WO2022168062A1

WO2022168062A1 - Variants of hyperthermophilic carboxylesterase for polymer synthesis

Info

Publication number: WO2022168062A1
Application number: PCT/IB2022/051111
Authority: WO
Inventors: Alexandra Teresa PIRES CARVALHO; Beatriz LOPES COLUMBANO MARQUES ALMEIDA; Pedro Miguel REIS FIGUEIREDO; Daniel Fernando ANDRADE RIBEIRO DOURADO; Stephanie Paul; Derek John QUINN; Thomas S. Moody; Paula Andreia FERNANDES DE SOUSA; Armando Jorge Domingues Silvestre DOMINGUES SILVESTRE
Original assignee: Universidade De Coimbra; Universidade De Aveiro
Priority date: 2021-02-08
Filing date: 2022-02-08
Publication date: 2022-08-11

Abstract

The present invention discloses enzymatic variants comprising a sequence comprising at least one amino acid substitution at a position selected from the group consisting of mutations located close to the oxyanion hole region, mutations close to the catalytic His-Asp pair and/or mutations close to residues that interact with the lactone-ring and mutations outside the active site. A process for the synthesis of polymers comprising the step of using an enzymatic variant according to the invention is also disclosed, as well as the use of the enzymatic variants of the invention in biotechnology, in particular in polymer synthesis, in material industry and/or for biomedical applications.

Description

DESCRIPTION

VARIANTS OF HYPERTHERMOPHILIC CARBOXYLESTERASE FOR POLYMER SYNTHESIS

FIELD OF THE INVENTION

The present invention is enclosed in the area of biochemistry and biomedicine, namely in the area of biocatalysis and biopolymers.

PRIOR ART

Archaeoglobus members are hyperthermophiles that can be found in hydrothermal vents, oil deposits, and hot springs. They can produce biofilm when subjected to environmental stresses such as extreme pH or temperature, high concentrations of metal, or the addition of antibiotics, xenobiotics, or oxygen. These archaeons are known to cause the corrosion of iron and steel in oil and gas processing systems by producing iron sulphide. Their biofilms, however, may have industrial or research applications in the form of detoxifying metal contaminated samples or to gather metals in an economically recoverable form.

Polyesters such as polycaprolactone (PCL) and Polycaprolactone- Polyethylene Glycol (PCL-PEG) are widely used in several biomedical applications, e.g. systems for drug and gene delivery, but has also extended to include proteins, peptides, vaccines and other bioactive molecules (antigens, antibodies, ribozymes, nerve growth factor, heparin, steroids, hormones and vitamins, among others); coatings in implant materials for tissue engineering (bone, cartilage, cardiovascular, blood vessel, skin, nerve, tendon, dental and ligament engineering, among others); orthopaedic devices, resorbable sutures; contraceptive devices; fixation devices; cell culture and others.^1-5 Polyester synthesis is mainly performed by chemical approaches but enzyme-catalyzed ring-opening polymerization (eROP) is considered one of the most promising approaches for the synthesis of polymeric biomaterials.⁶'⁷

Overall, enzymatic synthesis has several advantages over traditional chemical synthesis which make them better suited to obtain products for biomedical applications, namely by: 1) the use of milder/greener reaction conditions in terms of temperature and pressure conditions; 2) the type of solvents involved; 3) the high control of stereo-, chemo-, regio- and choro-selectivity; 4) the absence of toxic metal and/or orga no-catalysts.^7,8 Yet, enzymatic PCL and PCL-PEG synthesis is currently not conducted at industrial scale.

