Chemo-enzymatic approach for the synthesis of rare c3- sugars epimers using a glycoside 3-oxidase (engineered) enzymes

Abstract

The present disclosure relates to engineered bacterial glycoside-3-oxidases with improved catalytic and stability properties (and homologues) and their use in producing rare C3-sugar epimers. The present disclosure further relates to a method for the enzymatic oxidation of the C3-position of chemically protected saccharides at the C1-position, followed by a chemical reduction and deprotection steps.

Description

[0001] Chemo-enzymatic approach for the synthesis of rare C3- sugars epimers using a glycoside 3-oxidase (engineered) enzymes

[0002] This application claims the benefit of priority of Portuguese Application 20242006596861, filed 25 October 2024, which is incorporated by reference herein.

[0003] Field

[0004] The present disclosure relates to an engineered bacterial glycoside-3-oxidase (and homologues) with improved catalytic and stability properties and their use for producing rare C3-sugar epimers. The present disclosure further relates to a method for selective enzymatic oxidation of a protected saccharide and a method for epimerization of a saccharide, leading to naturally scarce sugars.

[0005] Background

[0006] Rare sugars, encompassing mono- and disaccharides and their derivatives, are defined by the International Society of Rare Sugars (ISRS) as carbohydrates found in low abundance in nature. These include twenty hexoses (e.g., D-allose, D-allulose and D-tagatose), nine pentoses (e.g., D-lyxose and L-xylulose) and disaccharides like turanose and isomaltulose. Rare sugars' ecological roles and physiological functions remain largely unexplored. Nevertheless, their unique functional properties as food additives, nutraceuticals, and active pharmaceutical ingredients have attracted considerable attention.

[0007] Various chemical and enzymatic methods have been developed to address their production. These include i) chemical isomerization, typically unselective, resulting in complex product mixtures; ii) selective oxidation of minimally protected sugars followed by reduction; and iii) site-selective radical epimerization reactions that produce rare sugars from biomass-derived carbohydrates. Enzymatic methods provide regio- and stereo-specificity under milder conditions, making them attractive, sustainable alternatives to traditional chemical synthesis. Over the past two decades, most studies on the enzymatic synthesis of rare sugars have utilized a strategy known as Izumoring: this approach enables the cyclical conversion of monosaccharides through enzymatic epimerization, isomerization and oxidation-reduction (Granstrom et al., J Biosci Bioeng, 97(2), 2004; Izumori et al., J Biotechnol, 124(4), 2006). Nevertheless, other non-Izumoring enzymatic techniques have also emerged. For example, pyranose-2-oxidases (P2Oxs) from the glucose-methanol-choline (GMC) family, by promoting the oxidation of hexoses at the C2 position, can synthesize D-tagatose and D-glucopyranosyl-β-1,6-D-mannopyranose from D-galactose and gentiobiosone, respectively, using P2Ox oxidation followed by a chemical reduction. (Giffhorn et al., Enzyme Microb Technol, 27(10), 2000) Based on the state of the art mentioned above, the objective of the present disclosure is to provide means and methods to epimerize saccharides at a defined position. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0008] Summary of the Invention

[0009] The inventors engineered G30x to improve its catalytic properties towards D-glucose using directed evolution, an approach used successfully to enhance the properties of various enzymes. The optimized enzyme can selectively oxidize the C3-OH group of partially protected D-glucose at improved rates. This variant facilitates a chemo-enzymatic approach for synthesizing the rare sugar D-allose in high yields. Given the enzyme's broad substrate specificity and potential for further engineering, this system is a promising platform for synthesizing rare sugars, creating economic opportunities for their industrial production and commercialization.

[0010] A first aspect of the disclosure relates to a polypeptide comprising an amino acid sequence of at least (>) 85% identity to SEQ ID NO 002, wherein the amino acid sequence comprises at least one of the following optimized residues (numbering concerning SEQ ID NO 002):

[0011] a. Q295 is histidine (H);

[0012] b. A327 is selected from the group of valine (V), leucine (L), and isoleucine (I); c. A335 is selected from the group of serine (S) and threonine (T);

[0013] d. P336 is selected from the group of serine (S) and threonine (T);

[0014] e. 1357 is selected from the group of alanine (A), and valine (V);

[0015] f. G366 is selected from the group of serine (S) and threonine (T).

[0016] A second aspect of the disclosure relates to an isolated nucleic acid encoding the polypeptide according to the first aspect.

[0017] A third aspect of the disclosure relates to a nucleic acid expression vector comprising a sequence encoding the polypeptide according to the first aspect.

[0018] A fourth aspect of the disclosure relates to a method for oxidation (oxidation of a secondary alcohol to a ketone) of a D-pyranoside or a D-furanoside comprising the steps:

[0019] a. Combining the D-pyranoside or the D-furanoside with the polypeptide, particularly using a polypeptide according to the first aspect of the solution.

[0020] A fifth aspect of the invention relates to a method for obtaining a pyranoside- or a furanoside-epimer comprising the steps:

[0021] a. performing the method according to the fourth aspect;

[0022] b. In a reduction step, the keto-pyranoside or keto-furanoside is reduced at its carbonyl position. A further aspect of the disclosure relates to using the polypeptide according to the first aspect for producing an oxidized (carbonyl group) saccharide.

[0023] A further aspect of the disclosure relates to using the polypeptide, according to the first aspect of producing a saccharide epimer.

[0024] An aspect of the present disclosure relates to a polypeptide comprising an amino acid sequence of at least ≥ 85%, ≥ 90%, ≥ 92%, ≥ 95%, ≥ 98%, ≥ 99% or 100% identity to SEQ ID NO 002, wherein the amino acid sequence comprises at least one of the following optimized residues: a. Q295 is histidine (H);

[0025] b. A327 is selected from the group of valine (V), leucine (L), or isoleucine (I);

[0026] c. A335 is selected from the group of serine (S) or threonine (T);

[0027] d. P336 is selected from the group of serine (S) or threonine (T);

[0028] e. I357 is selected from the group of alanine (A) or valine (V);

[0029] f. G366 is selected from the serine (S) or threonine (T) groups.

[0030] In some embodiments, the at least two of the optimized residues are present in the amino acid sequence of the polypeptide, particularly wherein at least three, more particularly at least four, even more particularly at least five, most particularly all six of the optimized residues are present in the amino acid sequence of the polypeptide.

[0031] In some embodiments, a. Q295 is histidine (H); b. A327 is valine (V); c. A335 is threonine (T); d. P336 is serine (S); e. I357 is valine (V); and f. G366 is serine (S).

[0032] In some embodiments, the amino acid sequence comprises an additional optimized residue: g. A206 is selected from the group of serine (S) or threonine (T), and / or h. D383 is selected from the group of asparagine (N), glutamine (Q), or serine (S).

[0033] In some embodiments, g. A206 is threonine (T) and / or h. D383 is asparagine (N).

[0034] In some embodiments, the polypeptide comprises or consists of SEQ ID NO 004.

[0035] In another aspect, the present disclosure relates to an isolated nucleic acid encoding the polypeptide according to the present disclosure.

[0036] In another aspect, the present disclosure relates to a nucleic acid expression vector comprising a sequence encoding the polypeptide according to the present disclosure.

[0037] In another aspect, the present disclosure relates to a cell comprising the polypeptide according to the present disclosure, isolated nucleic acid according to the present disclosure, nucleic acid expression vector according to the present disclosure, or any combination thereof.

[0038] In some embodiments, the cell is a prokaryotic or an eukaryotic cell.

[0039] In some embodiments, the cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida.

[0040] In another aspect, the present disclosure relates to a method for oxidation of a pyranoside or a furanoside comprising a step: a. combining the pyranoside or the furanoside with a polypeptide according to the present disclosure, or with a cell according to the present disclosure. In some embodiments, the pyranoside or the furanoside is unprotected at position C1 and oxidized at position C2.

[0041] In some embodiments, the furanoside is protected at position C1, C2 or both, and oxidized at position C3 or position C4.

[0042] In another aspect, the present disclosure relates to a method for oxidation of a pyranoside or a furanoside, comprising a step of combining the pyranoside or the furanoside with a glycoside 3-oxidase enzyme; wherein, the pyranoside is protected at position C1 and oxidized at position C3 and the furanoside is protected at position C1, C2 or both, and oxidized at position C3 or position C4.

[0043] In some embodiments, the glycoside 3-oxidase enzyme is selected from the group of:

[0044] a. PsG30x, AgCarA, MtCarA, CarA, or ScP2Ox; or

[0045] b. a homologue of PsG30x, AgCarA, MtCarA, CarA, or ScP2Ox; or

[0046] c. a variant of PsG30x, AgCarA, MtCarA, CarA, ScP2Ox, wherein the variant has at least 70% identity, >75%, >80%, >85%, >90%, or >95% identity with a polypeptide sequence of PsG30x, AgCarA, MtCarA, CarA, or ScP2Ox, particularly having a biological activity of at least 30%, at least 50% or at least 80% of a polypeptide of the sequence of PsG30x, AgCarA, MtCarA, CarA, or ScP2Ox.

[0047] In some embodiments, the glycoside 3-oxidase enzyme is selected from the PsG30x, AgCarA, MtCarA, CarA, and ScP2Ox group.

[0048] In some embodiments, the pyranoside or the furanoside is protected by a protecting group comprising an aromatic moiety, particularly with a substituted or unsubstituted benzyl group or benzoyl group, more particularly with an unsubstituted benzyl group or benzoyl group.

[0049] In some embodiments, the pyranoside is selected from the group of glucose, galactose, 2-deoxy-glucose, rhamnose, mannose, fucose and N-acetylglucosamine.

[0050] In some embodiments, the furanoside is selected from the group of arabinose, ribose and fructose. In some embodiments, the pyranoside or the furanoside is a D-enantiomer.

[0051] In some embodiments, the pyranoside is glucose or galactose.

[0052] In some embodiments, the pyranoside is in β or α configuration.

[0053] In some embodiments, the pyranoside or the furanoside is linked to at least one saccharide. In some embodiments, the method further comprises a reduction step at carbonyl position, affording a pyranoside- or a furanoside-epimer.

[0054] In some embodiments, the reduction step comprises addition of a compound selected from the group of NaBH4, NaBH4 / cerium chloride, LiAIH4, DIBAL, LS-selectride, K-selectride, L-selectride, Na(OMe)3BH, K(O-iPr)3BH, NaCNBH3, Na(OAc)3BH, LiBH4, Me2S. BH3, PhNEt2. BH3, NaNH2(BH3)2, NH3-BH3 / TiCI4, pinBH / NaOtBu, Sm / HCl / THF, Red-AI, particularly LS-selectride. In some embodiments, the pyranoside is D-glucose, and the pyranoside-epimer is D-allose.

[0055] In some embodiments, the pyranoside is D-galactose, and the pyranoside-epimer is D-gulose. In some embodiments, the method further comprises a step of deprotecting the pyranoside or the furanoside. In another aspect, the present disclosure relates to a use of the polypeptide according to the present disclosure for production of an oxidized saccharide.

[0056] In another aspect, the present disclosure relates to a use of the polypeptide according to the present disclosure for the production of a saccharide epimer.

[0057] In some embodiments, the epimer is D-allose or D-gulose.

[0058] Terms and definitions

[0059] General

[0060] For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0061] The terms “comprising,” “having,” “containing,” and “including,” and other similar forms and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C or can include components A, B, and C and one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of or “consisting of.”

[0062] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any expressly excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0063] Reference to “about” a value or parameter herein includes (and describes) variations directed to that value or parameter per se. For example, a description referring to “about X” consists of a description of “X.”

[0064] As used herein, including in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context dictates otherwise.

[0065] "And / or" where used herein is to be taken as a specific recitation of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0067] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0068] Sequences

[0069] Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the disclosure. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions) to complement the strand. The nucleic acids may be present in whole cells, cell lysate, partially purified or substantially pure form.

[0070] In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Smith and Waterman, Adv's local homology algorithm may be used to conduct sequence alignment for comparison. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0071] One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11, Extension 1; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1.-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the aboveidentified default parameters for protein and nucleic acid comparison, respectively.

[0072] Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e., the same sequence).

[0073] The term variant refers to a polypeptide that differs from a reference polypeptide but retains essential properties. A typical polypeptide variant differs in its primary amino acid sequence from another reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are similar overall and, in many regions, particularly at the substrate binding site or the FAD-binding site, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted amino acid residue may or may not be encoded by the genetic code. A variant of a polypeptide may be naturally occurring, or it may be a variant that is not known to occur naturally.

[0074] The term homologue in the context of the present specification relates to naturally occurring glycoside 3-oxidase polypeptide having a similar sequence, for example, at least 70% sequence identity and equivalent biological activity. A shared gene arrangement and regulatory structure might identify a homologous protein. Homologues may be identified, for example, by using a wildtype protein sequence (for example, SEQ ID NO 002) to query a freely available sequence similarity online search tool, as in the methods section headed Bioinformatic Analysis. Databases available at - Basic Local Alignment Search Tool = BLAST (NCBI; http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), Pfam (http: / / pfam.xfam.org, Mistry J. etal. 2020 Nucleic Ac. Res. doi: 10.1093 / nar / gkaa913), or STRING (https: / / string-db.org / cgi / about) may be used to identify conserved motifs in naturally occurring glycoside 3-oxidase homologues.

[0075] Particular embodiments use the sequences disclosed herein (i.e., 100% identical).

[0076] The term having at least a certain amount or substantially the same biological activity in the context of the present disclosure relates to the function of a glycoside-3-oxidase protein, i.e., the biological activity of oxidizing a pyranoside or a furanoside, as a non-limiting example, benzyl-O-glucoside. General Biochemistry: Peptides, Amino Acid Sequences

[0077] The term polypeptide in the context of the present specification refers to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The terms "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Amino acid residue sequences are given from an amino to a carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3rded. p. 21). Lower-case letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. Following standard nomenclature, amino acid residue sequences are denominated by either a three-letter or a single-letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

[0078] In general, and throughout this specification, the term “vector" refers to a DNA nucleic acid molecule capable of carry a foreign piece of DNA (gene coding for the protein of interest) nucleic acid to which it has been linked into a host cell. Its purpose is to facilitate the expression of the DNA inserted within the host cell. Such vectors are referred to herein as “expression vectors”. Common expression vectors of utility in recombinant DNA techniques are often plasmids.

[0079] The term "expression vector" as used herein refers to a nucleic acid molecule (e.g., a plasmid, phage, autonomously replicating sequence (ARS), artificial chromosome, yeast artificial chromosome (e.g., YAC)) that can be replicated in a host cell and be utilized to introduce a gene or genes into a host cell.

[0080] Organic Chemistry

[0081] The term carbohydrate according to the present disclosure refers to an organic compound comprising carbon, hydrogen and oxygen atoms, typically with the empirical formula (CH2O)n, and includes monosaccharides, disaccharides, oligosaccharides and polysaccharides. Carbohydrates exist in linear or cyclic forms and may be modified or substituted. The term encompasses naturally occurring and synthetic carbohydrates, as well as their stereoisomers, enantiomers, diastereoisomers, anomers and regioisomers.

[0082] A monosaccharide is a sugar comprising three to seven carbon atoms. Examples are glyceraldehyde (C3), erythrose or threose (C4), arabinose, ribose or xylose (C5), glucose, mannose, galactose or fructose (C6) or sedoheptulose (C7). The sugar alcohols and amino sugars of C3 to C7 monosaccharides are included in the group of monosaccharides according to the definition used herein.

[0083] The term aryl in the context of the present specification relates to a cyclic aromatic C5-C10 hydrocarbon. Examples of aryl include, without being restricted to, phenyl and naphthyl.

[0084] The term heteroaryl in the context of the present specification relates to a cyclic aromatic C2-C9 hydrocarbon that comprises at least one heteroatom (e.g., N, O, S). Examples of heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole.

[0085] The term oxidation or oxidized in the context of the present specification relates to forming a carbonyl (ketone or aldehyde) group at a saccharide's position comprising an OH group. In the context of the disclosure, oxidation refers to the modification of an OH group, resulting in an oxygen bound to a carbon through a double bond (carbonyl group). A keto position is the position of the saccharide, where the carbonyl group is located. In some embodiments, the OH group is positioned at C2 or at the C3 position. In some embodiments, the oxidized saccharide has a keto group at position C2 or C3.

