Method for hydroxylation of bile acids or analogues thereof

Abstract

The present disclosure relates to a method for hydroxylation of bile acids or analogues thereof. Aspects of the disclosure provide method for 7β-hydroxylation of Formula I compound or analogue or salt thereof catalyzed by an enzyme in the presence of a peroxide. Additional aspects also relate to method for converting Formula I compound or analogue or salt thereof to Formula II compound or analogue or salt thereof in the presence of an enzyme and a peroxide; a corresponding reaction mixture; and the associated use of the enzyme to catalyze 7β-hydroxylation of Formula I compound or analogue or salt thereof.

Description

[0001] “METHOD FOR HYDROXYLATION OF BILE ACIDS OR ANALOGUES THEREOF” FIELD OF THE INVENTION The present disclosure relates to hydroxylation of bile acids and / or analogues thereof. Particularly provided is the enzymatic production of Formula II compound or analogue or salt thereof by 7β- hydroxylation of Formula I compound or analogue or salt thereof. BACKGROUND Bile acids such as ursodeoxycholic acid (UDCA also known as Ursodiol) and derivatives thereof are powerful medicines used for the treatment of chronic liver disease and gallstones. Existing manufacturing of UDCA is based on animal derived cholic Acid (CA) with multi-step chemo- enzymatic processes. Cholic acid is recovered from bovine gallbladder at time of slaughter. As with any animal based raw materials, there are ethical concerns, availability / shortage issue and there is also an increased risk regarding the purity of the product. Alternatively, plant based raw materials (e.g., phytosterols) can be used for the synthesis of UDCA. However, it typically requires more chemical steps. A purely chemical process (either from animal or plant-based steroids) is therefore complex, environmentally unfriendly, and potentially costly. The main challenge is the (regio-, stereo-, and chemo-) selective hydroxylation of the starting steroids. Bile acids such as UDCA or its derivatives could be synthesized from lithocholic acid (LCA) or its derivatives by such a reaction, particularly by 7β-hydroxylation reaction of the LCA molecule. While enzymes can be used to perform such a reaction, the currently known enzymatic processes are complex, costly and resource intensive. Hence, there is a need for efficient, cost- effective, and simpler approaches to produce bile acids such as UDCA or its derivatives. SUMMARY Aspects of the present disclosure relate to a method for hydroxylating a compound of Formula I, or analogue or salt thereof, comprising contacting the compound of Formula I or analogue or salt thereof:

[0002] with an enzyme in the presence of a peroxide, to produce a compound of Formula II or analogue or salt thereof: wherein, R is independently selected from the group consisting of (CH2)nC=O(O)mR', alkyl, alkenyl, CH2OH, CHO, (CH2)2CONH(CH2)2SO3H, (CH2)2CONH(CH2)COOH, -CH=CH-CO2-alkyl, - CH=CH-CO2-aryl, -CH=CH-CO2H and -CH=CH-alkyl, wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRaRb, wherein Ra and Rb are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=O)-alkyl, -C(=O)-aryl and (CH2)2SO3H; R1 is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=O)-alkyl, -C(=O)-aryl, -S(=O)2-alkyl, -S(=O)2-aryl, -S(=O)2-OH and -P(=O)-(OR2)2, wherein R2is independently hydrogen or alkyl; n is 0 or an integer ranging from 1 to 5; m is 0 or 1; Y is hydrogen; X is hydrogen, hydroxy or oxygen; wherein R, R’, Ra, Rb, R1, R2 or X are optionally independently substituted; represents either a single bond or a double bond; and with the proviso that: both Rings A and B do not simultaneously contain double bond inside the rings; when is a double bond on the Ring A, R1 is absent; when is a single bond on the Ring A, then R1is present; when is a double bond inside either the Ring A or Ring B, Y is absent; and when is a single bond inside both the Rings A and B simultaneously, Y is present. Further aspects of the present disclosure relate to a method for converting the compound of Formula I or analogue or salt thereof to the compound of Formula II or analogue or salt thereof, comprising contacting the compound of Formula I or analogue or salt thereof with an enzyme in the presence of a peroxide, to produce the compound of Formula II or analogue or salt thereof. Additional aspects of the present disclosure relate to a method of manufacturing a compound of Formula II or analogue or salt thereof, the method comprising contacting the compound of Formula I or analogue or salt thereof with an enzyme in the presence of a peroxide, to produce said compound of Formula II or analogue or salt thereof. In some embodiments, said method comprises 7β-hydroxylation of the compound of Formula I or analogue or a salt thereof to produce the compound of Formula II or analogue or salt thereof which has a β-hydroxyl group at position C7. In some embodiments of the method, the enzyme is capable of using peroxide as a co-substrate, and the enzyme is a peroxygenase. In some embodiments of the method, the enzyme is unspecific peroxygenase (UPO). In some embodiments of the method, the peroxide is hydrogen peroxide (H2O2). In some embodiments, the method comprises converting lithocholic acid (LCA) or taurolithocholic acid (TLCA) into ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) respectively, comprising contacting the LCA or TLCA with an enzyme in the presence of a peroxide, to produce the UDCA or TUDCA. In some embodiments, the method comprises converting lithocholic acid (LCA) or taurolithocholic acid (TLCA) into ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) respectively, comprising contacting the LCA or TLCA with unspecific peroxygenase (UPO) in the presence of H2O2, to produce the UDCA or TUDCA. Further aspects of the disclosure relate to use of an enzyme for: i) 7β-hydroxylation of a compound of Formula I or analogue salt thereof to the compound of Formula II or analogue or salt thereof, or ii) converting a compound of Formula I or analogue or salt thereof to the compound of Formula II or analogue or salt thereof, wherein the enzyme requires peroxide as a co-substrate. In some embodiments of the use, the enzyme is unspecific peroxygenase (UPO). In some embodiments of the use, the Formula I or analogue or salt thereof is LCA or TLCA; the Formula II or analogue or salt thereof is UDCA or TUDCA; the enzyme is unspecific peroxygenase (UPO); and the peroxide is hydrogen peroxide (H2O2). Further aspects of the disclosure relate to a reaction mixture or reaction system comprising: (i) a compound of Formula I or analogue or salt thereof, (ii) an enzyme, and (iii) a peroxide. BRIEF DESCRIPTION OF FIGURES Figure 1 depicts a reaction scheme representing one embodiment of the present method including a method for hydroxylation of LCA to produce UDCA. Figure 2 shows HPLC results demonstrating the production of UDCA from LCA catalyzed by UPO in accordance with Example 1. Figure 3 shows HPLC results demonstrating the production of UDCA from LCA catalyzed by UPO in accordance with Example 2. Figure 4 shows HPLC results demonstrating the production of TUDCA from TLCA catalyzed by UPO in accordance with Example 3. Figure 5 shows HPLC results demonstrating the production of TUDCA from TLCA catalyzed by UPO in accordance with Example 4. DESCRIPTION OF THE INVENTION In view of the limitations discussed above, and to remedy the need in the art for an efficient way to produce bile acids or its derivatives, the present disclosure provides an efficient and viable enzymatic method. Particularly, the present disclosure describes enzymatic production of Formula II compound or analogue or salt thereof including bile acids such as ursodeoxycholic acid (UDCA) or analogues or salts thereof. Preferably, the disclosure provides enzymatic production of Formula II compound or analogue or salt thereof including bile acids such as ursodeoxycholic acid (UDCA) or its analogues or salts by 7β-hydroxylation of Formula I compound or analogue or salt thereof including bile acid precursors such as lithocholic acid (LCA) or its analogues or salts. Additionally provided are corresponding methods for conversion of Formula I compound or analogue or salt thereof to Formula II compound or analogue or salt thereof; a reaction mixture for production of Formula II compound or analogue or salt thereof; and use of enzyme(s) for production of Formula II compound or analogue or salt thereof. The present methods, the corresponding reaction mixture and associated use of the present disclosure are further described in greater detail in the following embodiments. For the sake of brevity, identical embodiments may not be repeated for each of the different methods, the reaction mixture and the use described herein. However, any combination of an embodiment captured anywhere in this disclosure with any other embodiment captured elsewhere in this disclosure, fall wholly within the ambit of the present disclosure. Such combinations can therefore be considered to derive complete meaning of the aspects described herein. The present disclosure provides a method for hydroxylating a compound of Formula I or analogue or salt thereof, comprising contacting the compound of Formula I or analogue or salt thereof: with an enzyme in the presence of a peroxide, to produce a compound of Formula II or analogue or salt thereof:

