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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/44—Preparation of O-glycosides, e.g. glucosides
  • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
  • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

• the present invention relates generally to improved methods for the cyclization and de-cyclization of long chain glycolipids, in particular sophorolipids.
• glycolipids especially those where the carbohydrate group is rhamnose or sophorose, are useful biosurfactants. They also possess other biological attributes and have been shown to be antimicrobial.
• a sophorolipid is a surface-active glycolipid compound that is synthesized by a selected number of non-pathogenic yeast species, such as Candida apicola and Starmerella bombicola.
• Sophorolipids typically consist of a hydrophobic fatty acid tail of 12-18 carbon atoms, and a hydrophilic sophorose carbohydrate head group; a glucose- derived disaccharide with an unusual b-1 ,2 glycosidic bond, that can be acetylated (Ac) on the 6’ and/or 6” positions.
• One terminal or sub-terminal hydroxylated fatty acid is b-glycosidically linked to the sophorose head at carbon 1 (T).
• the carboxylic end of this fatty acid is either free (acidic, or open, form) or internally esterified at the 4” or sometimes at the 6’ or 6” position (lactonic, or closed, form), examples of these are given below:
• the structures A and B are sophorolipids in their acidic, open form; C and D are sophorolipids in their lactonic form.
• (A) is a saturated, acidic sophorolipid
• (B) is an unsaturated, acidic sophorolipid
• (C) is a saturated, monomeric lactonic sophorolipid, esterified at 4” position
• (D) is a saturated, dimeric lactonic sophorolipid, esterified at the 4” position
• sophorolipids have the potential to disrupt biofilm formation and inhibit growth of a variety of clinically relevant organisms, such as bacteria, fungi, algae, mycoplasma and viruses.
• the proposed primary mechanism of action of these biosurfactants is membrane lipid order perturbation.
• sophorolipids are significantly influenced by the distribution of the lactone (closed, or cyclic) vs. acidic (open, or non- cyclic) form. Their use is somewhat limited in utility because a considerable proportion of the product made by fermentation is in both the acidic and lactonic forms, often in proportions of around 50:50 in the fermentative broth. Both forms have beneficial properties but it is generally desirable to have a substantially pure form of one or the other to make best use of such properties.
• the lactonic form is more efficient at reducing surface tension and has better antimicrobial properties.
• the acidic form displays better foam producing ability and solubility (Bacille, 2017). However, these beneficial properties of the lipids may be difficult to utilise.
• oral administration is often ineffective because the acidic form is unstable at low pH, making this form unsuitable for use where the product is designed to pass intact through the stomach and reach the lower parts of the gastrointestinal tract.
• their use in animal feed has been somewhat limited due to the fact that a considerable proportion of the product made by fermentation is in the ‘acidic’ form.
• a method for selective cyclization or de-cyclization of a mix of sophorolipids in the lactonic or acidic forms comprising selecting one or other of the following steps dependent upon the form required:
• the starting mix of sophorolipids preferably comprises at least 40:60 to 60:40 sophorolipids in the lactonic to acidic form.
• a second aspect of the present invention provides a method for producing a lactonic form of a sophorolipid from its acidic form, comprising reacting a sophorolipid composition that includes at least 40% w/w sophorolipids in their acidic form with a strong methylating agent to provide an end product having at least 80% w/w sophorolipids in their lactonic form.
• Any strong methylating agent may be used for cyclization of the acidic, open sophorolipid form to their lactonic, closed form but preferably diazomethane, or a derivative thereof, is used as the methylating agent, especially TMS-diazomethane.
• Other, strong methylating agents include methyl fluorosulfonate or methyl trifluoromethane sulfonate. Weaker methylating agents such as trimethylamine or iodomethane are not sufficient for cyclisation to occur.
• methylating agent may be added to the starting sophorolipid but preferably an equimolar amount of the methylating agent in a suitable solvent, such as hexane or methanol, is added. For example, 1M or 2M.
• the starting sophorolipid composition containing at least 40% w/w sophorolipids in their acidic form is dried prior to reacting with the methylating agent, more preferably being vacuum dried for at least one hour.