[0086] The term protected or protection group in the context of the present specification refers to a chemical moiety attached to (bound to) an OH group of the saccharide. Another chemical moiety then replaces the H of the OH group. The OH group with the chemical moiety attached has a lower chemical reactivity towards certain chemicals. The protection group contains an aromatic moiety or aromatic group in certain embodiments. In certain embodiments, the aromatic moiety or aromatic group is an aryl or a heteroaryl.

[0087] The term deprotected in the context of the present specification relates to removing a chemical moiety (the protection group) from the OH group of the saccharide.

[0088] Detailed Description of the Invention

[0089] The present disclosure relates to the selective epimerization of a carbohydrate using a chemoenzymatic conversion. According to some embodiments of the present disclosure, a carbohydrate is a monosaccharide. According to some embodiments of the present disclosure, a carbohydrate is a pyranoside or a furanoside. In some embodiments, the pyranoside or furanoside is modified or substituted. In some embodiments, the pyranoside or furanoside is bound to at least one saccharide. In some embodiments, a pyranoside or furanoside as described herein, is part of a di, tri- or higher saccharide.

[0090] The present disclosure is based, in part, on the finding that according to the present disclosure a disaccharide is used as a pyranoside or furanoside bound to another monosaccharide unit that acts as a protecting group, for example at the C1 position of a pyranoside.

[0091] The present disclosure relates to an enzyme created via mutagenesis which selectively and efficiently converts a carbohydrate such as pyranoside or a furanoside into an oxidized version at position C2, C3 or C4. The present disclosure is based, in part, on the finding that the oxidation position depends on whether the pyranoside or furanoside has an OH group at position C1, or whether the C1 oxygen atom is bound to another chemical moiety (particularly an aromatic protecting group). The present disclosure relates to a mutated enzyme, its use, and methods for substrate conversion to industrially applicable intermediate and end products. A first aspect of the disclosure relates to a polypeptide comprising an amino acid sequence of at least (>) 85% identity to SEQ ID NO 002, wherein the amino acid sequence comprises at least one of the following optimized residues (numbering SEQ ID NO 002):

[0092] a. Q295 is histidine (H);

[0093] b. A327 is selected from the group of valine (V), leucine (L), or isoleucine (I); c. A335 is selected from the group of serine (S) or threonine (T);

[0094] d. P336 is selected from the group of serine (S) or threonine (T);

[0095] e. I357 is selected from the group of alanine (A), or valine (V);

[0096] f. G366 is selected from the group of serine (S) or threonine (T).

[0097] In certain embodiments, the polypeptide comprises an amino acid sequence of at least (>) 90% identity to SEQ ID NO 002, wherein at least one of the optimized residues above is present. In certain embodiments, the polypeptide comprises an amino acid sequence of at least (>) 92% identity to SEQ ID NO 002, wherein at least one of the optimized residues above is present. In certain embodiments, the polypeptide comprises an amino acid sequence of at least (>) 95% identity to SEQ ID NO 002, wherein at least one of the optimized residues above is present. In certain embodiments, the polypeptide comprises an amino acid sequence of at least (>) 98% identity to SEQ ID NO 002, wherein at least one of the optimized residues above is present. In certain embodiments, the polypeptide comprises an amino acid sequence of at least (>) 99% identity to SEQ ID NO 002, wherein at least one of the optimized residues above is present. In certain embodiments, the polypeptide comprises an amino acid sequence of 100% identity to SEQ ID NO 002, wherein at least one of the optimized residues above is present.

[0098] In certain embodiments, the claimed polypeptide is limited to sequences exhibiting at 30%, particularly at least 50%, more particularly at least 80% of the biological activity of the polypeptide consisting of SEQ ID NO 004, wherein the biological activity is measured for the enzymatic oxidation of benzyl-glucoside according to the assay described below.

[0099] In certain embodiments, at least two of the optimized residues are present in the amino acid sequence of the polypeptide. In certain embodiments, at least three of the optimized residues are present in the amino acid sequence of the polypeptide. In certain embodiments, at least four of the optimized residues are present in the amino acid sequence of the polypeptide. In certain embodiments, at least five of the optimized residues are present in the amino acid sequence of the polypeptide. In certain embodiments, all six of the optimized residues are present in the amino acid sequence of the polypeptide.

[0100] In certain embodiments, Q295 is histidine (H). In certain embodiments, A327 is valine (V). In certain embodiments, A335 is threonine (T). In certain embodiments, P336 is serine (S). In certain embodiments, I357 is valine (V). In certain embodiments, G366 is serine (S).

[0101] In certain embodiments, the amino acid sequence comprises an additional optimized residue: g. A206 is selected from the serine (S) and threonine (T) groups.

[0102] In certain embodiments, the amino acid sequence comprises an additional optimized residue: h. D383 is selected from the group of asparagine (N), glutamine (Q) and serine (S).

[0103] In certain embodiments, the amino acid sequence comprises both additional optimized residues in g. and h.

[0104] In certain embodiments, A206 is threonine (T). In certain embodiments, D383 is asparagine (N). In certain embodiments, the polypeptide comprises or consists of SEQ ID NO 004.

[0105] In certain embodiments, the polypeptide comprises the following residues: G366S. In certain embodiments, the polypeptide comprises the following residues: G366S, A75T, A206T, Q295H. In certain embodiments, the polypeptide comprises the following residues: G366S, A75T, A206T, Q295H, P336S. In certain embodiments, the polypeptide comprises the following residues: G366S, A75T, A206T, Q295H, P336S, A327V, V416I, K420R. In certain embodiments, the polypeptide comprises the following residues: G366S, A75T, A206T, Q295H, P336S, A327V, D222G. In certain embodiments, the polypeptide comprises the following residues: G366S, A75T, A206T, Q295H, P336S, A327V, V416I, K420R, D222G. In certain embodiments, the polypeptide comprises the following residues: G366S, A206T, Q295H, P336S, A327V, A335T. In certain embodiments, the polypeptide comprises the following residues: G366S, A206T, Q295H, P336S, A327V, A335T, I357V, D383N.

[0106] A second aspect of the disclosure relates to an isolated nucleic acid encoding the polypeptide according to the first aspect.

[0107] A third aspect of the disclosure relates to a nucleic acid expression vector comprising a sequence that encodes the polypeptide as described in the first aspect.

[0108] According to some embodiments, the present disclosure relates to a cell comprising a polypeptide as described hereinabove, isolated nucleic acid as described hereinabove, nucleic acid expression vector as described hereinabove or any combination thereof.

[0109] In some embodiments, the cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida.

[0110] According to some embodiments, the present disclosure relates to a method for oxidation of a pyranoside or a furanoside comprising a step of combining the pyranoside or the furanoside with a cell as described hereinabove. The present disclosure is based, in part, on the finding that the use of whole-cell catalysis for the oxidation is an efficient alternative to the use of a free enzyme. According to the present disclosure, when using cells, there is no need for enzyme purification, and the cells are easily removed by centrifugation. In some embodiments, cells are used in free form. In some embodiments, cells are immobilized in alginate as described herein.

[0111] A fourth aspect of the disclosure relates to a method for oxidation of a pyranoside or a furanoside comprising the step:

[0112] a. combining the pyranoside or the furanoside with a polypeptide as described hereinabove in solution.

[0113] In certain embodiments, the pyranoside or the furanoside is unprotected at position C1 and is oxidized at position C2. In certain embodiments, the pyranoside or the furanoside is protected at position C1 and is oxidized at position C3.

[0114] In certain embodiments, the furanoside is oxidized at position C3 or C4. In certain embodiments, the furanoside is protected at position C1, C2 or both, and is oxidized at position C3 or C4. An alternative to the fourth aspect relates to a method for oxidation of a pyranoside or a furanoside, comprising a step of combining the pyranoside or the furanoside with a glycoside 3-oxidase enzyme;

[0115] the pyranoside is protected at position C1 and oxidized at position C3 and the furanoside is protected at position C1, C2 or both and oxidized at position C3 or C4. In certain embodiments, the glycoside 3-oxidase enzyme is selected from the group of:

[0116] a. PsG30x (Acession number A0A024H8G7), AgCarA (Acession number H0QPM2), MtCarA (Acession number A0A0M2HFA3), CarA (Acession number P0DXE4), or ScP2Ox (Acession number A0A117Q443); or

[0117] b. a homologue of PsG30x, AgCarA, MtCarA, CarA, or ScP2Ox; or

[0118] c. a variant of PsG30x, AgCarA, MtCarA, CarA, ScP2Ox, wherein the variant has at least 70% identity, particularly >75%, >80%, >85%, >90%, or >95% identity with a polypeptide sequence of PsG30x, AgCarA, MtCarA, CarA, or ScP2Ox, particularly having a biological activity of at least 30%, at least 50% or at least 80% of a polypeptide of the sequence of PsG30x, AgCarA, MtCarA, CarA, or ScP2Ox. In certain embodiments, the glycoside 3-oxidase enzyme is selected from the PsG30x, AgCarA, MtCarA, CarA, and ScP2Ox groups. The enzymes are further described in Kumano et al. PNAS 2021 or Kostelac et al. Appl Environ Microbiol 2024, which is incorporated by reference herein. The abbreviations refer to FAD-dependent pyranose oxidase (POx) and C-glycoside-3-oxidase (CG30x), CGOx from P. siccitolerans (PsG30x), FAD-dependent C-glycoside-3-oxidase (CGOx, EC 1.1.3.50) from Microbacterium 5-2b (CarA), M. trichothecenolyticum (MtCarA), and Arthrobacter globiformis (AgCarA), POx from Streptomyces canus (ScP20x).

[0119] Glycoside 3-oxidase, according to this aspect of the disclosure, are FAD-dependent glycoside 3-oxidase polypeptide with high specificity for glycoside substrates (Kumano et al PNAS 2021; Taborda et al. Nat Comm. 2023; Kostelac et al. Appl. Environ. Microbiol. 2024). Examples include but are not limited to glycoside 3-oxidase derived from bacterial sources Pseudarthrobacter siccitolerans (PsG30x), S. canus (ScP2Ox), Microbacterium 5-2b (CarA), M. trichothecenolyticum (MtCarA), and A. globiformis (AgCarA).

[0120] In certain embodiments, the glycoside 3-oxidase polypeptide is a Clade I enzyme derived from a species of bacteria in the genus Streptomyces or genus Amycolatopsis, for example, KaPOx derived from Kitasatospora aureofaciens (KaPOx). In other embodiments, it is a variant enzyme at least (>) 70% similar to KaPOx. In certain embodiments, the glycoside 3-oxidase polypeptide is a Clade II glycoside derived from a species of bacteria in the genus Microbacterium, for example, Car A of Microbacterium 5-2b. In other embodiments, it is a variant enzyme at least (>) 70% similar to CarA.

[0121] In certain embodiments, the glycoside 3-oxidase polypeptide is a Clade III enzyme derived from a bacterium in the genus Streptomyces, for example, ScPOx from S. canus. In other embodiments, it is a variant enzyme at least (>) 70% similar to ScPOx from S. canus.

[0122] In certain embodiments, the glycoside 3-oxidase polypeptide is a Clade IV enzyme derived from a species of bacteria in the genus Arthrobacter or Microbacterium, such as PsG30x from P. siccitolerans or MtCArA from M. trichothecenolyticum. In other embodiments, it is a variant enzyme at least (>) 70% similar to PsG30x from P. siccitolerans, or MtCArA from M. trichothecenolyticum. A pyranoside is a monosaccharide that can form a 6-membered ring (out of 5 C atoms and one O atom). A furanoside is a monosaccharide that can form a 5-membered ring (out of 4 C atoms and one O atom).

[0123] The numbering is exemplified here for D-glucose and D-fructose:

[0124]

[0125] According to either embodiment of the fourth aspect of the disclosure, it relates to a pyranoside or a furanoside substrate, which is protected at a position as described hereinabove by linkage to an aromatic group. In certain embodiments, the pyranoside or the furanoside is protected at a position as described hereinabove by a substituted or unsubstituted benzyl or benzoyl group. In certain embodiments, the pyranoside or the furanoside is protected by an unsubstituted benzyl group. Using a benzyl group is very practical, as one can simply evaporate the byproduct and obtain the pure product after cleaving it.

[0126] In some embodiments, a pyranoside or furanoside as described herein is protected at a position as described in the present disclosure. In some embodiments a protecting group as described herein comprises an aromatic moiety. In some embodiments a protecting group as described herein is a protecting group containing an aromatic group. Non-limiting examples of protecting groups as described herein are a substituted or unsubstituted benzyl group and a substituted or unsubstituted benzoyl group. The present disclosure is based, in part, on the finding that an aromatic moiety influences and drives the selective oxidation. In some embodiments, an aromatic moiety influences and drives the selective oxidation at position C3. In certain embodiments, the pyranoside is selected from the glucose, galactose, 2-deoxy-glucose, rhamnose, mannose, fucose and N-acetylglucosamine group.

[0127] In certain embodiments, the furanoside is selected from arabinose, ribose and fructose groups. In certain embodiments, the pyranoside or the furanoside is a D-enantiomer.

[0128] In certain embodiments, the pyranoside is glucose.

[0129] In certain embodiments, the pyranoside is in £ configuration.

[0130] In certain embodiments, the pyranoside or the furanoside is bound to (attached to) a saccharide, particularly via a £-(1-4) glycosidic bond.

[0131] A fifth aspect of the present disclosure relates to a method for obtaining a pyranoside- or a furanoside-epimer comprising the steps:

[0132] b. Performing the method according to the fourth aspect;

[0133] c. In a reduction step, the oxidized pyranoside or oxidized furanoside is reduced at its keto position (carbonyl position).

[0134] In certain embodiments, the reduction step (b) comprises addition of a compound or reagent mixtures selected from the group of NaBH4, NaBH4 / cerium chloride, LiAIH4, DIBAL, LS-selectride, K-selectride, L-selectride, Na(OMe)3BH, K(O-iPr)3BH, NaCNBH3, Na(OAc)3BH, LiBH4, Me2S. BH3, PhNEt2. BH3, NaNH2(BH3)2, NH3-BH3 / TiCI4, pinBH / NaOtBu, Sm / HCl / THF, Red-AI. In certain embodiments, the reduction step (b) comprises the addition of LS-selectride.

[0135] In certain embodiments, the D-pyranoside (educt) is D-glucose, and the pyranoside-epimer (product) is D-allose.

[0136] In some embodiments, the pyranoside is D-galactose, and the pyranoside-epimer is D-gulose. In certain embodiments, additionally the pyranoside or the furanoside is deprotected as a last step of the method.

[0137] A further aspect of the present disclosure relates to the use of polypeptide according to the first aspect for production of an oxidized saccharide.

[0138] A further aspect of the present disclosure relates to the use of polypeptide according to the first aspect for production of a saccharide epimer.

[0139] In certain embodiments, the epimer is D-allose. In some embodiments, the epimer is or D-gulose. Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such options may be combined freely to form discrete embodiments of the present disclosure disclosed herein. Thus, any of the alternative embodiments for an amino acid substitution may be combined with any of the alternative embodiments of a substrate and these combinations may be combined with any protection group mentioned herein.

[0140] The present disclosure is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the present disclosure but not to limit its scope. Description of the Figures

[0141] Fig. 1 shows the lineage of variants of PsG30x generated during the enzyme engineering.

[0142] The variants' names are highlighted in a filled box, and the mutations in the variants' genotypes are listed below. In the initial round, the variant screened with the highest activity, variant 1 A1, emerged with a single mutation (G366S). In the second round, the best variant, 5D5, showcases three additional mutations (A75T, A206T, Q295H). In the third round, the selected best variant, 41 G6, harbored an additional single mutation (P336S). The fourth round yielded two variants, 2G4 and 7E9, with comparable activities, and their genes underwent epPCR followed by DNA shuffling to retain mutually beneficial mutations. At this point, stability screening was introduced simultaneously with activity screening, leading to the selection of variant 2B9, which exhibited four-fold increased stability at 40 °C compared to variant 7E9. DNA shuffling between 2B9 and the wild-type was performed, eliminating putative deleterious mutations while preserving enzyme properties. The resulting variant, 6E3, bearing only six mutations, displayed similar activity and stability to 2B9 with ten mutations. In a final round of epPCR using 6E3 as a template, variant 16F10, due to the replacement of I357V and D383N, exhibited a 1.5-fold activity improvement. All screening details are summarized in Table 7.