[0003] wherein, R is independently selected from the group consisting of (CH2)nC=O(O)mR', alkyl, alkenyl, CH2OH, CHO, (CH2)2CONH(CH2)2SO3H, (CH2)2CONH(CH2)COOH, -CH=CH-CO2-alkyl, - CH=CH-CO2-aryl, -CH=CH-CO2H and -CH=CH-alkyl, wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRaRb, wherein Ra and Rb are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=O)-alkyl, -C(=O)-aryl and (CH2)2SO3H; R1is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=O)-alkyl, -C(=O)-aryl, -S(=O)2-alkyl, -S(=O)2-aryl, -S(=O)2-OH and -P(=O)-(OR2)2, wherein R2is independently hydrogen or alkyl; n is 0 or an integer ranging from 1 to 5; m is 0 or 1; Y is hydrogen; X is hydrogen, hydroxy or oxygen; wherein R, R’, Ra, Rb, R1, R2or X are optionally independently substituted; represents either a single bond or a double bond; and with the proviso that: both Rings A and B do not simultaneously contain double bond inside the rings; when is a double bond on the Ring A, R1 is absent; when is a single bond on the Ring A, then R1 is present; when is a double bond inside either the Ring A or Ring B, Y is absent; and when is a single bond inside both the Rings A and B simultaneously, Y is present. In some embodiments of the method, the alkyl in Formula I or Formula II is C1-C12alkyl, the alkenyl in Formula I or Formula II is C2-C12 alkenyl, the alkynyl in Formula I or Formula II is C2- C12alkynyl, the cycloalkyl in Formula I or Formula II is a hydrocarbon ring system radical comprising C3-C20 carbon atoms, and the aryl in Formula I or Formula II is a hydrocarbon ring system radical comprising C6-C18carbon atoms and at least one aromatic ring. In some embodiments of the method, the alkyl in Formula I or Formula II is C1-C6alkyl, the alkenyl in Formula I or Formula II is C2-C6 alkenyl, the alkynyl in Formula I or Formula II is C2- C6 alkynyl, the cycloalkyl in Formula I or Formula II is a hydrocarbon ring system radical comprising C3-C6 carbon atoms, and the aryl in Formula I or Formula II is a hydrocarbon ring system radical comprising C6-C12carbon atoms and at least one aromatic ring. In some embodiments of the method, the salt of Formula I or Formula II is selected from the group consisting of sodium salt, potassium salt, and quaternary ammonium salts (QAS). In some embodiments, the QAS is selected from the group consisting of tetramethylammonium, tetraethylammonium, n-tetrabutylammonium, and alkylbenzyldimethylammonium, or any combination thereof. As used in this disclosure, ‘analogs’, ‘analogues’, ‘derivative’ or ‘derivatives’ are used interchangeably referring to compounds in which one or more individual atoms have been replaced, either with a different atom, or with a different functional group; or a compound that is formed from a similar compound or a compound that can be imagined to arise from another compound, if one atom is replaced with another atom or group of atoms. As used in this disclosure, “7β-hydroxylation” or “hydroxylation at C7 position in beta- orientation” which are used interchangeably, refers to hydroxylating a compound (substrate) at C7 position of the molecule in beta orientation to obtain a compound (product) having hydroxyl group. Thus, in some embodiments, the product of the 7β-hydroxylation reaction comprises hydroxyl group in beta-orientation at the C7 position. As used in this disclosure, the numbering of atoms in the organic compounds (such as cyclopentanoperhydrophenanthrene / Sterane compounds or analogues or derivatives or salts thereof) follows the International Union of Pure and Applied Chemistry (IUPAC) numbering system. In some embodiments, the method comprises 7β-hydroxylation of the compound of Formula I or a salt thereof to produce the compound of Formula II or a salt thereof which has a β-hydroxyl group at position C7. As used in this disclosure, “enzyme capable of using peroxide” or “enzyme requiring presence of a peroxide” or the likes which are used interchangeably, refer to enzyme(s) which utilize peroxide or peroxide containing compounds as a source of oxygen and electrons for catalyzing an enzymatic reaction. In some embodiments, the present disclosure defines said enzyme(s) by their Enzyme Commission number (EC number). Such enzyme nomenclature based on EC number that classify the enzymes based on the enzyme-catalyzed reaction(s) is developed by the International Union of Biochemistry and Molecular Biology (IUBMB) and is well known to a person skilled in the art. In some embodiments of the method, the enzyme is capable of using the compound of Formula I or a salt thereof as a substrate and peroxide as a co-substrate for catalyzing the hydroxylation of the compound of Formula I or a salt thereof to produce the compound of Formula II or a salt thereof. In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is an enzyme from subclass EC 1.11 (subclass of oxidoreductases) which acts on peroxide as acceptor molecule. More particularly, the enzyme capable of using peroxide as a co-substrate is an enzyme from EC 1.11: Acting on a peroxide as acceptor. In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is a peroxygenase. In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is a peroxygenase from sub subclass EC 1.11.2. More particularly, the enzyme capable of using peroxide as a co-substrate is an enzyme from EC 1.11.2: Peroxygenases. In some embodiments, the peroxygenase of EC 1.11.2 comprises unspecific peroxygenase (EC 1.11.2.1), myeloperoxidase (EC 1.11.2.2), plant seed peroxygenase (EC 1.11.2.3), fatty-acid peroxygenase (EC 1.11.2.4), 3-methyl-L-tyrosine peroxygenase (EC 1.11.2.5), or L-tyrosine peroxygenase (EC 1.11.2.6). In some embodiments of the method, the peroxygenase is a fungal peroxygenase. In some embodiments of the method, the peroxygenase is a peroxygenase enzyme from Proto- fungi or pseudo-fungi. In some embodiments of the method, the enzyme is unspecific peroxygenase (UPO). In the present disclosure, the enzyme “unspecific peroxygenase” or “UPO” is defined based on the currently known classifications considering criteria such as their source, origin, size, EC number description etc. which is non-exhaustive, and thereby includes any UPO enzyme. In some embodiments, this also includes enzymes from other EC classes which are modified in such a way that they are covered by EC 1.11.2.1. In some embodiments, the present methods encompass any UPO enzyme which is capable of using peroxide as a co-substrate for catalyzing 7β-hydroxylation of the compound of Formula I or a salt thereof to produce the compound of Formula II or a salt thereof. In some embodiments of the method, the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1 belonging to the sub-subclass of oxidoreductases. More particularly, the enzyme of the present method is an enzyme from EC 1.11.2.1: Unspecific peroxygenase. In some embodiments of the method, the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1 that act with H2O2 or peroxide as acceptor molecule, one oxygen atom of which is incorporated into the product. In some embodiments of the method, the unspecific peroxygenase (UPO) is a wild-type UPO obtained from fungi or proto-fungi, or both. In some embodiments of the method, the unspecific peroxygenase (UPO) is a wild-type UPO obtained from fungi or proto-fungi. In some embodiments, the UPO enzyme obtained from fungi can be divided into two families: i) Family I UPOs – the ‘short’ UPO sequences which include representatives of UPO- containing phylogenetic groups of fungi selected from Ascomycota, Cryptomycota, Basidiomycota, Chytridiomycota, Mucoromycotina, Oomycetes, Zoopagomycota, and Peronosporomycetes; ii) Family II UPOs – the ‘long’ UPO sequences which include representatives of UPO- containing phylogenetic groups of fungi selected from Ascomycota and Basidiomycota. In some embodiments of the method, the UPO is Family I UPOs having a mean size of around 30 kDa. In some embodiments of the method, the UPO is Family II UPOs having a mean size of around 44.4 kDa. In some embodiments of the method, the UPO is a wild-type or native UPO obtained from the fungi selected from the group consisting of Agrocybe sp., Aspergillus sp., Coprinopsis sp., Chaetomium sp., Leptoxyphium sp., Coprinellus sp., Collariella sp., Daldinia sp., Humicola sp., Hypoxylon sp., Marasmius sp., Myceliophthora sp., Candolleomyces sp., Pestalotiopsis sp., Thielavia sp., fungi from the same family or class thereof, and combinations thereof. In some embodiments of the method, the UPO is a wild-type or native UPO obtained from the fungi selected from the group consisting of Agrocybe aegerita, Aspergillus niger, Coprinopsis cinerea, Coprinopsis verticillate, Chaetomium globosum, Leptoxyphium fumago, Coprinellus radians, Collariella virescens, Daldinia caldariorum, Humicola insolens, Hypoxylon sp. EC38, Marasmius rotula, Myceliophthora wettsteinii, Myceliophthora fergusii, Myceliophthora hinnulea, Candolleomyces aberdarensis, Pestalotiopsis virgatula, Thielavia hyrcaniae, and combinations thereof. In some embodiments of the method, the UPO is a wild-type or native UPO obtained from the proto-fungi selected from the group consisting of proto-fungi from class Oomycetes (or Peronosporomycetes), class Hyphochytriomycetes, and a combination thereof. As used in this disclosure, “wild-type UPO” or “native UPO” or “wild-type or native UPO” which are used interchangeably, refer to an UPO enzyme which is expressed or produced in the native organism (i.e, an organism naturally producing the UPO enzyme). For instance, the UPO gene or polynucleotide is expressed in a native organism (e.g., fungi or proto-fungi) and the produced UPO enzyme is employed in the present methods. In some embodiments, said UPO gene or polynucleotide is not modified or mutated before expression in the native organism. In some embodiments, said UPO gene or polynucleotide is modified or mutated before expression in the native organism. In some embodiments of the method, the unspecific peroxygenase (UPO) is a recombinant UPO enzyme expressed in a host cell. In some embodiments, the host cell is selected from fungi and bacteria. UPO enzymes are for example provided by Aminoverse BV (for example in its Article No. reference AVUP07700 UPO enzyme kit) and EvoEnzyme SL (for example in its EVO UPOkit). In some embodiments, the unspecific peroxygenase (UPO) forms part of the Aminoverse’s Article No. reference AVUP07700 UPO enzyme kit. In some embodiments, the unspecific peroxygenase (UPO) forms part of the EvoEnzyme’s EVO UPOkit. As used in this disclosure, “fungi” refers to organisms of the Fungi kingdom which includes yeasts, rusts, smuts, mildews, molds, and mushrooms. In some embodiments of the method, the host cell is yeast. As used in this disclosure, “proto-fungi” and “pseudo-fungi” are used interchangeably and refer to organisms from phylum Oomycota and phylum Hyphochytriomycota. As used in this disclosure, “recombinant UPO” or “recombinant unspecific peroxygenase” which are used interchangeably, refer to an UPO enzyme which is expressed or produced in a non-native organism (also referred as host cell) using recombinant DNA techniques. For instance, the UPO gene or polynucleotide sourced from a native organism (e.g., fungi or proto-fungi) is subjected to heterologous expression in the host cells to produce recombinant UPO enzyme(s). In some embodiments, said UPO gene or polynucleotide is not modified or mutated before expression in host cells. In some embodiments, said UPO gene or polynucleotide is modified or mutated before expression in host cells. In another instance, the UPO gene or polynucleotide is a synthetic or artificially synthesized UPO which is subjected to heterologous expression in the host cells to produce recombinant UPO enzyme(s). As used in this disclosure, “host cell” refers to a cell or organism that can be used to express heterologous polynucleotide or gene, such as the peroxygenase described herein. In some embodiments of the method, the UPO is a recombinant UPO expressed in a host cell which is yeast. In some embodiments, the yeast host cell is an industrial strain. Numerous industrial strains of yeast are known and are suitable for expressing or producing the recombinant UPO described herein. In some embodiments of the method, the UPO is a recombinant UPO expressed in a host cell which is a yeast selected from the group consisting of Pichia sp., Saccharomyces sp., Leptoxyphium sp., Agrocybe sp., Candida sp., Hansenula sp., Schizosaccharomyces sp., Kluyveromyces sp., and Yarrowia sp.. In some embodiments of the method, the UPO is a recombinant UPO expressed in a host cell which is a yeast selected from the group consisting of Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Leptoxyphium fumago, Agrocybe aegerita, Schizosaccharomyces pombe, Hansenula polymorpha, Kluyveromyces lactis, Candida albicans, and Yarrowia lipolytica. In some embodiments of the method, the UPO is a recombinant UPO expressed in Pichia pastoris. In some embodiments of the method, the UPO is a recombinant UPO expressed in Pichia pastoris, which is Komagataella pastoris or Komagataella phaffii. In some embodiments of the method, the UPO is a recombinant UPO expressed in all industrial strains of Pichia pastoris, which is Komagataella pastoris or Komagataella phaffii. In some embodiments of the method, the UPO is a recombinant UPO expressed in Saccharomyces cerevisiae. In some embodiments of the method, the UPO is a recombinant UPO expressed in all industrial strains of Saccharomyces cerevisiae. In some embodiments of the method, the UPO is a recombinant UPO expressed in a fungal host cell. In some embodiments, the fungal host cell is an industrial strain. Numerous fungal industrial strains are known and are suitable for expressing or producing the recombinant UPO described herein. In some embodiments of the method, the UPO is a recombinant UPO expressed in a host cell which is a fungi selected from the group consisting of Aspergillus sp., Penicillium sp., Fusarium sp., Myceliophthora sp., Rhizopus sp., Acremonium sp., Neurospora sp., Sordaria sp., Magnaporthe sp., Allomyces sp., Ustilago sp., Botrytis sp., and Trichoderma sp.. In some embodiments of the method, the UPO is a recombinant UPO expressed in a host cell which is a fungi selected from the group consisting of Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Fusarium venenatum, Fusarium graminearum and Myceliophthora thermophila. In some embodiments of the method, the UPO is a recombinant UPO expressed in bacterial host cell. In some embodiments, the bacterial host cell is an industrial strain. Numerous bacterial industrial strains are known and are suitable for expressing or producing the recombinant UPO described herein. In some embodiments of the method, the UPO is a recombinant UPO expressed in bacteria selected from the group consisting of Escherichia sp., Streptomyces sp., Lactobacillus sp., and Bacillus sp. In some embodiments of the method, the UPO is a recombinant UPO expressed in bacteria which is Escherichia coli. In some embodiments of the method, the UPO is a recombinant UPO expressed in all industrial strains of Escherichia coli. In some embodiments of the method, the UPO is a recombinant UPO expressed in bacteria which is Bacillus subtilis. In some embodiments of the method, the UPO is a recombinant UPO expressed in all industrial strains of Bacillus subtilis. In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is any wild-type enzyme that can use peroxide as a source of oxygen and electrons and catalyze 7β- hydroxylation of the compound of Formula I or a salt thereof. In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is any variant or mutant of the UPO enzyme that can use peroxide as a source of oxygen and electrons and catalyze 7β-hydroxylation of the compound of Formula I or a salt thereof enzyme. In some embodiments of the method, the enzyme capable of using peroxide as a co-substrate is any variant or mutant enzyme that can use peroxide as a source of oxygen and electrons and catalyze 7β-hydroxylation of the compound of Formula I or a salt thereof. Mutations can be made in a nucleotide or amino acid sequence of enzyme by a variety of methods known to one of ordinary skill in the art. For example, mutations can be made by PCR-directed mutation, site-directed mutagenesis, by chemical synthesis of a gene encoding a polypeptide, by gene editing tools, by any-gene-any-plasmid (AGAP) cloning, by codon optimization, or by insertions, such as insertion of a tag (e.g., a HIS tag or a GFP tag). Mutations can include, for example, substitutions, deletions, and translocations, generated by any method known in the art. Methods for producing mutations may be found in in references such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., 4thEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York, 2010. As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code. As will be understood by one of ordinary skill in the art, a mutation within a codon may or may not change the amino acid that is encoded by the codon due to degeneracy of the genetic code. In some embodiments, the one or more mutations in the coding nucleotide sequence do not alter the amino acid sequence and / or activity of the enzyme mutant relative to the reference enzyme before mutation. In some embodiments, the one or more mutations alter the amino acid sequence and / or activity of the enzyme mutant relative to the reference enzyme before mutation. In some embodiments of the method, the enzyme described herein is provided in the form of an enzyme powder or enzyme solution. In some embodiments of the method, the peroxygenase enzyme described herein is provided in the form of an enzyme powder or enzyme solution. In some embodiments of the method, the UPO enzyme described herein is provided in the form of an enzyme powder or enzyme solution. In some embodiments of the method, the enzyme described herein is provided in the form of a lyophilized enzyme powder. In some embodiments of the method, the enzyme described herein is provided in the form of an immobilized enzyme. In some embodiments of the method, the peroxygenase enzyme described herein is provided in the form of an immobilized enzyme. In some embodiments of the method, the UPO enzyme described herein is provided in the form of an immobilized enzyme. Immobilization techniques which result in imprisonment of whole cells or enzymes in a support or matrix are known to an ordinary skilled in the art and any such immobilization techniques can be employed herein. For instance, immobilization techniques can include one or more of adsorption, covalent, anionic, or cationic binding, entrapment, copolymerization, encapsulation, active inclusion bodies and cross-linked enzyme aggregates (CLEA). For the sake of brevity, each of these immobilization techniques are not being detailed herein. In some embodiments, the enzyme can be put on a matrix, synthetic or natural, via absorption, covalent, anionic, or cationic binding, or immobilized via encapsulation, active inclusion bodies or CLEA, or any other immobilization method known to an ordinary skilled in the art. In some embodiments of the method, the enzyme described herein is provided in the form of whole cells expressing the enzyme. In some embodiments, the whole cells are organisms (e.g., fungi or proto-fungi) expressing wild-type or native peroxygenase. In some embodiments, the whole cells are organisms (e.g., fungi or proto-fungi) expressing wild-type or native UPO. In some embodiments, the whole cells are host cells (e.g., fungi, yeast, or bacteria) expressing recombinant peroxygenase. In some embodiments, the whole cells are host cells (e.g., fungi, yeast, or bacteria) expressing recombinant UPO. In some embodiments of the method, the enzyme described herein is provided in the form of an extract or a lysate of the whole cells expressing the enzyme. Preparation of an extract or lysate from whole cells obtained by culturing or fermentation process is known to an ordinary skilled in the art and any such preparation methods can be employed herein to obtain a cell extract or cell lysate comprising the enzyme described herein. For the sake of brevity, such methods for preparing cell extract or lysate are not being detailed herein. In some embodiments, the extract or lysate is prepared from whole cells which are organisms (e.g., fungi or proto-fungi) expressing wild-type or native peroxygenase. In some embodiments, the extract or lysate is prepared from whole cells which are organisms (e.g., fungi or proto-fungi) expressing wild-type or native UPO. In some embodiments, the extract or lysate is prepared from whole cells which are host cells (e.g., fungi, yeast, or bacteria) expressing recombinant peroxygenase. In some embodiments, the extract or lysate is prepared from whole cells which are host cells (e.g., fungi, yeast, or bacteria) expressing recombinant UPO. In some embodiments of the method, the enzyme described herein is provided in the form of an enzyme extract which is prepared by processing the excrete of cell culture to obtain a supernatant and a pellet, wherein the supernatant is the enzyme extract. In some embodiments of the method, the enzyme described herein is provided in the form of an enzyme extract which is prepared by subjecting the excrete of cell culture to solid liquid separation to obtain a supernatant and a pellet, wherein the supernatant is the enzyme extract. In some embodiments, the solid liquid separation is performed by methods selected from the group consisting of centrifugation, filtration, sedimentation and decantation, distillation, other methods known to an ordinary person skilled in the art, and combinations thereof. In some embodiments, said supernatant is directly used as biocatalyst. In some embodiments, said supernatant is directly used as immobilized enzyme. In some embodiments, said supernatant is further purified via techniques such as chromatography (for instance size exclusion chromatography or ion exchange chromatography), or precipitation, or adsorption of other compounds in the mixture. In some embodiments of the method, the enzyme is provided in the form of any combination of i) an enzyme powder or solution, ii) immobilized enzyme, iii) whole cells expressing the enzyme, and iv) an extract or lysate of the whole cells expressing the enzyme. In some embodiments of the method, the peroxide is an inorganic peroxide, an organic peroxide, or both. In some embodiments of the method, the peroxide is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H2O2), persulfate (SO^^ ^^ ^ or S^O^), potassium peroxymonosulfate (KHSO5), oxone (2KHSO5·KHSO4·K2SO4), peroxydisulfuric acid (H2S2O8), sodium peroxide (Na2O2), potassium peroxide (K2O2), barium peroxide (BaO2), calcium peroxide (CaO2), magnesium peroxide (MgO2), lithium peroxide (Li2O2), and combinations thereof. In some embodiments of the method, the peroxide is hydrogen peroxide (H2O2). In some embodiments of the method, the peroxide is an organic peroxide selected from the group consisting of peracetic acid (CH3CO3H), tert-butyl hydroperoxide [(CH3)3COOH], magnesium monoperoxyphthalate (C16H10MgO10), 3-chloroperoxybenzoic acid (3-ClC6H4CO3H), urea hydrogen peroxide (NH2CONH2∙H2O2), benzoyl peroxide (C14H10O4), tert-butyl peroxybenzoate (C6H5CO3C(CH3)3), benzoyl peroxide (C14H10O4), di-tert-butyl peroxide [(CH3)3COOC(CH3)3], methyl ethyl ketone peroxide (C8H18O6), diacetyl peroxide ((CH3CO2)2), cumene hydroperoxide (C9H12O2), trifluoroperacetic acid (CF3CO3H), and combinations thereof. In some embodiments of the method, all the peroxide is provided or dosed at the start of the reaction. In some embodiments of the method, the peroxide is provided or dosed at various time points including at the start of the reaction followed by dosing the peroxide at different time-points of the reaction. In some embodiments of the method, all the peroxide is provided or dosed during the progress of the reaction. In some embodiments of the method, the peroxide is provided as a solution. In some embodiments of the method, the peroxide is provided in a pure peroxide form. In some embodiments of the method, the peroxide is generated by in situ peroxide generation system. In situ peroxide generation using techniques such as biocatalytic, photochemical, or electrochemical methods or any other method are known to an ordinary skilled in the art. For the sake of brevity, such methods of in situ peroxide generation are not being detailed herein. In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(a) to produce a compound of Formula II(a)

