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Enzyme selectivity as a tool in analytical chemistry
KTH, School of Biotechnology (BIO).
2007 (English)Licentiate thesis, comprehensive summary (Other scientific)
Abstract [en]

Enzymes are useful tools as specific analytical reagents. Two different analysis methods were developed for use in the separate fields of protein science and organic synthesis. Both methods rely on the substrate specificity of enzymes. Enzyme catalysis and substrate specificity is described and put in context with each of the two developed methods.

In paper I 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.

In paper II and III, 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 conversion and enantiomeric excess of the sample could be calculated from the relative differences in absorbance.

Place, publisher, year, edition, pages
Stockholm: KTH , 2007. , viii, 35 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2007:5
Keyword [en]
substrate specificity, competing substrates, enantiomeric excess analysis, library screening, ladder sequencing, carboxypeptidase Y, 2-pyridylmethylamine, MALDI, Candia antarctica lipase B, pig liver esterase, horse liver alcohol dehydrogenase, acylated cyanohydrins
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-4377ISBN: 978-91-7178-674-6 (print)OAI: oai:DiVA.org:kth-4377DiVA: diva2:12022
Presentation
2007-06-01, FB54, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20101108Available from: 2007-05-15 Created: 2007-05-15 Last updated: 2010-11-08Bibliographically approved
List of papers
1. 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
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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
2. 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
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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
3. High Throughput Synthesis and Analysis of Acylated Cyanohydrins
Open this publication in new window or tab >>High Throughput Synthesis and Analysis of Acylated Cyanohydrins
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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

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