[0143] Fig. 2 shows the visual representation and molecular details of the mutations introduced in the hit variant 16F10. (A) Cartoon representation of wild-type PsG30x structure (PDB 7QF8) with all mutations present in 16F10 represented by the Ca as spheres (Fig. 1). All mutations are located distal to FADN5 (B) Detailed view, close to FAD, with D-GIc docked and the two mutations (G366S, Q295H) closer to the active site. Fig. 3 shows chemo-enzymatic synthesis of D-allose from 1 -O-benzyl-P-glucopyranoside.

[0144] a) 1 U mL'1of 16F10 enzyme, 0.1 mg mL'1of catalase at pH 7.5, 25 °C for 6h30min under aerobic conditions, 100% yield; b) LS-Selectride, DMSO: THF (2:1), 0°C, 2h, 86%; c) Pd / C 10%, 50 psi, MeOH, overnight, 94%.

[0145] Fig. 4 shows the distance matrix among the mutations introduced in the hit variant 16F10 and between them and FAD. The distances are colored with black (lower distances) and greyscale (greater distances).

[0146] Fig. 5 shows an example of apparent steady-state kinetics of wild-type and 16F10 variants. Kinetics were obtained for (A) D-GIc, (B) 1-O-Benzyl-a / p-D- glucopyranoside and (C) 1-O-benzyl-P-D-glucopyranoside. The data in black correspond to the wild-type and light grey to the 16F10 variant. All measurements were performed at 37 °C in 100 mM sodium phosphate buffer at optimal pH using the HRP-AAP / DCHBS coupled assay. The dots represent the experimental enzymatic activities measured, and the lines are the fit of the experimental data to the Michaelis-Menten equation using OriginLab software.

[0147] Fig. 6 shows the transglycosylation of benzyl alcohol using disaccharides as donors to obtain 1-O-benzyl-P-D-glucopyranoside 1 and 1-O-benzyl-P-D-galactopyranoside 5. a) 13 U mL'1of almond p-glucosidase, at pH 5, 25 °C for 10 hours; b) 6 U mL'1of A. oryzae p-galactosidase, at pH 5, 25 °C for 6 hours.

[0148] Fig. 7 shows the Mitsunobu benzoylation of glucose and galactose to afford the 1-0- benzoyl protected substrates 6 and 7. a) 1,4-dioxane, benzoic acid, PhsP, diisopropyl azodicarboxylate, 1 hour, room temperature.

[0149] Fig. 8 shows the oxidation of benzyl and benzoyl glycosides 5, 6 and 7 to the corresponding keto products 8-10 using purified PsG30x variant, a) 3 U mL'1of 16F10 enzyme variant, 0.1 mg mL'1of catalase at pH 7.6, 25 °C under aerobic conditions.

[0150] Fig. 9 shows the oxidation of model substrate 1 catalysed by whole cells producing PsG30x (variant 16F10) in 50 mM phosphate buffer pH 7.6, room temperature and 500 rpm. a) 104 mg / ml wet cells of 16F10 enzyme variant, pH 7.6, 25 °C under aerobic conditions.

[0151] Fig. 10 shows the regioselective reduction of keto derivatives 8-10 using LS-Selectride®, leading to reduced products 11, 12, 13 and deprotection using hydrogenation for benzyl glycosides and hydrolysis catalysed by lipase for benzoylated derivatives affording rare sugars 4 and 14. a) LS-Selectride, DMSO: THF (2:1), 0°C, 2h, b) Pd / C 30%, 60 psi, EtOH, 48 hours, c) 10 mg Burkholderia cepacian lipase, pH 8, 50 °C, 24 hours.

[0152] Description of the Tables

[0153] Table 1 shows optimal pH and apparent steady-state catalytic parameters for D-GIc. Activity was measured using the HRP-AAP / DCHBS coupled assay. All reactions were performed in 100 mM sodium phosphate buffer at the optimal pH and 37 °C. The kinetic parameters were determined by fitting the data directly on the Michaelis- Menten equation using OriginLab.

[0154] Table 2 shows a comparison of activities between 16F10 and wild-type for a range of substrates. Activity was measured using the HRP-AAP / DCHBS coupled assay. All reactions were performed with 100 mM substrate in 100 mM sodium phosphate buffer at pH 7.5 and 25 °C. Nd - not detected Table 3 shows a summary of the stability properties of the evolutionary intermediates. Apparent melting temperatures (TmApp) were measured by steady-state fluorescence following the intrinsic fluorescence of the enzyme, aggregation temperature (Tagg) was monitored by static light scattering and half-life time at 40 °C (ti / 240°c) was measured by following the loss activity over time. The activity at each time point was measured using the HRP-AAP / DCHBS system.

[0155] Table 4 shows apparent steady-state catalytic parameters for D-GIc and 1-O-benzyl- glucoside. Activity for D-GIc, 1-O-benzyl-a / p-D-glucopyranoside and 1-O-benzyl-£- D-glucopyranoside was measured using the HRP-AAP / DCHBS coupled assay. All reactions were performed in 100 mM sodium phosphate buffer at the optimal pH and 37 °C. The kinetic parameters were determined by fitting the data directly on the Michaelis-Menten equation using OriginLab.

[0156] Table 5 shows reaction conditions for the stereoselective reduction of 1-O-benzyl-3-keto-£- D-glucopyranoside 2. All reactions were performed at 0°C. The ratio of diastereomers 1 and 3 obtained after reduction with LS-Selectride was determined by1H NMR.

[0157] Table 6 shows the bacterial strains, plasmids, and primers used in this study. F indicates forward primers, and R is reverse primers.

[0158] Table 7 shows a summary of the variants obtained by directed evolution. The concentration of the mutagenic agent (MnC ) in the epPCR is mentioned for each round of evolution performed. The concentration of substrate (D-GIc) used in the screenings, the number of variants screened in both applied screening approaches (‘Activity- on-plate / 96-well plate screenings) and the relative activity and stability values to the respective parent are presented. N. D. means not determined.

[0159] Table 8 shows a summary of production yields for all directed evolution intermediates. The Bradford method was used to determine the total concentration of the enzyme. Functional enzyme concentration was quantified by absorbance at 450 nm.

[0160] Table 9 shows the apparent steady-state catalytic parameters for D-GIc in pyranose oxidases described in the literature, nd - not detected.

[0161] Table 10 shows a summary of the stability properties of the evolutionary intermediates.

[0162] Apparent melting temperatures (TmApp) were measured by steady-state fluorescence following the intrinsic fluorescence of the enzyme, aggregation temperature (Tagg) was monitored by static light scattering and half-life time at 40°C (ti / 240°c) was measured by following the loss activity over time. The activity at each time point was measured using the HRP-AAP / DCHBS system. Table 11 shows optimal pH and apparent steady-state catalytic parameters for D-GIc of variants where mutation P336S was inserted (wild-type) or removed (2G4, 7E9 and 2B9) considered in this study. Activity was measured using the HRP-AAP / DCHBS coupled assay. All reactions were performed in 100 mM sodium phosphate buffer at the optimal pH and 37 °C. The kinetic parameters were determined by fitting the data directly on the Michaelis-Menten equation using OriginLab.

[0163] Table 12 shows optimal pH and apparent steady-state catalytic parameters for D-GIc variants constructed using site-directed mutagenesis to assess the role of mutations G366S, A75T, A206T and Q295H. Activity was measured using the HRP-AAP / DCHBS coupled assay. All reactions were performed in 100 mM sodium phosphate buffer at the optimal pH and 37 °C. The kinetic parameters were determined by fitting the data directly on the Michaelis-Menten equation using OriginLab.

[0164] Table 13 summarizes the methods used for reactions involved in allose synthesis, r.t. - room temperature; n.i. - not indicated; n.d. - not detected. * The Izumoring strategy was extensively used for the biological production of D-allose from D-allulose (D- psicose) or D-fructose using one or two enzymes, respectively, produced by different systems and under various conditions. D-altrose (or other sugars) are obtained as byproducts. However, as D-allose was not isolated, it was impossible to determine the yield of the process, and it was not easy to quantify the presence and amount of byproducts. These byproducts are epimeric sugars, which are difficult to separate from allose by chromatographic methods.

[0165] Examples

[0166] Example 1: Laboratory evolution of PsG30x and kinetic characterization

[0167] Directed evolution allows for fine-tuning enzyme properties, yielding tailored enzymes with superior performance while circumventing the potentially ineffective rational design. The promiscuous activity of the PsG30x for various monosaccharides and C-glycosides (Taborda et al., 2023 ibid; Mendes etal., J Mol Catal B Enzym, 133, 2016) indicates that this enzyme can serve as the starting point (template) for an enzyme engineering strategy to enhance its catalytic efficiency towards D-glucose (D-GIc) and other mono and disaccharides. Directed evolution using error-prone PCR (epPCR) and DNA shuffling techniques followed by activity screening for D-GIc (Table 7). After seven iterative rounds of directed evolution, approximately 50,000 variants were screened, and one hit variant (16F10) was identified with improved activity for D-GIc (Fig. 1 and Table 1).

[0168] All enzyme variants were purified (Table 8), and the percentage of the functional enzyme (i.e., with a full complement of FAD) ranged from 20 to 60 %. The optimal pH (7.5) of wild-type for D-GIc was up-shifted by 0.5 to 1 unit in the second round (variant 5D5). The catalytic performance of the evolutionary intermediates and the hit variant was assessed. The inventors show that throughout the engineering process, the turnover number (kcat) significantly improved, representing the most notable difference in catalytic parameters, positively impacting the catalytic efficiency (kcat / Km) observed for D-GIc among the variants in the later generations (Table 1). In contrast, the Km, a parameter associated with substrate affinity, remained unchanged during the evolutionary pathway. Previous studies have emphasized that these enzymes' active site topology and oligomeric state are key factors affecting substrate specificity within P2Oxs (tetramer) / G3Oxs (monomer) subfamilies despite their high sequence similarity. Indeed, despite the observed improvements in variants of PsG3Ox, the kcat for D-Glc of the hit variant 16F10 remains 10- to 20-fold lower than those observed for D-Glc in fungal P2Oxs such as TmP2Ox and PcP2Ox, whereas the Km value is three orders of magnitude higher (Table 9).

[0169] The activity of variant 16F10 was also tested against a diverse array of substrates, including different mono-, di- and trisaccharides, as well as one aminosugar, D-glucosamine (Table 2) and remarkably, it exhibited increased activity when compared to the wild-type for most of the tested substrates. The highest enhancements were measured for D-maltose and D-galactose, with activity increasing ~ 35-fold compared to the wild-type; D-GIc and lactose followed, demonstrating around a 20-fold increased enzymatic activity.

[0170] Example 2 Thermostability of variants

[0171] Thermal unfolding profiles were performed to calculate the mid-point thermal temperatures, i.e., melting temperatures (Tm). The unfolding is described as a two-state process where the folded and unfolded states seem to be the only ones that accumulate significantly (Fig S1). All variants exhibited slightly lower melting temperatures than the wild-type (49°C); for example, the hit variant showed a Tmaround 44 °C (Table 3). However, protein aggregation, assessed by static light scattering (data not shown), reveals the onset of aggregation (Tagg) at similar temperatures. Since the aggregation process irreversibly removes unfolded proteins from the equilibrium between the folded and unfolded states, it may significantly affect the melting temperature measurements, leading to its designation as an apparent value, TmApp.

[0172] Regarding the kinetic, so-called, long-term or operational stability, which quantifies the amount of enzyme that loses activity irreversibly during incubation at a specific temperature, the first two variants (1A1 and 5D5) exhibited a remarkable 10-fold improvement in the stability as compared to wild-type (27 min at 40 °C). In contrast, variant 41 G6, in the 3rd round, showed a substantial tradeoff between activity and stability, with a slight increase in kcat(Table 2) but at a high cost of kinetic stability: ~100-fold decrease in half-life time (5 min at 40 °C) (Table 3). The kinetic stability quantifies the amount of enzyme that denaturates irreversibly owing to protein aggregation, misfolding, and covalent changes. (Sanchez-Ruiz et al., Biophys Chem, 148(1-3), 2010) The wildtype and different variants deactivate according to a first-order process, which the classical Lumry can describe-Eyring model applied to the majority of enzymes (N - - U <-> D, where N, U and D are the native, the reversible unfolded, and the irreversible denatured enzyme), pointing to a simple pathway of unfolding and denaturation. Since this effect was due to a single mutation, P336S, The inventors have introduced and removed this mutation in different genetic backgrounds. The replacement of P336S in wild-type has not affected activity but led to a pronounced decrease in the half-life time at 40 °C (Tables 11 and 10); in contrast, its removal from subsequent variants 2G4, 7E9, and 2B9 (4th and 5th round) result in a significant increase in stability but at expenses of a 2-fold decreased activity (Table 11 and 10). These observations suggest that P336S has influenced the evolutionary trajectory of the enzyme towards improved activity for D-glucose and indicate that it exhibits genotype-dependent effects, hinting at epistatic interactions. (Miton et al., Curr Opin Struct Biol, 69, 2021) The mutations introduced in subsequent rounds of evolution compensated for the negative impact of P336S, and all subsequent variants show increased stability, retaining 50% of activity after 80 to 290 min at 40 °C (Table 3). It is important to mention that during the first four generations, the screening of variant libraries during the directed evolution was based solely on activity measurements, which led to the selection of variant 41 G6 with unexpectedly low kineticstability after purification. From the fourth generation onward, the inventors simultaneously screened for activity and stability, selecting only those variants that performed well in both properties. Notably, as mentioned, the hit variant 16F10 exhibited a kcat approximately 20-fold higher and a half-life time at 40 °C, almost 10-fold higher than the wild-type enzyme (Table 1 and 3).

[0173] Example 3: Molecular details of variants

[0174] All mutations present in the hit variant 16F10 and introduced during the evolution are distal to the FADN5 (>10 A) (Fig. 2A, Fig. 4). It is now well known that mutations located distantly from the active site can also enhance enzyme performance through allosteric effects, thereby inducing changes at the active site via a network of interactions. By the combination of site-directed mutagenesis and the analysis of enzyme structures, the inventors hypothesize that the improvement of enzyme activity resulted from the following mutations: G366S (1st round), Q295H (2nd round), P336S (3rd round), A327V (4th round), A335T (5th round), and I357V (7th round); excluding mutations D383N and A206T (Fig 2A). The visual inspection of the enzyme structure with D-GIc docked in a catalytic competent orientation (PDB 7QFD) (Taborda et al., 2023 ibid) reveals that the G366S mutation is nearby (~ 3.4 A) to the C4-OH group of D-GIc, arguably forming an additional hydrogen bond through Ser-OH group (Fig. 2B). This mutation is also close to Q297 (~ 2.3 A) and Q340 (~ 5.1 A), which are critical residues for the substrate orientation in the active (Taborda et al., 2023 ibid). Mutation Q295H is at ~ 4.0 A to the catalytic residue H440 and also close to Q297 (Fig. 2B). Single mutants A75T, A206T, and Q295H (introduced in the 2nd round) were constructed whereas A75T and A206T showed similar catalytic properties to wild-type, revealing its minor or null role, the mutation Q295H exhibited a 2-fold higher kcat (Table 12). The type of amino acid transition Q / H can conceivably change the microenvironment surrounding the residue: the optimal pH (7.5) of wild-type for D-Glucose was up-shifted by 0.5 to 1 unit in the second round (variant 5D5), most likely due to the introduction of the Q295H mutation. Interestingly, the combination of Q295H with G366S exhibits an increased activity than the activity sum of individual variants and attains the activity of variant 5D5 (Table 12). These results demonstrate that the effect of these mutations (Q295H with G366S) is also dependent on the genetic background, i.e., of epistatic interactions (Miton et al., 2021 ibid), previously mentioned concerning P336S (Table 11). This residue is positioned in a loop interconnecting β-strands with residues of the active site, and its introduction has caused a drastic decrease in kinetic stability (Table 3), likely due to increased loop flexibility (due to the removal of the rigid proline), resulting in a more rapid and irreversible denaturation. The introduction of the adjacent A335T mutation in variant 2B9 may have counteracted the destabilizing effect of the P336S, as observed by the increased half-life at 40 °C (from 100 minutes to 290 minutes; Table 3). A327V mutation is close to the β-strand where the substrate-interacting Q340 (at ~ 6 A) belongs and can supposedly induce changes in the positioning or dynamics of this non-catalytic residue. Lastly, I357V is in a critical structural element, the substrate loop. This loop can adopt different conformations throughout the catalytic cycle (Taborda et al., 2023 ibid) and replacing a less hydrophobic residue may presumably modulate loop dynamics, impacting catalysis.