[0004] Formula II(a) wherein, R" is OH, NH(CH2)2SO3H, NHCH2CO2H, O-alkyl, or O-aryl; comprising contacting the compound of Formula I(a) with an enzyme in the presence of a peroxide, to produce said compound of Formula II(a). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(a) to produce a compound of Formula II(a) Formula II(a) wherein, R" is OH, NH(CH2)2SO3H, NHCH2CO2H, O-alkyl, or O-aryl; comprising contacting the compound of Formula I(a) with unspecific peroxygenase (UPO) in the presence of a peroxide, to produce said compound of Formula II(a). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(a) to produce a compound of Formula II(a) Formula II(a) wherein, R" is OH, NH(CH2)2SO3H, NHCH2CO2H, O-alkyl, or O-aryl; comprising contacting the compound of Formula I(a) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2), to produce said compound of Formula II(a). In some embodiments, when R" is OH in Formula I(a), the compound is LCA and when R" is OH in Formula II(a), the compound is UDCA. Accordingly, in some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA

[0005] comprising contacting the LCA with an enzyme in the presence of a peroxide, to produce said UDCA. In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA

[0006] comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said UDCA. In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA

[0007] comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said UDCA. In some embodiments, when R" is NH(CH2)2SO3H in Formula I(a), the compound is taurolithocholic acid (TLCA) and when R" is NH(CH2)2SO3H in Formula II(a), the compound is tauroursodeoxycholic acid (TUDCA). Accordingly, in some embodiments, the present disclosure provides a method for hydroxylation of TLCA to produce TUDCA

[0008] comprising contacting the TLCA with an enzyme in the presence of a peroxide to produce said TUDCA. In some embodiments, when R" is NH(CH2)2SO3H in Formula I(a), the compound is taurolithocholic acid (TLCA) and when R" is NH(CH2)2SO3H in Formula II(a), the compound is tauroursodeoxycholic acid (TUDCA). Accordingly, in some embodiments, the present disclosure provides a method for hydroxylation of TLCA to produce TUDCA comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said TUDCA. In some embodiments, when R" is NH(CH2)2SO3H in Formula I(a), the compound is taurolithocholic acid (TLCA) and when R" is NH(CH2)2SO3H in Formula II(a), the compound is tauroursodeoxycholic acid (TUDCA). Accordingly, in some embodiments, the present disclosure provides a method for hydroxylation of TLCA to produce TUDCA comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said TUDCA. In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(b) to produce a compound of Formula II(b)

[0009] Formula II(b) wherein, R is as defined according to Formula I or Formula II above; comprising contacting the compound of Formula I(b) with an enzyme in the presence of a peroxide, to produce said compound of Formula II(b). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(b) to produce a compound of Formula II(b) comprising contacting the compound of Formula I(b) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(b). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(b) to produce a compound of Formula II(b) comprising contacting the compound of Formula I(b) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(b). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(c) to produce a compound of Formula II(c)

[0010] wherein, R is as defined according to Formula I or Formula II above; comprising contacting the compound of Formula I(c) with an enzyme in the presence of a peroxide to produce said compound of Formula II(c). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(c) to produce a compound of Formula II(c) comprising contacting the compound of Formula I(c) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(c). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(c) to produce a compound of Formula II(c) comprising contacting the compound of Formula I(c) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(c). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(d) to produce a compound of Formula II(d)

[0011] wherein, R and X are as defined according to Formula I or Formula II above; comprising contacting the compound of Formula I(d) with an enzyme in the presence of a peroxide to produce said compound of Formula II(d). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(d) to produce a compound of Formula II(d) comprising contacting the compound of Formula I(d) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(d). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(d) to produce a compound of Formula II(d) comprising contacting the compound of Formula I(d) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(d). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(e) to produce a compound of Formula II(e)