The most tested enzymes for polyester synthesis are the immobilized forms of Candida antarctica lipase B (CalB). The most common form is immobilized on Lewatit VP OC 1600 (Novozyme 435).^9-12 This form was previously shown to produce these polymers at a wide range of sizes. For example, PCL (number average of molecular weight (Mn) of 9,480 g/mol in toluene at 60 °C for 88 hr), PCL-PEG (63-70% yield at 70 °C, Mn of 12,500-17,600 g/mol¹³ and in a latter work, Mn of 11,900-19,000 g/mol at 70 °C, 1.28-1.59 polydispersity index¹⁴). Controlling the size of the polyesters is crucial for the applications, since the Mn and polydispersity of the polymers affect the stability and diameter of nanoparticles that can be obtained from them. The nanoparticles diameter is then related to their permeability and retention for drug delivery applications and other physical-chemical properties. Other limitations include cost issues of enzyme immobilization at large scale and the use of petroleum-based carriers for enzyme immobilization is not truly green. Thermophilic enzymes are easier to purify when expressed in mesophilic hosts and have a higher resistance to chemical denaturants. Reactions at higher temperatures also provide fewer risks for microbial contamination.¹⁵ Finally, and specifically regarding polymerization reactions, high temperature decreases the viscosity of the medium and aggregation of the resulting polymer products, allowing for the enzyme to more easily access the polymer units, which is very promising for large-scale polyesters synthesis. Notwithstanding these attractive features, wild-type (WT) hyperthermostable enzymes are still not particularly adequate for these polyesterification reactions. For example, the hyperthermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus (AfEST) in the free and immobilized forms allows the formation of polymeric chains with similar Mn values between 670-1,580 g/mol and monomer conversion ratios between 45-100% at 80 °C.^16-18 These Mn values are low and there is a large variation in their sizes, meaning that the enzyme forms small polymer chains of variable length. As stated before, controlling the size of the polyesters is crucial for the applications.

Compared with CalB, AfEST displayed a better Michaelis-Menten constant ( M) for E-caprolactone (E-CI), but an inferior rate constant (k_cat).¹⁶ Both enzymes have an a/|3 hydrolase fold composed by a Ser-His-Asp catalytic triad and an oxyanion hole region responsible for the stabilization of the negative charge developed on the oxygen atom of the tetrahedral intermediate structures.¹⁹'²⁰ The catalytic serine residue act as the nucleophile and the histidine as an acid/base (transferring protons between the catalytic serines and the substrate), stabilized by the aspartate residue.^19,21'²² These enzymes' active sites differ on the residues that make the oxyanion hole and on the sizes and orientation of the acyl- and alcohol-binding pockets.²³'²⁴ Additionally, AfEST has a cap domain composed of five helices from two separate regions (residues 1-54 and 188-246), ²⁴ whereas CalB active site is flanked by two highly mobile short a-helixes, a5 (residues 142-146) and alO (residues 268-287) helixes, where the former acts as the putative lid.²⁵

Protein engineering has been essential to better understand proteins' function, enzyme dynamics, and active site architectures.²⁶ AfEST has been engineered to improve affinity with organophosphorus compounds²⁷ and in the resolution of ibuprofen esters.²⁸ Here, this enzyme was used for the first time, as a starting point and as a proof-of-concept, to become more similar in terms of polyester synthesis activity to CalB, without compromising its stability. The rational approach was based on a detailed characterization of the reaction profiles for the acylation step of ROP reaction (Figure 1). A computational design and experimental validation for AfEST mutants was developed to improve PCL and triblock of PCL-b-PEG-b-PCL synthesis. However, until now, Archaeoglobus fulgidus has not been engineered for polymerization reactions, which is the focus of the present invention. The present solution intended to innovatively overcome such issues.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to reengineer the thermophilic carboxylesterase, namely from Archaeoglobus fulgidus for a more efficient synthesis of aliphatic polyesters, particularly PCL or PCL-PEG.

The reengineering approach of the present invention is based on the detailed mechanistic characterization of the eROP for PCL and PCL-PEG copolymers synthesis by the CalB lipase and AfEST carboxylesterase enzymes. By changing the active site of AfEST in such a way to optimize the active site arrangement and by achieving the best compromise possible between first transition state (TSi) and second transition state (TS2) stabilization, the inventors were able to design new variants for the eROP reactions with E-CI.

The present invention provides enzymatic variants of Archaeoglobus fulgidus or Candida antarctica that allow for the more efficient synthesis (in terms of product yield and size) of the aliphatic polyesters (PCL and PCL-b-PEG-b-PCL) that are interesting biomaterials.

In a main embodiment, the present invention discloses an enzymatic variant comprising a sequence comprising at least one amino acid substitution at a position selected from the group consisting of: mutations located close to the oxyanion hole region; mutations close to the catalytic His-Asp pair; and/or mutations close to residues that interact with the lactone-ring and mutations outside the active site.