[0175] Example 4: Chemo-enzymatic synthesis of D-allose using the engineered enzyme

[0176] D-allose synthesis involves the regioselective oxidation of D-GIc at the C3 position. Therefore, the inventors have synthesized protected 1-O-benzyl-a / p-D-glucopyranoside (mixture cd 2:1) and tested the enzymatic activity of wild-type and engineered variant 16F10, which shows higher selectivity for C3 over C2 (Table 4; Fig. 5). Comparing the catalytic parameters obtained, 16F10 showed a 10-fold increase in the turnover number (kcat). At the same time, the Km remained unchanged as compared to the wild-type enzyme. Interestingly, regarding the Km values, a 25-fold decrease was observed for the substrate 1-O-benzyl-a / p-D-glucopyranoside compared to D-GIc (Table 4). These results are consistent with previous findings, which highlight the crucial role of an aromatic aglycone in facilitating substrate access and binding to the active site. (Taborda et al., 2023 ibid) The oxidation product of 1-O-benzyl-a / p-D-glucopyranoside was characterized by NMR, confirming the presence of the ketone at C3. However, a partial conversion (36% yield) was obtained, even after 24 h reaction. Interestingly, the p-anomerwas the predominant isomer present in the NMR spectra, which indicated some stereoselectivity for this isomer. Therefore, the p-isomer (1-O-benzyl-P-D-glucopyranoside) was synthesized (Ye et al., Eur J Med Chem, 264, 2024), and the enzyme's activity was tested towards this substrate. The obtained catalytic parameters confirmed that the p-anomer is more efficiently oxidized than the a-anomer, as observed by the increase of the kcat parameter and also reflected in catalytic efficiency (kcat / Km) (Table 4). A bioconversion reaction using the 1-O-benzyl-P-D-glucopyranoside 1 as substrate (20 mg of the substrate in a volume of 1.5 mL) was performed, and a total conversion (100% yield) was obtained, affording 1-O-benzyl-3-keto-P-D-glucopyranoside 2 (Fig. 3) with complete regioselectivity and without the need for further purification. The following step involved the stereoselective reduction of the resulting compound 2 to afford 1-O-benzyl-P-D-allopyranoside 3 (Fig. 3). The allose-derived product has an inverted configuration at the C3 position compared to D-GIc, with the 3-OH group positioned cis to the vicinal 2-OH and 4-OH groups. Different reducing agents were studied to obtain the best yield and cis selectivity (Table 5).

[0177] Reduction with NaBH₄ afforded a mixture of the two diastereomeric alcohols 1 and 3 with very low selectivity towards the cis (alloside 3) product. K- and LS-Selectride provided exclusively the corresponding C3 c / s-alcohol 3 (Fig. 3); however, LS-selectride afforded the highest yield (86%, Table 5). The high 1,2-c / s stereoselectivity of Selectride is well documented. It is attributed to the steric bulk of the reducing agent-inducing attack at the side opposite to a vicinal group in cyclohexanones, which normally forms the axial alcohol predominantly. (Jumde et al., J Org Chem, 81 (22), 2016; Brown et al., J Am Chem Soc, 94(20), 1972; Krishnamurthy et al., J Am Chem Soc, 98(11), 1976) To finally obtain D-allose, the removal of the anomeric benzyl protecting group was easily accomplished by hydrogenation, affording exclusively D-allose 4 (Fig. 3) in 94% yield, without the need for purification. In conclusion, a new efficient regio- and stereoselective chemo-enzymatic method was developed to synthesize the rare sugar D-allose in 81 % overall yield.

[0178] Example 5: Optimization of the protection strategy for benzyl-protected substrates and synthesis of benzoyl-protected monosaccharides

[0179] Using disaccharides as glycoside donors reduces the expenses and eases the large-scale application of the process. Therefore, a biocatalytic method utilizing cellobiose and the enzyme β-glucosidase was first employed to enhance the D-GIc protection strategy, enabling the synthesis of benzyl-P-D-glucupyranosides in high yields under mild, aqueous conditions, thereby reducing both energy input and solvent waste. Benzyl alcohol was used as an acceptor in the glycosylation reaction and subsequently recycled. Transglycosylation using disaccharides has been successfully applied in the one-step synthesis not only to synthesize 1-O-P-benzyl-glucoside 1 but also 1-O-β-benzyl-galactoside 5, when lactose, instead of cellobiose, was used as substrate (Fig. 6). The compounds 1 and 5 were successfully isolated in 24% and 26% yields and are substrates for variant 16F10. Compound 1 serves as a precursor of D-allose, whereas compound 5 is the starting material for the synthesis of D-gulose, another important rare sugar. Alternatively, Mitsunobu benzoylation of glucose and galactose was used to afford compounds 6 and 7 carrying benzoyl protecting groups instead of benzyl protecting groups (Fig. 7). Compound 6 was synthetized in 74% yield (the p anomer was the major product (a:p 1:50) and compound 7 was obtained in 16% yield.

[0180] Example 6: Enzymatic oxidation of glycosides 5, 6 and 7 with engineered PsG30x Oxidations (Fig. 8) were performed with substrates 5, 6 and 7 using the variant 16F10. Reactions were carried out with 3 U mL'1of purified enzyme (Fig. 8). Complete conversion was achieved within 24 - 29 h at room temperature. The oxidised glycosides were isolated in high purity and used for downstream transformations. However, the oxidation product 8 exhibited limited stability and was prone to hydration in aqueous solution. NMR analysis confirmed the presence of both hydrated and non-hydrated forms of the oxidised species at C3. Accordingly, the product was either stored under frozen conditions or used immediately in subsequent reduction reactions. When substrate 6 was subjected to identical oxidation conditions as 5, complete conversion was also observed within 24 h. Overall, these studies demonstrate the enzymatic system is highly effective for the selective oxidation of benzyl glycosides derived from glucose and galactose (substrates 1 and 5), and moreover can oxidise the benzoylated glucose derivative 6 with high regioselectivity and efficiency.

[0181] Example 7 Oxidation using whole cell overproducing 16F10 variant

[0182] Whole-cell catalysis offers several advantages, such as increased stability of the enzyme, which eliminates the need for enzyme purification, thereby reducing operational costs and increasing robustness, ultimately simplifying the entire process. Tested conditions (40 mM 1, 20 OD₆₀₀nm of cells (104 mg mL'1of WCW) in 20 mM sodium phosphate buffer, pH 7.6, in a total volume of 10 mL) were sufficient for the complete oxidation of model substrate 1 in 4 h with a relatively high yield of 81 % of 2. When the reaction was completed, cells were easily removed by membrane filtration. The crude reaction underwent purification, and the obtained product was subjected to stereoselective reduction and deprotection, yielding 79% of the final product, 4. Hence, the whole cell catalysis proved to be a cost-effective alternative. Whole cell reactions were not possible to perform for the oxidation of 5 due to lacZ gene present in the used vector. Due to this, benzoylated sugar derivatives 6 and 7 seem as proper candidates for whole cell catalysis. Oxidation of 6 was conducted successfully with 62 % yield of oxidized product 9, which was a noticeably improvement in comparison with purified enzyme catalysis with a promising applicability.

[0183] Example 8: Selective reduction and deprotection of compounds 8, 9 and 10 yielding rare sugars The stereoselective reduction of oxidized benzyl glycosides was performed following the conditions previously reported for substrate 2, which afforded the corresponding reduced product in high yield and with excellent stereoselectivity. These earlier results served as a benchmark for comparison with newly tested substrates. For compound 8, the reduction proceeded under analogous conditions (Fig. 10), but the isolated yield of compound 11 was considerably lower (41%). This reduced efficiency is likely due to the lower stability of substrate 8, which leads to partial decomposition before or during the reduction step. Following reduction, deprotection of the intermediate product 11 was successfully achieved, affording the D-gulopyranose 14 in 79% yield. Ester protected oxidation product 9 was effectively reduced to 12 in 45% yield, using LS-Selectride® under the same reaction conditions. The reducing agent was able to simultaneously deprotect the benzoylated substrate leading to 47% of 4 (Fig. 10). For the further deprotection of 12 hydrolysis with amano lipase (Burkholderia cepacia) was performed, affording D-allopyranose 4 in additional 48 % yield. Example 9: Discussion

[0184] Carbohydrates are central biomolecules in numerous biological fields, with their natural availability highly dependent on their chemical structure. Low-abundance sugars, often called 'rare sugars,' hold significant potential as nutraceuticals and food additives, yet their synthesis poses some challenges. Carbohydrate oxidases, with their high regio- and stereospecificity, enable the selective oxidation of specific hydroxyl groups in carbohydrates into carbonyl groups. They use molecular oxygen as a clean oxidant and produce hydrogen peroxide as a by-product. Traditional methods often involve lengthy synthetic routes and complex purification processes, and they can yield low amounts or mixtures of products. The novel chemo-enzymatic approach developed in this study efficiently synthesizes D-allose from minimally protected D-glucose, achieving an overall yield of 81% using an engineered glycoside-3-oxidase variant that catalyzes the initial regioselective oxidation at the C3 of the substrate. Diastereoselective chemical reduction, followed by subsequent hydrogenolysis, yielded the D-allose sugar. This chemo-enzymatic strategy is efficient and rapid and facilitates the isolation of pure products in excellent yields with minimal and straightforward product isolation procedures. The method was also adapted to synthesize other C3 epimers, such as the rare sugars D-gulose starting from protected D-galactose. Additionally, the 3-keto group formed during the process offers opportunities for further functionalization into various derivatives, an area of ongoing research.

[0185] Example 10: Material and Methods

[0186] Bacterial strains, plasmids and cultivation medium

[0187] Escherichia coli strains, plasmids, and primers used in this work are summarized in Table 6. E. coli strain DH5a (Novagen) and E. cloni 10G elite (Lucigen corporation-Biosearch technologies) propagated and amplified plasmid constructs. E. coli Rosetta pLysS (DE3, Novagen) was used to express the wild-type PsG30x and its variants cloned in the pET-15b plasmid (Novagen). The library screenings used E. coli KRX (Promega) as an expression strain. Luria Bertani medium (LB) was used for cell cultivation, supplemented with 100 pg mL'1of ampicillin (NZYTech) and, in the case of Rosetta pLysS, also with 20 pg mL'1of chloramphenicol (NZYTech, Lisbon, Portugal). Construction of mutant libraries by error-prone PCR

[0188] The error-prone PCR (epPCR) of the psg3ox gene was performed using the primers PsG30x_GA_FW and PsG30x_GA_RV (Table 6) in a total volume of 50 pL reaction containing 100 ng of DNA template, 1 pM of each primer, 200 pM of dNTPs, 1.5 mM MgCI2, Taq polymerase buffer, 50 pM (or 100 pM) MnCI2 and 2.5 U of Taq polymerase (Thermo Scientific). The PCR program included an initial denaturation of 2 min at 95°C followed by 30 cycles of 1 min at 95°C, 1 min at 69°C and 2 min at 72°C and a final step of 10 min at 72°C. and purified using lllustra GFX PCR DNA kit (GE Healthcare). The PCR of the vector (pET-15b) was performed with the primer pET15b_GA_FW and pET15b_GA_RV (Table 6). The PCR was performed in a final volume of 50 pl reaction containing 50 ng of DNA template (pET-15b), 0.5 pM of each primer, 200 pM of dNTPs, 1 U of Q5 High-fidelity DNA polymerase (New England Biolabs) in a program that include an initial denaturation of 30 sec at 98°C followed by 30 cycles of 10 sec at 98°C, 30 sec at 71 °C and 3min at 72°C and a final step of 2 min at 72°C. The PCR product was purified. A digestion of 6 h at 37 °C with Dpnl (New England Biolabs) was performed to avoid the presence of the DNA template in the PCR products. The ligation was performed with a ratio gene: vector 1:10 using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). The ligation was performed at 50 °C for 1 h, and afterward, it was used to transform E. cloni 10 G elite cells, allowing the propagation of the library. The plasmid library was extracted to transform electrocompetent KRX cells in a Gene Pulser Xcell™ (Bio-Rad Laboratories) under defined conditions (C = 10 pF, PC = 600 Q, V = 1.8 kV). The cell suspension was spread on solid media containing 100 pg mL'1of ampicillin and 0.1 % rhamnose.

[0189] Construction of DNA shuffling libraries

[0190] The amplification of the psg3ox genes selected to be shuffled was conducted using the primers pET21_FW and pET21_RV (Table 6). PCR proceeded in a program that include the following steps: 5 min at 95°C followed by 20 cycles of 1 min at 95°C, 1 min at 55°C, 2 min at 72°C and a final step of 10 min at 72°C. The DNA from each amplified gene variant was pooled in the same ratio, totalizing 1400 ng and digested with DNase I (0.15 U mL'1) in 20 pL of 200 mM Tris-HCI, pH 7.0, with 80 mM MnCI2 at 15°C for 10 min and was stopped by adding 6 pL of 0.5 M of EDTA. The obtained fragments were analyzed in 1 % agarose gel, dialyzed using a nitrocellulose membrane and subsequently reassembled in a primerless PCR containing 5 pL of DNA fragments, 10 pM of dNTPs, and 0.5 U of NZYProof DNA Polymerase. The PCR was performed in a program that start with 3min at 96 °C followed by 45 cycles of 1 min 94 °C, 1.5 min 59 °C, 1.5 min 56 °C, 1.5 min 53 °C, 1.5 min 50 °C, 1.5 min 47 °C, 1.5 min 44 °C, 1.5 min 41 °C and 1 min + 5s / cycle at 72 °C, and finalized with an extention of 10 min 72°C. The final PCR amplification was performed using the primers PsG30x_GA_FW and PsG30x_GA_RV (Table 6). The amplification was carried out in a total volume of 20 pL, including 1 pL of the reassembled product and with similar conditions and program than the 25 pM of each primer, 10 mM of dNTPs, 200 pM of buffer, and 0.5 U of NZYProof DNA Polymerase. The PCR program included an initial denaturation of 2 min at 95°C followed by 30 cycles of 1 min at 95°C, 1 min at 69°C and 2 min at 72°C and a final step of 10 min at 72°C. The PCR products were purified using the previously mentioned kit from the agarose gel. The resulting product was ligated in the vector backbone described above for constructing mutant libraries by epPCR, transformed in E. cloni 10 G elite cells to propagate the library and transformed in KRX strain for further screening after plasmid extraction.

[0191] Library screenings

[0192] For the 'Activity-on-plate' screening, the colonies resulting from the transformation in KRX strain were transferred onto Whatman chromatography paper, and the plates were re-incubated at 37 °C until the colonies reappeared. Subsequently, the filter papers were soaked in a solution of 20 mM Tris-HCI, pH 7.6, and subjected to 3 cycles of freezing (40 min at -20 °C) followed by thawing (20 min at 37 °C). Following the lysis cycles, the filter papers were soaked in a mixture containing 1 mM 4-Aminoantipyrine (AAP, Acros Organics), 10 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid sodium salt (DCHBS, Alfa Aesar), 8 U mL'1horseradish peroxidase (HRP, Sigma-Aldrich), and 0.5 M D-glucose (D-GIc, PanReac Applichem) in 100 mM phosphate buffer, pH 7.5. The active variants were identified by developing the pink chromogenic compound N-(4-antipyryl)-3-chloro-5-sulfonate-p-benzoquinone-monoimine. The active variants were picked to 96-deep well plates (VWR) containing 400 pl of LB supplemented with the appropriate antibiotic and left to grow overnight at 37 °C with agitation at 750 rpm (pre-culture). New 96-deep well plates, containing 900 pL of LB supplemented with ampicillin, were inoculated with 100 pl of the pre-culture and incubated for 3 h at 37 °C, 750 rpm. Next, the expression of the target gene was induced with 10 mM of rhamnose, and cultures proceeded overnight at room temperature at 750 rpm. Afterwards, the cells were harvested by centrifugation at 4000 rpm (Eppendorf 5810 R centrifuge). The 96-well plates containing the cell pellets underwent three lysis cycles by submersion in liquid nitrogen and thawing at room temperature for 10 minutes. Then, the pellets were resuspended in 200 pl of 20 mM Tris-HCI buffer, pH 7.6, supplemented with 2 mg mL'1of lysozyme, 1 U mL'1DNase, 5 mM MgCh, and protease inhibitors cocktail (3.5 pM antipain and 5 pM leupeptin). This suspension was incubated for 20 minutes at room temperature under agitation at 750 rpm. Subsequently, the lysate was clarified by centrifugation at 4000 rpm. The supernatants (cell crude extracts) were collected for enzymatic activity assays in a reaction mixture containing 0.1 mM of AAP, 1 mM of DCHBS, 8 U mL'1of HRP and 50-500 mM of D-GIc (depending on the round of evolution) in phosphate buffer pH 7.5 at 25 °C. The formation of a pink chromogen at 515 nm (£515= 26 000 M'1cm'1) was measured using a Synergy2 microplate reader (BioTek).