[0012] Formula II(e) wherein, R, R1and X are as defined according to Formula I or Formula II above; comprising contacting the compound of Formula I(e) with an enzyme in the presence of a peroxide to produce said compound of Formula II(e). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(e) to produce a compound of Formula II(e) comprising contacting the compound of Formula I(e) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(e). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(e) to produce a compound of Formula II(e) comprising contacting the compound of Formula I(e) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(e). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(f) to produce a compound of Formula II(f)

[0013] Formula II(f) comprising contacting the compound of Formula I(f) with an enzyme in the presence of a peroxide to produce said compound of Formula II(f). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(f) to produce a compound of Formula II(f) comprising contacting the compound of Formula I(f) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(f). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(f) to produce a compound of Formula II(f) comprising contacting the compound of Formula I(f) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(f). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(g) to produce a compound of Formula II(g)

[0014] comprising contacting the compound of Formula I(g) with an enzyme in the presence of a peroxide to produce said compound of Formula II(g). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(g) to produce a compound of Formula II(g) comprising contacting the compound of Formula I(g) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(g). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(g) to produce a compound of Formula II(g) comprising contacting the compound of Formula I(g) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(g). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h) Formula I(h)

[0015] Formula II(h) comprising contacting the compound of Formula I(h) with an enzyme in the presence of a peroxide to produce said compound of Formula II(h). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h) comprising contacting the compound of Formula I(h) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(h). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h) comprising contacting the compound of Formula I(h) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(h). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(i) to produce a compound of Formula II(i)

[0016] Formula II(i) comprising contacting the compound of Formula I(i) with an enzyme in the presence of a peroxide to produce said compound of Formula II(i). In some embodiments, the present disclosure provides a method for hydroxylation a compound of Formula I(i) to produce a compound of Formula II(i) comprising contacting the compound of Formula I(i) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(i). In some embodiments, the present disclosure provides a method for hydroxylation a compound of Formula I(i) to produce a compound of Formula II(i) comprising contacting the compound of Formula I(i) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(i). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(j) to produce a compound of Formula II(j)

[0017] wherein, R3is hydrogen, alkyl, or aryl; comprising contacting the compound of Formula I(j) with an enzyme in the presence of a peroxide to produce said compound of Formula II(j). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(j) to produce a compound of Formula II(j) comprising contacting the compound of Formula I(j) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(j). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(j) to produce a compound of Formula II(j) comprising contacting the compound of Formula I(j) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(j). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(k) to produce a compound of Formula II(k)

[0018] comprising contacting the compound of Formula I(k) with an enzyme in the presence of a peroxide to produce said compound of Formula II(k). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(k) to produce a compound of Formula II(k) comprising contacting the compound of Formula I(k) with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said compound of Formula II(k). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I(k) to produce a compound of Formula II(k) comprising contacting the compound of Formula I(k) with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said compound of Formula II(k). In some embodiments, the present disclosure provides a method for hydroxylation of a compound of Formula I to produce a compound of Formula II comprising contacting the compound of Formula I with an enzyme in the presence of a peroxide to produce said compound of Formula II, wherein the compound of formula I is selected from the group consisting of 12-Ketolithocholic acid (CAS reference 5130-29-0), (5β)-3,12-Dioxocholan-24-oic acid (CAS reference 2958-05-6), Dehydrolithocholic acid (CAS reference 1553-56-6), lithocholic acid (LCA, CAS reference 434- 13-9) and taurolithocholic acid (TLCA; CAS reference 516-90-5). In some embodiments, the method is carried out at a temperature of about 5°C to about 80°C. In some embodiments, the method is carried out at a temperature of about 10°C to about 60°C. In some embodiments, the method is carried out at a temperature of about 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or 80°C, including any values therebetween. In some embodiments, the method is carried out for a time period ranging from about 15 minutes to about 150 hours. In some embodiments, the method is carried out for a time period ranging from about 30 minutes to about 48 hours. In some embodiments, the method is carried out for a time period ranging from about 15 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hour, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 75 hours, 80 hours, 85 hours, 90 hours, 95 hours, 100 hours, 105 hours, 110 hours, 115 hours, 120 hours, 125 hours, 130 hours, 135 hours, 140 hours, 145 hours, or 150 hours, including any values or ranges therebetween. In some embodiments, the method is carried out at a pH of about 2 to about 10. In some embodiments, the method is carried out at a pH of about 4 to about 9. In some embodiments, the method is carried out at a pH of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10, including any values therebetween. In some embodiments of the method, the molar ratio of the compound of Formula I or analogue or a salt thereof and the enzyme is from about 100:1 to about 10,000,000:1. In some embodiments of the method, the molar ratio of the compound of Formula I or analogue or a salt thereof and the enzyme is about 1000:1 to about 10,000,000:1. In some embodiments of the method, the molar ratio of the compound of Formula I or analogue or a salt thereof and the peroxide is from about 1:0.1 to about 1:10. In some embodiments of the method, the molar ratio of the compound of Formula I or analogue or a salt thereof and the peroxide is from about 1:0.8 to about 1:2. In some embodiments of the method, the molar ratio of the enzyme and the peroxide is from about 1:100 to about 1:100,000,000. In some embodiments of the method, the molar ratio of the enzyme and the peroxide is from about 1:1000 to about 1: 2,000,000. In some embodiments, the method is carried out in the presence of one or more of: i) a solvent, ii) a surfactant, iii) solubilizing agent, iv) an antioxidant, v) a buffer, and vi) a monovalent salt. In some embodiments of the method, the solvent is a polar solvent, or a non-polar solvent, or a mixture thereof. In some embodiments, the solvent is selected from the group consisting of water, dimethyl sulfoxide, methanol, ethanol, acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethylformamide, propylene carbonate, isopropanol, butanol, octanol, dimethylacetamide, hexane, chloroform, diethyl ether, cyclopentyl methyl ether, ethyl laurate, phthalates, toluene, petroleum ether, carbon tetrachloride, benzene, cyclohexane, xylene, heptane, pentane, decane, dodecane, isopropyl ether, dichloromethane, methyl tert-butyl ether, oleates, and combinations thereof. In some embodiments of the method, the surfactant is selected from the group consisting of cationic surfactant, anionic surfactant, non-ionic surfactant, and combinations thereof. In some embodiments, the cationic surfactant is selected from the group consisting of cetyltrimethylammonium bromide, benzalkonium chloride, cetrimonium chloride, lauryl trimethyl ammonium chloride, dodecylbenzenesulfonic acid, octadecyltrimethylammonium bromide, stearalkonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecylpyridinium chloride, dodecylpyridinium chloride, cetylpyridinium chloride, and combinations thereof. In some embodiments, the anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, sodium lauryl sulfate, sodium alkylbenzenesulfonate, sodium dioctyl sulfosuccinate, sodium oleate, sodium stearate, sodium palmitate, sodium cocoyl isethionate, sodium lauroyl sarcosinate, sodium dodecylbenzenesulfonate, sodium naphthalenesulfonate, sodium decyl sulfate, sodium lauroyl lactylate, and combinations thereof. In some embodiments, the non-ionic surfactant is selected from the group consisting of octylphenol ethoxylate (Triton X-100), octylphenol ethoxylate (Triton X-100), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), sorbitan monooleate (Span 80), polyethylene glycol dodecyl ether (Brij 35), polyethylene glycol cetyl ether (Brij 58), polyethylene glycol-block- polypropylene glycol-block-polyethylene glycol (Pluronic F68), sorbitan monooleate ethoxylated (Polysorbate 80), octylphenol ethoxylate (Nonidet P-40), octylphenol ethoxylate (octylphenol ethoxylate), poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Poloxamer 188), poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Poloxamer 407), nonylphenol ethoxylate (Tergitol NP-10), nonylphenol ethoxylate (Tergitol NP- 40), octylphenol ethoxylate (Igepal CO-630), and combinations thereof. In some embodiments of the method, the solubilizing agent is selected from the group consisting of cyclodextrins, cucurbit[n]urils, pillararenes, lecithin, and combinations thereof. In some embodiments of the method, the cyclodextrins is selected from the group consisting of α (alpha)-cyclodextrin, β (beta)-cyclodextrin, γ (gamma)-cyclodextrin, modified cyclodextrin, and combinations thereof. In some embodiments, the modified cyclodextrins are native cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin) which serve as scaffolds on which functional groups and / or other substituents can be assembled. In some embodiments, the modified cyclodextrin is methyl-β- cyclodextrin. In some embodiments of the method, the antioxidant is selected from the group consisting of ascorbic acid, tocopherol, polyphenol, flavonoids, and combinations thereof. In some embodiments of the method, the buffer is selected from the group consisting of potassium phosphate buffer, sodium phosphate buffer, citric acid buffer, acetic acid buffer, 2-(N- morpholino)ethanesulfonic acid [MES] buffer, BisTris buffer, piperazine-N,N′-bis(2- ethanesulfonic acid) [PIPES] buffer, N,N-Bis(hydroxyethyl)-2aminoethanesulfonic acid [BES] buffer, (3-(N-morpholino)propanesulfonic acid) [MOPS] buffer, (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) [HEPES] buffer, tris(hydroxymethyl)aminomethane [Tris] buffer, Tris-acetate-EDTA [TAE] buffer, carbonate buffer, and combinations thereof. In some embodiments of the method, the monovalent salt is selected from the group consisting of sodium salt, potassium salt, lithium salt, quaternary ammonium salt (QAS), and combinations thereof. In some embodiments, the method comprises agitation of the reaction mixture comprising the compound of Formula I or analogue or a salt thereof, the enzyme and the peroxide. In some embodiments, the agitation is performed by techniques selected from the group consisting of stirring, shaking, bubbling of gas, overhead shaking, and combinations thereof. In some embodiments, the method comprises agitation of the reaction mixture to increase mass transfer rates. In some embodiments, agitation is performed for enhancing or lowering the reaction rates as desirable and understood by an ordinary skilled in the art. In some embodiments, agitation is performed until mass transfer rates are achieved as desirable and understood by an ordinary skilled in the art. In some embodiments, the method comprises agitation of a reaction mixture comprising the LCA or TLCA, the UPO and the H2O2 to achieve desirable reaction rate or mass transfer rate. In some embodiments of the method, the compound of Formula II or analogue or salt thereof is recovered and / or purified from an extract or lysate using techniques selected from the group consisting of single or multiple solvent extractions, precipitation, chromatography, any other downstream method known to an ordinary skilled in the art, and combinations thereof. In some embodiments of the method, the conversion of Formula I, or analogues or salts thereof to Formula II, or analogues or salts thereof ranges from about 1% to 100%. In some embodiments of the method, the conversion of Formula I, or analogues or salts thereof to Formula II, or analogues or salts thereof ranges from about 80% to 100%. In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA

[0019] comprising contacting the LCA with an enzyme in the presence of a peroxide to produce said UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the enzyme is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the peroxide is from about 1:0.1 to about 1:10, and the molar ratio of the enzyme and the peroxide is from about 1:100 to about 1:100,000,000. In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA as represented by Figure 1. In some embodiments, the present disclosure provides a method for hydroxylating LCA to produce

[0020] comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce said UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the peroxide is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the peroxide is from about 1:100 to about 1:100,000,000. In some embodiments, the present disclosure provides a method for hydroxylation of LCA to produce UDCA