For the purposes of interpreting the term "close" of the claims, it is intended to encompass a 5-15 A radius, particularly 10.0 A radius.

In a further embodiment, the present disclosure provides an enzymatic variant wherein the sequence comprises at least 90% homology with SEQ. ID 1, preferably at least 95% homology, even preferably at least 97% homology and even more preferably at least 99% homology. For the purposes of establishing homology any webserver tool for sequence alignments can be used, as for example web.expasy.org/sim.

In a further embodiment, the present disclosure provides an enzymatic variant wherein the mutations located close to the oxyanion hole region comprise the amino acid substitutions G89T, G89A, G89V, G89S, G88S, F90P and/or A161V.

In a further embodiment, the present disclosure provides an enzymatic variant wherein the mutations close to the catalytic His-Asp pair comprise the amino acid substitutions L257P, L257A, L284F, L284W, Y188N, Y188A and/or I209W.

In a further embodiment, the present disclosure provides an enzymatic variant wherein mutations close to residues that interact with the lactone-ring comprise V190Q, V190N, V190T, V190D, F218A, F218N, M215A, M215L, D211G, L210A and/or L210N.

In a further embodiment, the present disclosure provides an enzymatic variant wherein mutations outside the active site comprise N44S, N289W, I288F, 1288V, G206E, F17A, F23L, del2-27, del2-27/l 209F, del2-27/l209W and/or del2-27/L210F.

In a further embodiment, the present disclosure provides an enzymatic variant comprising a carboxylesterase, preferably a thermophilic carboxylesterase, even more preferably a hyperthermophilic carboxylesterase.

In a further embodiment, the present disclosure provides an enzymatic variant wherein the carboxylesterase is from Archaeoglobus fulgidus or Candida antarctica.

In a further embodiment, the present disclosure provides an enzymatic variant wherein it is Candida antarctica lipase B.

In a particular embodiment, the present invention further discloses a process for the synthesis of polymers comprising the step of using an enzymatic variant according to any embodiment of the present disclosure.

In a particular embodiment, the present invention further discloses a polymer obtained by the method of the previous claim, particularly polymers of aliphatic nature with ester linkages, namely polycaprolactone or polycaprolactone-polyethylene glycol or tri-block of PCL-b-PEG-b-PCL.

In a particular embodiment, the present invention further discloses a material comprising the polymer of the previous claim.

In a particular embodiment, the present invention further discloses the use of the enzymatic variant of the present invention in biotechnology, in particular in polymer synthesis, in material industry and/or for biomedical applications.

DESCRIPTION OF FIGURES

Figure 1 shows the first half part of the catalytic cycle, which concerns the nucleophilic attack of the serine side-chain oxygen to the carbonyl carbon of the E-CI substrate, which occurs concomitantly with proton transfer from the serine side-chain oxygen to the histidine residue forming the first tetrahedral intermediate structure (I NT-1).

Figure 2 shows the primary amino acid sequence of WT-AfEST and WT-CalB.

Figure 3 shows the dot plots for all expressed enzymatic variants 1. AfEST WT; 2. AfEST G89T; 3. AfEST G89A; 4. AfEST G89V; 5. AfEST G89S; 6. AfEST G88S; 7. AfEST F90P; 8. AfEST V190Q; 9. AfEST V190N; 10. AfEST V190T; 11. AfEST V190D; 12. AfEST L257P; 13. AfEST L257A; 14. AfEST L284F; 15. AfEST L284W; 16. AfEST N44S; 17. AfEST N289W; 18. AfEST I288F; 19. AfEST 1288V; 20. AfEST Y188N; 21. AfEST Y188A; 22. AfEST G206E; 23. AfEST D211G; 24. AfEST L210A; 25. AfEST L210N; 26. AfEST A161V; 27. AfEST I209W; 28. AfEST F17A; 29. AfEST F23L; 30. AfEST F218A; 31. AfEST F218N; 32. AfEST M215A; 33. AfEST M215L; 34. AfEST L2_S27.del ; 35. AfEST L2-S27.del J209F; 36. AfEST L2-S27.del J209W; 37. AfEST L2-S27.del _L210F; 38. AfEST G89V_V190Q; 39. AfEST G89V_L284F; 40. AfEST F17L_G89V; 41. AfEST F23L_G89V; 42. AfEST M215L_L257A; 43. AfEST F23L_G89V_V190Q; 44. CalB WT; 45. CalB T40G; 46. CalB T40A; 47. CalB T40S; 48. CalB V149G; 49. CalB W104A; 50. CalB D187A. DETAILED DESCRIPTION