[0193] Site-directed mutagenesis

[0194] The primers used to construct SDM variants are listed in Table 6. PCRs were performed in a thermal cycler in 50 pL reaction volumes containing 100 ng of DNA template, 1 pM of primers, 200 pM of dNTPs (NZYTech), and 1.5 U of NZYProof polymerase (NZYTech). The PCR program included an initial denaturation of 4 min at 95°C followed by 20 cycles of 1 min at 95 °C, 1.5 min at 68 °C and 8 min at 72 °C and a final step of 10 min at 72 °C. The amplified product was digested, purified, and transformed into a cloning strain. DNA sequencing confirmed the presence of the desired mutation(s).

[0195] PsG30x production, purification and kinetic characterization

[0196] The production and purification of PsG30x variants were performed as described previously. (Taborda et al., 2023 ibid) The optimal pH was determined using the Britton-Robinson buffer system (100 mM phosphoric acid, 100 mM boric acid and 100 mM acetic acid mixed with 1 M NaOH to the desired pH) in the pH range 5 to 9. The reaction mixture contains 0.1 mM of AAP, 1 mM of DCHBS, 8 U mL'1of HRP and 0.3 M of D-GIc. The activity was followed spectrophotometrically at 515 nm, as mentioned above. For steady-state kinetics, the reaction mixture contained 0.1 mM AAP, 1 mM DCHBS, 8 U mL'1HRP and 0-1.5 M of D-GIc (or 0-50 mM of 1-O-benzyl-D-glucopyranoside or 0-90 mM of 1-O-benzyl-D-galactopyranoside) in 100 mM sodium phosphate buffer at the optimal pH and 37 °C. The activity was followed spectrophotometrically at 515 nm. Apparent steady-state kinetic parameters were determined by fitting data directly into the Michaelis-Menten equation using Origin-Lab software. All catalytic parameters were calculated using three independent assays in triplicate. The total protein concentration was determined by Bradford assay using bovine serum albumin as standard. Purified preparations' absorption at 450 nm (E450nmFAD = 11 300 M'1cm’1) was measured to assess the functional fraction of enzyme preparations (e.g., for kinetic measurements).

[0197] Substrate screening

[0198] The substrate screening was performed with 100 mM of D(+)Glc (PanReac ApplieChem), D(+)galactose (Sigma-Aldrich), L(+)arabinose (PanReac ApplieChem), 2-deoxy-D-glucose (Sigma-Aldrich), D(-)ribose (VWR), L(+)rhamnose (PanReac ApplieChem), D(+)mannose (Alfa Aesar), D(-)frutose (Sigma-Aldrich), L(-)fucose (Carl Roth), D-lactose (Sigma-Aldrich), D(+)maltose (Sigma-Aldrich), D(+)trehalose (Sigma-Aldrich), D(+)raffinose (Alfa aesar) and D(+)glucosamine (Sigma-Aldrich) in a reaction mixture containing 0.1 mM AAP, 1 mM DCHBS, 8 U mL'1HRP in 100 mM sodium phosphate buffer at the optimal pH and 25 °C. The activity was followed spectrophotometrically at 515 nm, as mentioned above.

[0199] Stability of enzyme variants

[0200] The thermostability of the enzymes was determined using steady-state fluorescence in a Cary Eclipse spectrofluorometer (Agilent Technologies). Enzyme preparations of 0.2 mg mL'1in 20 mM Tris-HCI pH 7.6 with 0.2 M NaCI were placed in a thermostatically controlled block, and a temperature gradient with an increment of 1 °C min'1was applied up to 70 °C. Structural denaturation was monitored using an excitation wavelength of 296 nm, and the emission was recorded at 340 nm. Protein aggregation was monitored by measuring static light scattering at 500 nm as excitation and emission wavelengths. A pure enzyme preparation was incubated at 40 °C for the kinetic stability assays. Aliquots were withdrawn over time, cooled on ice, and assayed for activity in a reaction containing 0.5 M of D-GIc, 0.1 mM of AAP, 1 mM of DCHBS, 8 U mL'1of HRP in 100 mM sodium phosphate buffer, pH 7.5. The half-life time, corresponding to the time required for the enzyme to lose half of its initial activity, was estimated using the formula t1 / 2= ln(2) / k (where k represents the linear regression slope between In(activity) and time).

[0201] Enzymatic oxidation of benzyl-glucoside by 16F10 variant

[0202] The compounds 1-O-benzyl-a / p-D-glucopyranoside and 1-O-benzyl-P-glucopyranoside were synthesized from D-GIc using a reaction procedure described in the literature. The enzymatic oxidation of benzyl-glucoside was performed using 20 mg of 1-O-Benzyl-a / p-D-glucopyranoside, 5 U mL'1of the 16F10 variant and 0.1 mg mL'1of catalase (Sigma-Aldrich) in a total volume of 1.5 mL in water (adjusted to pH 7.5-8.0). The reaction proceeded for 24 h at 25 °C under magnetic stirring to facilitate oxygenation of the solution. The reaction progress was monitored by TLC in Merck 60 F254 silica gel plates (90 / 10 ethyl acetate / methanol). At the end of the reaction time, the enzymes were removed by ultrafiltration using a 30 kDa Amicon (Merck) at 4000 rpm in an Eppendorf centrifuge. The reaction product was lyophilized and purified by flash column (Kieselgel 60, 0.032-0.063mm) with 100% ethyl acetate to afford 1-O-benzyl-3-keto-a / p-D-glucopyranoside (2a and 2P; 1.4:1 ct / p) in 36% yield as a viscous colourless syrup.1H NMR (400 MHz, MeOH-d4): 5 7.46-7.27 (m, 10H, Ar-H), 5.28 (d, J = 4.40 Hz, 1H, H1-a), 5.00 (d, J = 11.97 Hz, 1H, CH2-Bn), 4.76 (dd, J = 12.09 Hz, J = 3.00 Hz, 2H, CH2-Bn), 4.61 (d, J = 11.92 Hz, 1H, CH2-Bn), 4.48 (d, J = 7.94 Hz, 1 H, H 1 -p), 4.45 (dd, J = 4.38 Hz, J = 1.24 Hz, 1 H, H2-a), 4.29-4.25 (m, 2H, H4-a and H4-p), 4.22 (dd, J = 7.90 Hz, J = 1.65 Hz, 1H, H2-p), 3.99 (dd, J = 12.20 Hz, J = 2.03 Hz, 1H, H6-p), 3.88-3.79 (m, 3H, H6-p and H6-a), 3.75 (ddd, J = 9.70 Hz, J = 4.50 Hz, J = 2.30 Hz, 1 H, H5-a), 3.38-3.34 (m, 1 H, H5-p).13C NMR (100 MHz, MeOH-d4): 5205.7 (C3-a), 205.6 (C3-p), 137.2 (Ar-C-p), 137.0 (Ar-C-a), 128.0, 127.9, 127.8, 127.7, 127.5, 127.4 (Ar-C), 103.3 (C1-p), 100.6 (C1-a), 77.0 (C5-p), 76.9 (C2-p), 75.7 (C5-a), 74.7 (C2-a), 72.3 (C4-a), 72.0 (C4-p), 70.6 (CH2-Bn-p), 69.2 (CH2-Bn-a), 61.2 (C6-p), 60.0 (C6-a) ppm. HRMS: m / z: [M+H₂O]⁺ calcd for C₁₃H₂₀O₇ 286.1053; Found 286.1287.

[0203] For the conversions with the p anomer, the oxidation reaction was performed using 20 mg of 1-O-benzyl-P-glucopyranoside, 1 U mL'1ofthe 16F10 variant, 0.1 mg mL'1of catalase in a total volume of 1.5 mL in water (adjusted to pH 7.5-8.0). The reaction proceeded for 6h30m at 25°C with magnetic stirring. The reaction was monitored as above, and the enzyme was removed by ultrafiltration. The 1-O-Benzyl-3-keto-P-D-glucopyranoside 2p was lyophilized overnight and obtained in quantitative yield (100%) as a viscous colorless syrup. [a]D20= -75.61 (c 1,2, MeOH).1H NMR (400 MHz, MeOH-d4): 57.45-7.43 (m, 2H, Ar-H), 7.37-7.28 (m, 3H, Ar-H), 5.0 (d, J = 11.93 Hz, 1 H, CH2-Bn), 4.75 (d, J = 11.77 Hz, 1 H, CH2-Bn), 4.48 (d, J = 7.89 Hz, 1 H, H1 ), 4.27 (dd, J = 10.19 Hz, J = 1.66 Hz, 1H, H4), 4.22 (dd, J = 7.88 Hz, J = 1.73 Hz, 1H, H2), 3.99 (dd, J = 12.01 Hz, J = 2.05 Hz, 1H, H6), 3.85 (dd, J = 12.05 Hz, J = 4.89 Hz, 1H, H6), 3.36 (dd, J = 4.96 Hz, J = 2.08 Hz, 1H, H5) ppm.13C NMR (100 MHz, MeOH-d4): 5205.8 (C-3), 137.3 (Ar-C), 128.0, 127.8, 127.5 (Ar-C), 103.3 (C1), 76.9 (C2), 76.8 (C5), 72.3 (C4), 70.7 (CH2-Bn), 61.2 (C6) ppm. HRMS: m / z: [M+H2O]+calcd for C₁₃H₂₀O₇ 286.1053; Found 286.1050.

[0204] Selective reduction of benzyl-keto-glucoside and removal of the protecting group

[0205] LS-selectride 1.0 M in THF (0.47 mmol, 0.5 mL) was added to compound 2p (0.157 mmol, 42 mg) in DMSO: THF (2:1, 2 mL) and the reaction was stirred for 2 h at 0°C. The excess LS-selectride was removed by adding acidic ion exchange resin (Dowex DVB WX8 H+-form), and the mixture was filtered and concentrated under a vacuum. The crude product was purified by flash column (Kieselgel 60, 0.032-0.063mm) with 90:10 ethyl acetate / methanol to afford the 1-O-Benzyl-P-D- allopyranoside 3 (36 mg) in 86% yield as a viscous colorless syrup. [a]D20= -66.36 (c 1.2, MeOH).

[0206] 1H NMR (400 MHz, MeOH-d4): 5 7.45-7.43 (m, 2H, Ar-H), 7.36-7.26 (m, 3H, Ar-H), 4.95 (d, J = 11.74 Hz, 1 H, CH2-Bn), 4.76 (d, J = 7.78 Hz, 1 H, H1 ), 4.67 (d, J = 11.74 Hz, 1 H, CH2-Bn), 4.07 (t, J = 2.92 Hz, 1H, H3), 3.92-3.87 (m, 1H, H6) 3.74-3.68 (m, 2H, H5 and H6), 3.53 (dd, J = 9.37 Hz, J = 3.01 Hz, 1 H, H4), 3.40 (dd, J = 7.99 Hz, J = 3.01 Hz, 1 H, H2) ppm.13C NMR (100 MHz, MeOH-d4): 5 137.9 (Ar-C), 127.9, 127.8, 127.2 (Ar-C), 99.6 (C1), 74.1 (C5), 71.6 (C3), 71.0 (C2), 70.3 (CH2-Bn), 67.6 (C4), 61.8 (C6) ppm. HRMS: m / z: [M+Na]+calcd for C₁₃H₁₈NaO₆ 293.0996; Found 293.0996.

[0207] The compound 3 (24 mg, 0.089 mmol) in MeOH (2 mL) was hydrogenated at 50 psi in the presence of Pd / C 10% (0.08 g) overnight. Then the reaction mixture was filtered through celite and washed with MeOH; the solvent evaporated to afford D-allose 4 (15 mg) in 94% yield as a viscous colorless syrup, major anomer p.1H NMR (400 MHz, MeOH-d4): 55.06 (d, J = 3.45 Hz, 1 H, H1 -a), 4.85 (d, J = 7.89 Hz, 1 H, H1-p), 4.06 (t, J = 3.00 Hz, 1 H, H3-p), 3.85 (dd, J = 11.62 Hz, J = 2.26 Hz, 1 H, H6-p), 3.78-3.71 (m, 1H, H5-p), 3.68-3.63 (m, 1H, H6-p), 3.54-3.48 (m, H4-p), 3.26 (dd, J = 7.99 Hz, J = 2.99 Hz, 1H, H2) ppm.13C NMR (100 MHz, MeOH-d4): 5 94.0 (C1-p), 93.8 (C1-a), 74.1 (C5-p), 72.2 (C2-p), 71.6 (C3-p), 67.6 (C4-p), 61.9 (C6-p) ppm. HRMS: m / z: [M+Na]+calcd for C₆H₁₂NaO₆ 203.0526; Found 203.0528.

[0208] Alternative synthesis of protected monosaccharides using glycosidase-mediated transglycosylation

[0209] To support sustainable enzymatic oxidation studies, aromatic glycosides 1-O-benzyl-P-D-glucopyranoside 1, 1-O-benzyl-P-D-galactopyranoside 5, were synthesised enzymatically using glycosidase-mediated transglycosylation (Fig. 6), replacing conventional multi-step chemical synthesis. This green approach used a single enzymatic step to achieve selective formation of the desired p-anomeric linkage, avoiding the need for protecting groups, harsh reagents, or purification of multiple intermediates. Compounds 1 and 5 were synthetised via enzymatic transglycosylation using cellobiose and lactose as the donor substrates and benzyl alcohol as the acceptor. The reactions were carried out in a two-phase system consisting of buffer and benzyl alcohol. Cellobiose (3.6 g) or lactose (7.2 g) were dissolved in 30 mL of sodium acetate buffer pH 5. Glycosidase (100 mg of p-glucosidase; 23 mg of p-galactosidase) was added, followed by benzyl alcohol (30 mL, >99 %). The reaction was stirred at room temperature for 10 or 6 h, respectively, and the progress was monitored by TLC on silica gel plates using DCM-MeOH (5:1, v / v) as the eluent. The reaction was terminated at the point of maximal glycoside formation by heating in a boiling water bath for 10 min under continuous stirring. After the termination of reaction, the biphasic mixture was concentrated under vacuum. The crude material was purified by flash column chromatography on silica gel 60 (230-400 mesh). The eluent was MeOH / DCM (from 10:1 to 5:1, v / v). 1-P-O-Benzyl-galactopyranoside 5: 26% yield.1H NMR (400 MHz, DMSO-de) 5 7.44 - 7.24 (m, 5H, Ph), 4.95 (d, J = 4.8 Hz, 1H, 2-OH), 4.81 (d, J= 12.2 Hz, 1H, Ph-CH2), 4.70 (d, J = 5.6 Hz, 1H, 4-OH), 4.61 - 4.54 (m, 2H, 6-OH, Ph-CH2), 4.37 (d, J = 4.6 Hz, 1H) (3-OH), 4.19 (d, J = 7.6 Hz, 1H, H1), 3.64 (s, 1 H, H5), 3.54 (q, J = 5.6 Hz, 2H, 2xH6), 3.42 - 3.23 (m, 3H, H2, H3, H4).13C NMR (101 MHz, DMSO-de) 5 138.7, 128.5, 128.0, 127.7 (Ph), 103.2 (C1), 75.7 (C4), 73.8 (C3), 71.1 (C2), 69.8 (Ph-CH2), 68.6 (C5), 60.9 (C6).

[0210] Oxidation of benzyl-galactoside using 16F10 variant

[0211] Reactions with the 1-O-benzyl galactoside were performed using 129 mg of compound 5 dissolved in 50 mM phosphate buffer, pH 7.6, in a total volume of 3 mL, with 3 U mL-1 of the 16F10 variant and 0.1 mg mL-1 of catalase, at 700 rpm and room temperature under aerobic conditions. The reaction was monitored by TLC till complete conversion of the starting material to product 8 (Fig.