[0021] comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the H2O2 is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H2O2is from about 1:100 to about 1:100,000,000. In some embodiments of the method, UDCA is recovered and / or purified from an extract or lysate using techniques selected from the group consisting of single or multiple solvent extractions, precipitation, chromatography, any other downstream method known to an ordinary skilled in the art, and combinations thereof. In some embodiments of the method, the conversion of LCA to UDCA ranges from about 1% to 100%. In some embodiments of the method, the conversion of LCA to UDCA ranges from about 80% to 100%. In some embodiments, the present disclosure provides a method for hydroxylation of TLCA to produce TUDCA

[0022] comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said TUDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the TLCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the TLCA and the H2O2 is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H2O2 is from about 1:100 to about 1:100,000,000. In some embodiments of the method, TUDCA is recovered and / or purified from an extract or lysate using techniques selected from the group consisting of single or multiple solvent extractions, precipitation, chromatography, any other downstream method known to an ordinary skilled in the art, and combinations thereof. In some embodiments of the method, the conversion of TLCA to TUDCA ranges from about 1% to 100%. In some embodiments of the method, the conversion of TLCA to TUDCA ranges from about 80% to 100%. Biocatalysts / enzymes such as cytochrome P450 monooxygenases (P450) which can catalyse 7β- hydroxylation of substrates such as bile acid or their analogues (e.g., LCA) often rely on complex regeneration systems to deliver the required redox equivalents to the active site to form the active ‘Compound I’, activating molecular oxygen and performing the reaction. Required redox equivalents come in the form of NAD(P)H and are often transferred using other proteins like cytochrome P450 reductase (CPR). Furthermore, especially in fungi, these reaction systems are membrane bound. This restricts the use of such biocatalysts to whole cells, introducing transfer limitations over the cell wall for both oxygen and substrate, potential side reactions and extra need of substrates. Additional issues with enzymes such as P450 are uncoupling of activated oxygen, the slow dissolution of molecular oxygen in the aqueous phase and the relative instability of these proteins. In general, employing enzymes such as P450 in the reaction is considered complex and there is no or very limited industrial application yet of such enzyme classes towards production of bile acids or their analogues (e.g., UDCA) due to the limitations discussed above. Therefore, using enzymes such as P450 is expected to be resource and time intensive and increase the overall cost of the process. On the other hand, without wishing to be bound by any theory, the present inventors surprisingly found that employing enzyme(s) capable of using peroxide as the source of both oxygen and electrons such as peroxygenases (e.g., UPO) would circumvent the above discussed issues, thereby allowing for a simple cost-effective process for 7β-hydroxylation of substrates such as bile acid or their derivatives. For instance, UPO are heme containing enzymes able to perform various oxyfunctionalization reactions. The actual reaction takes place via an oxoferryl-heme species at the active site called ‘Compound I’. However, unlike enzymes such as P450, for UPO, the oxygen and electrons required to form the active compound I do not come from molecular oxygen and expensive cofactors like NAD(P)H. Nor are there complicated regeneration systems required for said biocatalyst. Instead, UPO can directly accept H2O2 or other inorganic / organic peroxides as the source of oxygen and electrons to form the active intermediate. Additionally, UPO also conveniently function in solution or as immobilized biocatalyst. Altogether, compared to P450 based systems, employing enzymes capable of using peroxide, such as unspecific peroxygenases (e.g., UPO) result in much less complicated reaction schemes, more stable proteins and most importantly, the additional flexibility to apply these enzymes conveniently outside a living organism. The ability to apply these enzymes independently of their host cells or native organisms, and their general higher stability as compared to enzymes such as P450 along with being economical are great advantages in large scale applications in production of bile acids or their analogues. The present disclosure further relates to a method for converting the compound of Formula I or analogue or a salt thereof to the compound of Formula II or analogue or a salt thereof, comprising contacting the compound of Formula I or analogue or a salt thereof: with an enzyme in the presence of a peroxide, to produce a compound of Formula II or analogue or salt thereof:

[0023] wherein, R is independently selected from the group consisting of (CH2)nC=O(O)mR', alkyl, alkenyl, CH2OH, CHO, (CH2)2CONH(CH2)2SO3H, (CH2)2CONH(CH2)COOH, -CH=CH-CO2-alkyl, - CH=CH-CO2-aryl, -CH=CH-CO2H and -CH=CH-alkyl, wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRaRb, wherein Ra and Rb are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=O)-alkyl, -C(=O)-aryl and (CH2)2SO3H; R1is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=O)-alkyl, -C(=O)-aryl, -S(=O)2-alkyl, -S(=O)2-aryl, -S(=O)2-OH and -P(=O)-(OR2)2, wherein R2is independently hydrogen or alkyl; n is 0 or an integer ranging from 1 to 5; m is 0 or 1; Y is hydrogen; X is hydrogen, hydroxy or oxygen; wherein R, R’, Ra, Rb, R1, R2or X are optionally independently substituted; represents either a single bond or a double bond; and with the proviso that: both Rings A and B do not simultaneously contain double bond inside the rings; when is a double bond on the Ring A, R1 is absent; when is a single bond on the Ring A, then R1 is present; when is a double bond inside either the Ring A or Ring B, Y is absent; and when is a single bond inside both the Rings A and B simultaneously, Y is present. The present disclosure additionally relates to a method for converting the compound of Formula I(a) or analogue or a salt thereof to the compound of Formula II(a) or analogue or a salt thereof, comprising contacting the compound of Formula I(a) or analogue or a salt thereof: Formula I(a) with an enzyme in the presence of a peroxide, to produce the compound of Formula II(a) or analogue or a salt thereof:

[0024] Formula II(a) wherein, R" is OH, NH(CH2)2SO3H, NHCH2CO2H, O-alkyl, or O-aryl. In some embodiments, the enzyme is capable of using peroxide as a co-substrate and is an enzyme from class EC 1.11 which acts on peroxide as acceptor. In some embodiments, the enzyme is peroxygenase. In some embodiments, the enzyme is a peroxygenase from class EC 1.11.2. In some embodiments, the peroxygenase is a fungal peroxygenase or a peroxygenase obtained from proto-fungi. In some embodiments, the enzyme is unspecific peroxygenase (UPO). In some embodiments, the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1 belonging to the sub-subclass of oxidoreductases. More particularly, the enzyme in the present method is an enzyme from EC 1.11.2.1: Unspecific peroxygenase. In some embodiments, the UPO is a wild-type UPO obtained from fungi, or proto-fungi, or a combination thereof. In some embodiments, the UPO is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. In some embodiments, the fungi is yeast. In some embodiments, the enzyme is in the form of: i) an enzyme powder or solution, ii) immobilized enzyme, iii) whole cells expressing the enzyme, iv) an extract or lysate of the whole cells expressing the enzyme, or v) any combination of i to iv. In some embodiments, the peroxide is an organic peroxide, an inorganic peroxide, or both. In some embodiments of the method, the peroxide is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H2O2), persulfate (SO^^ ^^ ^ or S^O^), potassium peroxymonosulfate (KHSO5), oxone (2KHSO5·KHSO4·K2SO4), peroxydisulfuric acid (H2S2O8), sodium peroxide (Na2O2), potassium peroxide (K2O2), barium peroxide (BaO2), calcium peroxide (CaO2), magnesium peroxide (MgO2), lithium peroxide (Li2O2), and combinations thereof. In some embodiments of the method, the peroxide is hydrogen peroxide (H2O2). In some embodiments of the method, the peroxide is an organic peroxide selected from the group consisting of peracetic acid (CH3CO3H), tert-butyl hydroperoxide [(CH3)3COOH], magnesium monoperoxyphthalate (C16H10MgO10), 3-chloroperoxybenzoic acid (3-ClC6H4CO3H), urea hydrogen peroxide (NH2CONH2∙H2O2), benzoyl peroxide (C14H10O4), tert-butyl peroxybenzoate (C6H5CO3C(CH3)3), benzoyl peroxide (C14H10O4), di-tert-butyl peroxide [(CH3)3COOC(CH3)3], methyl ethyl ketone peroxide (C8H18O6), diacetyl peroxide ((CH3CO2)2), cumene hydroperoxide (C9H12O2), trifluoroperacetic acid (CF3CO3H), and combinations thereof. In some embodiments of the method, the conversion of Formula I, or analogues or salts thereof to Formula II, or analogues or salts thereof ranges from about 1% to 100%. In some embodiments of the method, the conversion of Formula I, or analogues or salts thereof to Formula II, or analogues or salts thereof ranges from about 80% to 100%. In some embodiments, the method comprises converting lithocholic acid (LCA): to ursodeoxycholic acid (UDCA):

[0025] In some embodiments, the method comprises converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA), comprising contacting the LCA with an enzyme in the presence of a peroxide to produce the UDCA. In some embodiments, the method comprises converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA), comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce the UDCA. In some embodiments, the method comprises converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA), comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2), to produce the UDCA. In some embodiments, the method comprises converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA), comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2), to produce the UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the H2O2is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H2O2 is from about 1:100 to about 1:100,000,000. In some embodiments of the method, the conversion of LCA to UDCA ranges from about 80% to 100%. In some embodiments, the method comprises converting taurolithocholic acid (TLCA):