The more general and advantageous configurations of the present invention are described in the Summary of the invention above. Such configurations are detailed below in accordance with other advantageous and/or preferred embodiments of implementation of the present invention.

A preferred embodiment of the present invention relates to engineered variants of hyperthermophilic carboxylesterase from the archaeon AfEST sequence of WT AfEST SEQ ID 1 (Figure 2) that improve product yield and the polymers size in the synthesis of poly(E-caprolactone) (PCL) and tri-block of PCL-b-poly(ethylene glycol)-b- PCL (PCL-b-PEG-b-PCL). Each variant comprises at least one amino acid substitution at a position selected from the group consisting of: mutations located near the oxyanion hole region (including the amino acid substitutions G89T, G89A, G89V, G89S, G88S, F90P and A161V); mutations near the catalytic His-Asp pair (including the amino acid substitutions L257P, L257A, L284F, L284W, Y188N, Y188A, I209W); mutations near residues that interact with the lactone-ring (V190Q, V190N, V190T, V190D, F218A, F218N, M215A, M215L, D211G, L210A and L210N) and mutations outside the active site (N44S, N289W, I288F, 1288V, G206E, F17A, F23L, del2-27, del2-27/l209F, del2-27/l209W and del2-27/L210F). It also includes variations in SEQ ID 2 positions T40G, T40A, T40S, V149G, W104A, D187A (Figure 2).

The newly reported variants were designed and screened with computational methods, namely Molecular Dynamics and Quantum Mechanics/Molecular Mechanics. After a detailed characterization of the enzymes' landscapes, the genes for these mutants and WTs were synthetized and vector cloned. Plasmids were transformed into BL21 E. coli competent cells and grown on antibiotic selection plates at 37 °C. After treatment, enzyme expression was detected using a dot blot (Figure 3) and the enzymes isolated.

To test the performance of these mutants, PCL eROP reactions were carried out with E-CI and the mutant dissolved in toluene at 70 °C and 90 °C for 72 hr. In the case of the assays for PCL-PEG synthesis, PEG4000 were added as well. To evaluate the remaining E-CI, Gas Chromatography (GC) was applied to the isolated product samples. Furthermore, the synthesized PCL and PCL-PEG copolymers were extensively characterized by means of Attenuated total reflectance Fourier transform infrared (ATR FTIR) spectroscopy and by Proton Nuclear Magnetic Resonance (¹H NMR) to assess their main structural features.

Through the main FTIR spectroscopic features observed for all dried samples, notably: two main bands near 2,942 and 2,964 cm ¹ and a small shoulder at ca. 2,880 cm'¹, arising from the antisymmetric and symmetric stretching of C-H bond of methylene groups, characteristic of both PEG and PCL moieties; one very intense and sharp vibrational band near 1,719 cm , attributed to the carbonyl stretching mode (C=O) of an ester moiety, characteristic of the PCL moiety; a band at 1,469 cm⁴, attributed to the C-H bending vibration of PEG moiety; and another two high intensity bands at 1,237 and 1,173 cm ¹ attributed to the asymmetric and symmetric C-O-C stretching mode, also from an ester group, characteristic of PCL units.²⁹ Additionally, a broad band centered at around 3,450 cm ¹ arising from the terminal hydroxyl groups is also observed, which is consistent with the attainment of relatively low molecular weight polymers. Nevertheless, the relative intensity of this band compared with, for example those at 2,940 and 2,963 cm⁴, clearly decreases for those polymers obtained in higher amounts and compared with WT-CalB (e.g. L257A versus WT-CalB), thus, indicating higher molecular weights. All these attributions are in accordance with literature values for similar polymers and also with the polymers obtained with WT enzymes.¹⁴'²⁹'³⁰ These characterizations confirmed the success of the new AfEST-mutant mediated polymer synthesis, especially regarding the WT-AfEST and WT-CalB enzymes (Figures 3 and 4). Moreover, the main spectroscopic features of the polymers enzymatically synthesized, confirmed the predominant aliphatic nature with ester linkages, typical of PCL-PEG copolymers.

