Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • 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
Removing the Active-Site Flap in Lipase A from Candida antarctica Produces a Functional Enzyme without Interfacial Activation
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
Number of Authors: 4
2016 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 17, no 2, 141-145 p.Article in journal (Refereed) Published
Abstract [en]

A mobile region is proposed to be a flap that covers the active site of Candida antarctica lipase A. Removal of the mobile region retains the functional properties of the enzyme. Interestingly interfacial activation, required for the wild-type enzyme, was not observed for the truncated variant, although stability, activity, and stereoselectivity were very similar for the wild-type and variant enzymes. The variant followed classical Michaelis-Menten kinetics, unlike the wild type. Both gave the same relative specificity in the transacylation of a primary and a secondary alcohol in organic solvent. Furthermore, both showed the same enantioselectivity in transacylation of alcohols and the hydrolysis of alcohol esters, as well as in the hydrolysis of esters chiral at the acid part.

Place, publisher, year, edition, pages
2016. Vol. 17, no 2, 141-145 p.
Keyword [en]
Candida antarctia lipase A, enzyme catalysis, flap, interfacial activation, lid, protein engineering
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-127884DOI: 10.1002/cbic.201500471ISI: 000369963500005PubMedID: 26543016OAI: oai:DiVA.org:su-127884DiVA: diva2:940221
Available from: 2016-06-20 Created: 2016-03-14 Last updated: 2017-10-11Bibliographically approved
In thesis
1. Engineering Candida antarctica Lipase A for Enantioselective Transformations in Organic Synthesis: Design, Immobilization and Organic Solvent Screening of Smart Enzyme Libraries
Open this publication in new window or tab >>Engineering Candida antarctica Lipase A for Enantioselective Transformations in Organic Synthesis: Design, Immobilization and Organic Solvent Screening of Smart Enzyme Libraries
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of enzymes as catalysts in organic synthesis constitutes an attractive alternative to conventional chemical catalysis. Enzymes are non-toxic and biodegradable and they can operate under mild reaction conditions. Furthermore, they often display high chemo-, regio- and stereoselectivity, enabling specific reactions with single product outcome.

By the use of protein engineering, enzymes can be altered for the specific needs of the researcher. The major part of this thesis describes engineering of lipase A from Candida antarctica (CalA), for improved enantioselectivity in organic synthetic transformations.

The first part of the thesis describes a highly combinatorial method for the introduction of mutation sites in an enzyme library. By the simultaneous introduction of nine mutations, we found an enzyme variant with five out of the nine possible mutations. This quintuple variant had an enlarged active site pocket and was enantioselective and active for our model substrate, an ibuprofen ester. This is a bulky substrate for which the wild-type enzyme shows no enantioselectivity and very poor activity.

In the second part of the thesis, we continued our approach of combinatorial, focused enzyme libraries. This time we aimed at decreasing the alcohol pocket of CalA, in order to increase the enantioselectivity for small and medium-sized secondary alcohols. The enzyme library was bound on microtiter plates and screened by a transacylation reaction in organic solvent. This library yielded an enzyme variant with high enantioselectivity for the model substrate 1-phenyl ethanol, and high to excellent selectivity for other alcohols tested. Screening in organic solvent is advantageous since a potential hit is more synthetically useful.

In the third part of the thesis, we used manipulated beads of controlled porosity glass (EziG™) for enzyme immobilization, and demonstrated the generality of this carrier for several enzyme classes. EziG™ allowed fast enzyme immobilization with simultaneous purification and yielded active biocatalysts in all cases.

The last project describes the function of the proposed active site flap in CalA. In our study, we removed this motif. The engineered variant was compared to the wild-type enzyme by testing the amount of interfacial activation and the selectivity for certain alcohols. We showed that the motif is indeed controlling the entrance to the active site and that the flap is not part of the enantioselectivity determining machinery. 

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2015. 59 p.
Keyword
Candida antarctica Lipase A, protein engineering, enzyme libraries, enzyme immobilization, biocatalysis
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-116170 (URN)978-91-7649-168-3 (ISBN)
Public defence
2015-05-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2015-05-06 Created: 2015-04-12 Last updated: 2017-10-11Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMed

Search in DiVA

By author/editor
Bäckvall, Jan-E.
By organisation
Department of Organic Chemistry
In the same journal
ChemBioChem (Print)
Organic Chemistry

Search outside of DiVA

GoogleGoogle Scholar

Altmetric score

Total: 500 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • 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