Patent Description:
The present invention relates to a process to produce mono-rhamnolipids.

Rhamnolipids are carboxylic acid containing anionic surfactants that consists of one or more alkyl chains connected via a beta hydroxy group to a rhamnose sugar. They may be produced by various bacterial species. Rhamnolipids are desirable as natural green surfactants, offering both sustainability benefits as well as application benefits such as good foaming profile, mildness on the skin and excellent sensorial benefits.

Di-rhamnolipids rich mixtures can be produced from fermentation processes, for example disclosed in <CIT> and <CIT>. The di-rhamnolipids are much more easily accessible from fermentation compared to the mono-rhamnolipids. The mono-rhamnolipids are a particularly useful material in the field of detergent compositions because they produce improved cleaning in comparison to the di-rhamnolipids, see for example <CIT>.

One route to obtaining higher yields of mono-rhamnolipids is fermentation host strain engineering, but this is a costly and complex approach to generating mono-rhamnolipids.

Attempts have been made to enzymatically convert di-rhamnolipids to mono-rhamnolipids using an immobilised Naringinase enzyme (<NPL>). However, improvements are still desired.

There is therefore a desire to produce a simpler and cost-effective route to generate higher yields of mono-rhamnolipids.

This problem can be overcome by the process of the invention as described herein.

The invention relates to a process to convert di-rhamnolipid to mono-rhamnolipid, wherein the process comprises the following process steps:-.

wherein the α-L-rhamnosidase enzyme does not have β-D-glucosidase activity.

Preferably in the process to produce mono-rhamnolipids, the α-L-rhamnosidase enzyme is from the genus Aspergillus, more preferably from Aspergillus niger, Aspergillus terreus, or Aspergillus lentulus, most preferably from Aspergillus niger, Aspergillus terreus.

Preferably in the process to produce mono-rhamnolipids, the enzyme is immobilised on a support using a technique selected from adsorption, covalent bonding, entrapment and/or crosslinking.

Preferably the starting di-rhamnolipid material has a carbon alkyl length of from C<NUM> to C<NUM>, more preferably from C<NUM>-C<NUM>.

Preferably the resulting mono-rhamnolipid material has a carbon alkyl length of from C<NUM> to C<NUM>, more preferably from C<NUM>-C<NUM>.

Preferably the rhamnose by-product is removed from the enzymatic reaction mixture as the reaction progresses.

Preferably in the process to produce mono-rhamnolipids, the temperature during the reaction is from <NUM> to <NUM>, preferably from <NUM> to <NUM>, more preferably from <NUM> to <NUM>, most preferably from <NUM> to <NUM>.

Preferably in the process to produce mono-rhamnolipids, the pH during the reaction is from pH <NUM> to <NUM>, preferably from pH <NUM> to <NUM>, more preferably from pH <NUM> to <NUM>, most preferably from pH <NUM> to <NUM>.

By using α-L-rhamnosidase enzyme that has α-L-rhamnosidase activity but also does not have β-D-glucosidase activity, the di-rhamnolipid starting material can be selectively converted to mono-rhamnolipid material. This selective nature of the process using an α-L-rhamnosidase enzyme that has α-L-rhamnosidase activity but also does not have β-D-glucosidase activity, results in the desired mono-rhamnolipid material, by selective removal of one rhamnose moiety, in contrast to other enzymes which either do not covert the di-rhamnolipid material at all, or remove both rhamnose units, neither of which results in the desired mono-rhamnolipid material.

The indefinite article "a" or "an" and its corresponding definite article "the" as used herein means at least one, or one or more, unless specified otherwise.

% relates to the amount by weight of the ingredient based on the total weight of the composition. For anionic surfactants, wt. % is calculated based on the protonated form of the surfactant.

Rhamnolipid is a biosurfactant. These are a class of glycolipid. They are constructed of rhamnose combined with beta-hydroxy fatty acids. Rhamnose is a sugar. Fatty acids are ubiquitous in animals and plants.