• the starting sophorolipid contains at least 50% w/w sophorolipids in their acidic form, more preferably at least 75% w/w, especially 100% w/w.
• the methylating agent is preferably added to the starting sophorolipid composition at a reduced temperature, preferably being less than 10°C. Stirring may assist the reaction.
• the solution is preferably vacuum dried to provide the end product having at least 75%, more preferably at least 80% lactonic sophorolipids.
• a third aspect of the present invention provides a method for producing an acidic form of a sophorolipid from its lactonic form comprising reacting a sophorolipid composition that includes at least 40% w/w sophorolipids in their lactonic form with a genetically engineered enzyme having at least 50% homology with lipase B to provide an end product having at least 70% w/w sophorolipids in their acidic form.
• the starting sophorolipid composition contains at least 50% w/w sophorolipids in their lactonic form, more preferably at least 75% w/w, especially 100% w/w.
• the mixture is preferably dissolved in water, or another suitable solvent, and heated to at least 35°C, preferably above 37°C, preferably at least 40°C.
• the enzyme may be immobilised, for example on beads or a column.
• the mixture is stirred for at least 3 hours, preferably 4-6 hours.
• the end product is then removed from the enzyme, for example by filtration and dried, for example being vacuum dried.
• the end product has at least 75%, more preferably at least 85% acidic sophorolipids
• the method uses genetically engineered enzymes having the same functionality as a lipase, in particular lipase B, for conversion of the lactonic to the acidic form.
• the genetically engineered enzyme has at least 50% homology, more preferably at least 60% homology, more preferably at least 70% homology, especially at least 80% homology, ideally at least 90% homology with lipase B.
• the genetically engineered enzyme comprises an amino acid sequence selected from the following SEQ. ID No.s 1-5 or a functional variant or homologue thereof. More preferably the enzyme comprises SEQ ID No. 1 or the enzyme has at least the amino acid sequences SEQ. ID No.s 2 to 5.
• the nucleic acid coding sequence may be incorporated into a vector, preferably a plasmid.
• the vector comprising the nucleic acid may be operably linked to one or more regulatory nucleic acid sequences.
• the vector, or part thereof may be inserted into a host cell, such as bacteria or yeast, for expression of the enzyme. This may be episomal, or integrated into the host genome in a transient or stable manner.
• the host is selected from Pichia spp., Saccharomyces spp., Aspergillus spp. or Escherichia coii.
• the enzyme may be grown and expressed into the media, or the cell culture may be chemically, mechanically or physically disrupted to release the active enzyme.
• the enzyme in its pure form may be immobilised for flow-through systems or repeated use.
• Figure 1 is a ESI-ToF mass spectra for a sophorolipid mixture formed by fermentation prior to treatment with a methylating agent, specifically TMS-diazomethane;
• Figure 2 is a ESI-ToF mass spectra for the composition of Figure 1 following treatment with a methylating agent, specifically TMS-diazomethane; and
• Figure 3 is a ESI-ToF mass spectra for the composition of Figure 2 following treatment with a lipase, specifically Lipase B from C. antarctica.
• the present invention provides synthetic processes that enable the production of either substantially pure sophorolipid in its lactonic form or substantially pure sophorolipid in its acidic form.
• the present invention provides new methods for the cyclization of long chain glycolipids, in particular where the carbohydrate group is sophorose.
• Example 1 Single-step Preparation of the Lactonic form of a Sophorolipid from a Mixture of Acidic and Lactonic Forms.
• a sophorolipid mixture formed by fermentation is completely dried prior to carrying out the single step preparation of the lactonic form of the lipid.
• the vacuum is reduced to 20 mbar; and the vacuum is then left at 20 mbar for a further 60 minutes.
• the dried sophorolipid was then transferred to a round bottom flask and dissolved in the minimum amount of ethyl acetate. The flask was then placed in an ice bath with stirring.
• Figures 1 and 2 are electrospray ionisation time of flight (ESI-ToF) mass spectra showing the mixture pre- and post-treatment with the methylating agent. It is clear that post-treatment the product has a significantly higher proportion of lactonic forms of the sophorolipid compared to the acidic forms.