Importantly, the reactions carried out with the AfEST mutants provided in general a more efficient route to prepare PCL homopolymer and PCL-PEG copolymer, than simply using the WT or even compared to WT-CalB. For example, when comparing the PCL-PEG polymerization reaction carried out at 70 °C using WT-AfEST and L257A mutant, the yield increased from 12 to 49%. Another important observation was that PCL-PEG was typically isolated in higher yields than in the case of PCL, irrespective of the mutant used. This differs from the prior polymers synthesis methods because the engineered variants of the present invention are designed to improve the polyesters yield and size and present mutations in the positions reported on Table 1.

The designed variants were able to improve the yield of the products meaning that more substrate was converted into the polyesters, instead of the acid intermediate (6-hydroxycaprioc acid). FTIR analysis showed that the best enzymatic variants had lower intensity signals for hydroxyl ends, meaning that they formed very low amounts of the lower molecular weight polymers.

Table 1 below shows the product yields (in percentage) for the enzyme variants when compared with the WT enzymes, for the synthesis of PCL and PCL-PEG at 70 and 90 °C. Enzyme 1 is the WT-AfEST; Enzymes 44 and 51 are the WT-CalB; Enzymes 2- 43 are the AfEST mutants; Enzymes 45-50 and 52-57 are the CalB mutants.

Table 1 (continues in the next page)

PCL PCL-PEG

70 °C 90°C 70 °C 90°C

1 AfEST WT 9 12 9 44

2 G89T 45 34 2 39

3 G89A 32 47 26 56

4 G89V 0 19 15 18

5 G89S 0 18 16 51

6 G88S 7 9 13 16

7 F90P 0 15 5 9

8 V190Q 30 53 16 47

9 V190N 19 53 8 52

10 V190T 16 52 39 64

11 V190D 25 59 29 50

12 L257P 4 38 7 33

13 L257A 29 48 27 55

14 L284F 40 55 34 57

15 L284W 34 55 70 66

16 N44S 15 48 59 61

17 N289W 7 43 28 50

18 I288F 24 61 57 57

19 1288V 10 54 41 53 20 Y188N 0 44 15 47

21 Y188A 10 34 5 31

22 G206E 10 47 68 63

23 D211G 12 48 60 103

24 L210A 22 52 64 55

25 L210N 10 45 46 56

26 A161V 0 24 27 35

27 I209W 8 47 39 55

28 F17A 14 53 38 44

29 F23L 4 36 21 56

30 F218A 8 42 31 67

31 F218N 2 29 21 46

32 M215A 5 45 32 60

33 M215L 12 48 33 43

34 X2-27 0 31 7 26

35 X2-27, 1209F 4 26 1 31

36 X2-27, 1209W 0 23 13 29

37 X2-27, L210F 0 27 9 1

38 G89V, V190Q 0 8 0 8

39 G89V, L284F 0 18 9 8

40 F17L, G89V 5 4 0 3

41 F23L, G89V 4 9 0 0

42 M215L, L257A 60 42 9 28

43 F23L, G89V,

0 0 8 2

V190Q

44 CALB WT-1 0 9 0 8

45 T40G-1 0 0 0 0

46 T40A-1 0 0 0 0

47 T40S-1 0 0 0 0

48 V149G-1 0 0 0 0

49 W104A-1 0 0 0 0

50 D187A-1 0 0 0 0

51 CALB WT-2 0 9 0 8

52 T40G-2 0 0 0 0

53 T40A-2 0 0 0 0

54 T40S-2 0 0 0 0

55 V149G-2 0 0 0 0

56 W104A-2 0 0 0 0

57 D187A-2 0 0 0 0

As will be clear to one skilled in the art, the present invention should not be limited to the embodiments described herein, and a number of changes are possible which remain within the scope of the present invention. Of course, the preferred embodiments shown above are combinable, in the different possible forms, being herein avoided the repetition all such combinations.

REFERENCES

(1) Woodruff, M. A.; Hutmacher, D. W. The Return of a Forgotten Polymer— Polycaprolactone in the 21st Century. Progress in Polymer Science 2010, 35 (10), 1217-1256. https://doi.Org/10.1016/j.progpolymsci.2010.04.002.

(2) Kumari, A.; Yadav, S. K.; Yadav, S. C. Biodegradable Polymeric Nanoparticles Based Drug Delivery Systems. Colloids and Surfaces B: Biointerfaces 2010, 75 (1), 1-18. https://doi.Org/10.1016/j.colsurfb.2009.09.001.

(3) Dash, T. K.; Konkimalla, V. B. Poly-e-Caprolactone Based Formulations for Drug Delivery and Tissue Engineering: A Review. Journal of Controlled Release 2012, 158 (1), 15-33. https://doi.Org/10.1016/j.jconrel.2011.09.064.

(4) Sinha, V. R.; Bansal, K.; Kaushik, R.; Kumria, R.; Trehan, A. Poly-e-Caprolactone Microspheres and Nanospheres: An Overview. International Journal of Pharmaceutics 2004, 278 (1), 1-23. https://doi.Org/10.1016/j.ijpharm.2004.01.044.

(5) Seyednejad, H.; Ghassemi, A. H.; van Nostrum, C. F.; Vermonden, T.; Hennink, W. E. Functional Aliphatic Polyesters for Biomedical and Pharmaceutical Applications. Journal of Controlled Release 2011, 152 (1), 168-176. https://doi.Org/10.1016/j.jconrel.2010.12.016.

(6) Jerome, C.; Lecomte, P. Recent Advances in the Synthesis of Aliphatic Polyesters by Ring- Opening Polymerization. Advanced Drug Delivery Reviews 2008, 60 (9), 1056-1076. https://doi.Org/10.1016/j.addr.2008.02.008.

(7) Zhang, J.; Shi, H.; Wu, D.; Xing, Z.; Zhang, A.; Yang, Y.; Li, Q. Recent Developments in Lipase-Catalyzed Synthesis of Polymeric Materials. Process Biochemistry 2014, 49 (5), 797-806. https://doi.Org/10.1016/j.procbio.2014.02.006.

(8) Kadokawa, J.; Kobayashi, S. Polymer Synthesis by Enzymatic Catalysis. Curr Opin Chem Biol 2010, 14 (2), 145-153. https://doi.Org/10.1016/j.cbpa.2009.ll.020.

(9) Poojari, Y.; Beemat, J. S.; Clarson, S. J. Enzymatic Synthesis of Poly(e-Caprolactone): Thermal Properties, Recovery, and Reuse of Lipase B from Candida Antarctica Immobilized on Macroporous Acrylic Resin Particles. Polymer Bulletin 2013, 70 (5), 1543-1552. https://doi.org/10.1007/s00289-013-0916-l.

(10) Polloni, A. E.; Veneral, J. G.; Rebelatto, E. A.; de Oliveira, D.; Oliveira, J. V.; Araujo, P. H.

H.; Sayer, C. Enzymatic Ring Opening Polymerization of co-Pentadecalactone Using Supercritical Carbon Dioxide. The Journal of Supercritical Fluids 2017, 119, 221-228. https://doi.Org/10.1016/j.supflu.2016.09.019.

(11) Zhao, H.; Nathaniel, G. A.; Merenini, P. C. Enzymatic Ring-Opening Polymerization (ROP) of Lactides and Lactone in Ionic Liquids and Organic Solvents: Digging the Controlling Factors. RSC Adv. 2017, 7 (77), 48639-48648. https://doi.org/10.1039/C7RA09038B. (12) Pellis, A.; Comerford, J. W.; Weinberger, S.; Guebitz, G. M.; Clark, J. H.; Farmer, T. J.

Enzymatic Synthesis of Lignin Derivable Pyridine Based Polyesters for the Substitution of Petroleum Derived Plastics. Nature Communications 2019, 10 (1), 1762. https://doi.org/10.1038/s41467-019-09817-3.

(13) He, F.; Li, S.; Vert, M.; Zhuo, R. Enzyme-Catalyzed Polymerization and Degradation of Copolymers Prepared from e-Caprolactone and Polyethylene Glycol). Polymer 2003, 44 (18), 5145-5151. https://doi.org/10.1016/S0032-3861(03)00562-7.

(14) Huang, Y.; Li, L.; Li, G. An Enzyme-Catalysed Access to Amphiphilic Triblock Copolymer of PCL-b-PEG-b-PCL: Synthesis, Characterization and Self-Assembly Properties. Designed Monomers and Polymers 2015, 18 (8), 799-806. https://doi.org/10.1080/15685551.2015.1078113.

(15) Vieille, C.; Zeikus, G. J. Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability. Microbiol. Mol. Biol. Rev. 2001, 65 (1), 1-43. https://doi.Org/10.1128/MMBR.65.l.l-43.2001.

(16) Ma, J.; Li, Q..; Song, B.; Liu, D.; Zheng, B.; Zhang, Z.; Feng, Y. Ring-Opening Polymerization of E-Caprolactone Catalyzed by a Novel Thermophilic Esterase from the Archaeon Archaeoglobus Fulgidus. Journal of Molecular Catalysis B: Enzymatic 2009, 56 (2), 151-157. https://doi.Org/10.1016/j.molcatb.2008.03.012.

(17) Li, G.; Li, Q. Thermophilic Esterase from the Archaeon Archaeoglobus Fulgidus Physically Immobilized on Hydrophobic Macroporous Resin: A Novel Biocatalyst for Polyester Synthesis. Biotechnol Bioproc E 2011, 16 (6), 1201-1207. https://doi.org/10.1007/sl2257-011-0260-y.

(18) Ren, H.; Xing, Z.; Yang, J.; Jiang, W.; Zhang, G.; Tang, J.; Li, Q. Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(e-Caprolactone) Synthesis. Molecules 2016, 21 (6). https://doi.org/10.3390/molecules21060796.

(19) Bezborodov, A. M.; Zagustina, N. A. Lipases in Catalytic Reactions of Organic Chemistry. Appl Biochem Microbiol 2014, 50 (4), 313-337. https://doi.org/10.1134/S0003683814040024.

(20) Simon, L.; Goodman, J. M. Enzyme Catalysis by Hydrogen Bonds: The Balance between Transition State Binding and Substrate Binding in Oxyanion Holes. J. Org. Chem. 2010, 75 (6), 1831-1840. https://doi.org/10.1021/jo901503d.

(21) Douka, A.; Vouyiouka, S.; Papaspyridi, L.-M.; Papaspyrides, C. D. A Review on Enzymatic Polymerization to Produce Polycondensation Polymers: The Case of Aliphatic Polyesters, Polyamides and Polyesteramides. Progress in Polymer Science 2018, 79, 1-25. https://doi.Org/10.1016/j.progpolymsci.2017.10.001.

(22) Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E.; Dodson, G.; Tolley, S.; Turkenburg, J. P.; Christiansen, L.; Huge-Jensen, B.; Norskov, L.; Thim, L.; Menge, U. A Serine Protease Triad Forms the Catalytic Centre of a Triacylglycerol Lipase. Nature 1990, 343 (6260), 767. https://doi.org/10.1038/343767a0.

(23) Stauch, B.; Fisher, S. J.; Cianci, M. Open and Closed States of Candida Antarctica Lipase B: Protonation and the Mechanism of Interfacial Activation. J. Lipid Res. 2015, 56 (12), 2348- 2358. https://doi.org/10.1194/jlr.M063388. (24) De Simone, G.; Menchise, V.; Manco, G.; Mandrich, L.; Sorrentino, N.; Lang, D.; Rossi, M.; Redone, C. The Crystal Structure of a Hyper-Thermophilic Carboxylesterase from the Archaeon Archaeoglobus FulgidusllEdited by R. Huber. Journal of Molecular Biology 2001, 314 (3), 507-518. https://doi.org/10.1006/jmbi.2001.5152.

(25) Uppenberg, J.; Hansen, M. T.; Patkar, S.; Jones, T. A. The Sequence, Crystal Structure Determination and Refinement of Two Crystal Forms of Lipase B from Candida Antarctica. Structure 1994, 2 (4), 293-308. https://doi.org/10.1016/S0969-2126(00)00031-9.

(26) Lutz, S.; lamurri, S. M. Protein Engineering: Past, Present, and Future. Methods Mol. Biol. 2018, 1685, 1-12. https://doi.org/10.1007/978-l-4939-7366-8_l.

(27) Wang, Y.; Li, B.; Han, W.; Yang, G.; Zhang, Z.; Feng, Y. Redesigning the Active Site of a Carboxyl Esterase from the Archaeon Archaeoglobus Fulgidus to Improve Sensitivity to Organophosphorus Compounds. Process Biochemistry 2012, 47 (12), 2219-2226. https://doi.Org/10.1016/j.procbio.2012.08.021.

(28) Ma, F.; Chung, M. T.; Yao, Y.; Nidetz, R.; Lee, L. M.; Liu, A. P.; Feng, Y.; Kurabayashi, K.; Yang, G.-Y. Efficient Molecular Evolution to Generate Enantioselective Enzymes Using a DualChannel Microfluidic Droplet Screening Platform. Nature Communications 2018, 9 (1), 1030. https://doi.org/10.1038/s41467-018-03492-6.

(29) Ulker, C.; Gokalp, N.; Guvenilir, Y. Enzymatic Synthesis and Characterization of Polycaprolactone by Using Immobilized Lipase onto a Surface-Modified Renewable Carrier. Polish Journal of Chemical Technology 2016, 18 (3), 134-140. https://doi.org/10.1515/pjct- 2016-0060.

(30) Sousa, A. F.; Coelho, J. F. J.; Silvestre, A. J. D. Renewable-Based Poly((Ether)Ester)s from

2,5-Furandicarboxylic Acid. Polymer 2016, 98, 129-135. https://doi.Org/10.1016/j.polymer.2016.06.015.

Claims

1. Enzymatic variant comprising a sequence comprising at least one amino acid substitution at a position selected from the group consisting of: mutations located close to the oxyanion hole region; mutations close to the catalytic His-Asp pair; and/or mutations close to residues that interact with the lactone-ring and mutations outside the active site.

2. Enzymatic variant according to the previous claim wherein the sequence comprises at least 90% homology with SEQ. ID 1, preferably at least 95% homology, even preferably at least 97% homology and even more preferably at least 99% homology.

3. Enzymatic variant according to any of the previous claims, wherein the mutations located close to the oxyanion hole region comprise the amino acid substitutions G89T, G89A, G89V, G89S, G88S, F90P and/or A161V.

4. Enzymatic variant according to any of the previous claims, wherein the mutations close to the catalytic His-Asp pair comprise the amino acid substitutions L257P, L257A, L284F, L284W, Y188N, Y188A and/or I209W.

5. Enzymatic variant according to any of the previous claims, wherein mutations close to residues that interact with the lactone-ring comprise V190Q, V190N, V190T, V190D, F218A, F218N, M215A, M215L, D211G, L210A and/or L210N.

6. Enzymatic variant according to any of the previous claims wherein mutations outside the active site comprise N44S, N289W, I288F, 1288V, G206E, F17A, F23L, del2-27, del2-27/l 209F, del2-27/l209W and/or del2-27/L210F.

7. Enzymatic variant according to any of the previous claims comprising a carboxylesterase, preferably a thermophilic carboxylesterase, even more preferably a hyperthermophilic carboxylesterase.

8. Enzymatic variant according to any the previous claims wherein the carboxylesterase is from Archaeoglobus fulgidus or Candida antarctica.

9. Enzymatic variant according to any of the preceding claims wherein it is Candida antarctica lipase B.

10. Process for the synthesis of polymers comprising the step of using an enzymatic variant according to any of the previous claims.

11. Polymer obtained by the method of the previous claim, particularly polymers of aliphatic nature with ester linkages, namely polycaprolactone or polycaprolactone-polyethylene glycol or tri-block of PCL-b-PEG-b-PCL.

12. Material comprising the polymer of the previous claim.

13. Use of the enzymatic variant of any one of claims 1-9 in biotechnology, in particular in polymer synthesis, in material industry and/or for biomedical applications.