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One-pot biocatalytic amine transaminase/acyl transferase cascade for aqueous formation of amides from aldehydes or ketones
KTH, School of Biotechnology (BIO), Industrial Biotechnology. (AlbaNova University Center)ORCID iD: 0000-0003-3073-5641
KTH, School of Biotechnology (BIO), Industrial Biotechnology. (AlbaNova University Center)ORCID iD: 0000-0001-9001-9271
KTH, School of Biotechnology (BIO), Industrial Biotechnology. (AlbaNova University Center)ORCID iD: 0000-0002-2993-9375
KTH, School of Biotechnology (BIO), Industrial Biotechnology. (AlbaNova University Center)ORCID iD: 0000-0002-9577-832X
2016 (English)In: catalysis science & technology, ISSN 2044-4753, Vol. 6, 2897-2900 p.Article in journal (Refereed) Published
Resource type
Text
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. Vol. 6, 2897-2900 p.
National Category
Biocatalysis and Enzyme Technology
Identifiers
URN: urn:nbn:se:kth:diva-185329DOI: 10.1039/C6CY00435KISI: 000375545600004Scopus ID: 2-s2.0-84967261237OAI: oai:DiVA.org:kth-185329DiVA: diva2:920159
Note

QC 20160422

Available from: 2016-04-16 Created: 2016-04-16 Last updated: 2016-11-24Bibliographically approved
In thesis
1. Amine Transaminases in Biocatalytic Amine Synthesis
Open this publication in new window or tab >>Amine Transaminases in Biocatalytic Amine Synthesis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of enzymes, nature´s own catalysts, both isolated or as whole cells to perform chemical transformations is called biocatalysis. As a complement to classical chemical catalysis, biocatalysis can be an environmentally friendly and more economical option in the production and synthesis of chemicals. Research on the application of amine transaminases in synthesis of chiral amines have exploded over the last two decades and interest from the industry is increasing. Amine transaminases are promising catalysts due to their ability to perform reductive amination of ketones with excellent enantioselectivity.

For a process to be efficient, high substrate specificity of the applied enzyme is an important factor. A variant of Chromobacterium violaceum amine transaminase that was obtained through rational design has an increased specific activity toward (S)-1-phenylethylamine and a set of 4´-substituted acetophenones. This result makes this variant a promising catalyst for the asymmetric synthesis of similar amines.

Amine transaminase catalyzed asymmetric synthesis of amines generally suffers from unfavorable equilibrium. Two methods that include spontaneous tautomerization and biocatalytic amidation for equilibrium displacement have therefore been developed.

Efficient assays and screening methods are demanded for the discovery and development of novel amine transaminases. For this purpose, a sensitive fluorescence-based assay that holds promise as a high-throughput screening method was developed.

One of the major obstacles for application of enzymes in industrial processes is the instability of the enzyme toward harsh conditions. The stability of Chromobacterium violaceum amine transaminase was investigated and improved using co-solvents and other additives. Co-lyophilization with surfactants was also applied to improve the performance of the same enzyme in organic solvents.

Place, publisher, year, edition, pages
Stockholm: Henrik Land, 2016. 101 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:18
Keyword
Amine Transaminase, Biocatalysis, Transamination, Reductive Amination, Enzyme, Enzyme Engineering, Equilibrium Displacement, Screening, Enzyme Stability
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-194112 (URN)978-91-7729-164-0 (ISBN)
Public defence
2016-11-25, Kollegiesalen, Brinellvägen 8, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20161017

Available from: 2016-10-17 Created: 2016-10-17 Last updated: 2016-10-17Bibliographically approved
2. 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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Publisher's full textScopushttp://dx.doi.org/10.1039/C6CY00435K

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Land, HenrikMartinelle, MatsBerglund, Per

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