[0212] 8) 1-O-Benzyl-3-keto-P-D-galactopyranoside 8: Mixture of oxidised and hydrated forms ratio 10:4.

[0213] 86% yield.1H NMR (400 MHz, DMSO-d6) 57.45 - 7.22 (m, 6H, Ph), 4.90 - 4.76 (m, 1 H, Ph-CH2), 4.41 (s, 2H, H1, Ph-CH2), 4.26 - 4.16 (m, 2H Ph-CH2, H4), 3.91 (d, J = 1.4 Hz, 1 H, H2), 3.69 - 3.52 (m, 2H, 2xH6), 3.49-3.41 (m, 1H, H5).13C NMR (101 MHz, DMSO-de) 5206.5 (C3), 138.1, 128.6, 128.0, 127.9 (Ph), 103.9 (C1), 75.6 (C5), 73.5 (C2), 73.1 (C4), 70.3 (Ph-CH2), 59.3 (C6).

[0214] Protection of monosaccharides with benzoyl groups and selective oxidation by 16F10 variant 1-O-Benzoyl-P-D-glucopyranoside 6 was prepared by Mitsunobu benzoylation according to Takagi et al. (Figure 7) using 0.450 g of benzoic acid (3.7 mmol), 2 g of D-GIc (11.1 mmol), 4.82 g of triphenylphosphine (18.4 mmol) in 220 mL of 1,4-dioxane under anhydrous conditions (argon) by adding 4.95 mL of diisopropyl azodicarboxylate (DIAD, 25.8 mmol) dropwise and vigorously stirred for 1 hour at room temperature. The reaction was then quenched by slowly adding 200 mL of MeOH, and the product was isolated by chromatography on silica gel using a DCM: MeOH gradient (9:1 to 5:1) as the eluent. Purified product 6 was isolated as a white solid (770 mg, 74%, a:£ 1: 100).

[0215] 1-O-Benzoyl-P-D-glucopyranoside 6:1H NMR (400 MHz, DMSO-de) 58.05-7.97 (m, 2H, Ph), 7.73 - 7.63 (m, 1H, Ph), 7.55 (t, J = 7.7 Hz, 2H) (Ph), 5.63 - 5.53 (m, 1H, H1), 5.42 - 5.36 (m, 1H, 2-OH), 5.17-5.11 (m, 1H.3-OH), 5.03 (d, J = 5.5 Hz, 1 H, 4-OH), 4.57 (t, J= 5.9 Hz, 1 H, 6-OH), 3.76 - 3.62 (m, 1H, H6), 3.51-3.42 (m, 1H, H6), 3.38- 3.22 (m, 3H, H2, H3, H5), 3.20-3.12 (m, 1H, H4).

[0216] 13C NMR (101 MHz, DMSO-de) 5165.1, 134.2, 130.0, 129.6, 129.2 (Ph), 95.4 (C1), 78.4 (C5), 76.8 (C3), 72.9 (C2), 69.9 (C4), 61.0 (C6).

[0217] Reactions with the benzoylated substrates 6 were performed with 129 mg of 6 dissolved in 50mM phosphate buffer pH 7.6 in a total volume of 3 mL with 3U mL'1of 16F10 variant, 0.1 mg mL'1of catalase under 700 rpm, room temperature, aerobic conditions. TLC monitored the reaction till complete conversion of the starting material 6 (Fig. 8) The reaction mixture was concentrated under vacuum and was subsequently applied on a silica gel column and the product 9 was purified by elution with DCM: MeOH 20:1. 1-O-Benzoyl-3-keto-P-D-glucopyranoside 9: 50 % Yield.1H NMR (400 MHz, DMSO-de) 58.10 - 7.99 (m, 2H, Ph), 7.81 - 7.69 (m, 1 H, Ph), 7.64 - 7.52 (m, 2H, Ph), 5.83 (d, J= 6.3 Hz, 1H, 2-OH), 5.73 (d, J= 8.3 Hz, 1H, H1), 5.52 (d, J= 6.1 Hz, 1H, 4-OH), 4.92 (t, J= 5.9 Hz, 1H, 6-OH), 4.41 (ddd, J = 8.1, 6.2, 1.7 Hz, 1H, H2), 4.23 (ddd, J= 10.2, 6.0, 1.7 Hz, 1H, H4), 3.75 (ddd, J= 12.2, 5.4, 1.9 Hz, 1H, H6), 3.61 (ddd, J= 11.8, 6.3, 4.7 Hz, 1H, H6), 3.48 (ddd, J = 10.2, 4.7, 1.9 Hz, 1H, H5).13C NMR (101 MHz, DMSO-d6) 5205.8 (C3), 164.7, 134.5, 130.0, 129.4, 129.1 (Ph), 96.2 (C1), 78.1 (C5), 75.9 (C2), 72.2 (C4), 60.7 (C6).

[0218] Oxidation of protected sugars using whole cells carrying the variant 16F10

[0219] For whole-cell catalysis experiments (Fig. 9), E. coli BL21 Rosetta cells were cultured to overexpress the variant 16F10, following a protocol similar to the one mentioned previously. The correlation between the wet cell weight (WCW) and optical density (ODeoonm) was determined. For oxidation, 40 mM 1 (110 mg), 20 OD₆₀₀nm of cells (104 mg / mL of WCW) in 20 mM sodium phosphate buffer pH 7.6 in total volume of 10 mL were used. The reaction was stirred at 500 rpm and room temperature under aerobic conditions for around 24h till complete conversion (monitored by TLC mixture DCM: EA: MeOH 2:8:1). Cells were removed by centrifugation (4,000 g), reaction mixture concentrated under vacuum and isolated on silica gel column as previously described. Testing the whole cell catalysis for oxidation of the benzoylated analogues

[0220] For testing of benzoylated glycosides, 52 mM 6 (44 mg) were used under the same conditions. Oxidation of protected sugars using immobilized whole cells carrying the variant 16F10

[0221] Cells overproducing 16F10 variant were also immobilized in alginate to avoid contaminants in the reaction mixture, for that the recombinant E. coli cells were harvested, washed twice with sterile 0.9% NaCI solution in order to remove residual growth medium and resuspended in distilled water to an optical density (OD600) of approximately 30. Aliquots of 2 mL were prepared in microcentrifuge tubes, pelleted by centrifugation at 13,400 rpm for 5 min, and stored at -20 °C until further use. For immobilisation, 2 % (w / v) sodium alginate solution were prepared in distilled water. One frozen cell aliquot (corresponding to OD 30) was resuspended in 3 mL of the alginate solution at OD600® 30. The suspension was centrifuged at 4,000 g for 5 min to remove trapped air bubbles. A 0.15 M CaCI2solution was freshly prepared and placed under continuous stirring in a beaker. The alginate-cell suspension was loaded into a syringe fitted with a 23G needle (0.5 mm diameter) and extruded dropwise into the CaCI2solution. The resulting alginate beads were left to solidify for at least 30 min in fresh 0.15 M CaCI2solution. After gelation, the beads were collected and washed thoroughly with distilled water (3-4 times) to remove residual salts. Beads were stored in distilled water for no longer than 3-5 days before use. All beads corresponding to a cell suspension of OD600® 30 were used for the experiment. Oxidation reactions were typically conducted with 20 mg of the substrate 1 and 6 dissolved in 2 mL of 50 mM phosphate buffer (pH 7.6). The bead suspension was incubated at 26 °C with shaking at 300 rpm. Reaction progress and substrate conversion were monitored by TLC until completion or no further conversion was observed.

[0222] Selective reduction of 1-O-benzyl-3-keto-galactoside, 1-O-benzoyl-3-keto-glucoside and 1-O-benzoyl-3-keto-galactoside and removal of the protecting groups

[0223] Reduction of the C3 oxidised 1-O-benzyl glycoside 9 (Fig. 10) was carried out following previously described procedures for the preparation of 1-O-benzyl-P-D-allopyranoside from its oxidised precursor. Briefly, a solution of the oxidised compounds in DMSO: THF (2:1, 3 mL) under innert atmosphere at 0°C was treated with LS-Selectride® (1.0 M solution in THF, 3 equivalents). The reaction mixture was stirred for 2 h at 0°C, after which the reaction was quenched by addition of acidic ion-exchange resin (Dowex 50WX8, H+form). The suspension was filtered and the filtrate concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel 60, particle size 0.032-0.063 mm) with dichloromethane:methanol (20:1, v / v) as the eluent to afford 1-O-benzyl-p-D-gulopyranoside 11, as a colourless syrup. A slightly modified protocol in comparison with the previously published for obtaining free allose 4 was applied for the preparation of D-gulopyranose 14 (Fig. 10), employing Pd / C (10%) and 60 psi hydrogen pressure for 48h yielding 33 mg (79%) as a colourless syrup (predominantly p-anomer). The stereoselective reduction of benzoylated analogue 9 (Fig. 10) was also accomplished using LS-Selectride® to afford 12 in 45% yield and 4 in 47% yield. The final deprotection of 12 was carried out by enzymatic hydrolysis of the ester using Burkholderia cepacia lipase resulting in 48 % additional yield of 4. 1-O-Benzyl- p-D-gulopyranoside 11 41% yield.1H NMR (400 MHz, DMSO-de) 5 7.43 - 7.23 (m, 5H) (Ph), 4.80 (d, J = 12.3 Hz, 1 H, Ph-CH2), 4.62 - 4.50 (m, 2H, H1, Ph-CH2), 3.74 (t, J = 3.5 Hz, 1 H, H6), 3.69 (ddd, J = 6.2, 6.2, 1.3 Hz, 1 H, H5), 3.58 - 3.45 (m, 4H, H2, H3, H4, H6).13C NMR (101 MHz, DMSO-de) 5 138.7, 128.5, 128.0, 127.7 (Ph), 100.5 (C1), 74.1 (C5), 72.3 (C3), 69.6 (Ph-CH2), 69.5 (C2), 68.2 (C4), 60.9 (C6). 1-O-Benzoyl-p-D-allopyranoside 12. 45 % yield.1H NMR (400 MHz, DMSO-de) 58.10 - 8.01 (m, 2H, Ph), 7.74 - 7.63 (m, 1H, Ph), 7.59- 7.53 (m, 3H, Ph), 5.92 (d, J= 8.2 Hz, 1H, H1), 5.16 (d, J= 6.8 Hz, 1H, 4-OH), 5.10 -4.97 (m, 1H, 3-OH), 4.75 - 4.62 (m, 1 H, 2-OH), 4.54 (t, J = 5.7 Hz, 1 H, 6-OH), 3.98-3.93 (m, 1 H, H3), 3.73 - 3.54 (m, 3H, H5, H6), 3.54 - 3.38 (m, 3H, H2, H4, H6).13C NMR (101 MHz, DMSO-de) 5 165.3, 134.1, 129.9, 129.8, 129.2 (Ph), 93.7 (C1), 75.8 (C5), 71.8 (C3), 70.1 (C2), 67.2 (C4), 61.2 (C6). D-Gulopyranose 14, 79 % Yield. a:p 1:10).1H NMR (400 MHz, D2O) 54.80 (d, J= 8.4 Hz, 1H, H1), 3.98 (t, J= 3.5 Hz, 1H, H5), 3.92 (ddd, J = 6.8, 5.4, 1.4 Hz, 1H, H3), 3.73 (dd, J= 3.8, 1.3 Hz, 1H, H2), 3.70 - 3.61 (m, 2H, 2xH6), 3.58 - 3.52 (m, 1H, H4). a:b = 1:10.13C NMR (101 MHz, D2O) 5 93.7 (C1), 73.7 (C3), 71.1 (C5), 69.3 (C2), 69.0 (C4), 60.9 (C6).13C NMR consistent with the literature.

[0224] Other methods

[0225] All1H NMR spectra were obtained at 400 MHz in MeOH-d4, DMSO-de or D2O using the residual solvent peak as standard, and13C NMR spectra were obtained at 100.61 MHz in MeOH-d4, DMSO-de or D2O. Assignments are supported by 2D correlation NMR studies. Specific rotations were measured using a Perkin-Elmer D241 automatic polarimeter and are reported as follows: [a]D20(c g / 100mL; solvent). ESI-MS determined the molecular mass, and the mass spectra of the samples were acquired in the positive mode in a Bruker micrOTOF. SEQUENCES:

[0226] In the event of discrepancies between the sequences shown in the present specification and those of the enclosed sequence protocol according to WIPO standard ST.26, the sequences shown herein shall prevail.

[0227] > PsG3Ox wild type DNA - Pseudarthrobacter siccitolerans, SEQ ID NO 001 ATGAGCGGTCACCGGTATCCCGCCGCAGTTGACGTCGCCATCGTCGGCAGCGGTCCCACGGCTTCGGCCTA TGCGCGGATCCTCAGCGAGGAAGCCCCCGGTGCCACGATCGCGATGTTCGAAGTGGGCCCGACTGTCAGCA ATCCGCCCGGCGCGCACGTCAAGAACATCGAGGACCCTGATAGCCGCAGCCTCGCCCAGCGGGCGTCGGAG GGTCCCGGTGCCGGTGCTGCAACAGTGAATTCGCCGGGCGCCGTCAAGAGCGGCGAACGCCGTGCGCGCCC TGGAACTTACCTGCTGCAGGACGGCTACGCCTTCCCGGGCGAGGACGGCATGCCCGTCGCGGCCATGTCCA GCAACGTGGGCGGGATGGCCGCCCACTGGACCGCCGCCTGCCCCCGCCCGGGCGGCAAGGAACGCATCCCG TTCCTGCCGGACCTGGAAGAGCTCCTTAACGACGCCGACCGCCTTTTGGGCGTCACCACGCACGCTTTTGA TGGTGCCCCGTTCTCGGACCTGGTCCGTGAACGACTCGCCGCGGTCGTGGATCAGGGCCGCACGCCTGCCT TTCGGGTCCAGCCCATGCCCCTTGCTGTACACCGGCGGCAGGATGGCGCCCTCGTATGGTCCGGCTCCGAC GTCGTCATGGGGGAGGCCACCCGCGATAATCCGCAGTTTGAACTGTTTGATGAATCGCTGGTGACCCGTGT GCTGGTGGAGGACGGCACTGCTGCCGGCGTCGAAGTCCAGGACCGCCGCAGCGGTGACACTTATCAGGTGG CGGCCCGTTACGTTGTGGTGGGGGCGGACGCCCTGCGCACACCGCAGCTGCTGTGGGCGTCCGGGATCCGG CCCGACGCCCTGGGCCGCTACCTGAACGACCAGGCGCAGGTGGTGTTCGCGAGCAGGCTCCGCGACGTCCA GCCCGAGGACGCGCCGGCAGCGGCCAATGGTGCCCTCAGTGAGCAGAGCGGAGTGGCCTGGGTTCCCTACA CGGACGAGGCGCCCTTCCACGGCCAGATCATGCAGCTCGATGCTTCCCCGGTTCCCCTGGCCGATGATGAT CCCATCGTCCCGGGCTCCATCGTGGGGCTGGGCCTGTTCTGCGCGAAAGACCTGCAGCGTGAGGACCGGGT GGCGTTCGACGACGATACCCGCGACTCCTACGGCCTGCCCGCCATGCGCATCCACTACCGGCTGACCGAGC GGGACCACGTGGTACTGGACCGGGCCAGGCAGGAAATTGTCCGTCTGGGCAAGGCGGTGGGCGAACCGCTG GACGAGCGGCCCTTCGTCCTGCCGCCGGGTGCGTCGCTGCACTACCAGGGCACCACGCGGATGGGTGAGAC GGACGACGGCGAGAGCGTCTGTTCGCCGGACAGCCAGGTGTGGCAGGTCCCCGGCCTCTTTGTGGCCGGCA ACGGCGTTATCCCCACCGCTACAGCATGCAATCCCACCCTGACGTCGGTGGCGCTCGCCGTGCGCGGCGCC CGGAAAATCGCTGAAGAAATCACCAGCTCTTTACTTATGTCCGAATCAGACAATAGACTGTCTAAATAA

