CHIMERIC ENZYME AND METHOD FOR PRODUCING TERMINAL ALKENES

Abstract

The present invention refers to a chimeric enzyme constructed for the production of terminal alkenes from fatty acids, so that the cofactor necessary for the reaction is produced in situ from industrial waste, favoring the circular economy. Furthermore, the present invention deals with the process of producing terminal alkenes using said chimeric enzyme. This invention belongs to the field of industrial biotechnology and finds application in the biofuels and renewable chemicals industry.

Claims

1. Chimeric enzyme for the production of terminal alkenes characterized by comprising the decarboxylase enzyme SEQ ID NO: 1 joined in its terminal portion to the beginning of the alditol oxidase enzyme SEQ ID NO: 2 by means of a linker sequence.

2. Chimeric enzyme according to claim 1, characterized by the fact that the binding sequence is selected from the group comprising SEQ ID NO: 3 and SEQ ID NO: 4.

3. Chimeric enzyme, according to claim 1, characterized by having SEQ ID NO: 5.

4. Chimeric enzyme, according to claim 1, characterized by having SEQ ID NO: 6.

5. Process for the production of terminal alkenes characterized by comprising the steps of: a) Contacting chimeric enzyme with a fatty acid in the presence of glycerol, forming a reaction medium; b) Keeping the reaction medium under heating and stirring for a period of time, and c) Collecting the product.

6. Process according to claim 5, characterized by the fact that the chimeric enzyme is the decarboxylase SEQ ID NO: 1 linked in its terminal portion to the beginning of the alditol oxidase SEQ ID NO: 2 by means of a linker sequence.

7. Process according to claim 6, characterized by the fact that the binding sequence is selected from the group comprising SEQ ID NO: 3 and SEQ ID NO: 4.

8. Process according to claim 5, characterized by the fact that the enzyme is selected from the group comprising SEQ ID NO: 5 and SEQ ID NO: 6.

9. Process according to claim 5, characterized by the fact that the fatty acid is selected from the group comprising capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1) or combinations thereof.

10. Process according to claim 5, characterized by the fact that the preferred ratio between chimeric enzyme and fatty acid in the medium is 1.0 Mol L.sup.-1 of enzyme for 0.5 mMol L.sup.-1 of fatty acid.

11. Process, according to claim 5, characterized by the fact that the concentration of glycerol in the medium is, preferably, between 0.5 and 10%.

12. Process, according to claim 5, characterized by the fact that the reaction medium must be heated to a temperature between 30 and 40 C., preferably to 37 C.

13. Process, according to claim 5, characterized by the fact that agitation imposed on the medium is up to 500 rpm, preferably 300 rpm.

14. Process according to claim 5, characterized by the fact that the pH of the reaction medium is maintained between 7.0 and 8.0, preferably 7.5.

15. Process, according to claim 5, characterized by the fact that the time is, preferably, 30 minutes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 graphically presents the percentage of conversion of myristic acid (C14:0) to its corresponding terminal alkene (1-tridecene) by means of the chimeric enzyme of the present invention, using said enzyme with flexible linking sequence (SEQ ID NO: 3) at 1 M, 0.5 mM myristic acid and concentrations between 0.5 and 10% of glycerol in the reaction medium (50 mM sodium phosphate buffer, pH 7.5). The reaction was carried out at 37 C. with slow stirring (300 rpm) for 30 min. Comparatively, the result of the conversion obtained using the decarboxylase enzyme (SEQ ID NO: 1) in the presence of hydrogen peroxide added to the medium (1 mM) is presented.

[0014] FIG. 2 graphically presents the percentage of conversion of myristic acid (C14:0) to its corresponding terminal alkene (1-tridecene) by means of the chimeric enzyme of the present invention, using said enzyme with a rigid binding sequence (SEQ ID NO: 4) at 1 M, 0.5 mM myristic acid and concentrations between 0.5 and 10% of glycerol in the reaction medium (50 mM sodium phosphate buffer, pH 7.5). The reaction was carried out at 37 C. with slow stirring (300 rpm) for 30 min. Comparatively, the result of the conversion obtained using the decarboxylase enzyme (SEQ ID NO: 1) in the presence of hydrogen peroxide added to the medium (1 mM) is presented.

DETAILED DESCRIPTION OF THE INVENTION

[0015] As previously stated, the present invention concerns a chimeric enzyme constructed for the production of terminal alkenes from fatty acids, as well as the process for the production of said alkenes using said enzyme. From this point onwards, aspects of the present invention are detailed.

[0016] The chimeric enzyme for the production of terminal alkenes of the present invention is characterized, firstly, by the fact that it performs the conversion of fatty acids using glycerol as a substrate for the in situ production of hydrogen peroxide.

[0017] The chimeric enzyme for the production of terminal alkenes of the present invention is characterized by comprising the decarboxylase enzyme SEQ ID NO: 1 linked in its terminal portion to the beginning of the alditol oxidase enzyme SEQ ID NO: 2, hydrogen peroxide generating enzyme, by means of a linker sequence. Said binding sequence is preferably selected from the group comprising SEQ ID NO: 3 and SEQ ID NO: 4.

[0018] Thus, an object of the present invention is a chimeric enzyme for the production of terminal alkenes characterized by having SEQ ID NO: 5, which is the decarboxylase enzyme SEQ ID NO: 1, linked in its terminal portion to the SEQ ID binding sequence NO: 3, in turn linked to the initial portion of the alditol oxidase enzyme SEQ ID NO: 2.