[0026] . In some embodiments, the method comprises converting taurolithocholic acid (TLCA) to tauroursodeoxycholic acid (TUDCA), comprising contacting the TLCA with an enzyme in the presence of a peroxide to produce the TUDCA. In some embodiments, the method comprises converting TLCA to TUDCA, comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of a peroxide to produce the TUDCA. In some embodiments, the method comprises converting TLCA to TUDCA, comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce the TUDCA. In some embodiments, the method comprises converting TLCA to TUDCA, comprising contacting the TLCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce the TUDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the TLCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the TLCA and the H2O2 is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H2O2is from about 1:100 to about 1:100,000,000. In some embodiments of the method, the conversion of TLCA to TUDCA ranges from about 80% to 100%. In all embodiments of the method for converting a compound of Formula I or analogue or salt thereof to a compound of Formula II or analogue or salt thereof, all the additional features are as described by any of the embodiments mentioned above under ‘Method for hydroxylation of Formula I or analogue or salt thereof’. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments, completely fall within the purview of the method for converting a compound of Formula I or a salt thereof to a compound of Formula II or a salt thereof. Reaction Mixture or Reaction System The present disclosure also provides a reaction mixture or reaction system comprising: i) a compound of Formula I or analogue or salt thereof, as described above; ii) an enzyme; and iii) a peroxide. In some embodiments, the reaction mixture or reaction system comprises the compound of Formula I(a) or analogue or a salt hereof which is LCA or TLCA. In some embodiments, the enzyme in the reaction mixture or reaction system is capable of using peroxide as a co-substrate and is an enzyme from class EC 1.11 which acts on peroxide as acceptor. In some embodiments, the reaction mixture or reaction system comprises the enzyme which is peroxygenase. In some embodiments, the reaction mixture or reaction system comprises the enzyme which is a peroxygenase from class EC 1.11.2. In some embodiments, the enzyme of the reaction mixture or reaction system is unspecific peroxygenase (UPO). In some embodiments, the enzyme of the reaction mixture or reaction system is unspecific peroxygenase (UPO) of EC 1.11.2.1 belonging to the sub-subclass of oxidoreductases. More particularly, the enzyme of the reaction mixture or reaction system is an enzyme from EC 1.11.2.1: Unspecific peroxygenase. In some embodiments, the UPO is a wild-type UPO obtained from fungi, or proto-fungi, or a combination thereof. In some embodiments, the UPO is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. In some embodiments, the fungi is yeast. In some embodiments, the enzyme of the reaction mixture or reaction system is in the form of: i) an enzyme powder or solution, ii) immobilized enzyme, iii) whole cells expressing the enzyme, iv) an extract or lysate of the whole cells expressing the enzyme, or v) any combination of i to iv. In some embodiments, the peroxide in the reaction mixture or reaction system is an organic peroxide, an inorganic peroxide, or both. In some embodiments, the peroxide in the reaction mixture or reaction system is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H2O2), persulfate (SO^^^or potassium peroxymonosulfate (KHSO5), oxone (2KHSO5·KHSO4·K2SO4), peroxydisulfuric acid (H2S2O8), sodium peroxide (Na2O2), potassium peroxide (K2O2), barium peroxide (BaO2), calcium peroxide (CaO2), magnesium peroxide (MgO2), lithium peroxide (Li2O2), and combinations thereof. In some embodiments of the method, the peroxide in the reaction mixture or reaction system is hydrogen peroxide (H2O2). In some embodiments, the peroxide in the reaction mixture or reaction system is an organic peroxide selected a group comprising peracetic acid (CH3CO3H), tert-butyl hydroperoxide [(CH3)3COOH], magnesium monoperoxyphthalate (C16H10MgO10), 3-chloroperoxybenzoic acid (3-ClC6H4CO3H), urea hydrogen peroxide (NH2CONH2∙H2O2), benzoyl peroxide (C14H10O4), tert- butyl peroxybenzoate (C6H5CO3C(CH3)3), benzoyl peroxide (C14H10O4), di-tert-butyl peroxide [(CH3)3COOC(CH3)3], methyl ethyl ketone peroxide (C8H18O6), diacetyl peroxide ((CH3CO2)2), cumene hydroperoxide (C9H12O2), trifluoroperacetic acid (CF3CO3H), and combinations thereof. In some embodiments, the reaction mixture or reaction system comprises: i) LCA; ii) unspecific peroxygenase (UPO); and iii) hydrogen peroxide (H2O2). In some embodiments, the reaction mixture or reaction system comprises: i) TLCA; ii) unspecific peroxygenase (UPO); and iii) hydrogen peroxide (H2O2). In all embodiments of the reaction mixture or reaction system, all the additional features are as described by any of the embodiments mentioned above under ‘Method for hydroxylation of Formula I or analogue or salt thereof’. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments, completely fall within the purview of the reaction mixture or reaction system. Use The present disclosure also relates to use of an enzyme for hydroxylation of a compound of Formula I or analogue or salt thereof, wherein the Formula I is as defined in the preceding embodiments, and wherein the enzyme requires peroxide as a co-substrate. In some embodiments of the use, the hydroxylation is 7β-hydroxylation. The present disclosure additionally relates to use of an enzyme for converting a compound of Formula I or analogue or salt thereof to the compound of Formula II or analogue or salt thereof, wherein the Formula I and Formula II are as defined in the preceding embodiments, and wherein the enzyme requires peroxide as a co-substrate. In some embodiments of the use, the enzyme is capable of using peroxide as a co-substrate and is an enzyme from class EC 1.11 which acts on peroxide as acceptor. In some embodiments of the use, the enzyme is peroxygenase. In some embodiments of the use, the enzyme is a peroxygenase from class EC 1.11.2. In some embodiments of the use, the peroxygenase is a fungal peroxygenase or a peroxygenase obtained from proto-fungi. In some embodiments of the use, the enzyme is unspecific peroxygenase (UPO). In some embodiments of the use, the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1 belonging to the sub-subclass of oxidoreductases. More particularly, the enzyme is an enzyme from EC 1.11.2.1: Unspecific peroxygenase. In some embodiments of the use, the UPO is a wild-type UPO obtained from fungi, or proto-fungi, or a combination thereof. In some embodiments of the use, the UPO is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. In some embodiments, the fungi is yeast. In some embodiments of the use, the hydroxylation or the conversion is carried out in the presence of a peroxide which is an organic peroxide, an inorganic peroxide, or both. In some embodiments of the use, the hydroxylation or the conversion is carried out in the presence of hydrogen peroxide (H2O2). In some embodiments, the present disclosure provides use of unspecific peroxygenase (UPO) for converting lithocholic acid (LCA) to ursodeoxycholic acid (UDCA). In some embodiments, the present disclosure provides use of UPO for converting taurolithocholic acid (TLCA) to tauroursodeoxycholic acid (TUDCA). In all embodiments of the use, all the additional features are as described by any of the embodiments mentioned above under ‘Method for hydroxylation of Formula I or analogue or salt thereof’. For the sake of brevity, and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the said embodiments, completely fall within the purview of the use described herein. Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well- known / conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating one or more of the above-described embodiments. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein. Examples The following examples were performed using the Aminoverse BV’s Article NO. reference AVUP07700 UPO enzyme kit. In all of them, the presence of a compound of Formula II which has a β-hydroxyl group at position C7 (UDCA in Examples 1 and 2, and TUDCA in Examples 3 and 4) in the reaction medium was detected by HPLC means as shown in the figures. Example 1 7β-hydroxylation of LCA to produce UDCA Fungal unspecific peroxygenase (UPO) was employed for UDCA production by 7β-hydroxylation of LCA. About 2 gm L-1 of substrate (LCA, approx. 5.0 mM), about 0.1% (w / v) methyl-β- cyclodextrin, about 4% (v / v) DMSO, about 10 mM ascorbic acid, about 3.0 mM H2O2and about 1 to 2 mg of lyophilized enzyme powder (fungal UPO) were added in about 100 mM KPi buffer pH 6.0 at about 0.5 mL reaction volume. The reaction mixture was kept at about 30°C at about 1000 rpm for about 65 hours. HPLC results demonstrating the production of UDCA from LCA catalyzed by UPO is provided as Figure 2. Example 2 7β-hydroxylation of LCA to produce UDCA Fungal unspecific peroxygenase (UPO) was employed for UDCA production by 7β-hydroxylation of LCA. About 1 gm L-1 of substrate (LCA, approx. 2.5 mM), about 0.1% (w / v) methyl-β- cyclodextrin, about 4% (v / v) DMSO, about 10 mM ascorbic acid, about 0.3 mM H2O2and about 1 to 2 mg of lyophilized enzyme powder (fungal UPO) were added in about 100 mM KPi buffer pH 6.0 at about 0.5 mL reaction volume. The reaction mixture was kept at about 30°C at about 1000 rpm for about 65 hours. HPLC results demonstrating the production of UDCA from LCA catalyzed by UPO is provided as Figure 3. Example 3 7β-hydroxylation of TLCA to produce TUDCA Fungal unspecific peroxygenase (UPO) was employed for TUDCA production by 7β- hydroxylation of TLCA. About 2 gm L-1 of substrate (TLCA, approx. 4.0 mM), about 0.1% (w / v) methyl-β-cyclodextrin, about 4% (v / v) DMSO, about 10 mM ascorbic acid, about 3.0 mM H2O2and about 1 to 2 mg of lyophilized enzyme powder (fungal UPO) were added in about 100 mM KPi buffer pH 6.0 at about 0.5 mL reaction volume. The reaction mixture was kept at about 30°C at about 1000 rpm for about 65 hours. HPLC results demonstrating the production of TUDCA from TLCA catalyzed by UPO is provided as Figure 4. Example 4 7β-hydroxylation of TLCA to produce TUDCA Fungal unspecific peroxygenase (UPO) was employed for TUDCA production by 7β- hydroxylation of TLCA. About 1 gm L-1of substrate (TLCA, approx.2.5 mM), about 0.1% (w / v) methyl-β-cyclodextrin, about 4% (v / v) DMSO, about 10 mM ascorbic acid, about 0.3 mM H2O2 and about 1 to 2 mg of lyophilized enzyme powder (fungal UPO) were added in about 100 mM KPi buffer pH 6.0 at about 0.5 mL reaction volume. The reaction mixture was kept at about 30°C at about 1000 rpm for about 65 hours. HPLC results demonstrating the production of TUDCA from TLCA catalyzed by UPO is provided as Figure 5. In Figures 2 to 5, LCA, TLCA and TUDCA are detected using a photodiode array (PDA) at 215 nm for LCA or at 210 nm for TLCA and TUDCA. UDCA is detected using a refractive index detector. In Figures 2 to 5, X-axis corresponds to the retention time of any given molecule in the system and the unit is minutes. Y-axis is either milli-absorbance units (mAU) or micro-Refractive Index Units (uRIU) depending on the detection method. The present invention is further defined by the following numbered embodiments: 1) A method for hydroxylating a compound of Formula I or analogue or a salt thereof, comprising contacting the compound of Formula I or analogue or salt thereof: with an enzyme in the presence of a peroxide, to produce a compound of Formula II or analogue or salt thereof:

[0027] wherein, R is independently selected from the group consisting of (CH2)nC=O(O)mR', alkyl, alkenyl, CH2OH, CHO, (CH2)2CONH(CH2)2SO3H, (CH2)2CONH(CH2)COOH, -CH=CH-CO2- alkyl, -CH=CH-CO2-aryl, -CH=CH-CO2H and -CH=CH-alkyl, wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRaRb, wherein Ra and Rb are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=O)-alkyl, -C(=O)-aryl and (CH2)2SO3H; R1is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=O)-alkyl, -C(=O)-aryl, -S(=O)2-alkyl, -S(=O)2-aryl, -S(=O)2-OH and -P(=O)-(OR2)2, wherein R2 is independently hydrogen or alkyl; n is 0 or an integer ranging from 1 to 5; m is 0 or 1; Y is hydrogen; X is hydrogen, hydroxy or oxygen; wherein R, R’, Ra, Rb, R1, R2or X are optionally independently substituted; represents either a single bond or a double bond; and with the proviso that: both Rings A and B do not simultaneously contain double bond inside the rings; when is a double bond on the Ring A, R1is absent; when is a single bond on the Ring A, then R1 is present; when is a double bond inside either the Ring A or Ring B, Y is absent; and when is a single bond inside both the Rings A and B simultaneously, Y is present. ) The method of embodiment 1, wherein the method comprises 7β-hydroxylation of the compound of Formula I or analogue or a salt thereof to produce the compound of Formula II or analogue or a salt thereof which has a β-hydroxyl group at position 7. ) The method of embodiment 1 or embodiment 2, wherein the enzyme is capable of using the compound of Formula I or analogue or a salt thereof as a substrate and peroxide as a co- substrate for catalysing the hydroxylation of said compound of Formula I or analogue or a salt thereof to produce the compound of Formula II or analogue or a salt thereof. ) The method of any of the embodiments 1 to 3, wherein the enzyme capable of using peroxide as a co-substrate is an enzyme from Enzyme Commission (EC) class 1.11 which acts on peroxide as acceptor. ) The method of any of the embodiments 1 to 4, wherein the enzyme capable of using peroxide as a co-substrate is a peroxygenase. ) The method of any of the embodiments 1 to 5, wherein the peroxygenase is a fungal peroxygenase or a peroxygenase obtained from proto-fungi. ) The method of any of the embodiments 1 to 6, wherein the enzyme is unspecific peroxygenase (UPO); or the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1. ) The method of any of the embodiments 5 to 7, wherein the unspecific peroxygenase (UPO) is a wild-type UPO obtained from fungi, or proto-fungi, or both. ) The method of any of the embodiments 5 to 8, wherein the unspecific peroxygenase (UPO) is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. 0) The method of any of the embodiments 5 to 9, wherein the unspecific peroxygenase (UPO) is a recombinant unspecific peroxygenase (UPO) expressed in fungi or yeast selected from the group consisting of Pichia sp., Saccharomyces sp., Aspergillus sp., Fusarium sp., and Myceliophthora sp.; or wherein the unspecific peroxygenase (UPO) is a recombinant unspecific peroxygenase (UPO) expressed in bacteria which is Escherichia sp., Streptomyces sp., Lactobacillus sp., and Bacillus sp. 1) The method of any of the embodiments 1 to 10, wherein the enzyme is in the form of: i) an enzyme powder or solution, ii) immobilized enzyme, iii) whole cells expressing the enzyme, iv) an enzyme extract or lysate of the whole cells expressing the enzyme, or v) any combination of i to iv. 2) The method of any of the embodiments 1 to 11, wherein the peroxide is an inorganic peroxide, or an organic peroxide, or both. 3) The method of embodiment 12, wherein the peroxide is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H2O2), persulfate (SO^^^or S^O^^^), potassium peroxymonosulfate (KHSO5), oxone (2KHSO5·KHSO4·K2SO4), peroxydisulfuric acid (H2S2O8), sodium peroxide (Na2O2), potassium peroxide (K2O2), barium peroxide (BaO2), calcium peroxide (CaO2), magnesium peroxide (MgO2), lithium peroxide (Li2O2), and combinations thereof. 4) The method of embodiment 12, wherein the peroxide is an organic peroxide selected from the group consisting of peracetic acid (CH3CO3H), tert-butyl hydroperoxide [(CH3)3COOH], magnesium monoperoxyphthalate (C16H10MgO10), 3-chloroperoxybenzoic acid (3- ClC6H4CO3H), urea hydrogen peroxide (NH2CONH2∙H2O2), benzoyl peroxide (C14H10O4), tert-butyl peroxybenzoate (C6H5CO3C(CH3)3), benzoyl peroxide (C14H10O4), di-tert-butyl peroxide [(CH3)3COOC(CH3)3], methyl ethyl ketone peroxide (C8H18O6), diacetyl peroxide ((CH3CO2)2), cumene hydroperoxide (C9H12O2), trifluoroperacetic acid (CF3CO3H), and combinations thereof. ) The method of any of the embodiments 1 to 14, wherein the peroxide is provided as a pure peroxide, a peroxide solution or peroxide generated by in situ peroxide generation system. ) The method of any of the embodiments 1 to 15, wherein the alkyl is C1-C12 alkyl, the alkenyl is C2-C12 alkenyl, the alkynyl is C2-C12 alkynyl, the cycloalkyl is a hydrocarbon ring system radical comprising C3-C20carbon atoms, and the aryl is a hydrocarbon ring system radical comprising C6-C18 carbon atoms and at least one aromatic ring. ) The method of any of the embodiments 1 to 16, wherein the salt of Formula I or Formula II is selected from the group consisting of sodium salt, potassium salt, and quaternary ammonium salt (QAS). ) The method of any of the embodiments 1 to 17, wherein the method comprises: a) hydroxylation of a compound of Formula I(a) to produce a compound of Formula II(a)

[0028] R" is OH, NH(CH2)2SO3H, NHCH2CO2H, O-alkyl, or O-aryl; or b) hydroxylation of a compound of Formula I(b) to produce a compound of Formula II(b)

[0029] wherein, R is as defined in embodiment 1; or c) hydroxylation of a compound of Formula I(c) to produce a compound of Formula II(c) Formula I(c)

[0030] Formula II(c) wherein, R is as defined in embodiment 1; or d) hydroxylation of a compound of Formula I(d) to produce a compound of Formula II(d)

[0031] Formula II(d) wherein, R and X are as defined in embodiment 1; or e) hydroxylation of a compound of Formula I(e) to produce a compound of Formula II(e) Formula I(e)

[0032] Formula II(e) wherein, R, R1and X are as defined in embodiment 1; or f) hydroxylation of a compound of Formula I(f) to produce a compound of Formula II(f)

[0033] 5 or g) hydroxylation of a compound of Formula I(g) to produce a compound of Formula II(g)10

[0034] Formula II(g) ; or h) hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h)

[0035] Formula II(h) ; or i) hydroxylation of a compound of Formula I(i) to produce a compound of Formula II(i)

[0036] or 5 j) hydroxylation of a compound of Formula I(j) to produce a compound of Formula II(j) Formula I(j)

[0037] Formula II(j) wherein, R3is hydrogen, alkyl, or aryl; or, k) hydroxylation of a compound of Formula I(k) to produce a compound of Formula II(k)

[0038] 19) The method of any of the embodiments 1 to 18, wherein the method is carried out at a temperature of from about 5°C to about 80°C. 20) The method of any of the embodiments 1 to 19, wherein the method is carried out for a time period ranging from about 15 minutes to about 150 hours. 21) The method of any of the embodiments 1 to 20, wherein the method is carried out at a pH of about 2 to about 10. 22) The method of any of the embodiments 1 to 21, wherein the molar ratio of the compound of Formula I or a salt thereof and the enzyme is from about 100:1 to about 10,000,000:1; or wherein the molar ratio of the compound of Formula I or a salt thereof and the peroxide is from about 1:0.1 to about 1:10 or wherein the molar ratio of the enzyme and the peroxide is from about 1:100 to about 1:100,000,000. 23) The method of any of the embodiments 1 to 22, wherein the method is carried out in the presence of one or more of: i) a solvent selected from the group consisting of water, dimethyl sulfoxide, methanol, ethanol, acetone, acetonitrile, ethyl acetate, tetrahydrofuran, dimethylformamide, propylene carbonate, isopropanol, butanol, octanol, dimethylacetamide, hexane, chloroform, diethyl ether, cyclopentyl methyl ether, ethyl laurate, phthalates, toluene, petroleum ether, carbon tetrachloride, benzene, cyclohexane, xylene, heptane, pentane, decane, dodecane, isopropyl ether, dichloromethane, methyl tert-butyl ether, oleates, and combinations thereof; ii) a surfactant selected from the group consisting of cationic surfactant, anionic surfactant, non-ionic surfactant, and combinations thereof, wherein the cationic surfactant is selected from the group consisting of cetyltrimethylammonium bromide, benzalkonium chloride, cetrimonium chloride, lauryl trimethyl ammonium chloride, dodecylbenzenesulfonic acid, octadecyltrimethylammonium bromide, stearalkonium chloride, dodecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, hexadecylpyridinium chloride, dodecylpyridinium chloride, cetylpyridinium chloride, and combinations thereof, the anionic surfactant is selected from the group consisting of sodium dodecyl sulfate, sodium lauryl sulfate, sodium alkylbenzenesulfonate, sodium dioctyl sulfosuccinate, sodium oleate, sodium stearate, sodium palmitate, sodium cocoyl isethionate, sodium lauroyl sarcosinate, sodium dodecylbenzenesulfonate, sodium naphthalenesulfonate, sodium decyl sulfate, sodium lauroyl lactylate, and combinations thereof, and the non-ionic surfactant is selected from the group consisting of octylphenol ethoxylate (Triton X-100), octylphenol ethoxylate (Triton X-100), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), sorbitan monooleate (Span 80), polyethylene glycol dodecyl ether (Brij 35), polyethylene glycol cetyl ether (Brij 58), polyethylene glycol- block-polypropylene glycol-block-polyethylene glycol (Pluronic F68), sorbitan monooleate ethoxylated (Polysorbate 80), octylphenol ethoxylate (Nonidet P-40), octylphenol ethoxylate (octylphenol ethoxylate), poly(ethylene oxide)-block- poly(propylene oxide)-block-poly(ethylene oxide) (Poloxamer 188), poly(ethylene oxide)- block-poly(propylene oxide)-block-poly(ethylene oxide) (Poloxamer 407), nonylphenol ethoxylate (Tergitol NP-10), nonylphenol ethoxylate (Tergitol NP-40), octylphenol ethoxylate (Igepal CO-630), and combinations thereof; iii)solubilizing agent is selected from the group consisting of cyclodextrins, modified cyclodextrins, cucurbit[n]urils, pillararenes, lecithin, and combinations thereof; iv)an antioxidant selected from the group consisting of ascorbic acid, tocopherol, polyphenol, flavonoids, and combinations thereof; v) a buffer selected from the group consisting of potassium phosphate buffer, sodium phosphate buffer, citric acid buffer, acetic acid buffer, 2-(N-morpholino)ethanesulfonic acid [MES] buffer, BisTris buffer, piperazine-N,N′-bis(2-ethanesulfonic acid) [PIPES] buffer, N,N-Bis(hydroxyethyl)-2aminoethanesulfonic acid [BES] buffer, (3-(N- morpholino)propanesulfonic acid) [MOPS] buffer, (4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid) [HEPES] buffer, tris(hydroxymethyl)aminomethane [Tris] buffer, Tris-acetate-EDTA [TAE] buffer, carbonate buffer, and combinations thereof; and vi)monovalent salt selected from the group consisting of sodium salt, potassium salt, lithium salt, quaternary ammonium salts (QAS) and combinations thereof. 24) The method of any of the embodiments 1 to 23, wherein the method comprises agitation of the reaction mixture comprising the compound of Formula I or a salt thereof, the enzyme and the peroxide by techniques selected from the group consisting of stirring, shaking, bubbling of gas, overhead shaking, and combinations thereof. 25) A method for converting a compound of Formula I(a) or analogue or salt thereof to a compound of Formula II(a) or analogue or salt thereof, comprising contacting the compound of Formula I(a) or analogue or salt thereof:

[0039] with an enzyme in the presence of a peroxide, to produce the compound of Formula II(a) or analogue or salt thereof: wherein, R" is OH, NH(CH2)2SO3H, NHCH2CO2H, O-alkyl, or O-aryl. ) The method of embodiment 25, wherein the method comprises converting lithocholic acid (LCA): UDCA . ) The method of embodiment 25, wherein the method comprises converting taurolithocholic acid (TLCA):

