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Rational engineering of esterases for improved amidase specificity in amide synthesis and hydrolysis
KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.ORCID-id: 0000-0001-9001-9271
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2016. , s. 76
Serie
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:21
Nyckelord [en]
Amidase, Biocatalysis, Enzyme, Esterase, Enzyme engineering, Lipase, Substrate specificity
Nationell ämneskategori
Biokatalys och enzymteknik
Forskningsämne
Bioteknologi
Identifikatorer
URN: urn:nbn:se:kth:diva-196892ISBN: 978-91-7729-210-4 (tryckt)OAI: oai:DiVA.org:kth-196892DiVA, id: diva2:1049539
Disputation
2016-12-16, FD5, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 10:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20161125

Tillgänglig från: 2016-11-25 Skapad: 2016-11-24 Senast uppdaterad: 2016-11-25Bibliografiskt granskad
Delarbeten
1. Esterases with an Introduced Amidase-Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity
Öppna denna publikation i ny flik eller fönster >>Esterases with an Introduced Amidase-Like Hydrogen Bond in the Transition State Have Increased Amidase Specificity
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2012 (Engelska)Ingår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 13, nr 5, s. 645-648Artikel i tidskrift (Refereegranskat) Published
Nyckelord
biocatalysis, chemoselectivity, enzyme catalysis, hydrogen bonds, protein engineering
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
urn:nbn:se:kth:diva-93931 (URN)10.1002/cbic.201100779 (DOI)000301532300006 ()2-s2.0-84858306550 (Scopus ID)
Anmärkning

QC 20120504

Tillgänglig från: 2012-05-04 Skapad: 2012-05-03 Senast uppdaterad: 2017-12-07Bibliografiskt granskad
2. Exploring water as building bricks in enzyme engineering
Öppna denna publikation i ny flik eller fönster >>Exploring water as building bricks in enzyme engineering
2015 (Engelska)Ingår i: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 51, nr 97, s. 17221-17224Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Royal Society of Chemistry, 2015
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
urn:nbn:se:kth:diva-180600 (URN)10.1039/c5cc07162c (DOI)000366954800004 ()26426706 (PubMedID)2-s2.0-84948408732 (Scopus ID)
Forskningsfinansiär
Vetenskapsrådet, 621-2013-5138
Anmärkning

QC 20160120

Tillgänglig från: 2016-01-20 Skapad: 2016-01-19 Senast uppdaterad: 2017-11-30Bibliografiskt granskad
3. One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones
Öppna denna publikation i ny flik eller fönster >>One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones
2016 (Engelska)Ingår i: catalysis science & technology, ISSN 2044-4753, Vol. 6, s. 2897-2900Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Royal Society of Chemistry, 2016
Nationell ämneskategori
Biokatalys och enzymteknik
Identifikatorer
urn:nbn:se:kth:diva-185329 (URN)10.1039/C6CY00435K (DOI)000375545600004 ()2-s2.0-84967261237 (Scopus ID)
Anmärkning

QC 20160422

Tillgänglig från: 2016-04-16 Skapad: 2016-04-16 Senast uppdaterad: 2016-11-24Bibliografiskt granskad
4. Engineering the esterase/acyltransferase from Mycobacterium smegmatis: extended substrate scope for amide synthesis in water
Öppna denna publikation i ny flik eller fönster >>Engineering the esterase/acyltransferase from Mycobacterium smegmatis: extended substrate scope for amide synthesis in water
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
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.

Nationell ämneskategori
Biokatalys och enzymteknik
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-196890 (URN)
Anmärkning

QC 20161129

Tillgänglig från: 2016-11-24 Skapad: 2016-11-24 Senast uppdaterad: 2017-08-23Bibliografiskt granskad
5. Rational engineering of an esterase/acyltransferase for improved amidase specificity in amide synthesis and hydrolysis
Öppna denna publikation i ny flik eller fönster >>Rational engineering of an esterase/acyltransferase for improved amidase specificity in amide synthesis and hydrolysis
(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
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.

Nationell ämneskategori
Biokatalys och enzymteknik
Forskningsämne
Bioteknologi
Identifikatorer
urn:nbn:se:kth:diva-196891 (URN)
Anmärkning

QC 20161129

Tillgänglig från: 2016-11-24 Skapad: 2016-11-24 Senast uppdaterad: 2016-11-29Bibliografiskt granskad

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