Enzymatic hydrolysis of acylated steryl glycosides and method for treating biofuel

Abstract

The present invention relates to the use of at least one glycosidase of the class EC 3.2.1.21, EC 3.2.1.4, EC 3.2.1.7, and/or EC 3.2.1.80 for hydrolyzing at least one glycosiding bond in at least one substrate selected from the group of acylated steryl glycosides of the general formula (I): R.sup.1COO[C.sub.mH.sub.nO.sub.x].sub.yOR.sup.2 wherein R.sub.1 is a saturated or non-saturated C.sub.1-C.sub.30 alkyl, R.sub.2 is a sterol moiety, m=5-7, n=9-14 and x=5-7, y=1-5. The present invention relates further to a method of for hydrolysing steryl gylcosides using said enzymes in biofuel, oil and/or fat.

Claims

1. A method for hydrolyzing at least one glycoside bond in at least one substrate selected from the group of acylated steryl glycosides of the general formula I
R.sup.1COO[C.sub.mH.sub.nO.sub.x].sub.yOR.sup.2 wherein R.sub.1is a saturated or non-saturated C.sub.1-C.sub.30 alkyl, R.sub.2is a sterol moiety, m=5-7, n=9-14 and x=5-7, y =1-5 using at least one glycosidase of the class EC 3.2.1.7 and/or EC 3.2.1.80.

2. The method according to claim 1, wherein at least one glycosidase of the class EC 3.2.1.7 is an endoinulinase derived from an organism, wherein the organism is selected from the group consisting of Aspergillus sp., Xanthomonas sp., Penicillium sp., Bacillus sp., Arthrobacter sp., Kluyveromyces sp., Pseudomonas sp., Geomyces sp., Meyerozyma sp., and Streptomyces sp.

3. The method according to claim 1, wherein at least one glycosidase of the class EC 3.2.1.80 is an exoinulinase derived from an organism, wherein the organism is selected from the group consisting of Aspergillus sp., Kluyveromyces sp., Arabidopsis thaliana, Triticum sp., Cichorium sp., Cryptococcussp., Penicillium sp., Actinomyces sp., Streptomyces sp., Lactobacilus sp., Bacillus sp., Streptococcus sp., and others.

4. The method according to claim 1, wherein R.sup.1 is a saturated or non-saturated C.sub.5-C.sub.26 alkyl.

5. The method according to claim 1, wherein R.sup.2 is selected from a group consisting of sitosterol, campesterol, stigmasterol, brassicasterol, stigmastadienol, dihydrositosterol, sitostanol and d5-avenasterol.

6. The method according to claim 1, wherein [C.sub.mH.sub.nO.sub.x].sub.yO is a sugar moiety selected from a group consisting of a glucose, galactose, glucuronic acid, mannose, xylose or arabinose.

7. The method according to claim 1, wherein the substrate is a mixture of different acylated steryl glycosides or a mixture of acylated steryl glycosides and steryl glycosides.

8. A method for hydrolyzing at least one acylated steryl glycoside and/or at least one steryl glycoside in biofuel, oil and/or fat by mixing at least one glycosidase of the class EC 3.2.1.7 and/or EC 3.2.1.80 with the biofuel, oil or fat.

9. The method according to claim 8, comprising a degumming step.

10. The method according to claim 9, wherein the hydrolysis of at least one acylated steryl glycoside and/or at least one steryl glycoside takes place before, during or after the degumming step.

11. The method according to claim 8, comprising at least one transesterification step.

12. The method according to claim 11, wherein the hydrolysis of the at least one acylated steryl glycosides and/or the at least one steryl glycosides takes place before the transesterification.

13. The method according to claim 8, wherein following the hydrolysis of the at least one acylated steryl glycosides and/or the at least one steryl glycosides the sugars and fatty acids are removed by means of a polar phase.

