Process for the preparation of sugar monoglycerides

Abstract

A method of making a glycolipid, the method comprising: (a) making a glycolipid via a single catalytic step by contacting a hydroxyl fatty acid with a glycosyl donor in the presence of an enzyme selected from: i) a glycosyltransferase and ii) a glycosidase; (b) optionally isolating the glycolipid made; wherein the glycolipid has the following Formula 1, wherein n is 0 to 15; wherein X is selected from the group consisting of alkyl, aryl, heteroalkyl, heteroaryl, unsaturated alkenyl, and unsaturated heteroalkenyl, and combinations and stereoisomers thereof; and wherein X has a carbon chain length of 4 to 22, wherein M is selected from one or both of glucose, sulfoquinovose, fructose, galactose, ribose, maltose, xylose, rhamnose, sophorose, mannose, arabinose, fucose, and combinations and stereoisomers thereof; and wherein R is selected from -H, alkyl selected from linear, branched, or cyclic alkyl, heteroalkyl, aryl, heteroaryl, heteroarylalkyl, arylalkyl, taurine, and all possible stereoisomers and combinations thereof.(1).

Description

[0001] References to sequence lists

[0002] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Technical Field

[0003] This invention relates to cells and methods for producing glycosaminoglycans, and more particularly to cells and methods for producing rhamnose monolipids. Background Technology

[0004] In personal care products, surfactants are used for purposes such as cleaning, foaming, thickening, emulsifying, solubilizing, and antimicrobial effects. However, many commonly used surfactants are made from starting materials such as petrochemicals, and consumers are seeking more natural and gentler materials in their products. Biosurfactants, which are microbial-derived surfactants, can provide improved efficacy without compromising performance. Summary of the Invention

[0005] A method for preparing glycosyl monoesters, the method comprising: (a) preparing glycosyl monoesters via a single catalytic step by contacting a hydroxy fatty acid with a glycosyl donor in the presence of an enzyme selected from: i) a glycosyltransferase and ii) a glycosidase; (b) optionally isolating the prepared glycosyl monoesters; wherein the glycosyl monoesters have the following formula 1:

[0006] Formula 1: Wherein n is 0 to 15; wherein X is selected from alkyl, aryl, heteroalkyl, heteroaryl, unsaturated alkenyl and unsaturated heteroalkenyl, and combinations and stereoisomers thereof; and wherein X has a carbon chain length of 4 to 22; wherein M is selected from glucose, sulfoquinoline, fructose, galactose, ribose, maltose, xylose, rhamnose, sophorose, mannose, arabinose, fucose, and combinations and stereoisomers thereof, or both thereof; and wherein R is selected from -H, and from alkyl, heteroalkyl, aryl, heteroaryl, heteroarylalkyl, arylalkyl, tauryl, and all possible stereoisomers and combinations thereof. Attached Figure Description

[0007] For a more complete understanding of this disclosure, reference should be made to the following detailed description of the invention and the accompanying drawings.

[0008] Figure 1 It is a rooted phylogenetic representation that compares 12 sequence IDs with Seq. ID No. 17 (the root sequence) using multiple sequence comparison (MUSCLE) alignment by logarithmic expectation.

[0009] Figure 2 An exemplary rhamnolipid structure is shown.

[0010] Figure 3 The chromatogram shows the formation of Rha-C10 from SEQ ID NO: 30 after a reaction of rhamnose and 3-OH-decanoic acid in 75% glycerol at 15°C for 72 hours. The formed Rha-C10 (rhamnolipid C10) was traced at a retention time of 2.820 min. Detailed Implementation

[0011] There is interest in non-traditional surfactants, such as glycolipid biosurfactants, because many palm-based and petroleum-based surfactants face sustainability, environmental, and socioeconomic challenges. Glycolipids consist of a different set of naturally occurring surfactant molecules with a range of structures composed of polar sugar groups and lipid groups. Two main commercial classes of glycolipids are rhamnolipids (produced via bacterial fermentation) and sophorolipids (produced via yeast fermentation with a mixed oil and sugar feed). In addition to being considered environmentally friendly chemicals and achieving green certification, these materials have many other potential beneficial effects, such as gentleness, moisturizing properties, and cleaning effectiveness.

[0012] Many current commercial glycolipids exhibit poor performance and high cost compared to existing surfactants. Known for the structures of certain high-performance rhamnolipids, the inventors sought to produce optimized glycolipids. The inventors hypothesized that altering the molecular structure by simplifying the surfactant head group and elongating it to a single-chain length (making it structurally more similar to a typical surfactant) would increase surface activity. Production strategies involve short-term semi-synthetic fermentation, in which commercial rhamnolipids are used as feedstocks and hydrolyzed to produce simplified monolipolipid homologue structures (as described in U.S. Patents 63 / 557,812 and 63 / 558,670, Attorney General's Files 16682P and 16682P2, which are incorporated herein by reference), or by hydrolyzing rhamnolipids using hydrolytic enzymes in a cell-free system, or by genetically engineering them using recombinant DNA technology for heterologous expression in host cells (Attorney General's File 16700P, US 63 / 641,630), also incorporated herein by reference.

[0013] This invention relates to a method for preparing biosurfactants via one-step enzymatic glycosylation of hydroxy fatty acids. Biosurfactants can be prepared using glycosyltransferases or glycosidases. In some embodiments, the glycosyltransferases or glycosidases can be engineered using recombinant DNA technology, such as through heterologous expression in host cells. All methods of this invention produce glycomonolipids [Formula 1], which can then provide commercially and consumer-beneficial effects.

[0014] This invention provides methods and cells for preparing glycomonolipids. In some embodiments, the method for producing glycomonolipids intracellularly includes expressing one or more recombinant polypeptides that catalyze the glycosylation of hydroxy fatty acids, in some embodiments glycosyltransferases or glycosidases, and culturing cells under conditions suitable for polypeptide production to produce glycomonolipids, in some embodiments rhamnosyl monolipids.

[0015] In some embodiments, a method for producing glycosaminoglycans intracellularly is provided, the method comprising expressing a polypeptide having glycosyltransferase or glycosidase activity intracellularly; and culturing the cells under conditions suitable for polypeptide production to produce rhamnosaminoglycans.

[0016] A method for producing rhamnosyl monolipids in cells, the rhamnosyl monolipids having a chain length of about 4 to 22 carbons, is also provided. The method includes modifying cells to increase carbon flux and culturing the cells under conditions suitable for increasing carbon flux, thereby producing monolipids with a chain length of about 4 to about 22 carbons.

[0017] It also provides Pseudomonas bacteria that produce rhamnosyl monoesters with chain lengths of approximately 4 to 22 carbons.

[0018] This invention provides a method for preparing glycosylated monoesters via a glycosylation reaction of hydroxy fatty acids with a glycosyl donor catalyzed by a glycosyltransferase or glycosidase; wherein the glycosylated monoester has the following formula 1:

[0019] Formula 1

[0020] Wherein n has a carbon chain length of 0 to 15, and X is selected from alkyl, aryl, heteroalkyl, heteroaryl, unsaturated alkenyl, and unsaturated heteroalkenyl, as well as combinations and stereoisomers thereof; and has a carbon chain length of 4 to 22, wherein M is selected from glucose, sulfoquinoline, fructose, galactose, ribose, maltose, xylose, rhamnose, sophorose, mannose, arabinose, fucose, and combinations and stereoisomers thereof, or both thereof; and wherein R is selected from -H, and from alkyl, heteroalkyl, aryl, heteroaryl, heteroarylalkyl, arylalkyl, tauryl, and all possible stereoisomers and combinations thereof. For any given embodiment, X, M, and R can be any combination thereof.

[0021] In some implementations, the hydroxy fatty acid is a straight-chain hydroxy fatty acid.

[0022] In some embodiments, in Formula 1, X can be selected from alkyl and unsaturated alkenyl groups and has a carbon chain length of 4 to 22. In some embodiments, R can be selected from -H and alkyl groups and has a straight-chain carbon length of 1 to 4.

[0023] In some embodiments, the glycosyltransferase comprises a polypeptide sequence having at least 60% identity with any of or a fragment of any of SEQ ID NO: 1-12, in some cases 75% identity, in some cases 90% identity, in some cases 95% identity, or in some cases 100% identity.

[0024] In some embodiments, the glycosyltransferase has amino acid substitutions compared to SEQ ID NO: 1. In some embodiments, SEQ ID NO: 1 may have the following amino acid substitutions: for SEQ ID NO: 63, V25A, V31I, N49K, I82V, A83G, M163L, Q182R, A187P, T189V, L235F, L238P, G263S, T268A, S288P, S292A, I377V, E388D, S417Y; or for SEQ ID NO: 64 are V8I, V25A, V31I, N49K, I82V, E100Q, H119R, V124I, L161M, M163V, A167T, S179R, Q182R, A187P, T189A, H216R, G234S, L235F, P237E, L238P, E243D, G 263S, T268A, S288P, S292T, S294A, S295T, H313Q, I337V, I377V, E388D, D398E, S401C, L406R, L411I, A412V, R413A, T414E, A415P, R416G, S417Y, A420R. In these embodiments, “V25A” indicates that the valine (V or Val) at position 25 in SEQ ID NO: 1 is replaced by alanine (A or Ala), as found at position 25 in SEQ ID NO: 63.

[0025] In some embodiments, the glycosidase comprises a polypeptide sequence having at least 60% identity with any one of SEQ ID NO: 30-62 and 80 or a fragment thereof, in some cases 75% identity, in some cases 90% identity, in some cases 95% identity, and in some cases 100% identity. Preferably, the polypeptide sequence has at least 60% identity with SEQ ID NO: 30 or 80, in some cases 75% identity, in some cases 90% identity, in some cases 95% identity, and in some cases 100% identity.

[0026] In some implementations, the enzyme may be encapsulated and / or immobilized.

[0027] In some embodiments, hydroxy fatty acid glycosylation enzymes, glycosyltransferases, or glycosidases can be prepared by heterologous expression in host cells. The host cell can be eukaryotic and / or prokaryotic. The host cell can be, but is not limited to, at least one of the following: *Escherichia coli* (…). Escherichia coli Bacillus subtilis ( Bacillus subtilis ), Streptomyces blue ( Streptomyces coelicolor ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Pichia pastoris () Pichia pastoris ), brewer's yeast ( Saccharomyces cerevisiae Kluyveromycin (lactic acid yeast) Kluyveromyces lactis Aspergillus niger ( ) Aspergillus niger Trichoderma reesei ( Trichoderma reesei Corynebacterium spp. Corynebacterium spp.), Pseudomonas putida ( Pseudomonas putida Burkholderia thamnoides ( ) Burkholderia thailandensis Burkholderia spp. Parabulkholderia Acinetobacter spp.), Acinetobacter spp. Acinetobacter spp.), genus *Bacillus* ( Alcanivorax spp.), genus *Antarcticus* ( ) Antarctobacter spp.), Bacillus spp. Bacillus spp.), Burkholderia ( Burkholderia spp.), Candida albicans ( Candida apicola ), Fibromosomal fibrinolyticus ( Cellulomonas cellulans ), Alcaligenes aureobacterium eutum ( Cupriavidus necator ), Enterobacteriaceae ( Enterobacter spp.), spp. of Halomonas ( Halomonas spp.), Lactobacillus spp. Lactobacilli spp.), Marinebacteria ( Marinobacter spp.), Myxococcus spp. Myxococcus spp.), Nocardia spp. ( Nocardioides spp.), Pseudoalteromonas ( Pseudoalteromonas spp.), Pseudomonas ( Pseudomonas spp.), *Pseudomonas* spp. Pseudoxanthomonas spp.), Salmonella nephritis ( Renibacterium salmoninarum ), Rhodococcus spp. ( Rhodoccus spp.), Bogoria red yeast ( Rhodotorula bogoriensis ), Korean tetrad cocci ( Tetragenococcus koreensis ), Methylobacterium ( Methylobacterium spp.) and methyl erythrobacter ( Methylorubrum spp.).

[0028] In some implementations, the monoglyceride prepared by glycosyltransferase or glycosidase may be rhamnose monoglyceride.

[0029] In some embodiments, the glycosyl donor may comprise at least one rhamnose. Rhamnose may be, but is not limited to, nucleoside monophosphate (NMP), nucleoside diphosphate (NDP), or nucleoside triphosphate (NTP).

[0030] In some embodiments, the resulting rhamnose monoester may be selected from Rha-C8, Rha-C10, Rha-C12, Rha-C14, RhaRha-C8, RhaRha-C10, RhaRha-C12, and RhaRha-C14.

[0031] In some embodiments, the resulting rhamnosyl monoester can be selected from Rha-C10, Rha-C12, and Rha-C14. Rhamnosyl monoester can be produced by reacting a cell-free glycosyltransferase or glycosidase with a rhamnosyl donor and 3-hydroxydecanoic acid, 3-hydroxylauric acid, or 3-hydroxymyristic acid under stirring in a buffer solution at pH 6 to 10, at a temperature of 25°C to 42°C, and in a buffer solution.

[0032] In some embodiments, the present invention provides recombinant cells comprising the following: (i) a glycosyltransferase gene containing SEQ ID NO: 18-29, or (ii) a glycosidase gene.

[0033] In some implementation schemes of recombinant cells, the recombinant cells are *Pseudomonas putida*, *Escherichia coli*, *Bacillus subtilis*, *Streptomyces cerevisiae*, *Pseudomonas aeruginosa*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Aspergillus niger*, *Trichoderma reesei*, *Corynebacterium*, *Burkholderia thamnip*, *Burkholderia spp.*, *Acinetobacter*, *Acinetobacter*, *Antarctica*, *Bacillus*, *Burkholderia*, *Candida*, *Fibrinosporium*, *Alcaligenes*, *Enterobacter*, *Halomycosis*, *Lactobacillus*, *Hymenobacter*, *Myxococcus*, *Nocardia*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Nephroticis*, *Rhodococcus*, *Borgonia bogoria*, *Tetracoccus koreanum*, *Methylobacterium*, and *Methylobacterium rubrum*.

[0034] In some embodiments of the recombinant cells, the glycosyltransferase may comprise a polypeptide sequence having at least 60%, and in some embodiments at least 70%, or 75%, 80%, or 90%, 95%, or 100% identity with any of SEQ ID NO: 1-12. In other embodiments of the recombinant cells, the glycosidase may comprise a polypeptide sequence having at least 60%, and in some embodiments at least 70%, or 75%, or 80%, or 90%, or 95%, or 100% identity with any of SEQ ID NO: 30-62 and 80.

[0035] In other embodiments, the present invention provides a method for preparing rhamnose monoester in recombinant cells, the method comprising the following steps: a) Culture recombinant cells to produce rhamnosyl monolipids; and b) Rhamnose monoesters obtained by selective separation; The recombinant cells described herein comprise (i) a glycosyltransferase comprising SEQ ID NO: 1-12 or (ii) a glycosidase comprising SEQ ID NO: 30-62 or 80. In some embodiments of the method for preparing rhamnosyl monolipids in recombinant cells, the glycosyltransferase may comprise a polypeptide sequence having at least 60% identity with SEQ ID NO: 1-12 or fragments thereof, in some embodiments at least 70% identity, in some embodiments at least 80% identity, in some embodiments at least 90% identity, in some embodiments at least 95% identity, and in some embodiments 100% identity. In some embodiments of the method for preparing rhamnosyl monolipids in recombinant cells, the glycosidase may comprise a polypeptide sequence having at least 60% identity with SEQ ID NO: 30-62 or 80, in some embodiments at least 70% identity, in some embodiments at least 80% identity, in some embodiments at least 90% identity, in some embodiments at least 95% identity, and in some embodiments 100% identity.

[0036] In some embodiments, the present invention provides a method for producing glycomonolipids, wherein the glycomonolipids are produced by the growth of engineered cells through any of the following: A.) Constitutive growth from a carbon source (such as a raw material or sole carbon source) to produce monosaccharides; B.) Growth cells derived from a carbon source (such as raw material or unique carbon) are then cleaved to produce a protein complex to react the starting material, thereby producing glycosaminoglycans in vitro or in vitro. Includes the following steps: a) Contacting fatty acids with glycosyl donors in the presence of glycosyltransferases or glycosidases. b) Optionally separate monosaccharide; Among them, monosaccharides have the following formula 1:

[0037] Formula 1

[0038] Where n = 0 to 15; X is selected from alkyl, aryl, heteroalkyl, heteroaryl, unsaturated alkenyl, and unsaturated heteroalkenyl; and has a carbon chain length of 4 to 22; M is selected from glucose, sulfoquinoline, fructose, galactose, ribose, maltose, xylose, rhamnose, sophorose, mannose, arabinose, fucose, and combinations thereof and stereoisomers thereof; and R is selected from -H, from alkyl, heteroalkyl, aryl, heteroaryl, heteroarylalkyl, arylalkyl, tauryl, and all possible stereoisomers thereof, of straight-chain, branched or cyclic alkyl groups.