• the mixture identified in Figure 1 is comprised of 75.6% of the acidic, open sophorolipid form, and 24.4% of the lactonic, closed sophorolipid form.
• Figure 2 following treatment with TMS- Diazomethane, the mixture is now comprised of 20.38% of the acidic, open sophorolipid form, and 79.62% lactonic, closed sophorolipid form.
• methylating agents may be used in place of TMS-diazomethane, such as diazomethane or methyl fluorosulfonate or methyl trifluoromethane sulfonate.
• diazomethane derivative is used, with TMS-diazomethane and diazomethane being the preferred candidates providing the most efficient conversion of the acidic form to the lactonic form.
• Example 2 Single-step Preparation of the Acidic form of a Sophorolipid from a Mixture of Lactonic and Acidic Forms.
• the lactonic sophorolipids prepared in Example 1 were transferred to a Round Bottom flask and dissolved in water. The solution was heated to 45°C and stirred. ⁇ 1% w/w of immobilised Lipase B enzyme was then added to this solution which was stirred for 4- 6 hours.
• the Lipase B was from Moesziomyces antarcticus, also referred to as Sporobolomyces antarcticus, Trichosporon oryzae, Pseudozyma antarctica and Candida antarctica.
• the immobilised enzyme was filtered off from the solution.
• the solution was then vacuum dried to yield a high portion (>80%) acidic sophorolipids. This is illustrated in the ESI-ToF mass spectra of Figure 3 which shows a higher proportion of acidic sophorolipids.
• the mixture is now comprised of 89.10% of the acidic, open sophorolipid form, and 10.90% lactonic, closed sophorolipid form.
• Example 1 combining the steps of Example 1 and 2 enables selection of either the lactonic or acidic form as represented by the following scheme:
• Example 3 Esterase Candidates for Preparation of the Acidic form of a Sophorolipid.
• Lipase B amino acid sequence from C. antarctica (SEQ ID No. 1) was taken and standard protein-protein BLAST (pBLAST) was performed. The FASTA sequence of all alignments was exported and imported into Jalview. MUSCLE alignment was performed on all sequences, with the constraint that they were not from the C. antarctica organism. Other Lipase B variants from C. antarctica have a homology of 397.14% to SEC ID No. 1. SEQ ID No. 1:
• SEQ ID No. 2-5 Four conserved regions of amino acids were identified, and are detailed as SEQ ID No. 2-5. These regions correspond to amino acid residues 63-66; 128-132; 202-218; 237-241 from SEQ ID No. 1. These regions are considered essential for Lipase B activity.
• xi corresponds to either S, T or G amino acid residues.
• X 2 corresponds to either T, N or S amino acid residues.
• X3 corresponds to either I, L or F amino acid residues.
• X 4 corresponds to either Y, F or W amino acid residues.
• X5 corresponds to either A, S or G amino acid residues.
• Cb corresponds to either T, F, L or S amino acid residues.
• X 7 corresponds to either E, D or Q amino acid residues.
• Xs corresponds to either I, V or F amino acid residues.
• Xg corresponds to either Q, E or K amino acid residues.
• X10 corresponds to either Q, E, N or M amino acid residues. (Corresponds to residues 202-218 on SEQ ID No. 1)
• xu corresponds to either V, A or L amino acid residues.
• Xi2 corresponds to either S, D or A amino acid residues.
• Xi3 corresponds to either V, Y or I amino acid residues. (Corresponds to residues 237-241 on Seq ID No. 1)
• a vector incorporating the sequence may be inserted into a host organism such as Saccharomyces spp., Aspergillus spp. or Escherichia coli, or other suitable host organisms.
• Expression vectors may include pET and pESC derivatives, which may be episomal, or integrated into the host genome. It may be grown and expressed into the media, or the cell culture may be chemically, mechanically or physically disrupted to release the enzyme. Purification can be performed, and the enzyme in its pure form may be immobilised for flow through systems or repeated use.
• the present invention provides new techniques for the production of either substantially pure sophorolipid in its lactonic form or substantially pure sophorolipid in its acidic form, significantly increasing the ability to use the compounds based on the beneficial properties of either the lactonic or acidic forms.

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