14. The method according to claim 4, wherein R.sup.1 is a saturated or non-saturated C.sub.6-C.sub.20 alkyl.

15. The method according to claim 6, wherein [C.sub.mH.sub.nO.sub.x].sub.yO is a glucose sugar moiety.

16. The method according to claim 9, wherein the degumming step is an enzymatic degumming step.

17. The method according to claim 11, wherein the transesterification step is an enzymatic transesterification step.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) FIG. 1 illustrates the chemical reactions taking place when treating a mixture of ASG and SG with an inulinase preparation. The enzyme hydrolyses the glycosidic bond in ASG and SG without hydrolysing the acyl chain. The hydrolysation using inulinase results in the formation of free sterols which are soluble. The released sugar and sugar fatty acid esters (acyl-sugar) are soluble in the polar phase of crude oil processing.

(2) FIG. 6 illustrates the main stages of biodiesel production process and the state of ASG and SG in each of the singular stages. According to the embodiment shown in FIG. 6 the hydrolysis of ASG and SG by inulinases is combined with the degumming step (phospholipase treatment). The inulinase treatment is thus effected before the transesterification step.

EXAMPLE 1

(3) The following enzyme preparations were used: Fructozyme? L: commercial enzyme preparation (liquid) consisting of exoinulinase (3.2.1.80) and endoinulinase (3.2.1.7) from Aspergillus niger, from Novozymes Denmark; Novozym? 960: commercial enzyme preparation (liquid) consisting of endoinulinase (3.2.1.7) from Aspergillus niger, from Novozymes Denmark; and Lyophilized inulinase from Aspergillus niger (mixture of exo- and endoinulinase), from Sigma-Aldrich Switzerland.
a) Hydrolysis of Commercially Available ASG

(4) A stock solution of pure ASG in hexane:isopropanom 85:15 (purity: 98+%, Matreya, US) was prepared and used for further experiments. Correct volume was added to 30 ml tube in order to have an initial amount of 200 ?g ASG. Solvent was evaporated and ASG were redissolved in 500 ?l ethanol. Further, 0.1 M sodium acetate buffer pH 4.5 was added followed by the addition of the enzyme (total volume of buffer and enzyme=3 ml). The incubation was carried out at 40? C. for 21 hours in a shaking device with controlled temperature. Hydrolysis was stopped by starting the extraction of ASG using the Bligh and Dyer method. Prior to extraction, 2 ml of internal standard solution (0.008 mg DHC /ml EtOH) was added. The lower phase was then collected and evaporated to dryness, redissolved in 2 ml EtOH and transferred to a fresh tube. For GC analysis of the free sterols released in the enzymatic reaction (procedure as in Nystr?m, Sch?r, & Lampi, 2012, European Journal Lipid Science and Technology, 114 (6), 656-669), 1 ml was withdrawn, dried and derivatised to TMS-derivatives. For HPLC of the possibly remaining intact substrates, the remaining ethanol solution was dried and redissolved in hexane:isopropanol 85:15 and analysed using hexane:isopropanol 85:15 as mobile phase (column: Luna HILIC from Phenomenex). Samples were analysed in duplicate.

(5) b) Hydrolysis of ASG in Samples Extracted from Soy Beans and Soy Lecithin

(6) Total lipid extraction of soy beans and soy lecithin (both 2 g) was done by accelerated solvent extraction (ASE). Lipids were further fractionated using solid phase extraction whereas the ASG fraction was collected, evaporated to dryness. ASG fraction from soybeans was redissolved in 500 ?l EtOH, fraction from lecithin was redissolved in 1.5 ml EtOH and devided into three 500 ?l volumes. Samples were then subjected to enzymatic hydrolysis as described above (soy bean: 1 sample, only 200 ?l Novozyme 960 added, lecithin: 2 samples, 200 ?l Novozyme 960 and 200 ?l Fructozyme L tested) and measured by GC and HPLC also described above.