[0039] References to "implementation" or similar methods in this specification mean that the specific materials, features, structures, and / or characteristics described in conjunction with that implementation are included in at least one implementation, or optionally multiple implementations, but do not imply that all implementations include the described materials, features, structures, and / or characteristics. Furthermore, materials, features, structures, and / or characteristics can be combined in any suitable manner in different implementations, and materials, features, structures, and / or characteristics can be omitted or replaced as described. Therefore, unless otherwise stated or declared as incompatible, although not expressly illustrated in the combination, the implementations and aspects described herein may include elements or components of other implementations and / or aspects, or may be combined with elements or components of other implementations and / or aspects.

[0040] Unless otherwise specified, all percentages of ingredients described herein are by weight of the cosmetic composition and may be specified as “weight %”. Unless otherwise specified, all ratios are by weight. All ranges are inclusive and combinable. Significant figures do not express limitations on the quantities shown or on the accuracy of the measurements. All numerical values ​​should be understood to be modified by the word “about” unless otherwise specified. Unless otherwise indicated, all measurements are understood to have been performed at approximately 25°C and ambient conditions, where “ambient conditions” means conditions at approximately 1 atmosphere and approximately 50% relative humidity. All numerical ranges are narrow ranges including the extreme values; the upper and lower limits of the described ranges are interchangeable to form additional ranges not explicitly described.

[0041] The compositions of the present invention may comprise, consist of, or be composed of the essential components described herein, as well as optional ingredients. As used herein, “consistently consisting of” means that a composition or component may contain additional ingredients, provided that the additional ingredients do not substantially alter the essential and novel features of the composition or method protected by the claims. As used in the specification and appended claims, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the (described)” are intended to also include the plural forms.

[0042] definition

[0043] When used in the context of chemical groups: "hydrogen" refers to -H; "hydroxyl" refers to -OH; "oxo" refers to =O; "carbonyl" refers to -C(=O)-; "carboxyl" and "carboxylate" refer to -C(=O)OH (also written as -COOH or -CO2H) or its deprotonated form; "amino" means -NH2; "hydroxyamino" means -NHOH; "nitro" refers to -NO2; "imino" refers to =NH; "amine oxide" refers to N + O - N has three covalent bonds with atoms other than O; "isohydroxamic acid" or "isohydroxamic acid ion" refers to -C(O)NHOH or its deprotonated form.

[0044] The term "cation" refers to an atom, molecule, or chemical group having a net positive charge, including single-charged and multi-charged substances. A cation can be a single atom such as a metal (non-limiting examples include Na). + or Ca +2 ), single molecule (non-limiting examples include (CH3)4N + ) or chemical groups (non-limiting examples include –N(CH3)3 + The term "amine cation" refers to NR4. + A specific molecular cation in which the four substituted R moieties can be independently selected from H and alkyl groups; non-limiting examples include NH4. + (Ammonium), CH3NH3 + (Methylammonium), CH3CH2NH-3 + (Ethylammonium), (CH3)2NH2 + (Dimethylammonium), (CH3)3NH + (trimethylammonium) and (CH3)4N + (Tetramethylammonium). In some embodiments, the cation may be selected from Na+, K+, Li+, Cs+, +NH3R2; +NH2R2R3; +NHR2R3R4, +NR2R3R4R5, wherein R2, R3, R4 and R5 are each independently selected from alkyl, branched alkyl and cyclic alkyl groups.

[0045] The term "anion" refers to an atom, molecule, or chemical group that has a net negative charge, including single-charged and multi-charged substances. Anions can be single atoms (e.g., but not limited to halogen F). - Cl - ,Br - ), single molecule (non-limiting examples include CO3) -2 H2PO4 - HPO4 -2 PO4 -3 HSO4 - SO4 -2 The chemical group (non-limiting examples include sulfate, phosphate, sulfonate, phosphonate, hypophosphonate, sulfonate, mercapto, carboxylate, amine oxide, isohydroxamate, and hydroxylamino) is considered an anionic group if the removal of a proton results in a net negative charge. In solution, the chemical group is capable of losing a proton and discharging according to the Henderson-Hasselbalch equation (pH = pKa + log₂). 10 ([A - ] / [HA]; where [HA] is the molar concentration of the undissociated acid, and [A - The pKa (molar concentration of the conjugate base of the acid) changes with pH and becomes anion. When the pH of the solution equals the pKa value of the functional group, 50% of the functional groups will be anions, while the remaining 50% will have protons. Generally, if the pH is at or above the pKa of the functional group, the functional group in the solution can be considered anion.

[0046] The term "salt" refers to an electrically neutral combination of one or more anions and cations. For example, when R represents a salt of a carboxylate group -COOR, it should be understood that the carboxylate group (-COO-) is an anion with a negative charge of -1, and R is a cation with a positive charge of +1 to form an electrically neutral entity with an anion with a charge of -1, or R is a cation with a positive charge of +2 to form an electrically neutral entity with two anions, both with a charge of -1.

[0047] As used herein, the term "saturated" means that a chemical compound or group so modified does not have a carbon-carbon double bond and does not have a carbon-carbon triple bond, unless described below. With regard to the substitution pattern of a saturated chemical group, one or more carbon-oxygen double bonds or carbon-nitrogen double bonds may be present. When such bonds are present, it is not excluded that the carbon-carbon double bond may occur as part of a keto-enol tautomerism or an imine / enamine tautomerism.

[0048] When used without the modifier "substitution," the term "aliphatic" indicates that the modified chemical compound / group is acyclic or cyclic, but not aromatic. In aliphatic chemical compounds / groups, carbon atoms can be linked together in straight-chain, branched, or non-aromatic (alicyclic hydrocarbon) form. Aliphatic chemical compounds / groups can be saturated, i.e., linked by single bonds (alkane / alkyl); or unsaturated, i.e., having one or more double bonds (alkene / alkenyl) or one or more triple bonds (alkynyl / alkynyl).

[0049] When used without the modifier "substituted," the term "alkyl" refers to a monovalent saturated aliphatic group with a carbon atom as the linking point, having a straight or branched, cyclic, cyclic, or acyclic structure, and containing no atoms other than carbon and hydrogen. Thus, as used herein, cycloalkyl is a subset of alkyl groups in which the carbon atom forming the linking point is also a member of one or more non-aromatic ring structures, wherein the cycloalkyl group does not contain atoms other than carbon and hydrogen. As used herein, the term does not exclude the presence of one or more alkyl groups (permitted by carbon number restrictions) attached to the ring or ring system. Non-limiting examples of alkyl groups including -CH3(Me), -CH2CH3(Et), -CH2CH2CH3 (n-Pr or propyl), -CH(CH3)2 (i-Pr, 'Pr or isopropyl), -CH(CH2)2 (cyclopropyl), -CH2CH2CH2CH3 (n-Bu), -CH(CH3)CH2CH3 (sec-butyl), -CH2CH(CH3)2 (isobutyl), -C(CH3)3 (tert-butyl, t-butyl, t-Bu or tBu), -CH2C(CH3)3 (neopentyl), cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl. The term "alkanediyl" when used without the modifier "substituted" refers to a divalent saturated aliphatic group with one or two saturated carbon atoms as connecting points, having a straight or branched, cyclic, cyclic or acyclic structure, without carbon-carbon double or triple bonds, and without atoms other than carbon and hydrogen. The groups -CH2 (methylene), -CH2CH2-, -CH2C(CH3)2CH2-, and -CH2CH2CH2- are non-limiting examples of alkyl diester groups. When used without the modifier "substituted," the term "alkylidene" refers to a divalent group =CRR', where R and R' are independently hydrogen, alkyl, or R and R' together represent an alkyl diester having at least two carbon atoms. Non-limiting examples of alkylidene groups include: =CH2, =CH(CH2CH3), and =C(CH3)2. "Alkane" refers to the compound HR, where R is an alkyl group as defined above.

[0050] When any of these terms is used with the modifier “substitution”, one or more hydrogen atoms have been independently substituted by -OH, -F, -Cl, -Br, -I, -NH2, -NO2, -CO2H, -CO2CH3, -CN, -SH, -OCH3, -OCH2CH3, -C(O)CH3, -NHCH3, -NHCH2CH3, -N(CH3)2, -C(O)NH2, -OC(O)CH3, -S(O)2NH2, -P(O)(OH)2, -P(O)(OH)OP(O)(OH)2, -OP(O)(OH)2, -OP(O)(OH)OP(O)(OH)2, -S(O)2(OH) or -OS(O)2(OH). The following groups are non-limiting examples of substituted alkyl groups: -CH2OH, -CH2Cl, -CF3, -CH2CN, -CH2C(O)OH, -CH2C(O)OCH3, -CH2C(O)NH2, -CH2C(O)CH3, -CH2OCH3, -CH2OC(O)CH3, -CH2NH2, -CH2N(CH3)2, -CH2CH2Cl, -CH2P(O)(OH)2, -CH2P(O)(OH)OP(O)(OH)2, -CH2S(O)2(OH), and –CH2OS(O)2(OH). The term "halogenated alkyl" is a subset of substituted alkyl groups in which one or more hydrogen atoms have been replaced by a halogen group, and no atoms other than carbon, hydrogen, and halogen are present. The group -CH2Cl is a non-limiting example of a haloalkyl group. The term "fluoroalkyl" is a subset of substituted alkyl groups in which one or more hydrogen atoms have been replaced by a fluorine group, and no atoms other than carbon, hydrogen, and fluorine are present. The groups -CH2F, -CF3, and -CH2CF3 are non-limiting examples of fluoroalkyl groups.

[0051] When used without the modifier "substitution," the term "alkenyl" refers to a monovalent unsaturated aliphatic group having one carbon atom as a connecting point, having a straight or branched, cyclic, cyclic or acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. Non-limiting examples of alkenyl groups include: -CH=CH2 (vinyl), -C(CH3)=CH2 (methyl vinyl), -CH=CHCH3, -CH=CHCH2CH3, -CH2CH=CH2 (allyl), -CH2CH=CHCH3, and -CH=CHCH=CH2. When used without the modifier "substitution," the term "alkendiyl" refers to a divalent unsaturated aliphatic group having two carbon atoms as connecting points, having a straight or branched, cyclic, cyclic or acyclic structure, at least one non-aromatic carbon-carbon double bond, no carbon-carbon triple bond, and no atoms other than carbon and hydrogen. C=CH2 (vinylidene), -CH=CH-, -CH=C(CH3)CH2-, and -CH=CHCH2- are non-limiting examples of alkenyl groups. It should be noted that although alkenyl groups are aliphatic, once connected at both ends, it does not preclude the group from forming part of an aromatic structure. The terms "alkene" or "olefin" are synonymous and refer to compounds having the formula HR, where R is an alkenyl group as defined above.

[0052] The term "aryl," when used without the modifier "substituted," refers to a functional group derived from a simple aromatic ring compound, where the attachment point is a carbon atom on the aromatic ring. An aromatic ring is a hydrocarbon having a cyclic structure and a delocalized electron system. An aryl group is formed by removing a hydrogen atom from the ring. The names of aryl groups are based on the names of aromatic rings with a -yl suffix, such as phenyl, naphthyl, indolyl, etc. Aryl groups can be substituted with alkyl and / or heteroalkyl chains and can have one or more heteroatoms within the aryl ring.

[0053] The term "arylalkyl" refers to an aryl group attached to an alkyl dimethyl group, wherein the attachment point is on the alkyl dimethyl group.

[0054] Tauryl / taurate is defined as aminoethylsulfonyl.

[0055] A heteroatom is defined as an atom other than carbon, including but not limited to nitrogen (N), oxygen (O), or sulfur (S). Heteroatoms can be attached to straight-chain or branched alkyl chains, or to non-aromatic or aromatic rings as part of the ring, or as a substituent adjacent to it. More than one heteroatom can be present in an alkyl chain or ring.

[0056] An unsaturated hydrocarbon chain is defined as having, but not limited to, at least one carbon-carbon double bond (C=C). An unsaturated chain can have one double bond (alkenyl), two double bonds (dienyl), multiple double bonds (polyalkenyl), and / or a carbon-carbon triple bond (alkynyl). Unsaturated bonds can be adjacent to each other (conjugated) or separated by other carbon atoms in the chain.

[0057] The International Union of Pure and Applied Chemistry (IUPAC) defines a "monomer molecule" as "a molecule that can undergo polymerization to contribute structural units to the basic structure of a macromolecule." Polymers are macromolecules.

[0058] As used, “separation” refers to the removal of enzymes from solution before or after reaction with glycolipids.

[0059] As used, "immobilization" refers to fixing a glycosyltransferase or glycosidase onto a solid support, thereby removing it from solution or stabilizing the enzyme. Some examples of solid supports among several other solid supports known in the art include silica, chitosan, and bacterial spores.

[0060] As used with respect to a composition, “treat or treating” means adding or applying materials to the composition.

[0061] The word “about” modifies a particular value by referring to a range of positive or negative 20% or less (e.g., positive or negative 15% or less, 10% or less, or even 5% or less).

[0062] As used in the reference composition, “application” means applying or spreading the composition onto human keratin surfaces, such as skin or hair.

[0063] "Charge density" ("CD") refers to the ratio of the positive charge on a polymer to the molecular weight of the polymer.

[0064] "Personal care composition" means a product that is applied to or comes into contact with a body surface during normal use to provide beneficial effects. A body surface includes skin, such as the epidermis or mucous membranes; it also includes structures associated with the body surface, such as hair, teeth, or fingernails / toenails. Examples of personal care compositions include products applied to the human body to improve appearance, cleanliness, odor control, or overall aesthetics. Non-limiting examples of personal care compositions include oral care compositions such as tooth powder, mouthwash, mousse, foam, mouth spray, lozenges, chewing tablets, chewing gum, teeth whitening strips, dental floss and floss coatings, breath freshener dissolving strips, or denture care products, denture adhesive products; aftershave gels and creams, preshave preparations, shaving gels, creams or foams, moisturizers and lotions; cough and cold compositions, gels, gel caps and throat sprays; leave-on lotions and creams, shampoos, shower gels, body scrubs such as Vicks VapoRub; hair conditioning products, hair coloring and bleaching compositions, mousse, shower gels, bar soaps, antiperspirants, deodorants, hair removal products, lipsticks, foundations, mascaras, tanning agents and sunscreens; feminine care compositions such as lotions and lotion compositions for absorbent articles; baby care compositions for absorbent or disposable articles; and oral cleaning compositions for animals such as dogs and cats. Other non-limiting examples include hand soaps, hand sanitizers, bath products, shower gels, shampoos, body lotions, feminine care products, foot care products, deodorants, pet care products, and combinations thereof. Other non-limiting examples include wipes for personal care and household cleaning purposes; toilet paper; towels for hand drying, household drying, and household cleaning; facial tissues; skin care compositions; first-aid or surgical antiseptics; diapers; sanitary napkins; and combinations thereof.

[0065] The term "detergent composition" refers to a composition or formulation designed for cleaning soiled surfaces. Such compositions include, but are not limited to, dishwashing compositions, laundry detergent compositions, fabric softening compositions, fabric strengthening compositions, fabric freshening compositions, laundry pre-wash agents, laundry pretreatment agents, laundry additives, spray products, dry cleaning agents or compositions, laundry rinsing additives, washing additives, post-rinse fabric treatment agents, ironing aids, hard surface cleaning compositions, unit-dose formulations, delayed delivery formulations, detergents contained on or within porous substrates or nonwoven sheets, and other suitable forms readily apparent to those skilled in the art from the teachings herein. Such compositions can be used as cleaning pretreatment agents, cleaning posttreatment agents, or can be added during rinsing or washing cycles in a cleaning process. Detergent compositions can be in the form of liquids, powders, single-phase or multi-phase unit-dose or sachets, tablets, gels, pastes, sticks, or flakes. Preferably, the composition is for manual washing. The detergent composition of the present invention can be a dishwashing agent. The composition can be in liquid form. Other non-limiting examples include hard surface cleaners, deodorizers, fabric care compositions, fabric cleaning compositions, manual dishwashing liquids, automatic dishwashing liquids, floor waxes, kitchen cleaners, bathroom cleaners, and combinations thereof.

[0066] "Cleaning composition" refers to a personal care composition or product intended for use in cleaning body surfaces, such as skin or hair. Some non-limiting examples of cleaning compositions are shampoos, conditioners, conditioning shampoos, body gels, liquid hand soaps, facial cleansers, etc.