[0228] > PsG3Ox wild type AA -Pseudarthrobacter siccitolerans, SEQ ID NO 002 MSGHRYPAAVDVAIVGSGPTASAYARILSEEAPGATIAMFEVGPTVSNPPGAHVKNIEDPDSRSLAQRASE GPGAGAATVNSPGAVKSGERRARPGTYLLQDGYAFPGEDGMPVAAMSSNVGGMAAHWTAACPRPGGKERIP FLPDLEELLNDADRLLGVTTHAFDGAPFSDLVRERLAAWDQGRTPAFRVQPMPLAVHRRQDGALVWSGSD WMGEATRDNPQFELFDESLVTRVLVEDGTAAGVEVQDRRSGDTYQVAARYVWGADALRTPQLLWASGIR PDALGRYLNDQAQVVFASRLRDVQPEDAPAAANGALSEQSGVAWVPYTDEAPFHGQIMQLDASPVPLADDD PIVPGS IVGLGLFCAKDLQREDRVAFDDDTRDSYGLPAMRIHYRLTERDHWLDRARQEIVRLGKAVGEPL DERPFVLPPGASLHYQGTTRMGETDDGESVCSPDSQVWQVPGLFVAGNGVIPTATACNPTLTSVALAVRGA RKIAEEITSSLLMSESDNRLSK > PsG30x 16F10 variant DNA -synthetic construct, SEQ ID NO 003 ATGAGCGGTCACCGGTATCCCGCCGCAGTTGACGTCGCCATCGTCGGCAGCGGTCCCACGGCTTCGGCCTA TGCGCGGATCCTCAGCGAGGAAGCCCCCGGTGCCACGATCGCGATGTTCGAAGTGGGCCCGACTGTCAGCA ATCCGCCCGGCGCGCACGTCAAGAACATCGAGGACCCTGATAGCCGCAGCCTCGCCCAGCGGGCGTCGGAG GGTCCCGGTGCCGGTGCTGCAACAGTGAATTCGCCGGGCGCCGTCAAGAGCGGCGAACGCCGTGCGCGCCC TGGAACTTACCTGCTGCAGGACGGCTACGCCTTCCCGGGCGAGGACGGCATGCCCGTCGCGGCCATGTCCA GCAACGTGGGCGGGATGGCCGCCCACTGGACCGCCGCCTGCCCCCGCCCGGGCGGCAAGGAACGCATCCCG TTCCTGCCGGACCTGGAAGAGCTCCTTAACGACGCCGACCGCCTTTTGGGCGTCACCACGCACGCTTTTGA TGGTGCCCCGTTCTCGGACCTGGTCCGTGAACGACTCGCCGCGGTCGTGGATCAGGGCCGCACGCCTGCCT TTCGGGTCCAGCCCATGCCCCTTGCTGTACACCGGCGGCAGGATGGCACCCTCGTATGGTCCGGCTCCGAC GTCGTCATGGGGGAGGCCACCCGCGATAATCCGCAGTTTGAACTGTTTGATGAATCGCTGGTGACCCGTGT GCTGGTGGAGGACGGCACTGCTGCCGGCGTCGAAGTCCAGGACCGCCGCAGCGGTGACACTTATCAGGTGG CGGCCCGTTACGTTGTGGTGGGGGCGGACGCCCTGCGCACACCGCAGCTGCTGTGGGCGTCCGGGATCCGG CCCGACGCCCTGGGCCGCTACCTGAACGACCATGCGCAGGTGGTGTTCGCGAGCAGGCTCCGCGACGTCCA GCCCGAGGACGCGCCGGCAGCGGCCAATGGTGCCCTCAGTGAGCAGAGCGGAGTGGTCTGGGTTCCCTACA CGGACGAGACGTCCTTCCACGGCCAGATCATGCAGCTCGATGCTTCCCCGGTTCCCCTGGCCGATGATGAT CCCGTCGTCCCGGGCTCCATCGTGGGGCTGAGCCTGTTCTGCGCGAAAGACCTGCAGCGTGAGGACCGGGT GGCGTTCGACAACGATACCCGCGACTCCTACGGCCTGCCCGCCATGCGCATCCACTACCGGCTGACCGAGC GGGACCACGTGGTACTAGACCGGGCCAGGCAGGAAATTGTCCGTCTGGGCAAGGCGGTGGGCGAACCGCTG GACGAGCGGCCCTTCGTCCTGCCGCCGGGTGCGTCGCTGCACTACCAGGGCACCACGCGGATGGGTGAGAC GGACGACGGCGAGAGCGTCTGTTCGCCGGACAGCCAGGTGTGGCAGGTCCCCGGCCTCTTTGTGGCCGGCA ACGGCGTTATCCCCACCGCTACAGCGTGCAATCCCACCCTGACGTCGGTGGCGCTCGCCGTGCGCGGCGCC CGGAAAATCGCTGAAGAAATCACCAGCTCTTTACTTATGTCCGAATCAGACAATAGACTGTCTAAATAA

[0229] > PsG3Ox 16F10 variant AA - synthetic construct, SEQ ID NO 004 MSGHRYPAAVDVAIVGSGPTASAYARILSEEAPGATIAMFEVGPTVSNPPGAHVKNIEDPDSRSLAQRASE GPGAGAATVNSPGAVKSGERRARPGTYLLQDGYAFPGEDGMPVAAMSSNVGGMAAHWTAACPRPGGKERIP FLPDLEELLNDADRLLGVTTHAFDGAPFSDLVRERLAAWDQGRTPAFRVQPMPLAVHRRQDGTLVWSGSD WMGEATRDNPQFELFDESLVTRVLVEDGTAAGVEVQDRRSGDTYQVAARYVWGADALRTPQLLWASGIR PDALGRYLNDHAQVVFASRLRDVQPEDAPAAANGALSEQSGVVWVPYTDETSFHGQIMQLDASPVPLADDD PVVPGS IVGLSLFCAKDLQREDRVAFDNDTRDSYGLPAMRIHYRLTERDHWLDRARQEIVRLGKAVGEPL DERPFVLPPGASLHYQGTTRMGETDDGESVCSPDSQVWQVPGLFVAGNGVIPTATACNPTLTSVALAVRGA RKIAEEITSSLLMSESDNRLSK

[0230] > PsG30x_A75T_FW- synthetic construct, SEQ ID NO 005 GGGTCCCGGTACCGGTGCTGCAACAGTG

[0231] > PsG30x_A75T_RV- synthetic construct, SEQ ID NO 006

[0232] CACTGTTGCAGCACCGGTACCGGGACCC > PsG3Ox_A206T_FW- synthetic construct, SEQ ID NO 007 GCAGGATGGCACCCTCGTATGGTCCGGCTC

[0233] > PsG3Ox_A206T_RV- synthetic construct, SEQ ID NO 008 GAGGCCGGACCATACGAGGGTGCCATCCTGC

[0234] > PsG30x_Q295H_FW- synthetic construct, SEQ ID NO 009 CTGAACGACCATGCGCAGGTGGTGTTCGCG

[0235] > PsG30x_Q295H_RV- synthetic construct, SEQ ID NO 010 CGCGAACACCACCTGCGCATGGTCGTTCAG

[0236] > PsG30x_P336S_FW- synthetic construct, SEQ ID NO 011 CGGACGAGGCGTCCTTCCACGGCCAGATC

[0237] > PsG30x_P336S_RV- synthetic construct, SEQ ID NO 012 GATCTGGCCGTGGAAGGACGCCTCGTCCG

[0238] > PsG30x_S336P_FW- synthetic construct, SEQ ID NO 013 CGGACGAGGCGCCCTTCCACGGCCAGATC

[0239] > PsG30x_S336P_RV- synthetic construct, SEQ ID NO 014 GATCTGGCCGTGGAAGGGCGCCTCGTCCG

[0240] > pET21_FW- synthetic construct, SEQ ID NO 015

[0241] CT T CCC C AT C GGT GAT GT C GGC GAT AT AG

[0242] > pET21_RV- synthetic construct, SEQ ID NO 016 CCAAGGGGTTATGCTAGTTATTGCTCAG

[0243] > PsG3Ox_GA_FW- synthetic construct, SEQ ID NO 017 GCAGCCGGATCCTCGAGCATTATTTAGACAGTCTATTGTCTGATTCGG

[0244] > PsG3Ox_GA_RV- synthetic construct, SEQ ID NO 018 TGGTGCCGCGCGGCAGCCATATGAGCGGTCACCGGTATC

[0245] > pET15b_GA_FW- synthetic construct, SEQ ID NO 019 ATGGCTGCCGCGCGGCAC

[0246] > pET15b_GA_RV- synthetic construct, SEQ ID NO 020

[0247] ATGCTCGAGGATCCGGCTG Table 1

[0248] Optimal pH kcat fs'1) Km(M) kcat / Km(M'-1s'-1)

[0249] Wild-type 7.5 0.2 ± 0.05 0.37 ± 0.13 0.5 ± 0.1 1A1 7.5 0.4 ± 0.1 0.54 ± 0.11 0.7 ± 0.1 5D5 8.5 1.0 ± 0.2 0.33 ± 0.05 3.2 ± 0.4 41G6 8.0 1.2 ± 0.2 0.24 ± 0.05 5.1 ± 0.2 2G4 8.0 1.9 ± 0.2 0.26 ± 0.05 6.7 ± 0.8 7E9 8.0 2.3 ± 0.3 0.27 ± 0.05 8.6 ± 0.4 2B9 8.0 2.6 ± 0.3 0.23 ± 0.06 10.6 ± 0.9 6E3 8.0 3.0 ± 0.2 0.26 ± 0.01 11.5 ± 0.6 16F10 8.0 4.8 ± 1.0 0.37 ± 0.05 11.4 ± 0.8

[0250] Table 2

[0251] Specific activity (nmol min'1mg'1) Wild-type 16F10 Monosaccharides

[0252]

[0253] D(+)-glucose 36.9 ± 5.7 645.4 ± 164.4 D(+)-galactose 2.9 ± 0.7 97.1 ± 21.6

[0254] L(+)-arabinose 5.3 ± 0.9 68.2 ± 4.8

[0255] 2-deoxy-glucose 3.2 ± 0.5 44.2 ± 7.7

[0256] D(-)-ribose 2.6 ± 1.8 24.3 ± 4.0

[0257] L(+)-rhamnose 1.4 ± 0.1 8.9 ± 1.7

[0258] D(+)-mannose 0.3 ± 0.2 3.2 ± 0.1

[0259] D(-)-fructose 0.3 ± 0.1 1.6 ± 0.5

[0260] L(-)-fucose 0.5 ± 0.1 3.6 ± 1.0 Disaccharides

[0261]

[0262] D-lactose 0.6 ± 0.4 10.8 ± 2.8

[0263] D(+)-maltose 0.2 ± 0.1 7.3 ± 1.5

[0264] D(+)-trehalose 0.2 ± 0.03 1.8 ± 1.0 Trisaccharides

[0265]

[0266] D(+)-raffinose 0.2 ± 0.1 Nd Aminosugars

[0267]

[0268] D(+)-glucosamine 1.3 ± 0.3 0.5 ± 0.3 Table 3

[0269] Variant TmApp(°C) Tagg(°C) ti / 240°c(min)

[0270] Wild-type 49 ± 2 50 27 ± 2

[0271] 1A1 47 ± 3 46 366 ± 46

[0272] 5D5 47 ± 3 49 374 ± 82

[0273] 41G6 44 ± 1 43 5 ± 1

[0274] 2G4 43 ± 1 43 82 ± 8

[0275] 7E9 44 ± 2 45 102 ± 18

[0276] 2B9 47 ± 2 46 291 ± 30

[0277] 6E3 45 ± 2 48 212 ± 41

[0278] 16F10 44 ± 2 48 187 ± 30

[0279] Table 4

[0280] Variant kcat fs'1) Km(mM) kcat / Km(M’1s’1) Wild-type 0.2 ± 0.05 370 ± 130 0.5 ± 0.1 D-GIc

[0281] 16F10 4.8 ± 1.0 370 ± 50 11.4 ± 0.8 1 -O-benzyl-a / p-D- Wild-type 0.06 ± 0.01 13.4 ± 3.8 5.0 ± 1.0 glucopyranoside16F10 0 57 ± 0 05 17 4 ± 1.3 33,O ±1.O

[0282] 1-O-benzyl-p-D-Wild'type 0 31 1 0 01 16 4 1 4 0 19 3 1 5 2glucopyranoside 16F10 1.26 ± 0.07 11.3 ± 1.0 111.5 ± 4.0

[0283] Table 5

[0284] Diastereomeric Reagent Solvent Yield (%) ratio

[0285] 1:3

[0286] NaBH₄U MeOH 63 1:1.5 K-Selectride® DMSO: THF (2:1 ) 64 0:1 LS-Selectride® DMSO: THF (2:1 ) 86 0:1 Strains, Plasmids, or Genotype, properly, or sequence Reference or Primers source Strains

[0287] E. coli DH5a of) fite^sopE44 k th l gyrA96 NWflO,

[0288] E GM 10G elite

[0289]

[0290] cX74 araD139 A(a )7697 g y os® corporation gaK®L(strR) tws IOJJA - Bteareh Technologies E. coli KRX [F‘, traD36, DompP,sp^T |^ D( / acZ)M15] Qsa^ endA1, re<^1, gyrA96 (^), t / 7M, hsoR17 (o$- Promega, Of), e14- (Bfct -), relM, st^E44, D(fec^^), DfflWg c 7 RNA polymerase

[0291] E. coli Rosetta(DE3) F MSOB ) gal dm (DE3) (C«B) tfefflfl® pLysS

[0292] Plasmids

[0293] pET15b Cloning vector with a T7 promoter with a N-terminal T7tag and C-terminal BxHis tag;

[0294]

[0295] teyageo,. pSM1 pET15b with psg3ox gene cloned into

[0296]

[0297] sites1pAT-3 pET 15b with psg3ox gene cloned into / ^g[and

[0298]

[0299] sites carrying the mutation A75T introduced in This work the by site-directed mutagenesis

[0300] pAT-4 pET 15b with psg3ox gene cloned into ^ and

[0301]

[0302] sites carrying the mutation A206T introduced in This work the by site-directed mutagenesis

[0303] pAT-5 pET15b with psg3ox gene cloned into and

[0304]

[0305] sites carrying the mutation Q295H introduced in This work the by site-directed mutagenesis

[0306] pAT-6 pAT-1 A1 with psg3ox gene cloned into g|, and

[0307]

[0308] sites carrying the mutation G366S and Q295H This work introduced in the by site-directed mutagenesis

[0309] pAT-9 pET15b with psg3ox gene cloned intoand

[0310]

[0311] sites carrying the mutation S336P introduced in This work the by site-directed mutagenesis

[0312] pAT-10 pAT-2G4 with psg3ox gene cloned into

[0313]

[0314] and XbQls!tescarrying the mutation P336S introduced in This work the by site-directed mutagenesis

[0315] pAT-11 pAT-7E9 with psg3ox gene cloned into ^< and

[0316]

[0317] sites carrying the mutation P336S Introduced in This work the by site-directed mutagenesis

[0318] pAT-12 pAT-2B9 with psg3ox gene cloned into ^glgj,and

[0319]

[0320] sites carrying the mutation P336S introduced in This work the by site-directed mutagenesis pAT-5D5 pET15b with psg3ox gene cloned into and

[0321]

[0322] sites carrying the mutation G366S, A75T, A206T, This work Q295H introduced in the second round of directed evolution

[0323] pAT-41G6 pET15b with psg3ox gene cloned into ^g^and sites carrying the mutation G366S, A75T, A206T, This work Q295H, P336S introduced in the third round of directed evolution

[0324] pAT-7E9 pET15b with psg3ox gene cloned into ^fe^and sites carrying the mutation G366S, A75T, A206T, This work Q295H, P336S, D222G, A327V introduced in the fourth round of directed evolution

[0325] pAT-2G4 pET15b with psg3ox gene cloned into and sites carrying the mutation G366S, A75T, A206T, This work Q295H, P336S, A327V, V416I, K420R Introduced in the fourth round of directed evolution

[0326] pAT-2B9 pET15b with psg3ox gene cloned into ^g^and sites carrying the mutation G366S, A75T, A206T, This work Q295H, P336S, D222G, A327V, V416l', K420R, A335T introduced in the fifth round of directed

[0327] evolution

[0328] pAT-6E3 pET15b with psg3ox gene cloned into gj,and sites carrying the mutation G366S, A206T, This work Q295H, P336S, A327V, A335T introduced in the sixth round of directed evolution

[0329] pAT-16F10 pET15b with psg3ox gene cloned into sfej,and sites carrying the mutation G366S, A206T, This work Q295H, P336S, A327V, A335T, D383N, I357V Introduced in the seventh round of directed evolution

