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Lipase Specificity and Selectivity: Engineering, Kinetics and Applied Catalysis
KTH, School of Biotechnology (BIO), Biochemistry.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The specificity and selectivity of the enzyme Candida antarctica lipase B (CALB) were studiedfor several substrates and applications.With help of molecular modeling, the active site of CALB was redesigned for the ring openingpolymerization of D,D‐lactide. Two mutants, with about 90‐fold increase in activity ascompared to the wild‐type enzyme, were created. Changing a glutamine into alanineaccounted for this increase in both mutants by creating a larger space in the acyl donorpocket. The new space made it possible to accommodate the bulky substrate and improvethe transition state‐active site complementarity during polymer chain propagation.The enantioselectivity of CALB towards secondary alcohols was engineered by rationalredesign of the stereoselectivity pocket in the enzyme active site. A larger space created by asingle point mutation resulted in an 8’300’000 times change in enantioselectivity towards 1‐phenylethanol and the enantiopreference was inverted into S‐preference. The activitytowards the S‐enantiomer increased 64’000 times in the mutant as related to the wild‐type.The solvent and temperature effects on the enantioselectivity were studied for severalsubstrates and revealed the importance of entropy in the change in enantioselectivity.Substrate selectivity is of great importance for the outcome of enzyme catalyzed polymersynthesis. Ring opening polymerization (ROP) of γ‐acyloxy‐ε‐caprolactones will result in apolyester chain with pendant functional groups. CALB was found to have activity not onlytowards the lactone but also towards the γ‐ester leading to rearrangement of the monomersyielding γ‐acetyloxyethyl‐γ‐butyrolactone. This selectivity between the lactone and the γ‐ester was dependent on the type of group in the γ position and determined the ratio ofpolymerization and rearrangement of the monomers. Molecular dynamics simulations wereused to gain molecular understanding of the selectivity between the lactone and γ‐ester.In order to obtain (meth)acrylate functional polyesters we investigated the use of 2‐hydroxyethyl (meth)acrylate (HEA and HEMA) as initiators for ring opening polymerization.We found that, in addition to the ring opening polymerization activity, CALB catalyzed thetransacylation of the acid moiety of the initiators. The selectivity of CALB towards thedifferent acyl donors in the reaction resulted in a mixture of polymers with different endgroups. A kinetic investigation of the reaction showed the product distribution with timewhen using HEA or HEMA with ε‐caprolactone or ω‐pentadecalactone.The high selectivity of CALB towards lactones over (meth)acrylate esters such as ethyleneglycol di(meth)acrylate was used to design a single‐step route for the synthesis ofdi(meth)acrylated polymers. By mixing ω‐pentadecalactone with the ethylene glycoldi(meth)acrylate and the enzyme in solvent free conditions, we obtained >95 % ofdi(meth)acrylated polypentadecalactone.Taking advantage of the high chemoselectivity of CALB, it was possible to synthesizepolyesters with thiol and/or acrylate functional ends. When using a thioalcohol as initiatorCALB showed high selectivity towards the alcohol group over the thiol group as acyl acceptorfor the ROP reaction. The enzymatic ability of catalyzing simultaneous reactions (ROP andtransacylation) it was possible to develop a single‐step route for the synthesis ofdifunctionalized polyesters with two thiol ends or one thiol and one acrylate end by mixingthe initiator, lactone and a terminator.

Place, publisher, year, edition, pages
Stockholm: KTH , 2010. , viii, 48 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:17
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-25039ISBN: 978-91-7415-729-1 (print)OAI: oai:DiVA.org:kth-25039DiVA: diva2:355255
Public defence
2010-10-22, F3, Lindstedtsvägen 26, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20101006Available from: 2010-10-06 Created: 2010-10-06 Last updated: 2010-10-06Bibliographically approved
List of papers
1. Rational redesign of Candida antarctica lipase B for the ring opening polymerization of D,D-lactide
Open this publication in new window or tab >>Rational redesign of Candida antarctica lipase B for the ring opening polymerization of D,D-lactide
2011 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 47, no 26, 7392-7394 p.Article in journal (Refereed) Published
Abstract [en]

Based on molecular modelling, the enzyme Candida antarctica lipase B was redesigned as a catalyst for the ring opening polymerization of D, D-lactide. Two mutants with 90-fold increased activity as compared to the wild-type enzyme were created. In a preparative synthesis of poly(D,D-lactide) the mutants greatly improved the rate and the degree of polymerization.

Keyword
LACTIDE, POLY(LACTIDE-CO-GLYCOLIDE), POLYLACTIDES, POLYESTERS
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-25043 (URN)10.1039/c1cc10865d (DOI)000291823400028 ()2-s2.0-79959407879 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, IRENE, contract no. 227279, FP7-KBBE-2008-2B
Note
QC 20101006 Updated from manuscript to article in journalAvailable from: 2010-10-06 Created: 2010-10-06 Last updated: 2017-12-12Bibliographically approved
2. gamma-Acyloxy-epsilon-Caprolactones: Synthesis, Ring-Opening Polymerization vs. Rearrangement by Means of Chemical and Enzymatic Catalysis
Open this publication in new window or tab >>gamma-Acyloxy-epsilon-Caprolactones: Synthesis, Ring-Opening Polymerization vs. Rearrangement by Means of Chemical and Enzymatic Catalysis
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2008 (English)In: Macromolecular Symposia, ISSN 1022-1360, E-ISSN 1521-3900, Vol. 272, 28-38 p.Article in journal (Refereed) Published
Abstract [en]

gamma-Acyloxy-epsilon-caprolactones (3a-d) were prepared in two steps starting from 4-hydroxy-cyclohexanone (1). in the first step acylation of the hydroxyl group occurs and in the second step ring enlargement by Baeyer-Villiger oxidation. if this order of reaction is inverted rearrangement occurs in the Baeyer-Villiger oxidation Of 4-hydroxy-cyclohexanone leading to gamma-hydroxyethyl-gamma-butyrolactone. Using the first procedure gamma-acetyloxy- (33), gamma-benzoyloxy-(3B), gamma-acryloyloxy-(3c), and gamma-methacryloyloxy-epsilon-caprolactone (3d) were prepared. These monomers and for comparison reasons epsilon-caprolactone and gamma-methyl-epsilon-caprolactone were polymerized by means of chemical and enzymatic catalysis. The results were different depending on the monomer structure and catalyst used. in the presence of a chemical catalyst, all the monomers, except gamma-acetyloxy-epsilon-caprolactone, undergo controlled ring-opening polymerization. gamma-Acetyloxy-epsilon-caprolactone (3a), however, rearranges to a large extent under polymerization conditions to give gamma-acetyloxyethyl-gamma-butyrolactone (6a). in the presence of an enzyme (Novozyme 435, Lipase B from Candida antarctica (CALB) immobilized on a macroporous resin) all gamma-acyloxy-epsilon-caprolactones partly rearrange to result the corresponding gamma-acyloxy-gamma-butyrolactones, while epsilon-caproiactone and gamma-methyl-epsilon-caprolactone yield the corresponding polymers, the latter even in a stereoselective manner as reported earlier in the literature. A molecular dynamic study was performed with 3a and 3b as substrates to gain information on the substrate recognition displayed by CALB. A mechanism for the chemically and enzymatically catalyzed reactions of gamma-acyloxy-epsilon-caprolactones is proposed.

Keyword
enzyme and chemical catalysis, ring-opening polymerization, gamma-Acyloxy-epsilon-caprolactones
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-24968 (URN)10.1002/masy.200851203 (DOI)000261674400003 ()2-s2.0-55849127376 (Scopus ID)
Conference
International Conference on (Bio)degradable Polymers from Renewable Resources Vienna, AUSTRIA, NOV 18-21, 2007
Note

QC 20101004 (International Conference on (Bio)degradable Polymers from Renewable Resources Vienna, AUSTRIA, NOV 18-21, 2007)

Available from: 2010-10-04 Created: 2010-10-04 Last updated: 2017-12-12Bibliographically approved
3. Lipase Catalyzed HEMA Initiated Ring-Opening Polymerization: In Situ Formation of Mixed Polyester Methacrylates by Transesterification
Open this publication in new window or tab >>Lipase Catalyzed HEMA Initiated Ring-Opening Polymerization: In Situ Formation of Mixed Polyester Methacrylates by Transesterification
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2008 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 9, no 2, 704-710 p.Article in journal (Refereed) Published
Abstract [en]

2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the enzymatic ring-opening polymerization (ROP) of ω-pentadecalactone (PDL) and ∈-caprolactone (CL). The lipase B from Candida antarctica was found to catalyze the cleavage of the ester bond in the HEMA end group of the formed polyesters, resulting in two major transesterification processes, methacrylate transfer and polyester transfer. This resulted in a number of different polyester methacrylate structures, such as polymers without, with one, and with two methacrylate end groups. Furthermore, the 1,2-ethanediol moiety (from HEMA) was found in the polyester products as an integral part of HEMA, as an end group (with one hydroxyl group) and incorporated within the polyester (polyester chains acylated on both hydroxyl groups). After 72 h, as a result of the methacrylate transfer, 79% (48%) of the initial amount of the methacrylate moiety (from HEMA) was situated (acylated) on the end hydroxyl group of the PPDL (PCL) polyester. In order to prepare materials for polymer networks, fully dimethacrylated polymers were synthesized in a one-pot procedure by combining HEMA-initiated ROP with end-capping using vinyl methacrylate. The novel PPDL dimethacrylate (>95% incorporated methacrylate end groups) is currently in use for polymer network formation. Our results show that initiators with cleavable ester groups are of limited use to obtain well-defined monomethacrylated macromonomers due to the enzyme-based transesterification processes. On the other hand, when combined with end-capping, well-defined dimethacrylated polymers (PPDL, PCL) were prepared.

Keyword
Catalysis, Lipases, Monomers, Polyesters, Transesterification
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8005 (URN)10.1021/bm7010449 (DOI)000253102100040 ()2-s2.0-39749139410 (Scopus ID)
Note
QC 20100921. Uppdaterad från In press till Published (20100921).Available from: 2008-02-20 Created: 2008-02-20 Last updated: 2017-12-14Bibliographically approved
4. Systematic Comparison of HEA and HEMA as Initiators in Enzymatic Ring-Opening Polymerizations
Open this publication in new window or tab >>Systematic Comparison of HEA and HEMA as Initiators in Enzymatic Ring-Opening Polymerizations
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2009 (English)In: Macromolecular Bioscience, ISSN 1616-5187, E-ISSN 1616-5195, Vol. 9, no 7, 713-720 p.Article in journal (Refereed) Published
Abstract [en]

Two initiators (HEA and HEMA) containing a cleavable ester bond were compared in the lipase-catalyzed ROP of CL and PDL. HEA and HEMA displayed similar reaction efficiencies as initiators (acyl acceptors) in the enzymatic ROP. However, transacylation reactions were found to be 15 times faster on the HEA-initiated polyesters as compared with the HEAAA initiated polyesters (HEA/HEMA moieties as acyl donors). While in both cases the amount of HEA- and HEMA-initiated polymers could be maximized by H short reaction times, a well-defined (meth)acrylation by this approach was not possible. Our results show that trans esterification reactions are present at high rates throughout the enzyme-catalyzed ROP.

Keyword
biocatalysis, enzymes, kinetics, polyesters, ring-opening polymerization, transfer radical polymerization, antarctica lipase-b, chemoenzymatic, synthesis, epsilon-caprolactone, graft-copolymers, polyesters, poly(epsilon-caprolactone), macromonomers, lactones, poly(omega-pentadecalactone)
Identifiers
urn:nbn:se:kth:diva-18601 (URN)10.1002/mabi.200800290 (DOI)000267962700011 ()2-s2.0-68649110083 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
5. An S-selective lipase was created by rational redesign and the enantioselectivity increased with temperature
Open this publication in new window or tab >>An S-selective lipase was created by rational redesign and the enantioselectivity increased with temperature
2005 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 44, no 29, 4582-4585 p.Article in journal (Refereed) Published
Abstract [en]

Higher activity with larger pockets: The figure shows a superposition of intermediates that occur in acyl transfer to (S)-1-phenylethanol catalyzed by Candida antarctica lipase B (CALB). Wild-type CALB cannot accomodate the phenyl group (gray) in the stereospecificity pocket and form all of the catalytically essential H bonds. The Trp 104 Ala mutation liberates the volume in yellow, the S enantiomer is easily fitted, and the specificity constant increases by a factor of 130 000.

Keyword
enantioselectivity, enzyme catalysis, hydrolases, protein engineering, thermodynamics, DYNAMIC KINETIC RESOLUTION, SECONDARY ALCOHOLS, STEREOSELECTIVITY, STEREOCHEMISTRY, RECOGNITION, ENANTIOMERS, CATALYSTS, ENTROPY, CEPACIA
Identifiers
urn:nbn:se:kth:diva-13009 (URN)10.1002/anie.200500971 (DOI)000230737500019 ()2-s2.0-22744445622 (Scopus ID)
Note
QC20100524Available from: 2010-05-24 Created: 2010-05-24 Last updated: 2017-12-12Bibliographically approved
6. Single-step, solvent-free enzymatic route to alpha,omega-functionalized polypentadecalactone macromonomers
Open this publication in new window or tab >>Single-step, solvent-free enzymatic route to alpha,omega-functionalized polypentadecalactone macromonomers
2008 (English)In: Macromolecules, ISSN 0024-9297, E-ISSN 1520-5835, Vol. 41, no 14, 5230-5236 p.Article in journal (Refereed) Published
Abstract [en]

A straightforward enzymatic single-step route toward the synthesis of alpha,omega-functionalized polypentadecalactone (PPDL) macromonomers containing dithiol, thiol-acrylate, diacrylate, or dimethacrylate end groups has been developed. Two solvent-free approaches, mixing all components at start, using Candida antarctica lipase B (CALB) as an efficient catalyst were demonstrated. In the first approach difunctionalized polymers (with dithiol or thiol-acrylate end groups) were synthesized by mixing lipase, lactone, and equimolar amounts of functional initiator (6-mercapto-1-hexanol) and terminator (11-mercapto-1-undecanoic acid or vinyl acrylate). Polymers with a high fraction (95%) of dithiol end groups or polymers with thiol-acrylate end groups (86% and 96%, respectively) were obtained. In the second approach, a functional diester (ethylene glycol diacrylate or ethylene glycol dimethacrylate) was mixed with lactone and lipase without predrying, using water as an initial initiator. Reduced pressure was applied after 2 h of incubation to evaporate water and push the equilibrium toward high functionalization. Polymers with >96% diacrylated or dimethacrylated end groups were achieved.

Keyword
Acrylic monomers, Alcohols, Ethylene, Ethylene glycol, Glycols, Mixing, Monomers, Polymers, Solvents
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8004 (URN)10.1021/ma800074a (DOI)000257665900022 ()2-s2.0-48949100426 (Scopus ID)
Note
QC 20100929. Uppdaterad från Submitted till Published (20100929). Tidigare titel:"One-Step, Solvent Free Enzymatic Route to α,ω-Functionalized Poly-pentadecalactone Macromonomers". Available from: 2008-02-20 Created: 2008-02-20 Last updated: 2017-12-14Bibliographically approved
7. One-Pot Difunctionalization of Poly-(ω-pentadecalactone) with Thiol-Thiol or Thiol-Acrylate Groups, Catalyzed by Candida antarctica Lipase B
Open this publication in new window or tab >>One-Pot Difunctionalization of Poly-(ω-pentadecalactone) with Thiol-Thiol or Thiol-Acrylate Groups, Catalyzed by Candida antarctica Lipase B
Show others...
2006 (English)In: Macromolecular rapid communications, ISSN 1022-1336, E-ISSN 1521-3927, Vol. 27, no 22, 1932-1936 p.Article in journal (Refereed) Published
Abstract [en]

An enzymatic one-pot procedure has been developed for the synthesis of difunetional polyesters containing terminal thiols and acrylates. Candida antarctica lipase B was used as a catalyst for the ring-opening polymerization of ω-pentadecalactone. The polymerization was initiated with 6-mercaptohexanol, then terminated with γ-thiobutyrolactone or vinyl acrylate to create two types of difunetional polyesters with a very high content of thiol-thiol or thiolacrylate end-groups.

Keyword
Biocatalysis, Enzymes, Polyesters, Ring-opening polymerization
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-8003 (URN)10.1002/marc.200600527 (DOI)000242616900008 ()2-s2.0-33845199474 (Scopus ID)
Note
QC 20100924Available from: 2008-02-20 Created: 2008-02-20 Last updated: 2017-12-14Bibliographically approved

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