[0067] "Cosmetic agent" means any substance and any component thereof intended to be applied, poured, sprayed, atomized, introduced, or otherwise applied to the body of a mammal or any part thereof to provide a cosmetic effect. Cosmetic agents may include food additives and substances generally considered safe by the U.S. Food and Drug Administration (GRAS).

[0068] A “gel network phase” or “dispersed gel network phase” refers to a layered or capsule-shaped solid crystalline phase comprising at least one fatty alcohol, at least one gel network surfactant, and a liquid carrier. The layered or capsule-shaped phase may be formed from alternating layers, one layer comprising a fatty alcohol and a gel network surfactant, and another layer comprising a liquid carrier.

[0069] "Solid crystallization" refers to a crystalline structure at ambient temperature consisting of a layered or cystic phase whose melting transition temperature is below that of the phase. For example, the melting transition temperature of a layered or cystic phase can be about 30°C or higher (i.e., slightly above about room temperature). The melting transition temperature can be measured by differential scanning calorimetry, a conventional measurement method known to those skilled in the art.

[0070] "Suitable for application to human hair" means that the personal care composition or its components can be used in contact with human hair, scalp and skin without undue toxicity, incompatibility, instability, allergic reactions, etc.

[0071] "Substantially free of" means that the composition or ingredient contains less than 3% (e.g., less than 2%, less than 1%, or even less than 0.5%) of the subject material by weight of the composition or ingredient. "Free of" means that the composition or ingredient contains 0% of the subject material.

[0072] "Sulfated surfactant" refers to a surfactant containing a sulfate moiety. Some non-limiting examples of sulfated surfactants are sodium lauryl sulfate, sodium lauryl polyoxyethylene ether sulfate, ammonium lauryl sulfate, and ammonium lauryl polyoxyethylene ether sulfate. "Sulfate-free surfactant" refers to a surfactant that does not have a sulfate moiety.

[0073] The above definitions supersede any conflicting definitions in any references incorporated herein by reference. However, the fact that some terms are defined should not be construed as indicating that any undefined term is indeterminate. Rather, all terms used are intended to describe the invention in a way that allows those skilled in the art to understand its scope and practice.

[0074] This is not an exhaustive list of sugars, and many other sugars with different structures exist: Glucose is a monosaccharide and the primary source of energy in living organisms. Its chemical formula is C6H12O6. Fructose is another monosaccharide and is commonly found in fruits and honey. Its chemical formula is also C6H12O6. Galactose is a monosaccharide found in milk and dairy products. Its chemical formula is C6H12O6. Sucrose, also known as sugar, is a disaccharide composed of glucose and fructose. Its chemical formula is C12H22O11. Lactose is a disaccharide found in milk and dairy products. It is composed of glucose and galactose. Its chemical formula is C12H22O11. Maltose is a disaccharide formed by the combination of two glucose molecules. It is commonly found in germinated grains. Its chemical formula is C12H22O11. Ribose is a monosaccharide that is an essential component of RNA (ribonucleic acid) and plays a role in energy metabolism. Its chemical formula is C5H10O5. Deoxyribose is a monosaccharide and a component of DNA (deoxyribonucleic acid). It is similar to ribose but lacks an oxygen atom. Its chemical formula is C5H10O4.

[0075] rhamnolipid surfactants

[0076] Rhamnose (Rha, Rham), with the molecular formula shown below, is a naturally occurring deoxyglycan. It can be classified as a methylpentose or a 6-deoxyhexose.

[0077]

[0078] Rhamnolipids are a class of glycolipids that can be used as bacterial surfactants. They have a glycosyl head group, in this case, the rhamnose moiety, and an acidic fatty tail. There are two main classes of rhamnolipids: monorhamnolipids and dirhamnolipids, which consist of one or two rhamnose groups, respectively. Furthermore, monorhamnose and dirhamnose lipids can each have one or two lipids, for example, as... Figure 2 The combination shown.

[0079] The rhamnolipid surfactants mentioned in this article can be produced by microorganisms (e.g., Pseudomonas aeruginosa). Pseudomonas aeruginosa ), Pseudomonas putida ( Pseudomonas putida ), Pseudomonas aeruginosa ( Pseudomonas chlororaphis Rhamnolipid surfactants can provide beneficial cleaning effects due to their amphiphilic nature, which allows the surfactant to decompose and micelles to form around oils and other contaminants. The "trapped" contaminants can then be more easily rinsed away with water. Descriptions of various types of rhamnolipids are disclosed in EP2410039. Certain methods for preparing, extracting, and blending naturally occurring rhamnolipids are known in the art. This invention includes the preparation of monorhamnose monolipide biosurfactants.

[0080] Glycosylation of hydroxy fatty acids

[0081] In embodiments of the invention, 3-hydroxy fatty acids are enzymatically glycosylated to produce rhamnose monoesters (Rha-CX). Here, Rha refers to a single rhamnose group, C is defined as a monoester, and X is the carbon chain length. For example, Rha-C10 is a rhamnose monoester containing ten carbons in its chain.

[0082] Glycosylases can be selected from, but are not limited to, the group consisting of glycosyltransferases or glycosidases.

[0083] Glycosyltransferase (EC 2.4.1): In the context of this application, a glycosyltransferase is an enzyme capable of catalyzing the transfer of a sugar moiety to a receptor molecule. Glycosyltransferases include, but are not limited to, the group consisting of rhamnosyltransferases and glucosyltransferases.

[0084] This invention includes rhamnosyltransferases (RhlB and RhlC) from bacterial species. RhlB adds a rhamnosyl group to the lipid, and RhlC adds a second rhamnosyl group.

[0085] Glycosidases (EC 3.2.1): These enzymes catalyze the hydrolysis of glycosidic bonds. They can also perform the reverse reaction, which reforms glycosidic bonds.

[0086] This invention also includes variants of enzymes. As used herein, variants of enzymes include sequences resulting from modifications of the wild-type protein of the corresponding protein with one or more amino acids (e.g., 1, 2, 5, or 10 amino acids) or at said one or more amino acids. This invention also includes variants derived from truncated forms of wild-type enzymes, such as proteins having a truncated N-terminus or a truncated C-terminus. Some enzymes may include an N-terminal signal peptide that may be removed during cellular secretion. This invention includes variants without an N-terminal signal peptide.

[0087] Bioinformatics tools, such as SignalP ver 4.1 (Petersen TN., Brunak S., von Heijne G., and Nielsen H. (2011), Nature Methods, 8:785-786), can be used to predict the presence and length of such signal peptides. This invention also includes variants derived by adding additional amino acid sequences to wild-type proteins, such as N-terminal tags, C-terminal tags, or insertions in the middle of the protein sequence. Non-limiting examples of tags include maltose-binding protein (MBP) tags, glutathione S-transferase (GST) tags, thioredoxin (Trx) tags, 6X histidine-tags, small peptide markers (DYKDDDDK) tags, and any other tags known to those skilled in the art. Tags can be used to improve solubility and expression levels during fermentation or as stalks for enzyme purification.

[0088] It is important that enzyme variants retain and preferably improve upon the ability of wild-type proteins to catalyze the conversion of unsaturated fatty acids. While some performance degradation in a given characteristic of a variant can be tolerated, variants should retain and preferably improve upon suitable characteristics for their intended application. Screening variants of one of the wild-types can be used to determine whether they retain and preferably improve upon appropriate properties.

[0089] Variants can have “conserved” substitutions. Suitable examples of conserved substitutions include a conserved substitution in an enzyme, such as the conserved substitution in SEQ ID NO: 1.

[0090] Enzymes containing one or more conserved substitutions can be produced by manipulating the nucleotide sequence encoding the enzyme using, for example, standard methods such as site-directed mutagenesis or PCR.

[0091] Examples of amino acids that can replace the original amino acid in an enzyme and are considered conserved substitutions include: Ser replacing Ala; Lys replacing Arg; Gln or His replacing Asn; Glu replacing Asp; Asn replacing Gln; Asp replacing Glu; Pro replacing Gly; Asn or Gln replacing His; Leu or Val replacing Ile; Ile or Val replacing Leu; Arg or Gln replacing Lys; Leu or Ile replacing Met; Met, Leu, or Tyr replacing Phe; Thr replacing Ser; Ser replacing Thr; Tyr replacing Trp; Trp or Phe replacing Tyr; and Ile or Leu replacing Val. Conservation and non-conservation can also refer to functional similarity and dissimilarity.

[0092] Variants can also have “non-conservative” substitutions, where the resulting amino acid has properties different from the original amino acid. Examples of amino acids that can replace the original amino acid in an enzyme and are considered non-conservative substitutions include: Tyr replacing Ala; Asp replacing Gly; and Lys replacing Pro.

[0093] Variants include “modified enzymes” or “mutant enzymes” that comprise proteins having at least one amino acid substitution, insertion, and / or deletion. Suitable examples of modified enzymes include modifications such as those to SEQ ID NO: 1. Therefore, enzymes of the present invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid modifications of SEQ ID NO: 1 (selected from substitutions, insertions, deletions, and combinations thereof), such as those found in SEQ ID NO: 63-70.

[0094] Compared to SEQ ID NO: 1, the modifications found in SEQ ID NO: 63 are substitutions of V25A, V31I, N49K, I82V, A83G, M163L, Q182R, A187P, T189V, L235F, L238P, G263S, T268A, S288P, S292A, I377V, E388D, and S417Y; in SEQ ID NO: The modifications found in 64 are V8I, V25A, V31I, N49K, I82V, E100Q, H119R, V124I, L161M, M163V, A167T, S179R, Q182R, A187P, T189A, H216R, G234S, L235F, P237E, L238P, E243D, and G2. Substitution of 63S, T268A, S288P, S292T, S294A, S295T, H313Q, I337V, I377V, E388D, D398E, S401C, L406R, L411I, A412V, R413A, T414E, A415P, R416G, S417Y, and A420R; in SEQ The modifications found in ID NO: 65 are substitutions for V8I, V25A, V31I, A146T, M163V, Q182R, A187P, E209D, L235F, L238V, E243D, G263S, S288P, S292T, A315S, L359V, T372A, I377V, E388D, and D398E;The modifications found in SEQ ID NO: 66 are substitutions of V8I, V25A, V31I, E61A, E100Q, V124I, A136P, A146T, V156L, A167T, Q182R, A187P, T189V, L235F, P237Q, L238P, G263S, T268A, S288P, S292T, A315S, M330I, I377V, D398E, S401C, and R416K, in SEQ ID NO: The modifications found in SEQ ID NO: are substitutions of V8I, V25A, V31I, N49K, E61A, I82V, A146T, M163F, Q182R, A187P, L235F, P237Q, L238T, E243D, G263S, T268A, S288P, S292T, A315S, L359V, T372A, I377V, E388D, I394M, S401C, R416T, and A419V. The modifications found in 68 are V8I, V25A, V31I, N49K, D60Q, E61A, S84G, E100D, A126G, F133I, A136P, S140P, T141I, V156L, K157R, M163V, R166H, A167T, F168V, V172L, A174K, Q182R, and A187. Replacement of P, T189V, E209D, H216Q, L235F, P237E, L238P, N262D, G263A, T268A, V271A, S288P, S292A, V293L, E296D, S314C, A315S, M330F, I377V, T392K, I394M, S401C and A419V;The modifications found in SEQ ID NO: 69 are V8I, V25A, V31I, N49K, R56A, D60E, E61A, I82V, Q95A, V124I, A138S, T141V, A146T, M163L, S179R, Q182R, A187P, R202G, H216S, L235F, P237Q, L238T, E243D, N247S, and T268A. The substitutions of L276A, S286R, S288P, S292T, S294A, S295A, E296A, R298Q, I337V, L359V, V361L, F362S, T372A, I377V, Q381P, S386N, E388A, T392A, S401C, I408VLS, A415K, R416T, and A419V, and in SEQ The modifications found in ID NO: 70 are substitutions for V8I, V25A, E61A, A136P, S140P, V156L, K157R, Q182R, T189V, E209D, L235F, L238P, T268A, V271A, S288P, S292A, V293L, S314C, I377V, and T392K.

[0095] Compared to SEQ ID NO: 1, the preferred modification is the substitution of V25A, V31I, N49K, I82V, Q182R, A187P, L235F, L238P, G263S, T268A, S288P, I377V, E388D, and S417Y found in SEQ ID NO: 63 and SEQ ID NO: 64. More preferred substitutions are the substitutions of V25A, V31I, Q182R, A187P, L235F, G263S, S288P, I377V, and E388D found in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65. The most preferred substitutions are V25A, V31I, Q182R, A187P, L235F, S288P and I377V found in SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 and SEQ ID NO: 68.

[0096] Enzymes can be modified using a variety of chemical techniques to produce derivatives that have substantially the same or preferably improved activity as the unmodified enzyme, and optionally possess other desired properties. For example, the carboxyl group of a protein, whether carboxyl-terminal or a side chain, can be provided in the form of a pharmaceutically acceptable cationic salt, or esterified, for example, to form a C1-C6 alkyl ester, or converted to an amide of the formula CONR1R2, wherein R1 and R2 are each independently H or a C1-C6 alkyl group, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. The amino group of an enzyme, whether amino-terminal or a side chain, can be in the form of a pharmaceutically acceptable acid addition salt, such as HCl, HBr, acetic acid, benzoic acid, toluenesulfonic acid, maleic acid, tartaric acid, and other organic salts, or can be modified to a C1-C20 alkyl or dialkylamino group or further converted to an amide. The hydroxyl group of the protein side chain can be converted to an alkoxy or ester group using known techniques, such as a C1-C20 alkoxy or C1-C20 alkyl ester. The phenyl and phenolic rings of the protein side chains may be substituted with one or more halogen atoms, such as F, CI, Br, or I, or with C1-C20 alkyl, C1-C20 alkoxy, carboxylic acid, or ester, or amides of such carboxylic acids. The methylene groups of the protein side chains may extend to homologous C2-C4 alkylene groups. Thiols may be protected with any of a number of known protecting groups, such as an acetamide group. Those skilled in the art will also recognize methods for incorporating cyclic structures into the proteins of this disclosure to selectively and provide conformational constraints on the structure, thereby resulting in enhanced stability.

[0097] The percentage of amino acid identity was obtained via log-expected multiple sequence comparison (MUSCLE) version 5.1 (Edgar, RC (2021). MUSCLE v5 enables improved estimates of phylogenetic tree confidence by ensemble bootstrapping. bioRxiv The comparison generated on June 20, 2021, was calculated using the Super5 algorithm.

[0098] For enzyme sequence comparison, the following settings can be used: Alignment algorithm: Needleman and Wunsch, J.Mol.Biol.1970, 48: 443-453. Use the Blosum62 matrix as the comparison matrix for amino acid similarity (Henikoff S. and Henikoff JG, PNASUSA 1992, 89: 10915-10919). Use the following gap scoring parameters: gap penalty: 12, gap length penalty: 2, no penalty for terminal gaps.