Rhamnolipids are discussed in <NPL>et al. Rhamnolipids are produced by Evonik, Stepan, Glycosurf, AGAE Technologies and Urumqi Unite Bio-Technology Co. Rhamnolipids may be produced by strains of the bacteria Pseudomonas aeruginosa. There are two major groups of rhamnolipids; mono-rhamnolipids and di-rhamnolipids.

Mono-rhamnolipids have a single rhamnose sugar ring. A typical mono-rhamnolipid produced by P. aeruginosa is L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RhaC<NUM>C<NUM>). It may be referred to as Rha-C<NUM>-C<NUM>, with a formula of C<NUM>H<NUM>O<NUM>. Mono-rhamnolipids have a single rhamnose sugar ring.

The IUPAC Name is <NUM>-[<NUM>-[(2R,3R,4R,5R,<NUM>)-<NUM>,<NUM>,<NUM>-trihydroxy-<NUM>-methyloxan-<NUM>-yl]oxydecanoyloxy]decanoic acid.

Di-rhamnolipids have two rhamnose sugar rings. A typical di-rhamnolipid is L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha2C<NUM>C<NUM>). It may be referred to as Rha-Rha-C-<NUM>-C-<NUM>, with a formula of C<NUM>H<NUM>O<NUM>.

The IUPAC name is <NUM>-[<NUM>-[<NUM>,<NUM>-dihydroxy-<NUM>-methyl-<NUM>-(<NUM>,<NUM>, <NUM>-tri hydroxy-<NUM>-methyloxan-<NUM>-yl)oxyoxan-<NUM>-yl]oxydecanoyloxy]decanoic acid.

In practice a variety of other minor components with different alkyl chain length combinations, depending upon carbon source and bacterial strain, exist in combination with the above more common rhamnolipids.

Throughout this patent specification, we use the terms mono- and di-rhamnolipid in order to avoid possible confusion. However, if abbreviations are used R1 is mono-rhamnolipid and R2 is di-rhamnolipid.

Preferably the rhamnolipid is a di-rhamnolipid of formula: Rha2C<NUM>-<NUM>C<NUM>-<NUM>. The preferred alkyl chain length is from C<NUM> to C<NUM>. The alkyl chain may be saturated or unsaturated.

The following rhamnolipids have been detected as produced by the following bacteria: (C12:<NUM>, C14:<NUM> indicates fatty acyl chains with double bonds).

Rhamnolipids produced by P. aeruginosa (di-rhamnolipids):
Rha-Rha-C8-C10, Rha-Rha-C8-C12:<NUM>, Rha-Rha-C10-C8, Rha-Rha-C10-C10, Rha-Rha-C10-C12:<NUM>, Rha- Rha-C-<NUM>-C-<NUM>, Rha-Rha-C-<NUM>-C-<NUM>, Rha-Rha-C-<NUM>:<NUM>-C-<NUM>, Rha-Rha-C-<NUM>-C14:<NUM>.

Rhamnolipids produced by Burkholderia pseudomallei (di-rhamnolipids): Rha-Rha-C14-C14.

Rhamnolipids produced by Burkholderia (Pseudomonas) plantarii (di-rhamnolipids): Rha-Rha-C14-C14.

A typical di-rhamnolipid is L-rhamnosyl-L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha<NUM>C<NUM>C<NUM> with a formula of C<NUM>H<NUM>O<NUM>).

Preferably the resulting mono-rhamnolipid is a mono-rhamnolipid of formula: RhaC<NUM>-<NUM>C<NUM>-<NUM>. The preferred alkyl chain length is from C<NUM> to C<NUM>. The alkyl chain may be saturated or unsaturated.

A preferred mono-rhamnolipid material is L-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RhaC<NUM>C<NUM> with a formula of C<NUM>H<NUM>O<NUM>).

