Change search
ReferencesLink to record
Permanent link

Direct link
Rational engineering of esterases for improved amidase specificity in amide synthesis and hydrolysis
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0001-9001-9271
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. , 76 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:21
Keyword [en]
Amidase, Biocatalysis, Enzyme, Esterase, Enzyme engineering, Lipase, Substrate specificity
National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-196892ISBN: 978-91-7729-210-4OAI: oai:DiVA.org:kth-196892DiVA: diva2:1049539
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
List of papers
1. Esterases with an Introduced Amidase-Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity
Open this publication in new window or tab >>Esterases with an Introduced Amidase-Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity
Show others...
2012 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, no 5, 645-648 p.Article in journal (Refereed) Published
Keyword
biocatalysis, chemoselectivity, enzyme catalysis, hydrogen bonds, protein engineering
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-93931 (URN)10.1002/cbic.201100779 (DOI)000301532300006 ()2-s2.0-84858306550 (ScopusID)
Note

QC 20120504

Available from: 2012-05-04 Created: 2012-05-03 Last updated: 2016-11-29Bibliographically approved
2. Exploring water as building bricks in enzyme engineering
Open this publication in new window or tab >>Exploring water as building bricks in enzyme engineering
2015 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 51, no 97, 17221-17224 p.Article in journal (Refereed) Published
Abstract [en]

A novel enzyme engineering strategy for accelerated catalysis based on redesigning a water network through protein backbone deshielding is presented. Fundamental insight into the energetic consequences associated with the design is discussed in the light of experimental results and computer simulations. Using water as biobricks provides unique opportunities when transition state stabilisation is not easily attained by traditional enzyme engineering.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2015
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-180600 (URN)10.1039/c5cc07162c (DOI)000366954800004 ()26426706 (PubMedID)2-s2.0-84948408732 (ScopusID)
Funder
Swedish Research Council, 621-2013-5138
Note

QC 20160120

Available from: 2016-01-20 Created: 2016-01-19 Last updated: 2016-11-24Bibliographically approved
3. One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones
Open this publication in new window or tab >>One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones
2016 (English)In: catalysis science & technology, ISSN 2044-4753, Vol. 6, 2897-2900 p.Article in journal (Refereed) Published
Abstract [en]

An efficient one-pot one-step biocatalytic amine transaminase/acyl transferase cascade for the formation of amides from the corresponding aldehydes and ketones in aqueous solution has been developed. N-benzyl-2-methoxyacetamide has been synthesized utlilizing the developed cascade in conversions up to 97%. The cascade was also evaluated for the synthesis of chiral amides.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2016
National Category
Biocatalysis and Enzyme Technology
Identifiers
urn:nbn:se:kth:diva-185329 (URN)10.1039/C6CY00435K (DOI)000375545600004 ()2-s2.0-84967261237 (ScopusID)
Note

QC 20160422

Available from: 2016-04-16 Created: 2016-04-16 Last updated: 2016-11-24Bibliographically approved
4. Engineering the esterase/acyltransferase from Mycobacterium smegmatis: extended substrate scope for amide synthesis in water
Open this publication in new window or tab >>Engineering the esterase/acyltransferase from Mycobacterium smegmatis: extended substrate scope for amide synthesis in water
(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:nbn:se:kth:diva-196890 (URN)
Note

QC 20161129

Available from: 2016-11-24 Created: 2016-11-24 Last updated: 2016-11-29Bibliographically approved
5. Rational engineering of an esterase/acyltransferase for improved amidase specificity in amide synthesis and hydrolysis
Open this publication in new window or tab >>Rational engineering of an esterase/acyltransferase for improved amidase specificity in amide synthesis and hydrolysis
(English)Manuscript (preprint) (Other academic)
Abstract [en]

The esterase/acyltransferase from Mycobacterium smegmatis, MsAcT, display high acyltransfer capacity in water media with demonstrations found for both ester and amide syntheses. However, it has recently been discovered that esterases in contrast to amidases lack a key hydrogen bond in the transition state, donated by the scissile NH-group of the substrate. Esterases with improved amidase performance have been achieved with the introduction of amino-acid side chains or water network as hydrogen bond acceptors. Using the esterase from Mycobacterium smegmatis, MsAcT, the influence of this hydrogen bond was studied in both amide hydrolysis and synthesis, using a rational engineering approach. Two positions were selected for mutagenesis and enzyme variants with improved performance in amide synthesis and hydrolysis were generated. Compared to the wild-type, variant F154A had the highest absolute increase in amidase specificity (11-fold) and I194Q had the greatest change in relative amidase versus esterase reaction specificity (160-fold). The relative reaction specificities for amide over ester synthesis followed a similar trend as that of hydrolysis and the best variant was I194Q with a 32-fold increase compared to wt. Based on MD-simulations water seems to play an important role in the transition state as a hydrogen bond bridge between the NH-group of the amide substrate and the enzyme.

National Category
Biocatalysis and Enzyme Technology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-196891 (URN)
Note

QC 20161129

Available from: 2016-11-24 Created: 2016-11-24 Last updated: 2016-11-29Bibliographically approved

Open Access in DiVA

fulltext(2297 kB)29 downloads
File information
File name FULLTEXT01.pdfFile size 2297 kBChecksum SHA-512
747a263cf51e744ef2d8b25d6f9b4bc78ba399ffe065431146d3728001a8495cdac7bc8969656202e71951ae76423bb82f284919d05a51ad228e685a7c1b0654
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Hendil-Forssell, Peter
By organisation
Industrial Biotechnology
Biocatalysis and Enzyme Technology

Search outside of DiVA

GoogleGoogle Scholar
Total: 29 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Total: 107 hits
ReferencesLink to record
Permanent link

Direct link