[0099] In some embodiments of the present invention, the method for preparing glycosaminoglycans by a single catalytic step of contacting free fatty acids with a glycosyl donor in the presence of an enzyme can be carried out enzymatically, wherein the enzyme is a glycosyltransferase containing at least one amino acid mutation selected from: SEQ ID NO: The amino acid sequence of the glycosyltransferase of 1 includes M1A, H2R, V3I, I4L, I4V, I5L, I5V, T6I, A7P, V8I, F17L, V18L, V18L, V18I, I20L, T23A, T23S, T23S, T23A, T23E, V25A, A26S, V31I, T32S, F33V, F33M, C34A, A35T, A35V, S36Y, S36N, S36F, A37P, A37EW, A37EF, A37PA, A37E, A37G, A37P, A38V, A38G, A38Y, A38R, and F39. L, F39A, A40K, A40R, A40P, A40E, A40S, P41E, P41S, P41A, L42S, L42A, I43A, I43V, I43L, E44D, E44G, R45L, R45G, R45E, C46S, C46A, C46P, C 46R, G47A, F48M, F48L, N49S, N49A, N49E, N49T, N49G, N49G, N49K, N49E, N49R, N49S, F50V, F50L, L51I, L51V, L51V, L51V, P52A, L53V, L53R , L53I, G54T, T55E, T55S, T55P, R56A, R56A, R56A, R56P, E57N, E57D, E58G, E58Q, Y59F, Y59I, D60E, D60N, D60H, D60A, D60Q, D60Q, E61A, E 61A, V62L, V62I, V62A, V62P, M63I, M63V, M63A, M63L, M63S, M63P, A64T, A64V, A64S, A64Y, D65S, D65N, A67R, H70N, H70D, P71S, R72K, R72L , R72E, T73K, T73A, T73G, T73D, S74A, L75F, P76R, P76G, T77V, L78M, K80R, K80R, T81A, T81W, T81V, I82V, I82V, A83G, S84G, S84P, S84T, S8 4G, T85S, T85A, L86I, E88D, E88P, E88A, Q89L, Q89H, Y90F, R91D, R91E, L92V, L92M, E94Q, E94A, Q95A, A96L, A96L, A96V, A96E, A96T, A96H,C97A、C97V、C97T、D98N、D99R、D99E、D99G、D99E、E100D、E100Q、V102I、M103L、L104I、L104V、L104I、L104V、L104V、G105A、S106H、S106N、S106R、L107S、L107Q、L107V、S111A、L115M、L115V、K118R、K118R、H119R、H119Y、I121A、I121V、V124A、V124I、T125S、A126G、A126V、A126G、Q127E、Q127H、V128A、V128L、V128I、V128A、S129T、S129Q、S129A、F131W、F131Q、F131H、G132N、G132Q、G132D、F133L、F133V、F133I、F133I、F133I、F133L、W134L、A136P、A136S、A137Q、A137K、A137F、A137L、A137R、A137H、A137M、A138S、A138D、S140A、S140P、S140P、S140P、T141V、T141I、T141V、H142L、H142S、P143-、P144A、P144K、P144K、P144L、G145P、G145R、A146T、A146F、A146T、A146L、A146S、A146L、N147T、N147P、N147S、L148I、L148V、A150E、A150T、A150G、F151S、F151E、F151D、F151T、F151W、F151W、V152L、V152L、P153T、P153S、Y154R、Y154D、Y154R、Y154V、Y154R、Y154L、P155E、V156I、V156P、V156D、V156Q、V156G、V156L、V156I、V156A、V156E、V156L、K157R、K157E、K157R、R158A、R158S、R158N、R158K、R158E、R158M、R158Q、L159E、L159D、L159G、L159R、L159A、L159K、L159T、L160F、L160I、L160A、L160V、L160Y、L161M、L161F、L161M、L161W、L161M、G162L、G162D、G162R、G162R、G162W、M163E、M163I、M163G、M163L、M163L、M163D、M163F、M163V、M163I、M163F、M163V、M163K、I164T、I164E、I164G、I164C、I164A、I164V、E165V、E165Y、E165D、E165A、E165I、R166K、R166L、R166E、R166V、R166Q、R166H、R166D、R166S、A167G、A167T、A167P、A167L、A167T、A167G、A167S、A167M、A167N、A167S、A167D、A167E、F168I、F168S、F168V、F168C、F168A、F168E、F168V、L169V、R171D、R171E、R171N、R171K、V172T、V172I、V172I、V172A、V172A、V172L、M173C、M173C、A174T、A174K、A174K、A174G、E176D、E176A、S179R、S179T、S179G、S179R、F180L、F180V、F180L、Q182S、Q182A、Q182R、Q182R、Q182G、Q182A、L186M、A187P、A187P、T189A、T189V、T189V、T189A、K190S、K190R、K190R、K190T、K190S、I192F、I193M、I193L、I193F、S194G、S194G、Q195E、Q195R、I197L、I197V、S198H、S198A、S198H、D201Q、R202G、R202K、R202G、E209D、E209D、V214P、V214T、V214A、H216T、H216R、H216P、H216Q、H216S、H216S、A220S、A220S、Q221H、Q221N、Q221R、T222A、T222I、V223S、V223A、F230Y、S233A、S233A、S233G、S233T、G234S、G234D、L235D、L235E、L235G、L235F、Q236H、Q236E、Q236L、Q236H、Q236R、Q236E、Q236L、P237T、P237T、P237Q、P237E、P237A、P237Q、P237Q、L238V、L238P、L238T、L238M、L238I、L238R、L238V、A240T、A240P、A240D、E243D、D244A、D244E、D244E、N247A、N247A、N247S、N247S, N247Q, N247R, E248G, E248A, E248A, A249G, A249E, A249T, A249G, A249S, S250P, S250P, S250P, V253I, T259A, T259S, M260N, V261R, V261I, V261 L、V261I、N262D、N262D、N262H、N262D、N262D、N262D、G263A、G263A、G263S、 G263P、G263S、G263E、G263E、E264G、E264P、E264Q、E264T、E264D、E264A、Q26 5A, Q265T, Q265R, Q265R, Q265S, Y266F, T268R, T268A, T268S, A269T, V271A, V271I, V271L, V271A, V271A, Q272E, Q272E, L274C, K275R, K275R, L276S, L 276R, L276K, L276A, L276R, I277T, I277L, I277A, I277L, I277M, N278G, N278G, V282I, F283L, T285A, S286G, S286R, S286K, S286R, S286R, Q287N, Q287D Q287E、Q287G、S288P、S288T、S288A、S288E、S288G、V289D、V289A、V289T、V2 89P、D290E、D290L、D290V、P291D、P291A、P291H、P291Q、S292V、S292P、S292 T、S292A、S292E、V293P、V293L、V293L、V293D、V293P、V293A、S294P、S294A、 S294L、S295R、S295T、S295A、S295L、E296S、E296A、E296S、E296D、E296G、E29 6P, G297S, G297S, G297D, G297S, G297P, G297D, G297P, R298Q, R298Q, R298N, R298D, R298E, I299V, V301L, R303S, R303P, V305A, S308T, R309K, R309L, R 309A, R309S, I310L, H313Q, H313R, S314T, S314A, S314A, S314A, S314C, S314C, A315S, A315T, L317I, V323I, V323I, T326A, T326V, L328Q, M330I, M330LM330I, M330L, M330F, M330L, V334I, I337L, I337V, I337L, A338V, A338V, F341L, F346L, M352L, M352V, M352I, E353Q, C357V, C357V, L359I, L359V, V361 L, V361I, V361L, V361I, V361L, F362S, F362D, F362L, F362R, F362R, F362L, F362K, F362L, F362S, F362P, F362D, P363G, P363A, P363R, P363S, P364S, P36 4R, P364K, A365V, S366T, S366Q, A367G, A367G, A367T, E368R, E368Q, S369R, S369P, S369A, L370I, A371S, A371T, A371T, T372A, T372R, D375Q, D375Q, D 375R, D375E, K376R, K376T, K376R, K376R, I377L, I377V, N379D, N379K, N379K, N379G, N379D, S380D, S380D, S380D, S380E, Q381P, E382A, E382Q, E382A V383I, V383I, R384S, R384A, R384T, A385E, A385E, S386N, S386A, S386N, S386K, S386R, S386A, E388T, E388A, E388A, E388D, E388Q, E388R, R389Q, R389 K, R389E, Y390M, Y390T, Y390M, Y390L, Y390I, R391A, T392Q, T392A, T392K, T392K, T392P, T392S, T392K, T392K, L393R, L393R, L393Q, I394V, I394M, I39 4L、I394F、I394M、P395A、S396T、G397S、G397A、D398P、D398E、D398E、D398E 、S399A、S399T、S399R、A400G、S401L、S401C、S401C、S401A、E402Q、A403S、A 403R、A403T、A403K、L406S、L406E、L406D、L406N、L406R、Q407F、Q407A、Q40 7F、I408V、A410T、A410S、L411V、L411M、L411F、L411I、A412V、A412S、R413S、R413P, R413T, R413A, T414R, T414P, T414L, T414S, T414E, A415P, A415K, A415R, A415S, R416A, R416I, R416T, R416K, R416G, S417R, S417Y, S417A, S417K, R418Q, R418T, A419T, A419T, A419V, and A420R.

[0100] In some embodiments of the invention, the method for preparing glycosyl monoesters by a single catalytic step of contacting free fatty acids with a glycosyl donor in the presence of an enzyme can be carried out enzymatically, which may be a glycosyltransferase containing the deletion of at least one amino acid selected from the glycosyltransferase amino acid sequence of SEQ ID NO: 1, namely E44, R45, C46, ​​G47, F39, N49, F48, N49, F50, L51, P52, L53, G54, T55, R56, E57, E58, Y59, D60, E61, V62, P143, I164, D290, A415, R416, S417, R418, A419, and A420.

[0101] In some embodiments of the invention, the method for preparing glycosaminoglycans by a single catalytic step of contacting free fatty acids with a glycosyl donor in the presence of an enzyme can be carried out enzymatically, which may be a glycosyltransferase comprising an insertion of at least one amino acid selected from the amino acid sequences of the glycosyltransferases in SEQ ID NO: 1: T392AV, I408VLS, S401CPA, E248AG, N247AG, R418APAG, E248GG, ​​A420RVAA, R418VAAA, A37PT, P71GGTF, P71CGPGEGWW, P71GGRF. In these embodiments, “T392AV” indicates that alanine (A) and valine (V) are subsequently inserted after the amino acid (T or Thr) at position 392 in SEQ ID NO: 1.

[0102] In some embodiments of the present invention, the method for preparing glycosaminoglycans by a single catalytic step of contacting free fatty acids with a glycosyl donor in the presence of an enzyme can be carried out enzymatically, which may be a glycosidase comprising amino acids selected from: E206, E387, H150, N205, R79, Q18, E432, W425, Y321, Y322, F222, W361, F441, V209, F359, 341, excluding D or E, wherein the positions are numbered with reference to SEQ ID NO: 83.

[0103] In some embodiments of the present invention, the method for preparing glycosaminoglycans by a single catalytic step of contacting free fatty acids with a glycosyl donor in the presence of an enzyme can be carried out enzymatically, which may be a glycosidase comprising amino acids selected from the following: E572, E841, D567, H862, N849, R858, D579, W864, W576, W679, W684, wherein the positions are numbered with reference to SEQ ID NO: 84.

[0104] In some embodiments, the present invention may be a glycosyltransferase variant comprising a mutation in at least one of the following amino acids: SEQ ID NO: The amino acid sequences of the glycosyltransferases of 1 are as follows: M1A, H2R, V3I, I4L, I4V, I5L, I5V, T6I, A7P, V8I, F17L, V18L, V18L, V18I, I20L, T23A, T23S, T23S, T23A, T23E, V25A, A26S, V31I, T32S, F33V, F33M, C34A, A35T, A35V, S36Y, S36N, S36F, A37P, A37EW, A37EF, A37PA, A37E, A37G, A37P, A38V, A38G, A38Y, A38R, F39L, F39A, A40K. A40R, A40P, A40E, A40S, P41E, P41S, P41A, L42S, L42A, I43A, I43V, I43L , E44D, E44G, R45L, R45G, R45E, C46S, C46A, C46P, C46R, G47A, F48M, F48 L, N49S, N49A, N49E, N49T, N49G, N49G, N49K, N49E, N49R, N49S, F50V, F5 0L, L51I, L51V, L51V, L51V, P52A, L53V, L53R, L53I, G54T, T55E, T55S, T5 5P, R56A, R56A, R56A, R56P, E57N, E57D, E58G, E58Q, Y59F, Y59I, D60E, D 60N, D60H, D60A, D60Q, D60Q, E61A, E61A, V62L, V62I, V62A, V62P, M63I, M63V, M63A, M63L, M63S, M63P, A64T, A64V, A64S, A64Y, D65S, D65N, A67R , H70N, H70D, P71S, R72K, R72L, R72E, T73K, T73A, T73G, T73D, S74A, L75F , P76R, P76G, T77V, L78M, K80R, K80R, T81A, T81W, T81V, I82V, I82V, A83 G, S84G, S84P, S84T, S84G, T85S, T85A, L86I, E88D, E88P, E88A, Q89L, Q8 9H, Y90F, R91D, R91E, L92V, L92M, E94Q, E94A, Q95A, A96L, A96L, A96V, A 96E, A96T, A96H, C97A, C97V, C97T, D98N, D99R, D99E, D99G, D99E, E100D,E100Q、V102I、M103L、L104I、L104V、L104I、L104V、L104V、G105A、S106H、S106N、S106R、L107S、L107Q、L107V、S111A、L115M、L115V、K118R、K118R、H119R、H119Y、I121A、I121V、V124A、V124I、T125S、A126G、A126V、A126G、Q127E、Q127H、V128A、V128L、V128I、V128A、S129T、S129Q、S129A、F131W、F131Q、F131H、G132N、G132Q、G132D、F133L、F133V、F133I、F133I、F133I、F133L、W134L、A136P、A136S、A137Q、A137K、A137F、A137L、A137R、A137H、A137M、A138S、A138D、S140A、S140P、S140P、S140P、T141V、T141I、T141V、H142L、H142S、P143-、P144A、P144K、P144K、P144L、G145P、G145R、A146T、A146F、A146T、A146L、A146S、A146L、N147T、N147P、N147S、L148I、L148V、A150E、A150T、A150G、F151S、F151E、F151D、F151T、F151W、F151W、V152L、V152L、P153T、P153S、Y154R、Y154D、Y154R、Y154V、Y154R、Y154L、P155E、V156I、V156P、V156D、V156Q、V156G、V156L、V156I、V156A、V156E、V156L、K157R、K157E、K157R、R158A、R158S、R158N、R158K、R158E、R158M、R158Q、L159E、L159D、L159G、L159R、L159A、L159K、L159T、L160F、L160I、L160A、L160V、L160Y、L161M、L161F、L161M、L161W、L161M、G162L、G162D、G162R、G162R、G162W、M163E、M163I、M163G、M163L、M163L、M163D、M163F、M163V、M163I、M163F、M163V、M163K、I164T、I164E、I164G、I164C、I164A、I164V、E165V、E165Y、E165D、E165A、E165I、R166K、R166L、R166E、R166V、R166Q、R166H、R166D、R166S、A167G、A167T、A167P、A167L、A167T、A167G、A167S、A167M、A167N、A167S、A167D、A167E、F168I、F168S、F168V、F168C、F168A、F168E、F168V、L169V、R171D、R171E、R171N、R171K、V172T、V172I、V172I、V172A、V172A、V172L、M173C、M173C、A174T、A174K、A174K、A174G、E176D、E176A、S179R、S179T、S179G、S179R、F180L、F180V、F180L、Q182S、Q182A、Q182R、Q182R、Q182G、Q182A、L186M、A187P、A187P、T189A、T189V、T189V、T189A、K190S、K190R、K190R、K190T、K190S、I192F、I193M、I193L、I193F、S194G、S194G、Q195E、Q195R、I197L、I197V、S198H、S198A、S198H、D201Q、R202G、R202K、R202G、E209D、E209D、V214P、V214T、V214A、H216T、H216R、H216P、H216Q、H216S、H216S、A220S、A220S、Q221H、Q221N、Q221R、T222A、T222I、V223S、V223A、F230Y、S233A、S233A、S233G、S233T、G234S、G234D、L235D、L235E、L235G、L235F、Q236H、Q236E、Q236L、Q236H、Q236R、Q236E、Q236L、P237T、P237T、P237Q、P237E、P237A、P237Q、P237Q、L238V、L238P、L238T、L238M、L238I、L238R、L238V、A240T、A240P、A240D、E243D、D244A、D244E、D244E、N247A、N247A、N247S、N247S、N247Q、N247R、E248G、E248A、E248A、A249G、A249E, A249T, A249G, A249S, S250P, S250P, S250P, V253I, T259A, T259S, M260N, V261R, V261I, V261L, V261I, N262D, N262D, N262H, N262D, N262D, N262 D, G263A, G263A, G263S, G263P, G263S, G263E, G263E, E264G, E264P, E264Q, E264T, E264D, E264A, Q265A, Q265T, Q265R, Q265R, Q265S, Y266F, T268R, T26 8A, T268S, A269T, V271A, V271I, V271L, V271A, V271A, Q272E, Q272E, L274C, K275R, K275R, L276S, L276R, L276K, L276A, L276R, I277T, I277L, I277A, I 277L、I277M、N278G、N278G、V282I、F283L、T285A、S286G、S286R、S286K、S28 6R、S286R、Q287N、Q287D、Q287E、Q287G、S288P、S288T、S288A、S288E、S288G、 V289D, V289A, V289T, V289P, D290E, D290L, D290V, P291D, P291A, P291H, P291Q, S292V, S292P, S292T, S292A, S292E, V293P, V293L, V293L, V293D, V293 P、V293A、S294P、S294A、S294L、S295R、S295T、S295A、S295L、E296S、E296A、 E296S、E296D、E296G、E296P、G297S、G297S、G297D、G297S、G297P、G297D、G29 7P、R298Q、R298Q、R298N、R298D、R298E、I299V、V301L、R303S、R303P、V305A 、S308T、R309K、R309L、R309A、R309S、I310L、H313Q、H313R、S314T、S314A、S 314A, S314A, S314C, S314C, A315S, A315T, L317I, V323I, V323I, T326A, T326V, L328Q, M330I, M330L, M330I, M330L, M330F, M330L, V334I, I337L, I337VI337L, A338V, A338V, F341L, F346L, M352L, M352V, M352I, E353Q, C357V, C357V, L359I, L359V, V361L, V361I, V361L, V361I, V361L, F362S, F362D, F362 L, F362R, F362R, F362L, F362K, F362L, F362S, F362P, F362D, P363G, P363A, P363R, P363S, P364S, P364R, P364K, A365V, S366T, S366Q, A367G, A367G, A36 7T, E368R, E368Q, S369R, S369P, S369A, L370I, A371S, A371T, A371T, T372A, T372R, D375Q, D375Q, D375R, D375E, K376R, K376T, K376R, K376R, I377L, I 377V, N379D, N379K, N379K, N379G, N379D, S380D, S380D, S380D, S380E, Q381P, E382A, E382Q, E382A, V383I, V383I, R384S, R384A, R384T, A385E, A385E S386N, S386A, S386N, S386K, S386R, S386A, E388T, E388A, E388A, E388D, E388Q, E388R, R389Q, R389K, R389E, Y390M, Y390T, Y390M, Y390L, Y390I, R391 A, T392Q, T392A, T392K, T392K, T392P, T392S, T392K, T392K, L393R, L393R, L393Q, I394V, I394M, I394L, I394F, I394M, P395A, S396T, G397S, G397A, D39 8P、D398E、D398E、D398E、S399A、S399T、S399R、A400G、S401L、S401C、S401C 、S401A、E402Q、A403S、A403R、A403T、A403K、L406S、L406E、L406D、L406N、L 406R、Q407F、Q407A、Q407F、I408V、A410T、A410S、L411V、L411M、L411F、L41 1I、A412V、A412S、R413S、R413P、R413T、R413A、T414R、T414P、T414L、T414S、T414E, A415P, A415K, A415R, A415S, R416A, R416I, R416T, R416K, R416G, S417R, S417Y, S417A, S417K, R418Q, R418T, A419T, A419T, A419V, and A420R.