By using α-L-rhamnosidase enzyme that has α-L-rhamnosidase activity but also does not have β-D-glucosidase activity, the di-rhamnolipid starting material can be selectively converted to mono-rhamnolipid material. This selective nature of the process using an α-L-rhamnosidase enzyme that has α-L-rhamnosidase activity but also does not have β-D-glucosidase activity, results in the desired mono-rhamnolipid material, by selective removal of one rhamnose moiety, in contrast to other enzymes which either do not convert the di-rhamnolipid material at all, or remove both rhamnose units, neither of which results in the desired mono-rhamnolipid material.

The α-L-rhamnosidase enzyme is preferably from the genus Aspergillus.

Preferably the α-L-rhamnosidase enzyme is from Aspergillus niger, Aspergillus terreus, or Aspergillus lentulus. More preferably the α-L-rhamnosidase enzyme is from Aspergillus niger, or Aspergillus terreus.

In the process of the invention, the enzyme may suitably be immobilised.

The invention relates to a process to convert di-rhamnolipid to mono-rhamnolipid comprising the following process steps:-.

Preferably the rhamnose by-product is removed from the enzymatic reaction mixture as the reaction progresses. This can result in higher conversion and/or yield or the mono-rhamnolipid product.

The α-L-rhamnosidase enzyme can potentially be immobilised to improve characteristics such as activity, selectivity and specificity, as well as enzyme stability. The immobilisation itself can be performed by a physical interaction between enzyme and matrix or through a chemical process such as covalent bond formation between the enzyme and support. The typical lab procedure may involve, but not be limited to adsorption, covalent bonding, entrapment, and crosslinking. These methods are described in more detail in <NPL>et al. as well as in<NPL>et al.

Preferred commercial enzyme immobilisation materials include Praesto™ and Lifetech™ available from Purolite.

The invention will be further described with the following non-limiting examples.

Three enzyme variants with α-L-rhamnosidase activity were considered in this study:.

Megazyme enzyme from A. niger was supplied as an ammonium sulphate suspension in <NUM>% (w/v) sodium azide. terreus rhamnosidase, secreted in P. pastoris, was supplied in form of supernatant at total protein concentration of <NUM>/mL, and the sample for analysis was purified on the nickel column using <NUM> sodium phosphate elution buffer containing <NUM> of imidazole, Sigma Aldrich (Gillingham, United Kingdom) at pH <NUM>, and dialysed against the same buffer without imidazole.

Rhamnolipid sample used in this study was obtained from Evonik (Witten, Germany), in form of <NUM>% (w/v) water solution. The sample is a blend consisting of the various rhamnolipid congeners, with the major congener, (Rha-Rha-C10-C10), <NUM>-O-alpha-L-rhamnosyl-alpha-L-rhamnosyl-<NUM>-hydroxydecanoyl-<NUM>-hydroxydecanoic acid and it will be referred hereafter as RL2. This sample also contains a minor amount of rhamnosyl-<NUM>-hydroxydecanoyl-<NUM>-hydroxydecanoate congener (Rha-C10:<NUM>/C10:<NUM>), which will be referred hereafter as RL1. The expected molecular weight of RL1 is <NUM>*mol-<NUM>, and the expected molecular weight of RL2 is <NUM>*mol-<NUM>.

All other chemicals were obtained from Sigma Aldrich (Gillingham, United Kingdom) unless otherwise stated in the methods below.

RL2 samples were diluted in acetonitrile to final concentration of <NUM>, filtered with <NUM> nitrocellulose syringe filter, and applied onto column for analysis. The fractions were collected in <NUM> sec rate per fraction and subjected to MALDI-TOF analysis. Next, RL2 were used as substrates for enzymes from A. terreus and A. niger, and naringinase.