(7) c) Samples with Commercial ASG

(8) Free sterols stigmasterol, campesterol, stigmasta-5,23-dienol, sitosterol, sitostanol and d5-avenasterol (see FIG. 2) were detected in all the samples incubated with either Novozym 960, Fructozyme L or lyophilized inulinase. Samples without enzyme (blank) only showed a peak for the internal standard (DHC), which was added prior to extraction. Therefore, all the inulinase preparations were able to effectively hydrolyse ASG into free sterols. Due to the absence of free sterols in the blank samples other hydrolysis pathways (eg acid hydrolysis) causing the breakdown of ASG can be excluded.

(9) FIG. 2 shows a 3D-overlay of GC-FIC chromtograms of enzymatically hydrolysed ASG samples; 1: blank (no enzyme added), 2: ASG+Fructozyme L, 3: ASG+Novozyme 960, 4: ASG+lyophilized inulinase; peak identification: A: DHC (internal standard), B: Campesterol, C: Stigmasterol, D: Sitosterol, E: Sitostanol, F: ?5-avenasterol, G: unknown peak (no sterol) , H: unknown peak (no sterol)

(10) Quantification based on internal standard showed that the total amount of free sterols was at similar levels for all inulinase preparation tested (FIG. 3). In case 200 ?g ASG (initial amount) are fully hydrolysed 100.6 pg free sterols should be released using a conversion factor of 0.503. Incubation with Fructozyme L resulted in 57.1 ?g free sterols representing 113.5 ?g hydrolysed ASG, with Novozym 960 in 53.6 ?g free sterols representing 106.5 ?g hydrolysed ASG and with lyopihilzed inulinase in 50.8 ?g free sterols representing 101.0 ?g hydrolysed ASG. Therefore, ASG (initially 200 ?g) were decreased by 56.8% using Fructozyme L, by 53.6% using Novozym 960 and by 50.8% using lyophilized inulinase. Higher percentage of decrease can be achieved by adapting the condition parameters with regards to amount of enzyme, pH, temperature and dispersant added as it was obtained within SG hydrolysis with inulinase (almost 100%).

(11) The HPLC results (FIG. 4) also demonstrated that ASG are not yet fully hydrolysed. However, compared to the blank sample the ASG amount could be reduced by 59.8% with Fructozyme L, by 83.7% with Novozym 960 and by 42.1% with lyophilized inulinase.

(12) d) Samples with ASG Extracted from Soy Beans and Soy Lecithin

(13) The incubation ASG fraction extracted from soy beans and soy lecithin with Novozyme 960 (and additionally Fructozyme L for lecithin) also resulted in free sterols demonstrating the potential of inulinases to hydrolyse ASG in more complex system (containing traces of contaminants coming from raw material). The same free sterol pattern was observed as with the pure ASG samples sitosterol, campesterol, stigmasterol, stigmasta-5-23-dienol, sitostanol, d5-avenasterol.

(14) As shown in FIG. 5, the addition of 200 ?l Novozym 960 to lecithin ASG resulted in 291.1 ?g free sterols (=579.4 ?g hydrolysed ASG) whereas with 200 ?l Fructozyme L 209.1 ?g free sterols were obtained (=415.6 ?g hydrolysed ASG). The incubation of soy bean ASG with 200 ?l Novozym 960 released 343.8 ug free sterols (=683.5 ?g hydrolysed ASG). In the lecithin sample with Novozym 960, 19.1 ?g of remaining ASG were quantified whereas in the one with Fructozyme L 11.0 ?g ASG were found. In the soy bean sample, 135.1 ?g ASG remained intact.

(15) e) Conclusion

(16) ASG either as pure mixture or extracted from soy beans or soy lecithin were subjected to enzymatic hydrolysis in a water-based system with three different commercial inulinase preparations. The products of the hydrolysis, free sterols, were detected in all of the incubated samples demonstrating the capability of inulinases to cleave the glycosidic bond in ASG.