[0105] In some embodiments, the present invention may be a glycosyltransferase variant comprising the deletion of at least one of the following amino acids: E44, R45, C46, ​​G47, F39, N49, F48, N49, F50, L51, P52, L53, G54, T55, R56, E57, E58, Y59, D60, E61, V62, P143, I164, D290, A415, R416, S417, R418, A419, and A420 of the glycosyltransferase amino acid sequence of SEQ ID NO: 1.

[0106] In some embodiments, the present invention may be a glycosyltransferase variant comprising an insertion of at least one amino acid selected from the following: T392AV, I408VLS, S401CPA, E248AG, N247AG, R418APAG, E248GG, ​​A420RVAA, R418VAAA, A37PT, P71GGTF, P71CGPGEGWW, and P71GGRF of the glycosyltransferase amino acid sequence of SEQ ID NO: 1.

[0107] As used herein, “amplify,” “amplified,” or “amplification” refers to any process or protocol used to copy a polynucleotide sequence into a larger number of polynucleotide molecules, such as by reverse transcription, polymerase chain reaction, and ligase chain reaction.

[0108] As used in this article, "antisense sequence" refers to a sequence that specifically hybridizes with a second polynucleotide sequence.

[0109] As used herein, “cDNA” refers to DNA that is complementary to or identical to mRNA in single-stranded or double-stranded form. As used herein, carbons in fatty acids are numbered with the first carbon being part of a carboxylic acid group, and the second carbon adjacent to the first carbon. Numbering continues until the highest-numbered carbon is furthest from the carboxylic acid group.

[0110] As used herein, “complementary” refers to a polynucleotide that can pair with a second polynucleotide base. For example, a polynucleotide having the sequence 5'-GTCCGA-3' is complementary to a polynucleotide having the sequence 5'-TCGGAC-3'.

[0111] As used in this article, "conservative substitution" refers to the replacement of amino acids in a polypeptide with functionally similar amino acids.

[0112] As used herein, “encoding” refers to the inherent property of a nucleotide to serve as a template for the synthesis of other polymers and macromolecules. Unless otherwise specified, “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate to each other and encode the same amino acid sequence.

[0113] As used in this article, "endogenous" refers to polynucleotides, polypeptides, or other compounds that are naturally expressed or originate in an organism or cell. In other words, endogenous polynucleotides, polypeptides, or other compounds are not exogenous.

[0114] As used herein, "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operatively linked to a nucleotide sequence to be expressed. For example, a suitable expression vector may be autonomously replicating or integrated into a chromosome.

[0115] As used herein, “exogenous” refers to any polynucleotide or polypeptide that is not naturally expressed in the specific cells or organisms in which expression is desired. Exogenous polynucleotides, polypeptides, or other compounds are not endogenous.

[0116] As used herein, “hybridization” includes any process by which a nucleic acid strand binds to a complementary nucleic acid strand through base pairing. Therefore, the term refers to the ability of a target sequence’s complementary sequence to bind to a test sequence, or vice versa.

[0117] As used herein, “hybridization conditions” are typically categorized by measuring the “strictness” of the hybridization conditions. The degree of strictness can be based on, for example, the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, “maximum strictness” typically occurs at approximately Tm - 5°C (5°C lower than the probe’s Tm); “high strictness” is approximately 5°C-10°C below Tm; “medium strictness” is approximately 10°C-20°C below the probe’s Tm; and “low strictness” is approximately 20°C-25°C below Tm. Alternatively or additionally, hybridization conditions can be based on the salt or ionic strength conditions of hybridization and / or one or more strict washes. For example, 6xSSC (sodium citrate saline) = very low strictness; 3xSSC = low to medium strictness; 1xSSC = medium strictness; and 0.5xSSC = high strictness. Functionally, the maximum stringency condition can be used to identify nucleic acid sequences that have strict or near-strict identity with the hybridization probe; while the high stringency condition is used to identify nucleic acid sequences that have approximately 80% or more sequence identity with the probe.

[0118] As used herein, in the context of two or more polynucleotide or polypeptide sequences, “identical” or “identity percentage” means two or more identical sequences or sequences having a specified percentage of identical nucleotide or amino acid residues when compared and aligned to achieve maximum correspondence, such as when using sequence comparison algorithms or measured visually.

[0119] As used in this article, "long-chain fatty acids" refers to fatty acids with an aliphatic tail longer than 14 carbons.

[0120] As used herein, "medium-chain fatty acid" refers to a fatty acid with an aliphatic tail having between 6 and 14 carbons. In some embodiments, medium-chain fatty acids may have 11 to 13 carbons.

[0121] As used in this article, “naturally occurring” or “wild-type” refers to an object that can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in an organism (including a virus) that can be isolated from natural sources and has not been intentionally modified by humans in a laboratory is considered naturally occurring or wild-type.

[0122] As used herein, when describing the relationship between two DNA regions or two polypeptide regions, “operably linked” simply means that they are functionally related to each other. For example, if a promoter controls transcription of a sequence, then the promoter is operably linked to the decoding sequence. If a ribosome-binding site is located to allow translation, then that site is operably linked to the decoding sequence; if the sequence functions as a signaling sequence, such as by participating in the secretion of the mature form of the protein, then that sequence is operably linked to the peptide.

[0123] As used herein, “polynucleotide” refers to a polymer composed of nucleotides. Polynucleotides can be in the form of individual fragments or as components of larger nucleotide sequence constructs, derived from nucleotide sequences that enable identification, manipulation, and recovery of the sequence and its component nucleotide sequences at least once using standard molecular biology methods (e.g., using cloning vectors). When the nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes RNA sequences in which “U” replaces “T” (i.e., A, U, G, C).

[0124] As used herein, “polypeptide” refers to a polymer composed of amino acid residues, which may or may not contain modifications such as phosphate esters and formyl groups.

[0125] As used herein, a “recombinant expression vector” refers to a DNA construct encoding a polynucleotide for expressing a desired polypeptide, and may include, for example, a transcription subunit containing, the assembly of genetic elements (e.g., promoters and enhancers) that play a regulatory role in gene expression, a structural or decoding sequence transcribed into mRNA and translated into protein, and appropriate transcription and translation initiation and termination sequences. Recombinant expression vectors can be constructed in any suitable manner. The nature of the vector is not critical, and any vector, including plasmids, viruses, bacteriophages, and transposons, can be used. Possible vectors used in this invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids; bacteriophage DNA; yeast plasmids; vectors derived from combinations of plasmids and bacteriophage DNA; and DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies.

[0126] As used in this article, a "primer" refers to a polynucleotide that, when placed under conditions of induced synthesis, can specifically hybridize with a specified polynucleotide template and provide an initiation point for the synthesis of complementary polynucleotides.

[0127] As used herein, “recombinant polynucleotide” refers to a polynucleotide having a sequence that is not naturally linked together. Recombinant polynucleotides can be contained in a suitable vector, and this vector can be used to transform a suitable host cell. A host cell containing a recombinant polynucleotide is called a “recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, for example, a “recombinant polypeptide.”

[0128] As used in this article, "specific hybridization" refers to the preferential binding, double-stranding, or hybridization of polynucleotides with specific nucleotide sequences under strict conditions.

[0129] As used in this article, “strict conditions” refer to conditions under which the probe will preferentially hybridize with its target subsequence and, to a lesser extent, hybridize with other sequences or not hybridize with other sequences at all.

[0130] As used in this article, "short-chain fatty acids" refers to fatty acids with an aliphatic tail of fewer than 6 carbons.

[0131] As used herein, in the context of two nucleic acids or polypeptides, "substantially homologous" or "substantially identical" generally means two or more sequences or subsequences that, when compared and aligned to achieve maximum correspondence, have at least 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide or amino acid residue identity, as measured using sequence comparison algorithms or by visual inspection. Substantially identical can be present in any suitable region of the sequence, such as, for example, a region of at least about 50 residues, at least about 100 residues, or at least about 150 residues in length. In some embodiments, the sequences are substantially identical along the entire length of either or both of the compared biopolymers.

[0132] As used in this article, “NMP” is defined as a nucleotide monophosphate, and “NDP” is defined as a nucleotide diphosphate, where N is any of the nucleotide bases in DNA or RNA, namely A (adenine), T (thymine), C (cytosine), G (guanine), or U (uracil).

[0133] The composition produced by the method of the present invention may contain rhamnolipids, wherein at least about 25% by weight of all rhamnolipids are monolipids. In some embodiments, the weight percentage of all rhamnolipids that are monolipids in the composition may be at least about 15%, at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%. In some embodiments, the weight percentage of all rhamnolipids that are monoesters in the composition may be about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, to about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%, and any combination of these percentages herein.

[0134] In some embodiments, the composition produced by the method of the present invention may comprise rhamnolipids, wherein monorhamnolipids constitute at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% of all rhamnolipids by weight, or in some embodiments about 20% to about 100% of all rhamnolipids by weight, or about 25% to about 95%, or any combination thereof.

[0135] Example

[0136] Example A

[0137] Preparation of E. coli expression system containing rhamnosyltransferase gene

[0138] Rhamnosyltransferase (SEQ ID NO: 1-12) glycosylates lipids into their corresponding rhamnolipides. Derived from *Pseudomonas thaliana* (… Pseudomonas mediterranea The rhamnosyltransferase (SEQ ID NO: 1) was further engineered on a computer to design mutants (SEQ ID NO: 63-70) with improved activity in glycosylation of 3-hydroxy fatty acids into corresponding rhamnosyl monoesters. Codon-optimized genes encoding the rhamnosyltransferase from uncultured bacterial clones were designed and synthesized (SEQ ID NO: 18-29 and mutant sequences, SEQ ID NO: 71-78). After gene synthesis, the complete synthetic gene sequence was independently subcloned into pCOLADuet-1 or pET30a vectors (GenScript, Piscataway, NJ, USA) for heterologous expression. The recombinant plasmid was transformed into *E. coli* BL21 Star (DE3) cells (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA).

[0139] Example B

[0140] Preparation of FLAG-labeled rhamnosyltransferase

[0141] Further codon-optimized genes encoding rhamnosyltransferases (SEQ ID NO: 18 and 21) were designed and synthesized to include the FLAG-tagged amino acid sequence (DYKDDDDK) to SEQ ID NO: 1 and 4. The resulting FLAG-tagged proteins (SEQ ID NO: 13 and 16) were expressed in Luria broth (Thermo Fisher Scientific, Waltham, MA, USA) containing kanamycin and induced with 1 mM IPTG at 30 °C and 225 rpm for 12 h. Cells were harvested by centrifugation (3000 rpm, 30 min), lysed, and the proteins in the supernatant were purified using an immobilized FLAG-tag-specific monoclonal antibody against the FLAG-tagged proteins and eluted with FLAG-peptides (GenScript, Piscataway, NJ, USA). The proteins were stored at -80 °C in a buffer containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 10% glycerol.

[0142] Embodiment C of the present invention

[0143] Rhamnose monoester prepared from recombinant host cells via rhamnosyltransferase :

[0144] Single colonies of transformed *E. coli* BL21 Star(DE3) (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) cells from Example A (SEQ ID NO: 18-29) were inoculated into Luria broth (Thermo Fisher Scientific, Waltham, MA, USA) containing kanamycin (Teknova, Hollister, California, USA). Magic medium (Thermo Fisher Scientific, Waltham, MA, USA) was inoculated with 1 mM of 3-hydroxydecanoic acid (Sigma-Aldrich), 3-hydroxylauric acid (Sigma-Aldrich, St. Louis, MO, USA), or 3-hydroxymyristic acid (TCIAmerica, Portland, OR, USA). The mixture was then inoculated with a 1:100 dilution of culture grown from Luria broth and incubated at 37°C and 225 rpm for 24 hours. The supernatant was separated from the cell pellet by centrifugation, and the resulting rhamnolipids were analyzed by test method 1. The results are shown in Table 1.

[0145] Table 1. Quantification of rhamnolipids produced by one-step conversion of rhamnosyltransferase from Escherichia coli

[0146] As shown in Table 1, when combined with Pseudomonas fluorescens (… Pseudomonas fluorescens Compared to the reference out-of-sequence of the standard rhamnosyltransferase (SEQ ID NO: 17), rhamnosyltransferase genes expressed in SEQ ID NO: 1, 2, 4, 6 or 7 produce rhamnosyl monoester.

[0147] Example D

[0148] like Figure 1 As shown, the Super5 algorithm is used via Multiple Sequence Comparison by Log Expectation (MUSCLE) version 5.1 (Edgar, RC (2021). MUSCLE v5 enables improved estimates of phylogenetic tree confidence by ensemble bootstrapping. bioRxiv Sequences were aligned on June 20, 2021, and the γ20 probability of 20 class sites was optimized using FastTree version 2.1.11. Phylogenetic relationships were inferred by comparing 12 sequences with our canonical RhlB reference sequence (WP_003092007.1, from *Pseudomonas fluorescens* - SEQ ID NO: 17). The numbers at branch points, bootstrap values, and the probability of branching (scale 0-1.0, where 1.0 represents 100% probability) indicate the likelihood of closer proximity toward that branch or the relationship between the two enzyme members of that associated branch. The scale bar at the bottom indicates the numerical differences in amino acids at each site. Table 2 shows the rhamnosyltransferase substances and their corresponding sequence IDs.

[0149] Table 2 . =Indicates the nearest BLASTp hit on NCBI

[0150] Example E

[0151] Compared to the wild type, the mutant rhamnosyltransferase (SEQ ID NO: 63-70) exhibits increased activity in producing rhamnosyl monolipids. Sexual enhancement .

[0152] The mutant rhamnosyltransferase was tested in recombinant cells. Briefly, single colonies of *E. coli* BL21 Star (DE3) cells (Invitrogen) transformed with SEQ ID NO: 71-78 from Example A, or competent *Pseudomonas putida* transformed with SEQ ID NO: 81 or SEQ ID NO: 82 as described in Test Method 3, were inoculated into a solution containing 50 µg / mL kanamycin (…). Teknova, Hollister, CA, USA The expression was carried out in Luria broth (Thermo Fisher Scientific, Waltham, MA, USA). Magic medium (Invitrogen) (for expression in *E. coli*) or Luria broth (for expression in *Pseudomonas putida*) was mixed with 3-hydroxydecanoic acid (Sigma Aldrich, St. Louis, MO, USA) or 3-hydroxylauric acid (Sigma Aldrich, St. Louis, MO, USA) or 3-hydroxymyristic acid (TCI America, Portland, OR, USA) at a final concentration of 1 mM. These mixtures were then inoculated with cultures grown from the above Luria broth containing SEQ ID NO: 71-78 (in *E. coli*) or SEQ ID NO: 81 or SEQ ID NO: 82 (in *Pseudomonas putida*) diluted 1:100, and incubated at 225 rpm at 30°C for 20 h (*Pseudomonas putida*) or at 37°C for 24 h (in *E. coli*). The supernatant was separated from the cell pellet by centrifugation, and rhamnosyl monoester production was analyzed using assay method 1. For *E. coli*, the results are shown as quantifications of Rha-C10 in Table 3, Rha-C12 in Table 4, and Rha-C14 in Table 5; and in *Pseudomonas putida*, the results are shown as quantifications of Rha-C10 in Table 6. The activity improvement was calculated by comparing the amount of rhamnosyl monoester produced by the mutant with the amount produced by the wild-type (SEQ ID NO: 1). Compared to the wild-type SEQ ID NO: 1, the rhamnosyl monoester production of the mutant enzyme (SEQ ID NO: 63-70) was significantly increased. When specifically using *Pseudomonas putida* as a host, the activity improvement was surprisingly greater than 80-fold (Table 6).

