WO2022200273A2 - Methods for cyclization and de-cyclization of long chain glycolipids - Google Patents

Methods for cyclization and de-cyclization of long chain glycolipids Download PDF

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WO2022200273A2
WO2022200273A2 PCT/EP2022/057351 EP2022057351W WO2022200273A2 WO 2022200273 A2 WO2022200273 A2 WO 2022200273A2 EP 2022057351 W EP2022057351 W EP 2022057351W WO 2022200273 A2 WO2022200273 A2 WO 2022200273A2
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sophorolipids
lactonic
sophorolipid
acidic
methylating agent
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WO2022200273A3 (en
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David Garnett
Lloyd Cooper
Nathaniel JOHNS
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Pathway Intermediates Limited
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Publication of WO2022200273A2 publication Critical patent/WO2022200273A2/en
Publication of WO2022200273A3 publication Critical patent/WO2022200273A3/en

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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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Abstract

A method for selective cyclization or de-cyclization of a mix of sophorolipids in the lactonic or acidic forms, wherein the lactonic form is produced by reacting a mixture of sophorolipids with a strong methylating agent to provide an end product having at least 70% w/w sophorolipids in their lactonic form; and wherein the acidic form is produced by reacting a mixture of sophorolipids with a genetically engineered enzyme having at least 50% homology to lipase B to provide an end product having at least 70% w/w sophorolipids in their acidic form.

Description

Methods for Cyclization and De-cyclization of Long Chain Glycolipids
Field of the Invention.
The present invention relates generally to improved methods for the cyclization and de-cyclization of long chain glycolipids, in particular sophorolipids.
Background
It is well known that 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:
Figure imgf000003_0001
Where: Ri, R2= H or Ac l
Figure imgf000004_0001
Where: Ri, R2= H or Ac
Figure imgf000005_0001
Where: Ri, R2= H or Ac.
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 and (D) is a saturated, dimeric lactonic sophorolipid, esterified at the 4” position (Kulakovskaya, E. & K. T., [2014] Extracellular glycolipids of Yeasts; Biodiversity, Biochemistry and Prospects. Oxford, UK: Academic Press, Elsevier Inc.).
As with many biosurfactants, 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.
The physiochemical and biological properties of 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. For example, 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. As a result, 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.
Prior art fermentation methods have been optimized to increase the proportion of one form to the other in the broth, but it would be desirable to be able to produce such forms synthetically, ideally in their substantially pure form.
It is the aim of the present invention to provide improved methods for the cyclization and de-cyclization of long chain glycolipids, in particular those where the carbohydrate group is sophorose, that overcome, or at least alleviate, the abovementioned problems.
Summary of the Invention
According to a first aspect of the present invention there is provided a method for selective cyclization or de-cyclization of a mix of sophorolipids in the lactonic or acidic forms, the method comprising selecting one or other of the following steps dependent upon the form required:
(i) reacting the mix of sophorolipids with a strong methylating agent to provide an end product having at least 70% w/w sophorolipids in their lactonic form; or
(ii) reacting the mix of sophorolipids 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 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.
Any suitable amount of 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.
Preferably, 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.
Preferably, 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.
Preferably, 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. Preferably, 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.
Preferably, 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. Preferably, 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.
Preferably, 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. Preferably 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.
Brief Description of the Drawings
For a better understanding of the present invention and to show more clearly how it may be carried into effect, reference will now be made by way of example only to the accompanying drawings in which:
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.
Detailed Description
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. In the context of this disclosure, preferably at least 80%, more preferably at least 90%, especially 100% of the sophorolipid is in the desired lactonic form or acidic form in the end product. This significantly increases the ability to use the compounds based on the beneficial properties of either the lactonic or acidic forms.
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.
In this example, 10-12g (w/w) of the sophorolipid was placed in a 250 mL evaporating flask and placed in a rotary evaporator above a water bath at 70°C and was set to rotate at 100 rpm. The following series of vacuums were then applied to the system:
Initial vacuum set at 200 mbar;
After 10 minutes, the vacuum is reduced to 160 mbar;
After 20 minutes, the vacuum is reduced to 140 mbar;
After 30 minutes, the vacuum is reduced to 120 mbar;
After 40 minutes, the vacuum is reduced to 100 mbar;
After 50 minutes, the vacuum is reduced to 70 mbar;
After 60 minutes, the vacuum is reduced to 50 mbar;
After 70 minutes, the vacuum is reduced to 30 mbar;
After 80 minutes, 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.
An equimolar amount of at least 1 M TMS Diazomethane (in hexane) solution was added dropwise to the flask. The flask was kept in the ice bath, with stirring, for 3-4 hours.
The solution was then vacuum dried to yield a high portion (>80%) of lactonic sophorolipids.
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. In particular, 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. In 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.
Other types of methylating agents may be used in place of TMS-diazomethane, such as diazomethane or methyl fluorosulfonate or methyl trifluoromethane sulfonate. However, preferably a 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. In particular, following treatment with Lipase B, the mixture is now comprised of 89.10% of the acidic, open sophorolipid form, and 10.90% lactonic, closed sophorolipid form.
Thus, combining the steps of Example 1 and 2 enables selection of either the lactonic or acidic form as represented by the following scheme:
Figure imgf000012_0001
Ri, 2, Rs, R4 = H or Ac
Example 3: Esterase Candidates for Preparation of the Acidic form of a Sophorolipid.
Once it had been determined that an esterase, particularly a lipase (EC 3.1.1.3), especially lipase B, could convert the lactonic sophorolipid to the acidic form, further investigations were carried out to identify other suitable candidates, including the identification of the region of the lipase responsible for enabling the conversion of the lactonic form to the acidic form.
Investigations were therefore carried out to determine which part of the lipase B protein is most conserved by comparing the amino acid sequences of multiple, related lipases that share homology with lipase B, from different species.
Method:
The 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:
MKLLSLTGVAGVLATCVAATPLVKRLPSGSDPAFSQPKSVLDAGLTCQGASPSSVSKPILLVPGTGTTG
PQSFDSNWIPLSTQLGYTPCWISPPPFMLNDTQWTEYMVNAITALYAGSGNNKLPVLTWSQGGLVAQW
GLTFFPSIRSKVDRLMAFAPDYKGTVLAGPLDALAVSAPSVWQQTTGSALTTALRNAGGLTQIVPTTNL
YSATDEIVQPQVSNSPLDSSYLFNGKNVQAQAVCGPLFVIDHAGSLTSQFSYWGRSALRSTTGQARSA
DYGITDCNPLPANDLTPEQKVAAAALLAPAAAAIVAGPKQNCEPDLMPYARPFAVGKRTCSGIVTP
MUSCLE alignment was repeated on the remaining sequences and percentage identity was determined. The consensus sequence for highly conserved regions was analysed, for example, highlighting regions of at least 70% identify across all proteins, especially noting amino acids with 100% identity.
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.
SEQ ID No. 2:
PGTG
(Corresponds to residues 63-66 on SEQ ID No. 1)
SEQ ID No. 3:
X1WSQG
Where: xi corresponds to either S, T or G amino acid residues.
(Corresponds to residues 128-132 on SEQ ID No. 1)
SEQ ID No. 4:
VPTT X2X3X4S X5X6D X7XeV X9 P X10
Where: X2 corresponds to either T, N or S amino acid residues.
X3 corresponds to either I, L or F amino acid residues.
X4 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.
X7 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)
SEQ ID No. 5:
Xu Q Xi Xi3 C
Where: 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.
Thus, 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.