For each reaction, the enzymes were used in concentration <NUM>*mL-<NUM> and substrate was used at <NUM>. Both reactions were performed in <NUM> MES buffer, pH <NUM>. The total volume of the reaction mix was <NUM>. Both samples were incubated at <NUM> and <NUM> rpm for <NUM> hours in incubator shaker (Infors HT, Surrey). <NUM>µL of reaction mixture was diluted in acetonitrile at ratio <NUM>:<NUM>, filtered with <NUM> nitrocellulose syringe filter, and applied onto the C18 column. The control sample (i.e. R2 benchmark) was not treated with any enzyme.

Rhamnolipid analysis was carried out using a Dionex UltiMate <NUM> system (Thermo Scientific, Dreieich, Germany) composed of an RS Dual Gradient pump, an RS Autosampler, an RS Column Compartment, an RS Diode Array Detector and a Corona Veo SD Charged-Aerosol Detector (CAD). The response in this type of detector is proportional to mass of analyte reaching the detector per unit time and it is expressed in picoampere (pA).

Chromatographic separation was performed using Accucore™ C18 LC Column (<NUM> X <NUM>, <NUM>) (Thermo Scientific, Dreieich, Germany) at ambient temperature. The flow rate was set to <NUM>/min. Mobile phase gradient consisting of <NUM>% acetic acid/water (v/v) (A) and <NUM>% acetonitrile (B) was used. The gradient was set to <NUM>% B from <NUM> to <NUM>, increased to <NUM>% B from <NUM> to <NUM>, remained constant for <NUM>, and increased to <NUM>% B from <NUM> to <NUM> before it immediately decreased to <NUM>% and remained constant for equilibration. Total run time was <NUM>. The injection volume was <NUM>µL. Charged-aerosol detection was performed at ambient temperature with an acquisition rate of <NUM> and a filter constant of <NUM>. CAD nitrogen gas pressure was <NUM> psi. Chromeleon V7. <NUM> software was used for general HPLC-control, data acquisition and analysis for CAD measurements.

MALDI-TOF was used for analysis RL2 fractions collected as described above. The matrix solution was <NUM>, <NUM>-dihydroxybenzoic acid dissolved in <NUM>% aqueous acetonitrile containing <NUM>% trifluoroacetic acid (TFA). <NUM>µL of HPLC fractionations, <NUM>µL of matrix solution and <NUM>µL of distilled water were mixed in an Eppendorf tube. <NUM>µL of the matrix-sample mixture was spotted onto the stainless-steel anchor chip. The spots were air-dried at room temperature and then analysed by MALDI TOF MS, using Ultraflexlll MALDI-TOF/TOF mass spectrometer (Bruker, Billerica, MA) operated with Smart beam laser system.

The rhamnolipid MS spectra were attained in a positive mode reflector; the used parameters for RLs detection were: ion source <NUM>, <NUM> kV and <NUM>, <NUM> kV; reflector, <NUM> kV and <NUM> kV; lens, <NUM> kV. The LIFT mode was applied for MS/MS analysis, using the following considerations: ion source <NUM>, <NUM> kV and <NUM>, <NUM> kV; reflector, <NUM> kV and <NUM>, <NUM> kV; lens, <NUM> kV; LIFT <NUM>, <NUM> kV and LIFT <NUM>, <NUM> kV. The used mass range for measurements was m/z <NUM>-<NUM> Da and a maximum MS deviation of <NUM> Da. <NUM> laser shots were accumulated with increments of <NUM> shot, to represent the final mass spectrum. External MS calibration was conducted using peptide calibration standard II (Bruker Daltonics), FlexControl V. <NUM> software was used for system control, where FlexAnalysis V. <NUM> and BioTools V. <NUM> software were used for data processing.