[0153] Table 3. In Escherichia coli, compared with the control wild type, improvements were achieved through mutant rhamnosyltransferase and activity. Quantitative analysis of Rha-C10 generated from a one-step conversion .

[0154]

[0155] Table 4. In Escherichia coli, compared with the control wild type, improvements were achieved through mutant rhamnosyltransferase and activity. Quantitative analysis of Rha-C12 generated from a one-step conversion .

[0156]

[0157] Table 5. In Escherichia coli, compared with the control wild type, improvements were achieved through mutant rhamnosyltransferase and activity. Quantitative analysis of Rha-C14 produced by one-step conversion .

[0158]

[0159] Table 6. In *Pseudomonas putida*, compared with the control wild type, the levels of mutant rhamnosyltransferase and activity... Improved quantification of Rha-C10 produced from a one-step conversion .

[0160]

[0161] Example F

[0162] Preparation of rhamnosyl monoesters via rhamnosyltransferase in a cell-free system : The purified rhamnosyltransferase (SEQ ID NO: 1 or SEQ ID NO: 4) was mixed with 0.1 M sodium phosphate buffer (Sigma Aldrich) at pH 7.4; 0.5 mg / mL TDP-rhamnose (BOC Sciences) and 1.0 mg / mL 3-hydroxydecanoic acid (Sigma Aldrich) were dissolved in DMSO (Thermo Fischer) to obtain a final concentration of 5%, with a reaction volume of 500 µL. The mixture was incubated at 25 °C with stirring at 300 rpm for 48 hours. The obtained Rha-C10 was analyzed using assay method 1. Table 7 shows a summary of the results of the quantitative Rha-C10 formed in SEQ ID NO: 1 and SEQ ID NO: 4.

[0163] Table 7. Quantification of Rha-C10 produced from rhamnosyltransferase in a cell-free system via one-step conversion.

[0164] Example G

[0165] Purification of glycosidase

[0166] SEQ ID NO: 30 and SEQ ID NO: 80 were expressed according to test method 4. Unless otherwise specified, all purification steps were performed on ice or at 4°C using a cobalt affinity resin that recognizes the 6X histidine tag. HisPur ™5 mL of cobalt resin (Thermo Fisher) was transferred to a 50 mL Falcon tube and centrifuged at 800 x g for 2 minutes at 4 °C, discarding the supernatant. After equilibration with 10 mL of wash buffer (50 mM sodium phosphate, 300 mM NaCl, and 10 mM imidazole, pH 7.4), 10 mL of cell-free extract mixed with an equal volume of wash buffer was added to the cobalt resin, and the mixture was incubated at 16 °C at 150 rpm for 60 minutes. The supernatant was discarded by centrifugation at 800 x g for 2 minutes. The resin was washed five times with 10 mL of wash buffer, and adsorbed proteins were eluted with 5 mL of elution buffer (50 mM sodium phosphate, 300 mM NaCl, and 100 mM imidazole, pH 7.4). The elution step was repeated twice, and the fractions were combined and concentrated to approximately 1 mL. The fractions were then diluted twice with 10 mL of 0.1 M phosphate buffer (pH 6) and the imidazole content was minimized using an Amicon ultra-0.5 centrifuge filter (Millipore) with a 30 kDa molecular weight cutoff membrane. In the final step, the purified enzyme was concentrated to 1 mL and stored at 4°C. SDS-PAGE analysis was used to determine the purity and concentration of the purified enzyme.

[0167] Example H

[0168] Production of glycosaminoglycans via glycosidases in a cell-free system

[0169] The purified glycosidase (SEQ ID NO: 30 or SEQ ID NO: 80) was mixed with 0.1 M sodium phosphate buffer (pH 7) or 75% glycerol, 1.5 mg / mL sugar, and 3 mg / mL 3-hydroxydecanoic acid (Sigma Aldrich, St. Louis, MO, USA) to a final volume of 300 µL. The mixture was incubated at 15 °C with stirring at 1300 rpm for 72–96 hours. The reaction products were then analyzed by achiral HPLC and ELSD according to test method 4 to determine the quantitative glycosaminoglycans formed. Figure 3 This is a chromatogram of Rha-C10 formed by SEQ ID NO: 30. Rhamnose and 3-OH-decanoic acid were reacted in 75% glycerol at 15°C for 72 hours. The formed Rha-C10 was traced at a retention time of 2.820 min. Table 8 summarizes the results of the ability of the glycosidase to glycosylate 3-hydroxydecanoic acid to form Rha-C10 in phosphate buffer at pH 7.0 or in 75% glycerol.

[0170] Table 8. Quantification of Rha-C10 generated from glycosidase in a cell-free system via one-step conversion.

[0171] Example I

[0172] Production of monorhamnose monoesters by immobilized glycosidase

[0173] Recombinant glycosidase (SEQ ID NO: 30) in 100 mM phosphate buffer (pH 7.4) was applied to an affinity resin (e.g., nickel affinity resin; Cytiva HisTrap HP). The mixture was incubated at 4 °C for 30 min with shaking to immobilize the enzyme. Unbound enzyme was eluted, and the immobilized enzyme was then treated with 3-hydroxydecanoic acid (final concentration 4 mg / mL) and α-rhamnose (final concentration 2.2 mg / mL) in 5 mL of 75% glycerol / 25% water. The mixture was reacted at 25 °C with shaking at 225 rpm for 3 days. The soluble mixture was collected, and rhamnose monoester formation was analyzed by assay method 1. Table 9 shows the quantitative results for Rha-C10.

[0174] Table 9. Quantification of Rha-C10 generated from one-step conversion by immobilized glycosidase

[0175] Test methods

[0176] 1. LC-MS / MS analysis

[0177] In liquid chromatography-mass spectrometry (LC-MS / MS), the "area under the curve" refers to the total area under the chromatographic peak on the mass spectrum. It directly corresponds to the amount of a specific analyte present in the sample and is used to determine its concentration for quantitative analysis by calculating the area under the peak.

[0178] The chromatographic method consisted of an ACQUITY BEH C18 Vanguard pre-column (130 Å, 1.7 µm, 2.1 mm × 5 mm, Waters, Milford, MA, USA), followed by an ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 100 mm, Waters, Milford, MA, USA). In ExionLC... ™ LC gradients were performed from 5% to 95% B (acetonitrile) over 10.5 minutes on an AD UPLC system (AB Sciex, Framingham, MA, USA), with a total run time of 15 minutes. Mobile phase A was a 4 mM ammonium acetate aqueous solution, and the flow rate was set to 0.4 mL / min. Samples were injected into the instrument using a PAL HTS-xt autosampler (CTC Analytics, Zwingen, Switzerland).

[0179] Rhamnose glycolipids eluted from a UPLC column were analyzed by tandem mass spectrometry on a Triple Quad 6500+ system (AB Sciex, Framingham, MA, USA). The instrument was operated in multiple reaction monitoring (MRM) and negatively charged spray ionization (ESI) modes.

[0180] All data processing was performed using Skyline software version 23.1.0.268 (MacCoss Lab, University of Washington, Seattle, WA, USA). The transformation results were exported to a CSV file for further analysis. For relative quantification, carbon-13 stable isotope-labeled rhamnolipides were prepared by fermentation using D-glucose (U-13C6) as the sole carbon source. A list of rhamnolipides and their corresponding transformations is included.

[0181] 2. Fermentation of microbial cultures

[0182] Suitable conditions for a fermentation broth containing a certain concentration of rhamnolipids include: a bacterial seed culture, preferably a strain of *Pseudomonas putida*; and a fermentation medium prepared in a suitable fermentation vessel equipped with paired fermentation broths for stirring and aeration. This fermentation medium contains a carbon source selected from the group consisting of: fatty distillates, used soybean oil, soybean soapstock, orange peel, distillation waste, wheat straw, sweet brine or bagasse oil, soybean oil, rapeseed oil, cocoa butter, olive oil, rice bran oil, palm oil, animal fats, glycerol, fatty acids, used edible oils, waste oils, waste fats, glucose, fructose, sucrose, lactose, maltose, corn syrup, corn molasses, soybean molasses, carbohydrates, carbohydrate-containing materials, glycerides, fatty acids, glycerol, and combinations thereof, including nitrogen and non-nitrogen sources and combinations thereof. Stirring and aeration of the fermentation broth are carried out at a temperature of 15°C to 40°C, more preferably 30°C.

[0183] 3. Preparation of competent cells of Pseudomonas putida

[0184] Incubate 5 mL of *Pseudomonas putida* (ATCC 47054) culture overnight in Luria broth (Thermo Fisher Scientific, Waltham, MA, USA). Wash the culture three times with sucrose by centrifugation, decantation, and resuspending in 1 mL of 300 mM sucrose. After final washing, the cell pellet was resuspended in 50 µL of 300 mM sucrose and transferred to a BioRad Gene Pulser 0.1 cm electroporation cuvette (Bio-Rad Laboratories, Hercules, CA, USA). Electroporation was performed at 2.5 kV, 200 ohms, and 25 μF. The pellet was then incubated in super-quality broth with catabolite inhibition (SOC) medium (Thermo Fisher Scientific, Waltham, MA, USA) at 30 °C and 225 rpm for 1 hour, and then plated on LB kanamycin-50 plates (Teknova, Hollister, CA, USA) overnight.

[0185] 4. Preparation of glycosidase lysates

[0186] Glycosidases were expressed according to methods commonly used in the art. Briefly, single colonies of transformed competent *E. coli* cells were inoculated into Luria broth (LB) or Terrific broth (TB) (TB; Research Products International, Mt. Prospect, IL, USA) containing appropriate antibiotics. LB or TB media were inoculated with a 1:100 dilution of the culture grown from LB or TB and incubated at 25°C until an appropriate optical density was achieved at 600 nm, followed by induction with 1 mM IPTG. Cells were harvested by centrifugation, and the pellet was lysed using procedures commonly used in the art. The supernatant was collected for enzyme assays; unless otherwise specified, no further purification was required.

[0187] 5. Chiral HPLC methods for the analysis of glycosidase reactions

[0188] The method was developed using the parameters shown in Table 8. ELSD (evaporative light for a scattering detector) was used to monitor glycosidase activity. The evaporator and nebulizer temperature was 40°C.

[0189] Table 7: Parameters of HPLC Method

[0190] Examples / Combinations

[0191] A. A method for preparing glycosaminoglycans, the method comprising: (a) Preparation of glycosaminoglycans via a single catalytic step by contacting hydroxy fatty acids with a glycosyl donor in the presence of an enzyme selected from the following: i. Glycosyltransferases; and ii. Glycosidase; (b) Optionally, the prepared glycosaminoglycans are separated; The glycosaminoglycan has the following formula:

[0192] Formula 1

[0193] Where n is between 0 and 15; Wherein X is selected from alkyl, aryl, heteroalkyl, heteroaryl, unsaturated alkenyl, and unsaturated heteroalkenyl; and wherein X has a carbon chain length of 4 to 22; M is selected from one or both of glucose, sulfoquinoline, fructose, galactose, ribose, maltose, xylose, rhamnose, sophorose, mannose, arabinose, fucose, and combinations thereof and stereoisomers thereof; And R is selected from -H, from alkyl, heteroalkyl, aryl, heteroaryl, heteroarylalkyl, arylalkyl, tauryl, and all their possible stereoisomers.

[0194] B. According to the method described in paragraph B, the hydroxy fatty acid is a straight-chain hydroxy fatty acid; And X is selected from alkyl and unsaturated alkenyl groups, and has a carbon chain length of 4 to 22.

[0195] C. The method according to paragraph A or B, wherein R is selected from -H and alkyl groups and has a straight-chain carbon length of 1 to 4.

[0196] D. The method described in any of paragraphs A through C, wherein the glycosyl donor comprises at least one rhamnose.

[0197] E. The method described in any of paragraphs A through D, wherein the rhamnose is a nucleoside monophosphate (NMP), a nucleoside diphosphate (NDP), or a nucleoside triphosphate (NTP).

[0198] F. The method described in any of paragraphs A through E, wherein the monosaccharide is rhamnosaccharide.

[0199] G. The method according to any one of paragraphs A to F, wherein the rhamnose monoester is selected from Rha-C8, Rha-C10, Rha-C12, Rha-C14, RhaRha-C8, RhaRha-C10, RhaRha-C12 and RhaRha-C14.

[0200] H. The method according to any one of paragraphs A to G, wherein the enzyme is a glycosyltransferase comprising a polypeptide sequence having at least 60% identity with SEQ ID NO: 1 to SEQ ID NO: 12 and fragments thereof.

[0201] I. The method according to any one of paragraphs A to H, wherein the glycosyltransferase comprises a polypeptide sequence having at least 75% identity with SEQ ID NO: 1 to SEQ ID NO: 12 and fragments thereof.

[0202] J. The method according to any one of paragraphs A to I, wherein the glycosyltransferase comprises a polypeptide sequence having at least 90% identity with SEQ ID NO: 1 to SEQ ID NO: 12 and fragments thereof.

