master mix and add to every position 1 in a 384 well transparent plate.

Using the platemaster, transfer 5 uL of enzyme from the fermentation plate and add to positions 1 the 384 well transparent plate.

Add a film to prevent evaporation.

Scan the absorbance profiles at 340, 385, 500 nm using a plate reader. Make sure the path length is adjusted to 55 uL. Scanning every 1 min, with 2 seconds of shaking between runs for 30 mins.

Leave the plate at RT for a further 1.5 hours.

Scan the absorbance at 340, 385, 500 nm using a plate reader. Make sure the path length is adjusted to 55 uL. Scanning once, make sure the path length is adjusted to 55 uL.

The same can be done for 1m1 parallel sample.

Figure 3 shows 50mL scale-up and 1mL parallel free enzyme specific yields for the homolog hits.

Screening for stability in Batch.

3. Prepare resins in 96 deep-well plate: 23 resins were evaluated to include 1 negative control.

In a 96 deep well plate, add 0.5 mL of each resin type (stock 50 mg per 1 mL where stated) to the appropriate columns using a P1000 pipettor.

For resin 24 (negative control) pipette 500uL 1xPBS.

4. Add enzyme and incubate (clicking step): Get a tip box and remove the 4th and 8th row of tips. This so as to not add enzyme to the 'no enzyme' controls.

Add 500uL of lysate to the resins using the platemaster multi-channel pipetting device.

For resin controls add 5000i..1xPES (this can be done manna 1y with a P1000 pipette or the platemaster).

Add lid and secure firmly.

Leave to click at RT for 120 mins whilst shaking on the thermomixer at 1400 r.p.m. with the lid on to ensure binding to bead.

In the meantime, prepare 1 L of assay material.

6. Supernatant activity assay: Centrifuge the plate for 1 min using the hencrtop centrifuge, Using the Platemaster, take 50 uL of the activity assay master mix and add a 384-well plate.

Using the platemaster, extract 5 uL of supernatant from each well of the 96 deep well plate supernatant and add to the 384-well plate in the position 1 wells.

Mix sample with mastermix by pipetting up and down several times.

Scan the absorbance profiles at 340, 385 and 450 nm using the ClarioStar plate reader capturing initial rates curves.

7. Washing steps: Wash the 96-well plate with 1mL of PBS 3X. Centrifuging between wash steps.

8. immobilized enzyme activity assay: Add 200uL x 5 volumes of reaction mixture to the plate, ensure the timer is initiated on addition of the first 200uL of reaction mixture. Add lid and secure firmly. Mix on thermomixer at 1400 r.p.m.

After 3, 10, 20, 30 mins, isolate 50 uL of supernatant and place in a 384-well plate using the Platemaster.

9. Perform UV scan in 384-well plate.

Scan the absorbance profiles at 340, 385 and 450 nm using the ClarioStar plate reader.

Make sure the path length is adjusted to 50 uL.

10. Multiple reuse cycles.

Repeat steps 7-9 to capture repetition cycles to determine stability of immobilized enzyme under batch conditions.

It was predicted that FN1096 (from the homologue search) would pair well with beads 12 and 13, where we would see imparted stability.

See Figures 4.1 to 4.8 for an example of reuse cycle data showing the enzyme stabilisation on a range of beads for each hit.

Flow reaction of FN1096 (stable hit indication) and FN667 1. Pour 10 mL (1g = 1mL) resin into a 50 mL falcon tube.

2. Add 4 mL of FN1096 to the falcon tube.

3. Leave mixing on the tube inverter for 15 mins at RT.

Assembling the cartridge: 1. Using a 2 mL disposable cartridge, transfer 2 mL of FN1096-bead 12 resin using a 1 mL pipettor. Total amount of resin loaded 2 mL assumed 2 g containing 0.8 mL of CFE = 8 mg of protein.

2. Assemble the cartridge and mount on FPLC.

3-HPA activity assay mastermix and FPLC: 1. Make 1L of assay reaction mixture in a duran bottle and connect to the Akta FPLC.

2. Switch on FPLC and computer, then load software.

3. Mount the cartridge on line of the FPLC, attach cis and trans 0.45 micron syringe filters to connect the cartridge.

4. Flow reaction mixture at 0.5 mL per min, measure wavelengths 340 nm, collect fractions in 1 mL wells using 96-deep well plate, stop after stated volumes mL of flow.

Then take the fractions and transfer 50 uL to a 384-well transparent plate. Scan the absorbance at 340, 385 and 500 nm. The plot the absorbance vs elution volume. 5. Follow the same steps for bead 3 for FN677 Figure 5 shows, in flow results at 0.5 mL per min. Bead 12 shows stability, constant amount of product formed over time, at two different reaction concentrations. Bead 3 on FN677 shows degradation over time and is not a stabilised. Control, no enzyme shows no product (yellow).

Clustering analysis K-modes analysis was performed as a route to predict the compatibility of 39 immobilization supports. The analysis aids prediction for suitability against desired process conditions, including clustering for the functional overlap of surface chemistries to be used downstream for pairing minimising the massive enzyme homologues space based on desired characteristics for stable enzyme immobilization.