α-I-Rhamnosidase activity of enzyme from A. terreus and enzyme from A. niger was determined by a colorimetric method using p-nitrophenyl- α-I-rhamnopyranoside (pNPR) (See the chemical formula equation shown in <FIG>). The pNPR substrate was first dissolved in DMSO, with concentration adjusted to <NUM> in <NUM> sodium phosphate buffer pH <NUM>. Enzymes were diluted to concentration of <NUM>µg/mL in <NUM> sodium phosphate buffer pH <NUM>. The reaction was prepared by mixing the substrate solution with enzyme solution in <NUM>:<NUM> ratio. The activity assay was performed on <NUM>-well Microtiter UV plate by Thermo Scientific (Waltham, MA USA). All reactions were done at pH <NUM> and in temperature <NUM>. The changes in absorbance were monitored at <NUM> for <NUM> minutes in Microplate reader Varioskan® Flash.

The analysis was performed only for α-I-rhamnosidase from A. The influence of temperature change and pH change on enzyme activity was investigated using the same colorimetric method as described above (See <FIG>). The pNPR substrate was first dissolved in DMSO, with concentration adjusted to <NUM> in a buffer of choice. Enzyme was diluted to concentration of <NUM>µg/mL in a buffer of choice. The reaction was prepared by mixing the substrate solution with enzyme solution in <NUM>:<NUM> ratio. The activity assay was performed on <NUM>-well Microtiter UV plate by Thermo Scientific (Waltham, MA USA). For analysis of the pH influence on enzyme activity, the reactions were performed in <NUM>, and buffers of choice were: <NUM> sodium phosphate buffer pH <NUM>, <NUM> Tris-HCl buffer pH <NUM>, <NUM> Tris-HCl buffer pH <NUM>, and <NUM> sodium carbonate buffer pH <NUM>. For analysis of the temperature influence on enzyme activity, the reactions were performed in <NUM> and <NUM>, in <NUM> sodium phosphate buffer at pH <NUM>. The changes in absorbance were monitored at <NUM> for <NUM> minutes in Microplate reader Varioskan® Flash. The standard curve for the p-nitrophenol (pNP) release was prepared by measurement of absorbance at <NUM> for series of pNP samples of different concentrations (in <NUM> of corresponding buffer).

The analysis was performed only for α-I-rhamnosidase from A. The influence of rhamnose excess on enzyme activity was investigated using the same colorimetric method as described above (See <FIG>). The pNPR substrate was first dissolved in DMSO, with concentration adjusted to <NUM> in <NUM> sodium phosphate buffer pH <NUM>. Enzymes were diluted to concentration of <NUM>µg/mL in <NUM> sodium phosphate buffer pH <NUM>. Following sequence of rhamnose concentrations were prepared in <NUM> sodium phosphate buffer pH <NUM>: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. <NUM>µL of the total reaction mixture was prepared by adding the substrate solution, the enzyme solution and the rhamnose solution in <NUM>:<NUM>:<NUM> ratio. The activity assay was performed on <NUM>-well Microtiter UV plate by Thermo Scientific (Waltham, MA USA). All reactions were done at pH <NUM> and in temperature <NUM>. The changes in absorbance were monitored at <NUM> for <NUM> minutes in Microplate reader Varioskan® Flash.

β-D-glucosidase activity of enzyme from A. terreus was determined by a colorimetric method using p-nitrophenyl-β-I-glucopyranoside (pNPG). The pNPG substrate was first dissolved in DMSO, with concentration adjusted to <NUM> in <NUM> sodium phosphate buffer pH <NUM>. To analyse the substrate preference, the pNPG was mixed with pNPR at <NUM>:<NUM> ratio, then dissolved in DMSO, and concentration of such mixture was adjusted to <NUM> in <NUM> sodium phosphate buffer pH <NUM>. As a control, pNPR, prepared as above, at concentration of <NUM> was used. Enzyme was diluted to concentration of <NUM>µg/mL in <NUM> sodium phosphate buffer pH <NUM>. The reactions were prepared by mixing the substrate solution with enzyme solution in <NUM>:<NUM> ratio. The activity assay was performed on <NUM>-well Microtiter UV plate by Thermo Scientific (Waltham, MA USA). All reactions were done at pH <NUM> and in temperature <NUM>. The changes in relative absorbance values were monitored at <NUM> for about <NUM> minutes in Microplate reader Varioskan® Flash.