[0330] Primers

[0331] PsG3Ox_A75T_FW 5’-GGGTCCCGGTAGCGGTGCTGCAACAGTG-3’ (SEQ ID NO 005) This work PsG3Ox_A75T_RV 5’-CACTGTTGCAGCACCGGTACCGGGACCC-3' (SEQ ID NO 006) This work PsG3Ox_A206T_FW 5’-GCAGGATGGCACCCTCGTATGGTCCGGCTC-3’ (SEQ ID NO 007) This work PsG3Ox_A206T_RV 5’-GAGGCCGGACCATACGAGGGTGCCATCCTGC-3’ (SEQ ID NO 008) This work PsG3Ox_Q295H_FW 5’-CTGAACGACCATGCGCAGGTGGTGTTCGCG-3’ (SEQ ID NO 009) This work PsG3Ox_Q295H_RV 5’-CGCGAACACCACCTGCGCATGGTCGTTCAG -3* (SEQ ID NO 010) This work PsG3Ox_P336S_FW 5'-CGGACGAGGCGTCCTTCCACGGCCAGATC-3’ (SEQ ID NO 011) This work PsG3Ox_P336S_RV S’-GATCTGGCCGTGGAAGGACGCCTCGTCCG-y (SEQ ID NO 012) This work PsG3Ox_S336P_FW 5'-CGGACGAGGCGCCCTTCCACGGCCAGATC-3’ (SEQ ID NO 013) This work PsG3Ox_S336P_RV 5’-GATCTGGCCGTGGAAGGGCGGCTCGTCCG-3’ (SEQ ID NO 014) This work pET21_FW 5’-CTTCCCCATCGGTGATGTCGGCGATATAG-3’ (SEQ ID NO 015) This work pET21_RV 5'-CCAAGGGGTTATGCTAGTTATTGCTCAG-3’ (SEQ ID NO 016) This work PsG3Ox_GA_FW 5’-GCAGCCGGATCCTCGAGCATTATTTAGACAGTCTATTGTCTGATTCGG-3' (SEQ ID NO 017) This work PsG3Ox_GA_RV 5’-TGGTGCCGCGCGGCAGCCATATGAGCGGTCACCGGTATC-3’ (SEQ ID NO 018) This work pET15b_GA_FW 5'-ATGGCTGCCGCGCGGCAC-3’ (SEQ ID NO 019) This work pET15b GA RV S’-ATGCTCGAGGATCCGGCTG-y (SEQ ID NO 020) This work

[0332]

[0333]

[0334] £.nnt£nn,,,,,,,,,n_ _ shuffling) 04 a0 5100 / 500 2B9D222G A335T1.0 ±0.2 3.7 ±06

[0335] 6E3 G366S, A206T, Q295H, P336S, A327V, A335T 1.3 ±0.2 1.2 ±0.1 1F3 G366S, A75T, A206T, Q295H, P336S, A327V, A335T 1.4 ±0.1 0.9 ±0.1 6th (Shuffling - ^289) - 50 2500 / 600 1D3 G366S, A206T, Q295H P336S, A327V, D222G A335T 1.3±0.1 1.3±0.2

[0336] 5F3 G366S, A206T, Q295H, P336S, A327V, A335T, V416I, K420R 1.0 ±0.2 1.1 ±0.1 11G7 G366S, A206T, Q295H, P336S, A327V, D222G, A335T, V416I 0.8 ±0.1 1.3 ±0.2

[0337] 7th0.1 50 11200 / 1750 16F10 G366S, A206T, Q295H, P336S, A327V, A335T, I357V, D383N 1.6 ±0.3 1.3 ± 0.1 Table 8

[0338] Productiono..,..

[0339] ....... Ratio functional /

[0340] Variant yield,,.

[0341] total enzyme

[0342] (mg / L culture)J

[0343] Wild-type 30 20 %

[0344] 1A1 62 25 %

[0345] 5D5 97 24 %

[0346] 41 G6 35 40 %

[0347] 2G4 20 50 %

[0348] 7E9 30 30 %

[0349] 2B9 35 55 %

[0350] 6E3 33 35 %

[0351] 16F10 37 37 %

[0352] Table 9

[0353] Origin Microorganism Enzyme cat (s'1) Km(M) kcat / Km(M'1s‘1)

[0354] Pseudarthrobacter siccitolerans PsG30x 0.2 0.4 0.5 Kitasatospora aureofaciens KaP2Ox 15.4 1.5 × 10-31.0 × 104n.., Microbacterium sp. 5-2b CarA nd - Bacterial

[0355] Arthrobacter globiformis AgCarA nd - M. trichotheecenolyticum MtCarA nd - Streptomyces canus ScP2Ox > 0.38 > 2.0 > 0.19 Phlebiopsis gigantea PhgP2Ox 40.5 1.2 × 10-33.4 × 104Trametes multicolor TmP2Ox 54.0 0.7 × 10-37.3 × 104Tricholoma matsutake TmaP2Ox 111.0 1.3 × 10-38.7 × 104Peniophora gigantea PegP2Ox 56 1.1 × 10-35.0 × 104Peniophora sp. PsP2Ox 9.4 5.0 × 10-31.8 × 103Fungal

[0356] Phanerochaete chrysosporium PcP2Ox 83.1 0.8 × 10-39.9 × 104Lyophyllum shimeji LsP2Ox 6.9 0.3 × 10-32.2 × 104Aspergillus nidulans AnP2Ox 35.4 1.8 × 10-32.0 × 104Aspergillus oryzae AoP2Ox 1.5 2.9 × 10-30.5 × 103Irpex lacteus IlP2Ox 33.8 0.7 x 10’34.6 x 104 Table 10

[0357] T App T

[0358] Variant(TC)J,agg ti / 240°c(min)

[0359] WT+P336S 47 ± 2 45 Not detected

[0360] 2G4-P336S 55 ± 2 53 990 ± 110 7E9-P336S 56 ± 2 54 2310 ± 260

[0361] 2B9-P336S 56 ± 3 55 990 ± 213

[0362] Table 11

[0363]

[0364] Table 12

[0365] Enzyme Mutation Optimal pH kcat (s'-1) Km(M) kcat / Km(M'-1s'-1) wild-type - 7.0 - 7.5 0.2 ± 0.05 0.37 ± 0.13 0.5 ± 0.1 1A1 G366S 7.5 0.4 ± 0.1 0.54 ± 0.11 0.7 ± 0.1 wild-type + A75T 7.5 0.2 ± 0.03 0.59 ± 0.14 0.4 ± 0.1 wild-type +A206T 7.5 0.3 ± 0.03 0.56 ± 0.05 0.5 ± 0.1 wild-type +Q295H 8.5 0.5 ± 0.02 0.28 ± 0.02 1.8 ± 0.1 wild-type +Q295H / G366S 8.5 0.9 ± 0.03 0.28 ± 0.02 3.2 ± 0.3 5D5 A75T; A206T;

[0366] 8.5 1.0 ± 0.2 0.33 ± 0.05 3.2 ± 0.4 Q295H; G366S

[0367]

[0368] Starting Temp. Time Steps (chemical Overall Enzymes Chemicals used (other than Byproducts Obs. References

[0369]

[0370] materials (°C) (h) or enzymatic) yield (%) solvents)

[0371]

[0372] glucose isomerase, D- allulose 3- epimerase, and Final compound

[0373] D-glucose* 84 One pot reaction No yield ribose 5-phosphate D-allulose, D- D-aJte. Not Zheng et al provided isomerase totose isolated. (2022)

[0374] (Fermentation ^Jz.coli}

[0375] D-allulose 3-epimerase Final compound D-fructose 60 2 One pot reaction 13 and.jibose 5-phosphate D-Allulose D-atoae, Not Lee et al (2018) Isomerase2 isolated.

[0376]

[0377] D-allulose Final compound Yeom et al 6 1 33 ribose 5-phosphate

[0378] nJ, D-aigee. Not (2011) isomerase 1 (D-nataae) isolated

[0379] Complex

[0380] D-altrose (D- purification step acetic acid and ammonium Bilik et al 2 1 (epimerization) 14-17 D-glucose 120 none rtaSft'D-altrose to remove molybdate (1988) 3 / 2) glucose and

[0381] mannose

[0382] glucose- 45-96 (C-3 Only C-3 derived. f(2,9-dimethyl-1,10- phenanthroline)- Jager et al 3 1 oxidation none n d. oxidation step starting (2013) Pd(m-QftG)}2(OH)2+benzoquinone reaction) reported compounds

[0383] Mixture of C-3 >98-92 (C-3 protected „ [(ngocuprpine)Pd- (QAcJHQTf): + and C-4 Only oxidation Chung et al oxidation none i fifii iiiiiifi monosaccha benzoquinone oxidation step reported (2016) reaction) products

[0384]

[0385] ytywac

[0386] D-glucose, 1 2 54 none KwawwoeJPd- ©A (QI + reduction step Final compound JlWrtfeet al benzoquinone. NaBH* (D-aitoJD- o-afcift (2016)

[0387] glucose 92 / 8)

[0388] D-glucose and other 42 N quinudidine. Ad-S nal compound Wang et al derivatives LED 4-CIOBzBuxN D-a|to (2020)

[0389]

[0390]

[0391]

[0392]

[0393] s

[0394]

[0395] 2 (Cyanohydrin

[0396] L-ribose reaction and O none sodium amalgam (reduction) reduction)

[0397]

[0398] 1.256-dl-O- 1) potassium metaperiodate, Isopropyhde potassium carbonate and rutheniu ne-a-D- dioxide 2) sodium borohydride 3 glucofurano Amberlite IR-120 (H+) ion-exchan

[0399]

[0400]

[0401] mono- and quinuclidine 130 mol %} MerNBF* (1 Only the

[0402] disaccharide 25 24 1 21-70 none HFIP (10 5 mL of. oxidation. 6-OH WftaR fe®1al s ACM, graphite electrodes, 5 mA ‘ ' needs to be (2023)

[0403] (constant current, 2.00 mA / cm2_) protected.

[0404] Raney nickel Final compound Pex«M et al

[0405]

[0406]

[0407] D-afcx< (1963)

[0408] . Herber et al D-glucose 1 7 none molybdenum - FinalcompoundUS5433793A*0SRpatent (1994)

Claims

Claims1. A polypeptide comprising an amino acid sequence of at least ≥ 85%, ≥ 90%, ≥ 92%, ≥ 95%, ≥ 98%, ≥ 99% or 100% identity to SEQ ID NO 002, wherein the amino acid sequence comprises at least one of the following optimized residues:a. Q295 is histidine (H);b. A327 is selected from the group of valine (V), leucine (L), or isoleucine (I);c. A335 is selected from the group of serine (S) or threonine (T);d. P336 is selected from the group of serine (S) or threonine (T);e. I357 is selected from the group of alanine (A) or valine (V);f. G366 is selected from the serine (S) or threonine (T) groups.

2. The polypeptide according to claim 1, wherein at least two of said optimized residues are present in the amino acid sequence of said polypeptide, particularly wherein at least three, more particularly at least four, even more particularly at least five, most particularly all six of said optimized residues are present in the amino acid sequence of said polypeptide.

3. The polypeptide according to any one of claims 1 to 2, whereina. Q295 is histidine (H);b. A327 is valine (V);c. A335 is threonine (T);d. P336 is serine (S);e. I357 is valine (V); andf. G366 is serine (S).

4. The polypeptide according to any one of claims 1 to 3, wherein the amino acid sequence comprises an additional optimized residue:g. A206 is selected from the group of serine (S) or threonine (T), and / or h. D383 is selected from the group of asparagine (N), glutamine (Q), or serine (S).

5. The polypeptide according to claim 4, whereing. A206 is threonine (T) and / orh. D383 is asparagine (N).

6. The polypeptide according to any one of claims 1 to 5, comprising or consisting of SEQ ID NO 004.

7. An isolated nucleic acid encoding the polypeptide according to any one of claims 1 to 6.

8. A nucleic acid expression vector comprising a sequence encoding the polypeptide according to any one of claims 1 to 6.

9. A cell comprising the polypeptide according to any one of claims 1 to 6, isolated nucleic acid according to claim 7, nucleic acid expression vector according to claim 8, or any combination thereof.

10. The cell according to claim 9, wherein said cell is a prokaryotic or eukaryotic cell.

11. The cell according to any one of claims 9 to 10, wherein said cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida.

12. A method for oxidation of a pyranoside or a furanoside comprising a step:a. combining the pyranoside or the furanoside with a polypeptide according to any one of claims 1 to 6, or with a cell according to any one of claims 9 to 11.

13. The method according to claim 12, wherein the pyranoside or the furanoside is unprotected at position C1 and oxidized at position C2.

14. The method according to claim 12, wherein the pyranoside is protected at position C1 and oxidized at position C3.

15. The method according to claim 12, wherein the furanoside is protected at position C1, C2 or both, and oxidized at position C3 or position C4.

16. A method for oxidation of a pyranoside or a furanoside, comprising a step of combining the pyranoside or the furanoside with a glycoside 3-oxidase enzyme;wherein, the pyranoside is protected at position C1 and oxidized at position C3, and the furanoside is protected at position C1, C2 or both and oxidized at position C3 or position C4.

17. The method according to claim 16, wherein said glycoside 3-oxidase enzyme is selected from the group of:a. PsG30x, AgCarA, MtCarA, CarA, or ScP20x; orb. a homologue of PsG30x, AgCarA, MtCarA, CarA, or ScP20x; orc. a variant of PsG30x, AgCarA, MtCarA, CarA, ScP20x, wherein said variant has at least 70% identity, >75%, >80%, >85%, >90%, or >95% identity with a polypeptide sequence of PsG30x, AgCarA, MtCarA, CarA, or ScP20x, particularly having a biological activity of at least 30%, at least 50% or at least 80% of a polypeptide of the sequence of PsG30x, AgCarA, MtCarA, CarA, or ScP20x.

18. The method according to any one of claims 16 to 17, wherein said glycoside 3-oxidase enzyme is selected from the PsG30x, AgCarA, MtCarA, CarA, and ScP20x group.

19. The method according to any one of claims 12 to 17, wherein said pyranoside or said furanoside is protected by a protecting group comprising an aromatic moiety, particularly with a substituted or unsubstituted benzyl group or benzoyl group, more particularly with an unsubstituted benzyl group or benzoyl group.

20. The method according to any one of claims 12 to 19, wherein said pyranoside is selected from the group of glucose, galactose, 2-deoxy-glucose, rhamnose, mannose, fucose and N-acetylglucosamine.

21. The method according to any one of claims from 12 to 19, wherein said furanoside is selected from the group of arabinose, ribose and fructose.

22. The method according to any one of claims 12 to 21, wherein said pyranoside or the furanoside is a D-enantiomer.

23. The method according to any one of claims 12 to 22, wherein said pyranoside is glucose or galactose.

24. The method according to any one of claims 12 to 23, wherein said pyranoside is in β or α configuration.

25. The method according to any one of claims 12 to 24, wherein said pyranoside or said furanoside is linked to at least one saccharide.

26. The method according to any one of claims 12 to 25, further comprising a reduction step at carbonyl position, affording a pyranoside- or a furanoside-epimer.

27. The method according to claim 26, wherein the reduction step comprises addition of a compound selected from the group of NaBH4, NaBH4 / cerium chloride, LiAIH4, DIBAL, LS- selectride, K-selectride, L-selectride, Na(0Me)3BH, K(O-iPr)3BH, NaCNBH3, Na(0Ac)3BH, LiBH4, Me2S·BH3, PhNEt2·BH3, NaNH2(BH3)2, NH3-BH3 / TiCl4, pinBH / NaOtBu, Sm / HCl / THF, Red-AI, particularly LS-selectride.

28. The method according to any one of claims 26 to 27, wherein said pyranoside is D-glucose, and said pyranoside-epimer is D-allose.

29. The method according to any one of claims 26 to 27, wherein said pyranoside is D- galactose, and said pyranoside-epimer is D-gulose.

30. The method according to any one of the claims 12 to 29, further comprising a step of deprotecting said pyranoside or said furanoside.

31. Use of the polypeptide according to any one of claims 1 to 6 for production of an oxidized saccharide.

32. Use of the polypeptide according to any one of claims 1 to 6 for the production of a saccharide epimer.

33. The use according to claim 32, wherein the epimer is D-allose or D-gulose.

Images