[0040] . ) The method of any of the embodiments 25 to 27, wherein the enzyme is peroxygenase. ) The method of any of the embodiments 25 to 28, wherein the enzyme is unspecific peroxygenase (UPO). 30) The method of any of the embodiments 25 to 29, wherein the peroxide is an organic peroxide, an inorganic peroxide, or both. 31) The method of any of the embodiments 25 to 30, wherein the peroxide is an inorganic peroxide selected from the group consisting of hydrogen peroxide (H2O2), persulfate (SO^^^or S^O^^^), potassium peroxymonosulfate (KHSO5), oxone (2KHSO5·KHSO4·K2SO4), peroxydisulfuric acid (H2S2O8), sodium peroxide (Na2O2), potassium peroxide (K2O2), barium peroxide (BaO2), calcium peroxide (CaO2), magnesium peroxide (MgO2), lithium peroxide (Li2O2), and combinations thereof; or the peroxide is an organic peroxide selected a group comprising peracetic acid (CH3CO3H), tert-butyl hydroperoxide [(CH3)3COOH], magnesium monoperoxyphthalate (C16H10MgO10), 3-chloroperoxybenzoic acid (3-ClC6H4CO3H), urea hydrogen peroxide (NH2CONH2∙H2O2), benzoyl peroxide (C14H10O4), tert-butyl peroxybenzoate (C6H5CO3C(CH3)3), benzoyl peroxide (C14H10O4), di-tert-butyl peroxide [(CH3)3COOC(CH3)3], methyl ethyl ketone peroxide (C8H18O6), diacetyl peroxide ((CH3CO2)2), cumene hydroperoxide (C9H12O2), trifluoroperacetic acid (CF3CO3H), and combinations thereof. 32) Use of an enzyme for 7β-hydroxylation of a compound of Formula I or analogue or salt thereof, or converting a compound of Formula I or analogue or salt thereof to the compound of Formula II or analogue or salt thereof, wherein the enzyme requires peroxide as a co-substrate, and wherein the Formula I and the Formula II are as defined in the preceding embodiments. 33) A reaction mixture or reaction system comprising: (i) a compound of Formula I or analogue or a salt thereof, or a compound of Formula I(a) or analogue or a salt thereof, as defined in the preceding embodiments; (ii) an enzyme; and (iii) a peroxide. 34) The use of embodiment 32 or reaction mixture of embodiment 33, wherein the Formula I or a salt thereof, or the compound of Formula I(a) or analogue or a salt thereof is LCA or TLCA. 35) The use of embodiment 32 or reaction mixture of embodiment 33, wherein the enzyme is peroxygenase. 36) The use or reaction mixture of any of the embodiments 32 to 35, wherein the enzyme is unspecific peroxygenase (UPO). 37) The use or reaction mixture of any of the embodiments 32 to 35, wherein the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1. 38) The use or reaction mixture of any of the embodiments 32 to 37, wherein the peroxide is an organic peroxide, an inorganic peroxide, or both. 39) The use or reaction mixture of any of the embodiments 32 to 38, wherein the inorganic peroxide selected from the group consisting of hydrogen peroxide (H ^^ 2O2), persulfate (SO^or S^O^^^), potassium peroxymonosulfate (KHSO5), oxone (2KHSO5·KHSO4·K2SO4), peroxydisulfuric acid (H2S2O8), sodium peroxide (Na2O2), potassium peroxide (K2O2), barium peroxide (BaO2), calcium peroxide (CaO2), magnesium peroxide (MgO2), lithium peroxide (Li2O2), and combinations thereof; or the organic peroxide selected from the group consisting of peracetic acid (CH3CO3H), tert-butyl hydroperoxide [(CH3)3COOH], magnesium monoperoxyphthalate (C16H10MgO10), 3-chloroperoxybenzoic acid (3-ClC6H4CO3H), urea hydrogen peroxide (NH2CONH2∙H2O2), benzoyl peroxide (C14H10O4), tert-butyl peroxybenzoate (C6H5CO3C(CH3)3), benzoyl peroxide (C14H10O4), di-tert-butyl peroxide [(CH3)3COOC(CH3)3], methyl ethyl ketone peroxide (C8H18O6), diacetyl peroxide ((CH3CO2)2), cumene hydroperoxide (C9H12O2), trifluoroperacetic acid (CF3CO3H), and combinations thereof. 40) The reaction mixture of any of the embodiments 32 to 39, comprising: (i) LCA or TLCA; (ii) unspecific peroxygenase (UPO); and (iii) hydrogen peroxide (H2O2). 41) The method of any of the embodiments 1 to 24, wherein the method comprises 7β- hydroxylation of LCA to produce UDCA

[0041] comprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the H2O2 is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H2O2 is from about 1:100 to about 1:100,000,000. The foregoing description of the embodiments reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “including” or “includes” or “such as” or “have” or “having” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Throughout this specification, the term ‘combinations thereof’ or ‘and combinations thereof’ or ‘any combination thereof’ or ‘any combinations thereof’ are used interchangeably and are intended to have the same meaning, as regularly known in the field of patents disclosures. As used herein, the term “comprising” when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and / or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c; a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b; the order of steps c, b, and a; and the order of steps c, a, and b, etc. As used in this specification and the appended claims, the singular forms “a,” “an” and “the” includes both singular and plural references unless the content clearly dictates otherwise. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein. With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The suffix “(s)” at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term. Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included. The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise. Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

Claims

CLAIMS 1) A method for hydroxylation of a compound of Formula I or a salt thereof, comprising contacting the compound of Formula I or a salt thereof:with an enzyme in the presence of a peroxide, to produce a compound of Formula II or a salt thereof:wherein, R is independently selected from the group consisting of (CH2)nC=O(O)mR', alkyl, alkenyl, CH2OH, CHO, (CH2)2CONH(CH2)2SO3H, (CH2)2CONH(CH2)COOH, -CH=CH-CO2- alkyl, -CH=CH-CO2-aryl, -CH=CH-CO2H and -CH=CH-alkyl, wherein R' is independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and NRaRb, wherein Ra and Rb are independently selected from the group consisting of hydrogen, alkyl, aryl, -C(=O)-alkyl, -C(=O)-aryl and (CH2)2SO3H; R1is independently selected from the group consisting of hydrogen, alkyl, acyl, mesityl, tosyl, -C(=O)-alkyl, -C(=O)-aryl, -S(=O)2-alkyl, -S(=O)2-aryl, -S(=O)2-OH and -P(=O)-(OR2)2, wherein R2 is independently hydrogen or alkyl; n is 0 or an integer ranging from 1 to 5; m is 0 or 1; Y is hydrogen; X is hydrogen, hydroxy or oxygen; wherein R, R’, Ra, Rb, R1, R2or X are optionally independently substituted; represents either a single bond or a double bond; with the proviso that: both Rings A and B do not simultaneously contain double bond inside the rings; when is a double bond on the Ring A, R1is absent; when is a single bond on the Ring A, then R1 is present; when is a double bond inside either the Ring A or Ring B, Y is absent; and when is a single bond inside both the Rings A and B simultaneously, Y is present; and wherein the enzyme is unspecific peroxygenase (UPO). 2) The method of claim 1, wherein the enzyme is unspecific peroxygenase (UPO) of EC 1.11.2.1.3) The method of claim 1 or claim 2, wherein the unspecific peroxygenase (UPO) is a wild- type UPO obtained from fungi or proto-fungi; or wherein the unspecific peroxygenase (UPO) is a recombinant unspecific peroxygenase (UPO) expressed in a host cell selected from fungi and bacteria. 4) The method of any of the claims 1 to 3, wherein the enzyme is in the form of: i) an enzyme powder or solution, ii) immobilized enzyme, iii) whole cells expressing the enzyme, iv) an enzyme extract or lysate of the whole cells expressing the enzyme, or v) any combination of i to iv. 5) The method of any of the claims 1 to 4, wherein the peroxide is an inorganic peroxide, or an organic peroxide, or both; or wherein the peroxide is hydrogen peroxide (H2O2); or wherein the peroxide is provided as a pure peroxide, a peroxide solution or a peroxide generated by in situ peroxide generation system. 6) The method of any of the claims 1 to 5, wherein the alkyl is C1-C12alkyl, the alkenyl is C2- C12alkenyl, the alkynyl is C2-C12alkynyl, the cycloalkyl is a hydrocarbon ring system radical comprising C3-C20 carbon atoms, and the aryl is a hydrocarbon ring system radical comprising C6-C18 carbon atoms and at least one aromatic ring; and wherein the salt of Formula I or Formula II is selected from the group consisting of sodium salt, potassium salt, and quaternary ammonium salt (QAS). 7) The method of any of the claims 1 to 6, wherein the method comprises: a) hydroxylation of a compound of Formula I(a) to produce a compound of Formula II(a)R" is OH, NH(CH2)2SO3H, NHCH2CO2H, O-alkyl, or O-aryl; or b) hydroxylation of a compound of Formula I(b) to produce a compound of Formula II(b)wherein, R is as defined in claim 1; or c) hydroxylation of a compound of Formula I(c) to produce a compound of Formula II(c)wherein, R is as defined in claim 1; or d) hydroxylation of a compound of Formula I(d) to produce a compound of Formula II(d)Formula II(d) wherein, R and X are as defined in claim 1; or e) hydroxylation of a compound of Formula I(e) to produce a compound of Formula II(e)Formula II(e) wherein, R, R1and X are as defined in claim 1; or f) hydroxylation of a compound of Formula I(f) to produce a compound of Formula II(f)or g) hydroxylation of a compound of Formula I(g) to produce a compound of Formula II(g)Formula II(g) ; or h) hydroxylation of a compound of Formula I(h) to produce a compound of Formula II(h)Formula II(h) ; or i) hydroxylation of a compound of Formula I(i) to produce a compound of Formula II(i)5 or j) hydroxylation of a compound of Formula I(j) to produce a compound of Formula II(j)wherein, R3 is hydrogen, alkyl, or aryl; or, k) hydroxylation of a compound of Formula I(k) to produce a compound of Formula II(k)8) The method of any of the claims 1 to 7, wherein the method is carried out at a temperature of from about 5 °C to about 80 °C and for a time period ranging from about 15 minutes to about 150 hours; or wherein the method is carried out at a pH of about 2 to about 10; or wherein the molar ratio of the compound of Formula I or a salt thereof and the enzyme is from about 100:1 to about 10,000,000:1, the molar ratio of the compound ofFormula I or a salt thereof and the peroxide is from about 1:0.1 to about 1:10, or the molar ratio of the enzyme and the peroxide is from about 1:100 to about 1:100,000,000. 9) The method of any one of claims 1-8, wherein the compound of Formula I is selected from the group consisting of 12-Ketolithocholic acid (CAS reference 5130-29-0), (5β)-3,12- Dioxocholan-24-oic acid (CAS reference 2958-05-6), Dehydrolithocholic acid (CAS reference 1553-56-6), lithocholic acid (LCA, CAS reference 434-13-9) and taurolithocholic acid (TLCA; CAS reference 516-90-5). 10) The method of any of the claims 1 to 9, wherein the method comprises 7β-hydroxylation of LCA to produce UDCAcomprising contacting the LCA with unspecific peroxygenase (UPO) in the presence of hydrogen peroxide (H2O2) to produce said UDCA, wherein the method is carried out at a temperature of about 5 °C to about 80 °C for a time period ranging from about 15 minutes to about 150 hours, and at a pH of about 2 to about 10, and wherein the molar ratio of the LCA and the UPO is from about 100:1 to about 10,000,000:1, the molar ratio of the LCA and the H2O2 is from about 1:0.1 to about 1:10, and the molar ratio of the UPO and the H2O2 is from about 1:100 to about 1:100,000,000. 11) A method for converting a compound of Formula I(a) or a salt thereof to a compound of Formula II(a) or a salt thereof, comprising contacting the compound of Formula I(a) or a salt thereof:Formula I(a) with an enzyme in the presence of a peroxide, to produce the compound of FormulaFormula II(a) wherein, R" is OH, NH(CH2)2SO3H, NHCH2CO2H, O-alkyl, or O-aryl; and wherein the enzyme is unspecific peroxygenase (UPO). 12) The method of claim 11, wherein:a) the method comprises converting lithocholic acid (LCA):UDCA ; or b) the method comprises converting taurolithocholic acid (TLCA):TUDCA ; or c) the enzyme is unspecific peroxygenase (UPO); or d) the peroxide is hydrogen peroxide (H2O2). 13) Use of an enzyme for 7β-hydroxylation of a compound of Formula I or a salt thereof, to produce a compound of Formula II or a salt thereof, wherein the enzyme is unspecific peroxygenase (UPO),and wherein the Formula I and the Formula II are as defined in claim 1.

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