Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Serine Hydrolase Selectivity: Kinetics and applications in organic and analytical chemistry
KTH, School of Biotechnology (BIO), Biochemistry.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The substrate selectivities for different serine hydrolases were utilized in various applications, presented in papers I-VI. The articles are discussed in the thesis in view of the kinetics of the enzyme catalysis involved.

In paper I the enantioselectivities towards a range of secondary alcohols were reversed for Candida antarctica lipase B by site directed mutagenesis. The thermodynamic components of the enantioselectivity were determined for the mutated variant of the lipase.

In papers II-III Candida antarctica lipase B was engineered for selective monoacylation using two different approaches. A variant of the lipase created for substrate assisted catalysis (paper II) and three different variants with mutations which decreased the volume of the active site (paper III) were evaluated. Enzyme kinetics for the different variants were measured and translated into activation energies for comparison of the approaches.

In papers IV and V three different enzymes were used for rapid analysis of enantiomeric excess and conversion of O-acylated cyanohydrins synthesized by a defined protocol. Horse liver alcohol dehydrogenase, Candida antarctica lipase B and pig liver esterase were sequentially added to a solution containing the O-acylated cyanohydrin. Each enzyme caused a drop in absorbance from oxidation of NADH to NAD+. The product yield and enantiomeric excess was calculated from the relative differences in absorbance.

In paper VI a method for C-terminal peptide sequencing was developed based on conventional Carboxypeptidase Y digestion combined with matrix assisted laser desorption/ionization mass spectrometry. An alternative nucleophile was used to obtain a stable peptide ladder and improve sequence coverage.

Place, publisher, year, edition, pages
Stockholm: KTH , 2010. , viii, 62 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:12
Keyword [en]
Candida antarctica lipase B, monoacylation of diols, kinetic resolution, thermodynamics in enzyme catalysis, enzyme engineering
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-12831ISBN: 978-91-7415-663-8 (print)OAI: oai:DiVA.org:kth-12831DiVA: diva2:319091
Public defence
2010-06-04, FD5, AlbaNova University Center, Roslagstullsbacken 21, Stockholm, 15:42 (English)
Opponent
Supervisors
Note
QC20100629Available from: 2010-05-24 Created: 2010-05-12 Last updated: 2010-06-29Bibliographically approved
List of papers
1. An S-selective lipase was created by rational redesign and the enantioselectivity increased with temperature
Open this publication in new window or tab >>An S-selective lipase was created by rational redesign and the enantioselectivity increased with temperature
2005 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 44, no 29, 4582-4585 p.Article in journal (Refereed) Published
Abstract [en]

Higher activity with larger pockets: The figure shows a superposition of intermediates that occur in acyl transfer to (S)-1-phenylethanol catalyzed by Candida antarctica lipase B (CALB). Wild-type CALB cannot accomodate the phenyl group (gray) in the stereospecificity pocket and form all of the catalytically essential H bonds. The Trp 104 Ala mutation liberates the volume in yellow, the S enantiomer is easily fitted, and the specificity constant increases by a factor of 130 000.