Figure 6 shows clustering of 39 immobilization supports into 3 clusters based on material types and immobilization chemistries. This analysis takes categorical factors and uses them to cluster the range of beads and can therefore be used as part of the prediction to assist in selection of the bead material for the specified process. The functional surface groups can also be layered into the analysis, to pair it with "predicted process conditions" and the minimisation of the enzyme-homologue space (i.e. charged enzyme surfaces for opposing charged beads) to select for the enzymes which are most likely to fit and results in stability gains.

It is understood that the Examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (19)

1. Claims 1. A method of identifying an enzyme and a solid support to which the enzyme can be immobilised in an active state for a desired period of time under specified conditions, comprising the steps of: (a) identifying two or more enzymes with different polypeptide sequences that catalyse a chemical reaction of interest; (b) identifying one or more solid supports; and (c) determining whether at least one of the enzymes can be immobilised to one or more of the solid supports in an active state for the desired period of time under the specified conditions, wherein the determination is made by comparing a characteristic of two or more of the enzymes to a characteristic of one or more solid supports, and selecting at least one enzyme and at least one solid support, wherein the selected enzyme is predicted to provide active immobilised enzyme when combined with the selected solid support under the specified conditions and over the desired period of time when the enzyme is immobilised to its paired support under the specified conditions.
2. 2. A method according to claim 1, wherein the specified conditions comprise one or more of a specified temperature, pH, substrate or cofactor to which the immobilised enzyme will be subjected to perform a catalytic process.
3. 3. A method according to claim 1 or claim 2, wherein the two or more enzymes comprise or more, 50 or more, 100 or more, 500 or more, or 5000 or more different polypeptide sequences that catalyse a chemical reaction of interest.
4. 4. A method according to any of claims 1 to 3, wherein the two or more enzymes comprise homologues of one enzyme from different species, optionally wherein the two or more enzymes comprise at least one enzyme from an extremophile species, optionally wherein the extremophile species is a thermophile, an acidophile or a halophile.
5. 5. A method according to any preceding claim, wherein the two or more enzymes comprise at least one enzyme having a mutation compared to the wild-type sequence, optionally wherein the mutation is engineered into the enzyme by a recombinant technique, optionally wherein the mutation increases or decreases the stability or the activity, or the stability and the activity, of the enzyme.
6. 6. A method according to any preceding claim, wherein step (a) comprises a bioinformatic search of known enzymes that catalyse the chemical reaction of interest, optionally wherein the search comprises a search for homologues of a reference enzyme, optionally wherein the search comprises a BLAST search of a reference sequence.
7. 7. A method according to any preceding claim, wherein the one or more solid supports comprises at least two or at least five different solid supports, optionally selected from controlled pore glass, polystyrene, sepharose, alginate, epoxy-resin, ion-exchange resin, amine-resin or adsorbent resin beads.
8. 8. A method according to preceding claim, wherein the period of time is the length of time taken to complete an industrial biocatalysis under the specified conditions.
9. 9. A method according to any preceding claim, wherein the period of time is at least 3 days, at least 1 week, at least 2 weeks, at least 1 month, at least 6 weeks, at least 2 months, at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months or at least 2 years, optionally wherein the period of time is at least one month or at least one year.
10. 10. A method according to any preceding claim, wherein the characteristic of the enzyme comprises one or more of the enzyme sequence, the proportion of charged residues, the proportion of surface charged residues, the secondary structure, the tertiary structure, or the pl.
11. 11. A method according to any preceding claim, wherein the characteristic of the solid support comprises one or more of the charge of the support, the chemistry of the conjugation site and the porosity of the support.
12. 12. A method according to any preceding claim, wherein more than one active enzyme-solid support pair is identified, and wherein the active enzyme-solid supports are ranked in terms of their expected activity over time under the specified conditions.
13. 13. A method according to any preceding claim, wherein the activity over time is scored as the mathematical product of the activity and the time over which the activity is predicted to be provided under the specified conditions.
14. 14. A method according to any preceding claim, wherein at least steps (a) and (c) are carried out by a computer, optionally wherein all steps are carried out by a computer.
15. 15. A method according to any preceding claim, further comprising the step of immobilising the at least one enzyme to its paired support, and optionally testing the activity of the at least one immobilised enzyme under the specified conditions.
16. 16. A method according to claim 15, wherein the activity of the at least one immobilised enzyme under the specified conditions is tested at a first time point (t1) and a second later time point (t2), optionally wherein the time between t1 and t2 is the desired period of time.
17. 17. A method according to claim 15 or claim 16, wherein the activity of the at least one immobilsied enzyme under the specified conditions is tested over multiple cycles of a batch reaction, and wherein the enzyme remains active for at least 2, 3, 4, 5 or 10 batch cycles.
18. 18. A method according to claim 15 or claim 16, wherein the activity of the at least one immobilsied enzyme under the specified conditions is tested in a flow reaction.
19. 19. A method of identifying a solid support to which an enzyme can be immobilised in an active state for a desired period of time in a specified reaction process, comprising the steps of: identifying one or more solid supports; and determining whether at least one of the solid supports can be used as a support for an enzyme in a specified reaction process for a desired period of time, wherein the determination is made by comparing characteristics of one or more solid supports to the conditions of the specified reaction process and selecting a solid support that can support an active enzyme in the specified reaction process.