[0203] K. The method according to any one of paragraphs A to J, wherein said enzyme is a glycosyltransferase containing at least one amino acid mutation selected from: SEQ ID NO: The amino acid sequences of the glycosyltransferases of 1 are as follows: M1A, H2R, V3I, I4L, I4V, I5L, I5V, T6I, A7P, V8I, F17L, V18L, V18L, V18I, I20L, T23A, T23S, T23S, T23A, T23E, V25A, A26S, V31I, T32S, F33V, F33M, C34A, A35T, A35V, S36Y, S36N, S36F, A37P, A37EW, A37EF, A37PA, A37E, A37G, A37P, A38V, A38G, A38Y, A38R, F39L, F39A, A40. K, A40R, A40P, A40E, A40S, P41E, P41S, P41A, L42S, L42A, I43A, I43V, I4 3L, E44D, E44G, R45L, R45G, R45E, C46S, C46A, C46P, C46R, G47A, F48M, F 48L, N49S, N49A, N49E, N49T, N49G, N49G, N49K, N49E, N49R, N49S, F50V, F50L, L51I, L51V, L51V, L51V, P52A, L53V, L53R, L53I, G54T, T55E, T55S , T55P, R56A, R56A, R56A, R56P, E57N, E57D, E58G, E58Q, Y59F, Y59I, D6 0E, D60N, D60H, D60A, D60Q, D60Q, E61A, E61A, V62L, V62I, V62A, V62P, M 63I, M63V, M63A, M63L, M63S, M63P, A64T, A64V, A64S, A64Y, D65S, D65N, A67R, H70N, H70D, P71S, R72K, R72L, R72E, T73K, T73A, T73G, T73D, S74A , L75F, P76R, P76G, T77V, L78M, K80R, K80R, T81A, T81W, T81V, I82V, I82 V, A83G, S84G, S84P, S84T, S84G, T85S, T85A, L86I, E88D, E88P, E88A, Q8 9L, Q89H, Y90F, R91D, R91E, L92V, L92M, E94Q, E94A, Q95A, A96L, A96L, A 96V, A96E, A96T, A96H, C97A, C97V, C97T, D98N, D99R, D99E, D99G, D99E,E100D、E100Q、V102I、M103L、L104I、L104V、L104I、L104V、L104V、G105A、S106H、S106N、S106R、L107S、L107Q、L107V、S111A、L115M、L115V、K118R、K118R、H119R、H119Y、I121A、I121V、V124A、V124I、T125S、A126G、A126V、A126G、Q127E、Q127H、V128A、V128L、V128I、V128A、S129T、S129Q、S129A、F131W、F131Q、F131H、G132N、G132Q、G132D、F133L、F133V、F133I、F133I、F133I、F133L、W134L、A136P、A136S、A137Q、A137K、A137F、A137L、A137R、A137H、A137M、A138S、A138D、S140A、S140P、S140P、S140P、T141V、T141I、T141V、H142L、H142S、P143-、P144A、P144K、P144K、P144L、G145P、G145R、A146T、A146F、A146T、A146L、A146S、A146L、N147T、N147P、N147S、L148I、L148V、A150E、A150T、A150G、F151S、F151E、F151D、F151T、F151W、F151W、V152L、V152L、P153T、P153S、Y154R、Y154D、Y154R、Y154V、Y154R、Y154L、P155E、V156I、V156P、V156D、V156Q、V156G、V156L、V156I、V156A、V156E、V156L、K157R、K157E、K157R、R158A、R158S、R158N、R158K、R158E、R158M、R158Q、L159E、L159D、L159G、L159R、L159A、L159K、L159T、L160F、L160I、L160A、L160V、L160Y、L161M、L161F、L161M、L161W、L161M、G162L、G162D、G162R、G162R、G162W、M163E、M163I、M163G、M163L、M163L、M163D、M163F、M163V、M163I、M163F、M163V、M163K、I164T、I164E、I164G、I164C、I164A、I164V、E165V、E165Y、E165D、E165A、E165I、R166K、R166L、R166E、R166V、R166Q、R166H、R166D、R166S、A167G、A167T、A167P、A167L、A167T、A167G、A167S、A167M、A167N、A167S、A167D、A167E、F168I、F168S、F168V、F168C、F168A、F168E、F168V、L169V、R171D、R171E、R171N、R171K、V172T、V172I、V172I、V172A、V172A、V172L、M173C、M173C、A174T、A174K、A174K、A174G、E176D、E176A、S179R、S179T、S179G、S179R、F180L、F180V、F180L、Q182S、Q182A、Q182R、Q182R、Q182G、Q182A、L186M、A187P、A187P、T189A、T189V、T189V、T189A、K190S、K190R、K190R、K190T、K190S、I192F、I193M、I193L、I193F、S194G、S194G、Q195E、Q195R、I197L、I197V、S198H、S198A、S198H、D201Q、R202G、R202K、R202G、E209D、E209D、V214P、V214T、V214A、H216T、H216R、H216P、H216Q、H216S、H216S、A220S、A220S、Q221H、Q221N、Q221R、T222A、T222I、V223S、V223A、F230Y、S233A、S233A、S233G、S233T、G234S、G234D、L235D、L235E、L235G、L235F、Q236H、Q236E、Q236L、Q236H、Q236R、Q236E、Q236L、P237T、P237T、P237Q、P237E、P237A、P237Q、P237Q、L238V、L238P、L238T、L238M、L238I、L238R、L238V、A240T、A240P、A240D、E243D、D244A、D244E、D244E、N247A、N247A、N247S、N247S、N247Q、N247R、E248G、E248A、E248A、A249G, A249E, A249T, A249G, A249S, S250P, S250P, S250P, V253I, T259A, T259S, M260N, V261R, V261I, V261L, V261I, N262D, N262D, N262H, N262D, N262 D, N262D, G263A, G263A, G263S, G263P, G263S, G263E, G263E, E264G, E264P, E264Q, E264T, E264D, E264A, Q265A, Q265T, Q265R, Q265R, Q265S, Y266F, T26 8R, T268A, T268S, A269T, V271A, V271I, V271L, V271A, V271A, Q272E, Q272E, L274C, K275R, K275R, L276S, L276R, L276K, L276A, L276R, I277T, I277L, I 277A、I277L、I277M、N278G、N278G、V282I、F283L、T285A、S286G、S286R、S28 6K、S286R、S286R、Q287N、Q287D、Q287E、Q287G、S288P、S288T、S288A、S288E、 S288G, V289D, V289A, V289T, V289P, D290E, D290L, D290V, P291D, P291A, P291H, P291Q, S292V, S292P, S292T, S292A, S292E, V293P, V293L, V293L, V293 D, V293P, V293A, S294P, S294A, S294L, S295R, S295T, S295A, S295L, E296S, E296A, E296S, E296D, E296G, E296P, G297S, G297S, G297D, G297S, G297P, G29 7D, G297P, R298Q, R298Q, R298N, R298D, R298E, I299V, V301L, R303S, R303P, V305A, S308T, R309K, R309L, R309A, R309S, I310L, H313Q, H313R, S314T, S 314A, S314A, S314A, S314C, S314C, A315S, A315T, L317I, V323I, V323I, T326A, T326V, L328Q, M330I, M330L, M330I, M330L, M330F, M330L, V334I, I337LI337V, I337L, A338V, A338V, F341L, F346L, M352L, M352V, M352I, E353Q, C357V, C357V, L359I, L359V, V361L, V361I, V361L, V361I, V361L, F362S, F362 D, F362L, F362R, F362R, F362L, F362K, F362L, F362S, F362P, F362D, P363G, P363A, P363R, P363S, P364S, P364R, P364K, A365V, S366T, S366Q, A367G, A36 7G, A367T, E368R, E368Q, S369R, S369P, S369A, L370I, A371S, A371T, A371T, T372A, T372R, D375Q, D375Q, D375R, D375E, K376R, K376T, K376R, K376R, I 377L, I377V, N379D, N379K, N379K, N379G, N379D, S380D, S380D, S380D, S380E, Q381P, E382A, E382Q, E382A, V383I, V383I, R384S, R384A, R384T, A385E A385E, S386N, S386A, S386N, S386K, S386R, S386A, E388T, E388A, E388A, E388D, E388Q, E388R, R389Q, R389K, R389E, Y390M, Y390T, Y390M, Y390L, Y390 I, R391A, T392Q, T392A, T392K, T392K, T392P, T392S, T392K, T392K, L393R, L393R, L393Q, I394V, I394M, I394L, I394F, I394M, P395A, S396T, G397S, G39 7A, D398P, D398E, D398E, D398E, S399A, S399T, S399R, A400G, S401L, S401C, S401C, S401A, E402Q, A403S, A403R, A403T, A403K, L406S, L406E, L406D, L 406N、L406R、Q407F、Q407A、Q407F、I408V、A410T、A410S、L411V、L411M、L41 1F、L411I、A412V、A412S、R413S、R413P、R413T、R413A、T414R、T414P、T414L、T414S, T414E, A415P, A415K, A415R, A415S, R416A, R416I, R416T, R416K, R416G, S417R, S417Y, S417A, S417K, R418Q, R418T, A419T, A419T, A419V, and A420R.

[0204] L. The method according to any one of paragraphs A to K, wherein the enzyme is a glycosyltransferase containing the deletion of at least one amino acid selected from: E44, R45, C46, ​​G47, F39, N49, F48, N49, F50, L51, P52, L53, G54, T55, R56, E57, E58, Y59, D60, E61, V62, P143, I164, D290, A415, R416, S417, R418, A419, and A420.

[0205] M. The method according to any one of paragraphs A to L, wherein the enzyme is a glycosyltransferase comprising an insertion of at least one amino acid selected from the group consisting of T392AV, I408VLS, S401CPA, E248AG, N247AG, R418APAG, E248GG, ​​A420RVAA, R418VAAA, A37PT, P71GGTF, P71CGPGEGWW, and P71GGRF.

[0206] N. The method described in any of paragraphs A through M, wherein the enzyme is a glycosyltransferase produced in a recombinant host cell.

[0207] O. The method described in any of paragraphs A through N, wherein the host cell is at least one of a eukaryote or a prokaryote.

[0208] P. According to the method described in any of paragraphs A to O, wherein the host cell is at least one of the following: *Escherichia coli*, *Bacillus subtilis*, *Streptomyces azureii*, *Pseudomonas aeruginosa*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Aspergillus niger*, *Trichoderma reesei*, *Corynebacterium*, *Pseudomonas putida*, *Burkholderia ts.*, *Burkholderia spp.*, *Acinetobacter*, *Acinetobacter*, *Antarctica*, *Bacillus spp.*, *Burkholderia*, *Candida*, *Fibrinosporium*, *Alcaligenes*, *Enterobacter*, *Halomyces*, *Lactobacillus*, *Hymenobacter*, *Myxococcus*, *Nocardia*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Nephroticis*, *Rhodococcus*, *Borgonia bogoria*, *Tetracoccus koreanum*, *Methylobacterium*, and *Methylrubia*.

[0209] Q. According to the method described in any of paragraphs A to P, the resulting monosaccharide is rhamnosaccharide.

[0210] R. The method according to any one of paragraphs A to Q, wherein the rhamnose monoester is produced in a cell-free system at a pH of 6 to 10, at a temperature of 25°C to 42°C, and optionally under stirring in a buffer solution.

[0211] S. The method according to any one of paragraphs A to R, wherein the enzyme is an encapsulated or immobilized glycosyltransferase.

[0212] T. The method according to any one of paragraphs A to S, wherein the enzyme is a glycosidase comprising a polypeptide sequence having at least 60% identity with SEQ ID NO: 30 to SEQ ID NO: 62 and 80 and fragments thereof.

[0213] U. The method according to any one of paragraphs A to T, wherein the glycosyltransferase comprises a polypeptide sequence having at least 75% identity with SEQ ID NO:30 to SEQ ID NO:62 and 80 and fragments thereof.

[0214] V. The method according to any one of paragraphs A to U, wherein the glycosyltransferase comprises a polypeptide sequence having at least 90% identity with SEQ ID NO:30 to SEQ ID NO:62 and 80 and fragments thereof.

[0215] W. The method described in any of paragraphs A through V, wherein the enzyme is a glycosidase produced in a recombinant host cell.

[0216] X. The method described in any of paragraphs A through W, wherein the host cell is at least one of a eukaryote or a prokaryote.

[0217] Y. The method described in any of paragraphs A through X, wherein the host cell is at least one of the following: *Escherichia coli*, *Bacillus subtilis*, *Streptomyces cerevisiae*, *Pseudomonas aeruginosa*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Aspergillus niger*, *Trichoderma reesei*, *Corynebacterium*, *Pseudomonas putida*, *Burkholderia ts.*, *Burkholderia spp.*, *Acinetobacter*, *Acinetobacter*, *Antarctica*, *Bacillus spp.*, *Burkholderia*, *Candida*, *Fibrinosporium*, *Alcaligenes*, *Enterobacter*, *Halomyces*, *Lactobacillus*, *Hymenobacter*, *Myxococcus*, *Nocardia*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Nephroticis*, *Rhodococcus*, *Borgonia bogoria*, *Tetracoccus koreanum*, *Methylobacterium*, and *Methylrubia*.

[0218] Z. The method according to any one of paragraphs A to Y, wherein the glycosidase comprises an amino acid selected from E206, E387, H150, N205, R79, Q18, E432, W425, Y321, Y322, F222, W361, F441, V209, F359, 341 that is not D or E, wherein the position is numbered with reference to SEQ ID NO: 83.

[0219] AA. The method according to any paragraph AZ, wherein the glycosidase comprises an amino acid selected from E572, E841, D567, H862, N849, R858, D579, W864, W576, W679, W684, wherein the position is numbered with reference to SEQ ID NO: 84.

[0220] The dimensions and values ​​disclosed herein should not be construed as strictly limited to the precise numerical values ​​cited. Rather, unless otherwise specified, each such dimension is intended to represent the stated value and the range surrounding its functional equivalent. For example, a dimension disclosed as “40 mm” is intended to represent “approximately 40 mm”.

[0221] Unless expressly excluded or otherwise limited, every reference cited herein, including any cross-references or related patents or applications, is incorporated herein by reference in its entirety. A reference to any document is not an admission that it is prior art concerning any invention disclosed or claimed herein, nor is it an admission that it, alone or in any combination with any other reference, teaches, suggests, or discloses any such invention. Furthermore, where any meaning or definition of a term in this invention conflicts with any meaning or definition of the same term in a referenced document, the meaning or definition given to that term in this invention shall prevail.

[0222] While specific embodiments of the invention have been illustrated and described by way of example, it will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. Therefore, it is intended that all such changes and modifications falling within the scope of the invention be covered by the appended claims.

Claims

1. A method for preparing glycosaminoglycans, the method comprising: (a) Preparation of glycosaminoglycans via a single catalytic step by contacting hydroxy fatty acids with a glycosyl donor in the presence of an enzyme selected from the following: i) Glycosyltransferases; and ii) Glycosidases; (b) Optionally, the prepared glycosaminoglycans are separated; The glycosaminoglycan has the following formula: Formula 1 Where n is between 0 and 15; Wherein X is selected from alkyl, aryl, heteroalkyl, heteroaryl, unsaturated alkenyl and unsaturated heteroalkenyl, as well as combinations and stereoisomers thereof; and wherein X has a carbon chain length of 4 to 22. M is selected from one or both of glucose, sulfoquinoline, fructose, galactose, ribose, maltose, xylose, rhamnose, sophorose, mannose, arabinose, fucose, and combinations thereof and stereoisomers thereof; And R is selected from -H, from alkyl, heteroalkyl, aryl, heteroaryl, heteroarylalkyl, arylalkyl, tauryl, and all their possible stereoisomers and combinations thereof.

2. The method according to claim 1, wherein the hydroxy fatty acid is a straight-chain hydroxy fatty acid; And X is selected from alkyl and unsaturated alkenyl groups, and has a carbon chain length of 4 to 22.

3. The method according to claim 2, wherein R is selected from -H and alkyl groups, and has a straight-chain carbon length of 1 to 4.

4. The method of claim 1, wherein the glycosyl donor comprises at least one rhamnose.

5. The method according to claim 4, wherein the rhamnose is nucleoside monophosphate (NMP), nucleoside diphosphate (NDP), or nucleoside triphosphate (NTP).

6. The method according to claim 1, wherein the monosaccharide is rhamnosaccharide.

7. The method according to claim 6, wherein the rhamnose monoester is selected from Rha-C8, Rha-C10, Rha-C12, Rha-C14, RhaRha-C8, RhaRha-C10, RhaRha-C12 and RhaRha-C14.

8. The method of claim 1, wherein the enzyme is a glycosyltransferase comprising a polypeptide sequence having at least 60% identity with any of SEQ ID NO: 1 to SEQ ID NO: 12 and fragments thereof.

9. The method of claim 1, wherein the glycosyltransferase comprises a polypeptide sequence having at least 75% identity with any of SEQ ID NO: 1 to SEQ ID NO: 12 and fragments thereof.