Keyword
enantioselectivity, enzyme catalysis, hydrolases, protein engineering, thermodynamics, DYNAMIC KINETIC RESOLUTION, SECONDARY ALCOHOLS, STEREOSELECTIVITY, STEREOCHEMISTRY, RECOGNITION, ENANTIOMERS, CATALYSTS, ENTROPY, CEPACIA
Identifiers
urn:nbn:se:kth:diva-13009 (URN)10.1002/anie.200500971 (DOI)000230737500019 ()2-s2.0-22744445622 (Scopus ID)
Note
QC20100524Available from: 2010-05-24 Created: 2010-05-24 Last updated: 2017-12-12Bibliographically approved
2. Selective monoacylation of diols by substrate assisted catalysis in T40A CALB
Open this publication in new window or tab >>Selective monoacylation of diols by substrate assisted catalysis in T40A CALB
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-13819 (URN)
Note
QC20100629Available from: 2010-06-29 Created: 2010-06-29 Last updated: 2010-06-29Bibliographically approved
3. Rational engineering of CALB for selective monoacylation of diols
Open this publication in new window or tab >>Rational engineering of CALB for selective monoacylation of diols
(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-13820 (URN)
Note
QC20100629Available from: 2010-06-29 Created: 2010-06-29 Last updated: 2010-06-29Bibliographically approved
4. High-Throughput Enzymatic Method for Enantiomeric Excess Determination of O-Acetylated Cyanohydrins
Open this publication in new window or tab >>High-Throughput Enzymatic Method for Enantiomeric Excess Determination of O-Acetylated Cyanohydrins
Show others...
2006 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 128, no 7, 2234-2235 p.Article in journal (Refereed) Published
Keyword
acetic acid derivative; cyanohydrin; enzyme; acetylation; article; catalyst; chemical modification; enantiomer; enzyme activity; high throughput screening; Acetylation; Animals; Benzaldehydes; Esterases; Hydrolysis; Lipase; Liver; NAD; Nitriles; Stereoisomerism; Swine
National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-6920 (URN)10.1021/ja058474r (DOI)000235562900040 ()2-s2.0-33644501771 (Scopus ID)
Note
QC20100809Available from: 2007-03-22 Created: 2007-03-22 Last updated: 2010-08-09Bibliographically approved
5. High Throughput Synthesis and Analysis of Acylated Cyanohydrins
Open this publication in new window or tab >>High Throughput Synthesis and Analysis of Acylated Cyanohydrins
Show others...
2007 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 13, no 15, 4334-4341 p.Article in journal (Refereed) Published
Abstract [en]

The yields and optical purities of products obtained from chiral Lewis acid/Lewis base-catalysed additions of alpha-ketonitriles to prochiral aldehydes could be accurately determined by an enzymatic method. The amount of remaining aldehyde was determined after its reduction to an alcohol, whilst the two product enantiomers were analysed after subsequent hydrolysis first by the (S)-selective Candida antarctica lipase B and then by the unselective pig liver esterase. The method could be used for analysis of products obtained from a number of aromatic aldehydes and aliphatic ketonitriles. Microreactor technology was successfully combined with high-throughput analysis for efficient catalyst optimization.

Keyword
asymmetric synthesis; cyanohydrins; enantioselectivity; enzymes; high-throughput screening; microreactors
National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-7108 (URN)10.1002/chem.200601638 (DOI)000246879100022 ()2-s2.0-34250318478 (Scopus ID)
Note
QC 20100809Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2010-08-09Bibliographically approved
6. C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests
Open this publication in new window or tab >>C-terminal ladder sequencing of peptides using an alternative nucleophile in carboxypeptidase Y digests
Show others...
2006 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 357, no 2, 167-172 p.Article in journal (Refereed) Published
Abstract [en]

 A method for improved sequence coverage in C-terminal sequencing of peptides, based on carboxypeptidase digestion, is described. In conventional carboxypeptidase digestions, the peptide substrate is usually extensively degraded and a full amino acid sequence cannot be obtained due to the lack of a complete peptide ladder. In the presented method, a protecting group is introduced at the C terminus of a fraction of the peptide fragments formed in the digest, and thereby further degradation of the C-terminally modified peptides are slowed down. The protecting group was attached to the C-terminal amino acid through a carboxypeptidase-catalyzed reaction with an alternative nucleophile, 2-pyridylmethylamine, added to the aqueous digestion buffer. Six peptides were digested by carboxypeptidase Y with and without 2-pyridylmethylamine present in the digest buffer, and the resulting fragments subsequently were analyzed with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Comparison of the two digestion methods showed that the probability of successful ladder sequencing increased, by more than 50% using 2-pyridylmethylamine as a competing nucleophile in carboxypeptidase Y digests.

Keyword
2-Pyridylmethylamine; MALDI; Matrix-assisted laser desorption/ionization mass spectrometry; Transpeptidation
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-7106 (URN)10.1016/j.ab.2006.07.025 (DOI)000241077800002 ()2-s2.0-33748784378 (Scopus ID)
Note
QC20100524Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2010-09-07Bibliographically approved

Open Access in DiVA

fulltext(945 kB)948 downloads
File information
File name FULLTEXT01.pdfFile size 945 kBChecksum SHA-512
779d98ac17cbae4746721d2921f476bfb86bce41054124b7ad00546260566f7d4fd0f5dad60b68c4558bc8b6b7511d997623081d098ea71789f24765b42be552
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Hamberg, Anders
By organisation
Biochemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 948 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

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 426 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf