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Engineering the esterase/acyltransferase from Mycobacterium smegmatis: extended substrate scope for amide synthesis in water
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0001-9001-9271
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0003-2644-0752
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0002-2993-9375
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Some esterases/lipases display high acyl transfer activity, favoring alcoholysis over hydrolysis, which make them valuable catalysts for synthesis reactions in aqueous media. An esterase from Mycobacterium smegmatis, MsAcT, has been characterized as an efficient catalyst for ester synthesis in water. The acyl donor specificity for MsAcT was however found to be very narrow and the enzyme displayed no activity towards esters with larger acyl group than butyrate. With rational engineering, the narrow acyl donor specificity of wild type MsAcT enzyme was altered and variants displaying extended substrate scope were generated. A double mutant, T93A/F154A, could accommodate methyl nonanoate as substrate, i.e. five carbons longer acyl group as compared to wild type, without compromising the acyl transfer capabilities. With similar selectivity towards a broad range of acyl donors (propionate to nonanoate) this is a more applicable catalyst than the wild type. Furthermore, the T93A/F154A variant was an efficient catalyst for synthesis of N-benzylhexanamide in water using methyl hexanoate as acyl donor, which is not a substrate for the wild type enzyme. The conversion reached 81% and the enzyme variant could potentially be used to produce amides in water with a wide variety of acyl donors.

National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-196890OAI: oai:DiVA.org:kth-196890DiVA, id: diva2:1049536
Note

QC 20161129

Available from: 2016-11-24 Created: 2016-11-24 Last updated: 2017-08-23Bibliographically approved
In thesis
1. Rational engineering of esterases for improved amidase specificity in amide synthesis and hydrolysis
Open this publication in new window or tab >>Rational engineering of esterases for improved amidase specificity in amide synthesis and hydrolysis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biocatalysis is an ever evolving field that uses enzymes or microorganisms for chemical synthesis. By utilizing enzymes that generally have evolved for specific reactions under mild conditions and temperatures, biocatalysis can be a more environmentally friendly option compared to traditional chemistry.

Amide-type chemistries are important and bond formation avoiding poor atom economy is of high priority in organic chemistry. Biocatalysis could potentially be a solution but restricted substrate scope is a limitation. Esterases/lipases usually display broad substrate scope and catalytic promiscuity but are poor at hydrolyzing amides compared to amidases/proteases. The difference between the two enzyme classes is hypothesized to reside in one key hydrogen bond present in amidases, which facilitates the transition state for nitrogen inversion during catalysis.

In this thesis the work has been focused on introducing a stabilizing hydrogen bond acceptor in esterases, mimicking that found in amidases, to develop better enzymatic catalysts for amide-based chemistries.

By two strategies, side-chain or water interaction, variants were created in three esterases that displayed up to 210-times increased relative amidase specificity compared to the wild type. The best variant displayed reduced activation enthalpy corresponding to a weak hydrogen bond. The results show an estimated lower limit on how much the hydrogen bond can be worth to catalysis.

MsAcT catalyze kinetically controlled N-acylations in water. An enzymatic one-pot one-step cascade was developed for the formation of amides from aldehydes in water that gave 97% conversion. In addition, engineered variants of MsAcT with increased substrate scope could synthesize an amide in water with 81% conversion, where the wild type gave no conversion. Moreover, variants of MsAcT displayed up to 32-fold change in specificity towards amide synthesis and a switch in reaction preference favoring amide over ester synthesis.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. p. 76
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:21
Keywords
Amidase, Biocatalysis, Enzyme, Esterase, Enzyme engineering, Lipase, Substrate specificity
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-196892 (URN)978-91-7729-210-4 (ISBN)
Public defence
2016-12-16, FD5, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20161125

Available from: 2016-11-25 Created: 2016-11-24 Last updated: 2016-11-25Bibliographically approved
2. Building blocks for polymer synthesis by enzymatic catalysis
Open this publication in new window or tab >>Building blocks for polymer synthesis by enzymatic catalysis
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The search for alternatives to oil-based monomers has sparked interest for scientists to focus on the use of renewable resources for energy production, for the synthesis of polymeric materials and in other areas. With the use of renewable resources, scientists face new challenges to first isolate interesting molecules and then to process them.

Enzymes are nature’s own powerful catalysts and display a variety of activities. They regulate important functions in life. They can also be used for chemical synthesis due to their efficiency, selectivity and mild reaction conditions. The selectivity of the enzyme allows specific reactions enabling the design of building blocks for polymers.

In the work presented here, a lipase (Candida antarctica lipase B (CalB)) was used to produce building blocks for polymers. An efficient route was developed to selectively process epoxy-functional fatty acids into resins with a variety of functional groups (maleimide, oxetane, thiol, methacrylate). These oligoester structures, based on epoxy fatty acids from birch bark and vegetable oils, could be selectively cured to form thermosets with tailored properties.

The specificity of an esterase with acyl transfer activity from Mycobacterium smegmatis (MsAcT) was altered by rational design. The produced variants increased the substrate scope and were then used to synthesize amides in water, where the wild type showed no conversion. A synthetic procedure was developed to form mixed dicarboxylic esters by selectively reacting only one side of divinyl adipate in order to introduce additional functional groups.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. p. 61
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:15
Keywords
Enzyme, Enzyme Engineering, Biocatalysis, Lipase, CalB, MsAcT, Substrate specificity, Selectivity, Polymer Chemistry, Polymer Synthesis
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-212499 (URN)978-91-7729-494-8 (ISBN)
Public defence
2017-09-22, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00
Opponent
Supervisors
Note

QC 20170823

Available from: 2017-08-23 Created: 2017-08-22 Last updated: 2017-08-23Bibliographically approved

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