10. The method of claim 1, wherein the enzyme is a glycosyltransferase comprising at least one amino acid mutation selected from: SEQ ID NO: The amino acid sequences of the glycosyltransferases of 1 are as follows: M1A, H2R, V3I, I4L, I4V, I5L, I5V, T6I, A7P, V8I, F17L, V18L, V18L, V18I, I20L, T23A, T23S, T23S, T23A, T23E, V25A, A26S, V31I, T32S, F33V, F33M, C34A, A35T, A35V, S36Y, S36N, S36F, A37P, A37EW, A37EF, A37PA, A37E, A37G, A37P, A38V, A38G, A38Y, A38R, F39L, F39A, A40. K, A40R, A40P, A40E, A40S, P41E, P41S, P41A, L42S, L42A, I43A, I43V, I4 3L, E44D, E44G, R45L, R45G, R45E, C46S, C46A, C46P, C46R, G47A, F48M, F 48L, N49S, N49A, N49E, N49T, N49G, N49G, N49K, N49E, N49R, N49S, F50V, F50L, L51I, L51V, L51V, L51V, P52A, L53V, L53R, L53I, G54T, T55E, T55S , T55P, R56A, R56A, R56A, R56P, E57N, E57D, E58G, E58Q, Y59F, Y59I, D6 0E, D60N, D60H, D60A, D60Q, D60Q, E61A, E61A, V62L, V62I, V62A, V62P, M 63I, M63V, M63A, M63L, M63S, M63P, A64T, A64V, A64S, A64Y, D65S, D65N, A67R, H70N, H70D, P71S, R72K, R72L, R72E, T73K, T73A, T73G, T73D, S74A , L75F, P76R, P76G, T77V, L78M, K80R, K80R, T81A, T81W, T81V, I82V, I82 V, A83G, S84G, S84P, S84T, S84G, T85S, T85A, L86I, E88D, E88P, E88A, Q8 9L, Q89H, Y90F, R91D, R91E, L92V, L92M, E94Q, E94A, Q95A, A96L, A96L, A 96V, A96E, A96T, A96H, C97A, C97V, C97T, D98N, D99R, D99E, D99G, D99E,E100D、E100Q、V102I、M103L、L104I、L104V、L104I、L104V、L104V、G105A、S106H、S106N、S106R、L107S、L107Q、L107V、S111A、L115M、L115V、K118R、K118R、H119R、H119Y、I121A、I121V、V124A、V124I、T125S、A126G、A126V、A126G、Q127E、Q127H、V128A、V128L、V128I、V128A、S129T、S129Q、S129A、F131W、F131Q、F131H、G132N、G132Q、G132D、F133L、F133V、F133I、F133I、F133I、F133L、W134L、A136P、A136S、A137Q、A137K、A137F、A137L、A137R、A137H、A137M、A138S、A138D、S140A、S140P、S140P、S140P、T141V、T141I、T141V、H142L、H142S、P143-、P144A、P144K、P144K、P144L、G145P、G145R、A146T、A146F、A146T、A146L、A146S、A146L、N147T、N147P、N147S、L148I、L148V、A150E、A150T、A150G、F151S、F151E、F151D、F151T、F151W、F151W、V152L、V152L、P153T、P153S、Y154R、Y154D、Y154R、Y154V、Y154R、Y154L、P155E、V156I、V156P、V156D、V156Q、V156G、V156L、V156I、V156A、V156E、V156L、K157R、K157E、K157R、R158A、R158S、R158N、R158K、R158E、R158M、R158Q、L159E、L159D、L159G、L159R、L159A、L159K、L159T、L160F、L160I、L160A、L160V、L160Y、L161M、L161F、L161M、L161W、L161M、G162L、G162D、G162R、G162R、G162W、M163E、M163I、M163G、M163L、M163L、M163D、M163F、M163V、M163I、M163F、M163V、M163K、I164T、I164E、I164G、I164C、I164A、I164V、E165V、E165Y、E165D、E165A、E165I、R166K、R166L、R166E、R166V、R166Q、R166H、R166D、R166S、A167G、A167T、A167P、A167L、A167T、A167G、A167S、A167M、A167N、A167S、A167D、A167E、F168I、F168S、F168V、F168C、F168A、F168E、F168V、L169V、R171D、R171E、R171N、R171K、V172T、V172I、V172I、V172A、V172A、V172L、M173C、M173C、A174T、A174K、A174K、A174G、E176D、E176A、S179R、S179T、S179G、S179R、F180L、F180V、F180L、Q182S、Q182A、Q182R、Q182R、Q182G、Q182A、L186M、A187P、A187P、T189A、T189V、T189V、T189A、K190S、K190R、K190R、K190T、K190S、I192F、I193M、I193L、I193F、S194G、S194G、Q195E、Q195R、I197L、I197V、S198H、S198A、S198H、D201Q、R202G、R202K、R202G、E209D、E209D、V214P、V214T、V214A、H216T、H216R、H216P、H216Q、H216S、H216S、A220S、A220S、Q221H、Q221N、Q221R、T222A、T222I、V223S、V223A、F230Y、S233A、S233A、S233G、S233T、G234S、G234D、L235D、L235E、L235G、L235F、Q236H、Q236E、Q236L、Q236H、Q236R、Q236E、Q236L、P237T、P237T、P237Q、P237E、P237A、P237Q、P237Q、L238V、L238P、L238T、L238M、L238I、L238R、L238V、A240T、A240P、A240D、E243D、D244A、D244E、D244E、N247A、N247A、N247S、N247S、N247Q、N247R、E248G、E248A、E248A、A249G, A249E, A249T, A249G, A249S, S250P, S250P, S250P, V253I, T259A, T259S, M260N, V261R, V261I, V261L, V261I, N262D, N262D, N262H, N262D, N262 D, N262D, G263A, G263A, G263S, G263P, G263S, G263E, G263E, E264G, E264P, E264Q, E264T, E264D, E264A, Q265A, Q265T, Q265R, Q265R, Q265S, Y266F, T26 8R, T268A, T268S, A269T, V271A, V271I, V271L, V271A, V271A, Q272E, Q272E, L274C, K275R, K275R, L276S, L276R, L276K, L276A, L276R, I277T, I277L, I 277A、I277L、I277M、N278G、N278G、V282I、F283L、T285A、S286G、S286R、S28 6K、S286R、S286R、Q287N、Q287D、Q287E、Q287G、S288P、S288T、S288A、S288E、 S288G, V289D, V289A, V289T, V289P, D290E, D290L, D290V, P291D, P291A, P291H, P291Q, S292V, S292P, S292T, S292A, S292E, V293P, V293L, V293L, V293 D, V293P, V293A, S294P, S294A, S294L, S295R, S295T, S295A, S295L, E296S, E296A, E296S, E296D, E296G, E296P, G297S, G297S, G297D, G297S, G297P, G29 7D, G297P, R298Q, R298Q, R298N, R298D, R298E, I299V, V301L, R303S, R303P, V305A, S308T, R309K, R309L, R309A, R309S, I310L, H313Q, H313R, S314T, S 314A, S314A, S314A, S314C, S314C, A315S, A315T, L317I, V323I, V323I, T326A, T326V, L328Q, M330I, M330L, M330I, M330L, M330F, M330L, V334I, I337LI337V, I337L, A338V, A338V, F341L, F346L, M352L, M352V, M352I, E353Q, C357V, C357V, L359I, L359V, V361L, V361I, V361L, V361I, V361L, F362S, F362 D, F362L, F362R, F362R, F362L, F362K, F362L, F362S, F362P, F362D, P363G, P363A, P363R, P363S, P364S, P364R, P364K, A365V, S366T, S366Q, A367G, A36 7G, A367T, E368R, E368Q, S369R, S369P, S369A, L370I, A371S, A371T, A371T, T372A, T372R, D375Q, D375Q, D375R, D375E, K376R, K376T, K376R, K376R, I 377L, I377V, N379D, N379K, N379K, N379G, N379D, S380D, S380D, S380D, S380E, Q381P, E382A, E382Q, E382A, V383I, V383I, R384S, R384A, R384T, A385E A385E, S386N, S386A, S386N, S386K, S386R, S386A, E388T, E388A, E388A, E388D, E388Q, E388R, R389Q, R389K, R389E, Y390M, Y390T, Y390M, Y390L, Y390 I, R391A, T392Q, T392A, T392K, T392K, T392P, T392S, T392K, T392K, L393R, L393R, L393Q, I394V, I394M, I394L, I394F, I394M, P395A, S396T, G397S, G39 7A, D398P, D398E, D398E, D398E, S399A, S399T, S399R, A400G, S401L, S401C, S401C, S401A, E402Q, A403S, A403R, A403T, A403K, L406S, L406E, L406D, L 406N、L406R、Q407F、Q407A、Q407F、I408V、A410T、A410S、L411V、L411M、L41 1F、L411I、A412V、A412S、R413S、R413P、R413T、R413A、T414R、T414P、T414L、T414S, T414E, A415P, A415K, A415R, A415S, R416A, R416I, R416T, R416K, R416G, S417R, S417Y, S417A, S417K, R418Q, R418T, A419T, A419T, A419V, and A420R.

11. The method of claim 1, wherein the enzyme is a glycosyltransferase produced in a recombinant host cell, wherein the host cell is at least one of a eukaryote or a prokaryote.

12. The method of claim 11, wherein the resulting glycosaminoglycan is produced in a cell-free system at a pH of 6 to 10, at a temperature of 25°C to 42°C, and optionally under stirring in a buffer solution.

13. The method of claim 1, wherein the enzyme is a glycosidase comprising a polypeptide sequence having at least 60% identity with SEQ ID NO: 30 to SEQ ID NO: 62 and 80 and fragments thereof.

14. The method according to claim 1, wherein the enzyme is a glycosidase produced in a recombinant host cell, wherein the host cell is at least one of the following: *Escherichia coli*, *Bacillus subtilis*, *Streptomyces cerevisiae*, *Pseudomonas aeruginosa*, *Pichia pastoris*, *Saccharomyces cerevisiae*, *Kluyveromyces lactis*, *Aspergillus niger*, *Trichoderma reesei*, *Corynebacterium*, *Pseudomonas putida*, *Burkholderia ts.*, *Burkholderia spp.*, *Acinetobacter*, *Acinetobacter*, *Acinetobacter*, *Antarctica*, *Bacillus*, *Burkholderia*, *Candida*, *Fibrinosporium*, *Alcaligenes*, *Enterobacter*, *Halomyces*, *Lactobacillus*, *Hymenobacter*, *Myxococcus*, *Nocardia*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Pseudomonas*, *Nephroticis*, *Rhodococcus*, *Borgonia bogoria*, *Tetracoccus koreanum*, *Methylobacterium*, and *Methylrubia*.

15. A glycosyltransferase variant comprising a mutation in at least one amino acid selected from: SEQ ID NO: The amino acid sequences of the glycosyltransferases of 1 are as follows: M1A, H2R, V3I, I4L, I4V, I5L, I5V, T6I, A7P, V8I, F17L, V18L, V18L, V18I, I20L, T23A, T23S, T23S, T23A, T23E, V25A, A26S, V31I, T32S, F33V, F33M, C34A, A35T, A35V, S36Y, S36N, S36F, A37P, A37EW, A37EF, A37PA, A37E, A37G, A37P, A38V, A38G, A38Y, A38R, F39L, F39A, A40K. A40R, A40P, A40E, A40S, P41E, P41S, P41A, L42S, L42A, I43A, I43V, I43L , E44D, E44G, R45L, R45G, R45E, C46S, C46A, C46P, C46R, G47A, F48M, F48 L, N49S, N49A, N49E, N49T, N49G, N49G, N49K, N49E, N49R, N49S, F50V, F5 0L, L51I, L51V, L51V, L51V, P52A, L53V, L53R, L53I, G54T, T55E, T55S, T5 5P, R56A, R56A, R56A, R56P, E57N, E57D, E58G, E58Q, Y59F, Y59I, D60E, D 60N, D60H, D60A, D60Q, D60Q, E61A, E61A, V62L, V62I, V62A, V62P, M63I, M63V, M63A, M63L, M63S, M63P, A64T, A64V, A64S, A64Y, D65S, D65N, A67R , H70N, H70D, P71S, R72K, R72L, R72E, T73K, T73A, T73G, T73D, S74A, L75F , P76R, P76G, T77V, L78M, K80R, K80R, T81A, T81W, T81V, I82V, I82V, A83 G, S84G, S84P, S84T, S84G, T85S, T85A, L86I, E88D, E88P, E88A, Q89L, Q8 9H, Y90F, R91D, R91E, L92V, L92M, E94Q, E94A, Q95A, A96L, A96L, A96V, A 96E, A96T, A96H, C97A, C97V, C97T, D98N, D99R, D99E, D99G, D99E, E100D,E100Q、V102I、M103L、L104I、L104V、L104I、L104V、L104V、G105A、S106H、S106N、S106R、L107S、L107Q、L107V、S111A、L115M、L115V、K118R、K118R、H119R、H119Y、I121A、I121V、V124A、V124I、T125S、A126G、A126V、A126G、Q127E、Q127H、V128A、V128L、V128I、V128A、S129T、S129Q、S129A、F131W、F131Q、F131H、G132N、G132Q、G132D、F133L、F133V、F133I、F133I、F133I、F133L、W134L、A136P、A136S、A137Q、A137K、A137F、A137L、A137R、A137H、A137M、A138S、A138D、S140A、S140P、S140P、S140P、T141V、T141I、T141V、H142L、H142S、P143-、P144A、P144K、P144K、P144L、G145P、G145R、A146T、A146F、A146T、A146L、A146S、A146L、N147T、N147P、N147S、L148I、L148V、A150E、A150T、A150G、F151S、F151E、F151D、F151T、F151W、F151W、V152L、V152L、P153T、P153S、Y154R、Y154D、Y154R、Y154V、Y154R、Y154L、P155E、V156I、V156P、V156D、V156Q、V156G、V156L、V156I、V156A、V156E、V156L、K157R、K157E、K157R、R158A、R158S、R158N、R158K、R158E、R158M、R158Q、L159E、L159D、L159G、L159R、L159A、L159K、L159T、L160F、L160I、L160A、L160V、L160Y、L161M、L161F、L161M、L161W、L161M、G162L、G162D、G162R、G162R、G162W、M163E、M163I、M163G、M163L、M163L、M163D、M163F、M163V、M163I、M163F、M163V、M163K、I164T、I164E、I164G、I164C、I164A、I164V、E165V、E165Y、E165D、E165A、E165I、R166K、R166L、R166E、R166V、R166Q、R166H、R166D、R166S、A167G、A167T、A167P、A167L、A167T、A167G、A167S、A167M、A167N、A167S、A167D、A167E、F168I、F168S、F168V、F168C、F168A、F168E、F168V、L169V、R171D、R171E、R171N、R171K、V172T、V172I、V172I、V172A、V172A、V172L、M173C、M173C、A174T、A174K、A174K、A174G、E176D、E176A、S179R、S179T、S179G、S179R、F180L、F180V、F180L、Q182S、Q182A、Q182R、Q182R、Q182G、Q182A、L186M、A187P、A187P、T189A、T189V、T189V、T189A、K190S、K190R、K190R、K190T、K190S、I192F、I193M、I193L、I193F、S194G、S194G、Q195E、Q195R、I197L、I197V、S198H、S198A、S198H、D201Q、R202G、R202K、R202G、E209D、E209D、V214P、V214T、V214A、H216T、H216R、H216P、H216Q、H216S、H216S、A220S、A220S、Q221H、Q221N、Q221R、T222A、T222I、V223S、V223A、F230Y、S233A、S233A、S233G、S233T、G234S、G234D、L235D、L235E、L235G、L235F、Q236H、Q236E、Q236L、Q236H、Q236R、Q236E、Q236L、P237T、P237T、P237Q、P237E、P237A、P237Q、P237Q、L238V、L238P、L238T、L238M、L238I、L238R、L238V、A240T、A240P、A240D、E243D、D244A、D244E、D244E、N247A、N247A、N247S、N247S、N247Q、N247R、E248G、E248A、E248A、A249G、A249E, A249T, A249G, A249S, S250P, S250P, S250P, V253I, T259A, T259S, M260N, V261R, V261I, V261L, V261I, N262D, N262D, N262H, N262D, N262D, N262 D, G263A, G263A, G263S, G263P, G263S, G263E, G263E, E264G, E264P, E264Q, E264T, E264D, E264A, Q265A, Q265T, Q265R, Q265R, Q265S, Y266F, T268R, T26 8A, T268S, A269T, V271A, V271I, V271L, V271A, V271A, Q272E, Q272E, L274C, K275R, K275R, L276S, L276R, L276K, L276A, L276R, I277T, I277L, I277A, I 277L、I277M、N278G、N278G、V282I、F283L、T285A、S286G、S286R、S286K、S28 6R、S286R、Q287N、Q287D、Q287E、Q287G、S288P、S288T、S288A、S288E、S288G、 V289D, V289A, V289T, V289P, D290E, D290L, D290V, P291D, P291A, P291H, P291Q, S292V, S292P, S292T, S292A, S292E, V293P, V293L, V293L, V293D, V293 P、V293A、S294P、S294A、S294L、S295R、S295T、S295A、S295L、E296S、E296A、 E296S、E296D、E296G、E296P、G297S、G297S、G297D、G297S、G297P、G297D、G29 7P、R298Q、R298Q、R298N、R298D、R298E、I299V、V301L、R303S、R303P、V305A 、S308T、R309K、R309L、R309A、R309S、I310L、H313Q、H313R、S314T、S314A、S 314A, S314A, S314C, S314C, A315S, A315T, L317I, V323I, V323I, T326A, T326V, L328Q, M330I, M330L, M330I, M330L, M330F, M330L, V334I, I337L, I337VI337L, A338V, A338V, F341L, F346L, M352L, M352V, M352I, E353Q, C357V, C357V, L359I, L359V, V361L, V361I, V361L, V361I, V361L, F362S, F362D, F362 L, F362R, F362R, F362L, F362K, F362L, F362S, F362P, F362D, P363G, P363A, P363R, P363S, P364S, P364R, P364K, A365V, S366T, S366Q, A367G, A367G, A36 7T, E368R, E368Q, S369R, S369P, S369A, L370I, A371S, A371T, A371T, T372A, T372R, D375Q, D375Q, D375R, D375E, K376R, K376T, K376R, K376R, I377L, I 377V, N379D, N379K, N379K, N379G, N379D, S380D, S380D, S380D, S380E, Q381P, E382A, E382Q, E382A, V383I, V383I, R384S, R384A, R384T, A385E, A385E S386N, S386A, S386N, S386K, S386R, S386A, E388T, E388A, E388A, E388D, E388Q, E388R, R389Q, R389K, R389E, Y390M, Y390T, Y390M, Y390L, Y390I, R391 A, T392Q, T392A, T392K, T392K, T392P, T392S, T392K, T392K, L393R, L393R, L393Q, I394V, I394M, I394L, I394F, I394M, P395A, S396T, G397S, G397A, D39 8P、D398E、D398E、D398E、S399A、S399T、S399R、A400G、S401L、S401C、S401C 、S401A、E402Q、A403S、A403R、A403T、A403K、L406S、L406E、L406D、L406N、L 406R、Q407F、Q407A、Q407F、I408V、A410T、A410S、L411V、L411M、L411F、L41 1I、A412V、A412S、R413S、R413P、R413T、R413A、T414R、T414P、T414L、T414S、T414E, A415P, A415K, A415R, A415S, R416A, R416I, R416T, R416K, R416G, S417R, S417Y, S417A, S417K, R418Q, R418T, A419T, A419T, A419V, and A420R.

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