We evaluated the ability of utilizing rhamnolipids as substrates by α-L-rhamnosidase using A. terreus α-L-rhamnosidase, A. niger α-L-rhamnosidase and naringinase.

The substrate sample was mixed with the studied enzyme at a concentration of <NUM>/mL and reactions were performed at <NUM>, in pH <NUM> for <NUM> hours. For the control, R2 sample was mixed with buffer only. Next, the reactions were analysed using RP-HPLC, and the control was additionally analysed using MAL-TOF, to confirm which peak corresponds to R1 and R2 congeners.

In <FIG>, the peak detected between <NUM> and <NUM> of retention time corresponds to void. The peak detected at <NUM> of the retention time corresponds to di-rhamnolipid fraction of the sample and the peak detected at <NUM> of the retention time corresponds to the more hydrophobic mono-rhamnolipid fraction. The fractions were collected, and their molecular masses were analysed by MALDI-TOF.

The chromatogram of the control sample presented two peaks at <NUM> of retention time and <NUM> of retention time (see <FIG>). The MALDI-TOF analysis revealed that MW of the substance eluted after <NUM> was corresponding to that of estimated MW for R2 and that MW of the substance eluted after <NUM> was corresponding to that of estimated MW for R1 (data not included). The analysis of the samples after <NUM> of enzymatic treatment revealed changes in the chromatogram for each reaction.

The chromatogram of the sample treated by enzyme from A. terreus revealed that only R1 peak was detected (see <FIG>). In the sample treated by enzyme from A. niger, both R2 and R1 peak were detected on chromatogram, however, peak R2 was much smaller in comparison to the control (see <FIG>). In sample treated by naringinase, none of these peaks were detected (see <FIG>). This suggests that naringinase, possesses both α-I-rhamnosidase and β-d-glucosidase activities, and fully trims rhamnose from rhamnolipid molecule. Due to this character of naringinase, the enzyme cannot be utilized for production of mono-rhamnolipids and it will be not further assessed.

The relative activity of α-L-rhamnosidase from A. terreus and α-L-rhamnosidase from A. niger were measured against the p-NPR. The reaction was monitored in time at <NUM> and pH <NUM> (See <FIG>). The enzymes caused release of the product - <NUM>-nitrophenol. Both enzymes showed different kinetic behaviour, with A. terreus enzyme acting faster, however, reaching plateau after <NUM> at <NUM>% of converted substrate. niger enzyme converted similar amount of substrate after <NUM>. , however, after that, the conversion did not reach plateau and this enzyme converted <NUM>% more substrate than A. terreus after <NUM>, with the kinetic curve suggesting that with longer time of reaction, the substrate could be fully converted. The <NUM>% conversion of R2 is plenty sufficient for test purposes, therefore the enzyme with faster kinetics was used for further analysis.

The relative activity of α-L-rhamnosidase from A. terreus was measured using pNPR substrate in the excess of rhamnose in the reaction mix. The analysis was performed in order to study product inhibition phenomenon. The monitoring of the enzyme activity in the reactions with various rhamnose concentrations has showed that the substrate conversion rate decreased in the reactions with increasing amount of rhamnose added (See <FIG>). This result suggests that the rhamnose generated during the reaction time might cause enzyme inhibition, which in turn hampers the full substrate conversion, so that selective removal of the rhamnose from the reaction medium will enable improved conversion to the desired mono-rhamnolipid material.

Claim 1:
A process to convert di-rhamnolipid to mono-rhamnolipid comprising the following process steps:-
(a) contact of a starting di-rhamnolipid material with an α-L-rhamnosidase enzyme which is immobilised on a support;
(b) separation of the produced mono-rhamnolipid from the reaction medium and/or side products;
wherein the α-L-rhamnosidase enzyme does not have β-D-glucosidase activity.