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Exploring water as building bricks in enzyme engineering
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0001-9001-9271
KTH, School of Biotechnology (BIO), Industrial Biotechnology.ORCID iD: 0000-0002-2993-9375
KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.ORCID iD: 0000-0002-4066-2776
2015 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 51, no 97, 17221-17224 p.Article in journal (Refereed) Published
Resource type
Text
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. Vol. 51, no 97, 17221-17224 p.
National Category
Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-180600DOI: 10.1039/c5cc07162cISI: 000366954800004PubMedID: 26426706Scopus ID: 2-s2.0-84948408732OAI: oai:DiVA.org:kth-180600DiVA: diva2:896177
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
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. 76 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:21
Keyword
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

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