Claims

CLAIMS:
1. 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 70% w/w sophorolipids in their lactonic form.
2. 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 agenetically engineered enzyme having at least 50% homology to Lipase B to provide an end product having at least 70% w/w sophorolipids in their acidic form.
3. A method for selective cyclization or de-cyclization of a mix of sophorolipids in the lactonic or acidic forms, the method comprising selecting one or other of the following steps dependent upon the form required:
(i) reacting the mix of sophorolipids with a strong methylating agent to provide an end product having at least 70% w/w sophorolipids in their lactonic form; or
(ii) reacting the mix of sophorolipids with a genetically engineered enzyme having at least 50% homology to Lipase B to provide an end product having at least 70% w/w sophorolipids in their acidic form.
4. The method according to claim 1 or 3 wherein the starting mix of sophorolipids comprises at least 40:60 to 60:40 sophorolipids in the lactonic to acidic form.
5. The method according to claim 1 , 3 or 4 wherein the strong methylating agent is diazomethane, or a derivative thereof, preferably TMS-diazomethane.
6. The method according to claim 1 , 3 or 4 wherein the strong methylating agent is methyl fluorosulfonate or methyl trifluoromethane sulfonate
7. The method according to claim 1 or any one of claims 3 to 6 wherein the methylating agent is added to the starting sophorolipid in an equimolar amount of the methylating agent, preferably in a solvent, especially wherein the solvent is selected from hexane or methanol.
8. The method of claim 1 or any one of claims 3 to 7 wherein the starting sophorolipid composition containing at least 40% w/w sophorolipids in their acidic form is dried prior to reacting with the methylating agent, preferably being vacuum dried for at least one hour.
9. The method of claim 1 or any one of claims 3 to 8 wherein 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.
10. The method of claim 1 or any one of claims 3 to 9 wherein the methylating agent is added to the starting sophorolipid composition at a reduced temperature, preferably being less than 10°C.
11. The method of claim 1 or any one of claims 3 to 10 wherein the solution is vacuum dried to provide the end product having at least 75%, preferably at least 80% lactonic sophorolipids.
12. The method of claim 2 or claim 3, wherein 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.
13. The method of claim 2, 3, or 12 wherein the mixture is dissolved in water, or another suitable solvent, and heated to at least 35°C, preferably above 37°C, preferably at least 40°C.
14. The method of claim 2, 3, 12 or 13 wherein the enzyme is immobilised on a support.
15. The method of claim 2, 3, 14 or 15 wherein the genetically engineered enzyme has an amino acid sequence having at least 60% homology, more preferably at least 70% homology, especially at least 80% homology, ideally at least 90% homology with lipase B.
16. The method of claim 2, 3 or any one of claims 13 to 15 wherein the genetically engineered enzyme comprises an amino acid sequence selected from SEQ. ID No.s 1 to 5 or a functional variant or homologue thereof.
17. The method of claim 16 wherein the genetically engineered enzyme comprises the amino acid sequence of SEQ. ID No. 1.
18. The method of claim 16 wherein the genetically engineered enzyme includes each of the amino acid sequences of SEQ ID. No. 2 to 5.
19. The method of claim 17 or claim 18 further comprising incorporating a nucleic acid sequence for any one of SEQ. ID No.s 1 to 5 into a vector, preferably a plasmid, for insertion into a host cell for expression of the genetically engineered enzyme.
20. The method of claim 19 wherein the host is selected from Pichia spp., Saccharomyces spp., Aspergillus spp. or Escherichia coli.
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