[0019] Also an object of the present invention is a chimeric enzyme for the production of terminal alkenes characterized by having SEQ ID NO: 6, which is the decarboxylase enzyme SEQ ID NO: 1, linked in its terminal portion to the binding sequence SEQ ID NO: 4, in turn linked to the initial portion of the enzyme alditol oxidase SEQ ID NO: 2.

[0020] The chimeric enzyme of the present invention has application in the conversion of fatty acids to alkenes. Therefore, a process for producing terminal alkenes is also an object of the present invention.

[0021] The process of the present invention is characterized by comprising the steps of: a) Contacting chimeric enzyme with a fatty acid in the presence of glycerol, forming a reaction medium; b) Keeping the reaction medium under heating and stirring for a period of time; and, c) Collecting the product.

[0022] In step a), the chimeric enzyme to be used is the decarboxylase SEQ ID NO: 1 joined in its terminal portion to the beginning of the alditol oxidase SEQ ID NO: 2 by means of a linker sequence. Said binding sequence is preferably selected from the group comprising SEQ ID NO: 3 and SEQ ID NO: 4. More specifically, the chimeric enzyme to be employed in step a) can be selected from the group comprising SEQ ID NO: 5 and SEQ ID NO: 6.

[0023] The fatty acid may be selected from the group comprising capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1) or combinations thereof.

[0024] Furthermore, the preferred ratio of chimeric enzyme to fatty acid in the medium is 1.0 Mol L.sup.-1 of enzyme for 0.5 mMol L.sup.-1 of fatty acid. Glycerol can be in concentrations preferably between 0.5 and 10% in the reaction medium.

[0025] In step b), the reaction medium must be heated to a temperature between 30 and 40 C., preferably 37 C.

[0026] The agitation imposed on the reaction medium at this stage of the process must be mild, up to 500 rpm, preferably 300 rpm.

[0027] The pH of the reaction medium must be maintained between 7.0 and 8.0, preferably 7.5.

[0028] Finally, the reaction medium must be maintained under the conditions set out for, preferably, 30 minutes.

[0029] In step c), the collected product can optionally be purified. For such a step, any separation and purification techniques known to one skilled in the art may be employed.

[0030] Below, as a basis, but without being restricted to them, examples of embodiments of the present invention are presented.

Example 1: Chimeric Enzyme Expression Procedure

[0031] The coding regions of the enzymes of the present invention (SEQ ID NO: 7 and SEQ ID NO: 8) were cloned individually into pET28a expression vectors, with a 6xHisTag tail to facilitate product purification. The recombinant vectors were transformed into E. coli BL21, and the expression of each of the chimeric enzymes was conducted in Terrific Broth (TB) medium. For expression, transformed bacteria were grown in 1 L of TB medium, at 30 C., containing the appropriate antibiotics. Upon reaching an optical density value equal to 1, the medium temperature was reduced to 18 C. and the induction of expression of the enzymes of the present invention was initiated with 0.2 mMol L.sup.-1 de IPTG, 20 Mol L.sup.-1 of hemin in 100 Mol L.sup.-1 of 5-aminolevulinate. After 24 h of induction, the cells were isolated and used to purify the chimeric enzymes. Purification was carried out on nickel resin, with 25 mMol L.sup.-1 sodium phosphate buffer (pH 7.5) and 500 mMol L.sup.-1 NaCl. Chimeric enzymes were recovered on an imidazole gradient, from 0-500 mMol L.sup.-1. Both constructions resulted in the production of soluble and functional chimeric enzymes (SEQ ID NO: 5 and SEQ ID NO: 6).

Example 2: Terminal Alkene Production Assay with Chimeric Enzymes

[0032] Conversion reactions of myristic acid to its corresponding terminal alkene (1-tridecene) were carried out with the chimeric enzymes separately, using a final volume of 1 mL. In all cases, the reaction medium was composed of the chimeric enzyme to be tested, at a concentration of 1 Mol L.sup.-1, the fatty acid, at a concentration of 0.5 mMol L.sup.-1, and 50 mM sodium phosphate buffer, pH 7.5. Different concentrations of glycerol (0.5, 1, 5 or 10%) were added to each reaction to evaluate the conversion rate (%) of myristic acid into 1-tridecene. All reactions were carried out at 37 C., with gentle stirring (300 rpm), for 30 min.

[0033] For comparison, a reaction with the traditional process of converting fatty acids to alkenes was carried out using the same process conditions, but replacing the chimeric enzymes with decarboxylase SEQ ID NO: 1 and glycerol with hydrogen peroxide at the concentration of 1 mM (final).

[0034] As a result, the traditional condition allowed a conversion of myristic acid to 1-tridecene of around 55% to be achieved. As can be seen in FIG. 1, the tests carried out with the chimeric enzyme SEQ ID NO: 5 of the present invention showed a conversion percentage between 40 and 50%, with the highest result being achieved with a concentration of 10% of glycerol. In FIG. 2, where the performance results of the chimeric enzyme SEQ ID NO: 6 are presented, a conversion between 40 and 55% can be seen, reaching these levels from 5% of glycerol in the reaction medium.

[0035] In this way, it is observed that the present invention makes it possible to generate peroxide in the reaction medium from glycerol. Instead of adding hydrogen peroxide to the medium, the addition of glycerol brings a new perspective to the disposal of this industrial waste in an effective way, using a single chimeric enzyme, without the need to use two enzyme systems to carry out the conversion reaction.