Ändra sökning
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Trametes versicolor laccase: random mutagenesis and heterologous expression in Pichia pastoris
Karlstads universitet, Institutionen för kemi.
2008 (Engelska)Självständigt arbete på avancerad nivå (magisterexamen), 20 poäng / 30 hpStudentuppsats
Abstract [en]

Laccase is a blue multi-copper oxidase. It has a broad biotechnical potential which increases the interest to study the enzyme further. A laccase-encoding gene from the white-rot fungus Trametes versicolor (lcc2) was mutated using two different methods for random mutagenesis: error-prone PCR and a method based on an E.coli strain (ES1301 mutS) that introduces random mutations. For the error-prone PCR reaction, the vector pPICZB with the lcc2 gene inserted was used as template. The E. coli strain ES1301 mutS was transformed with the vector pBluescript SKII with the lcc2 gene as insert. The mutagenesis products were cloned into the Pichia pastoris expression vector pPICZB for transformation of P. pastoris SMD1168. The transformants were spread on agar plates containing zeocin. Laccase-secreting transformants were selected by their ability to oxidize the substrates ABTS [2,2´-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] and syringaldazine [N,N´-bis(3,5-dimethoxy-4-hydroxybenzylidene)hydrazine], the products of which give green and purple colour, respectively. Around 20 transformants from each of the mutagenesis methods were transformed to plates containing 1 mM ABTS or 1 mM syringaldazine. None of the transformants produced any colour. Control transformants (pPICZB with unmutated lcc2) were also spread on plates with either ABTS or syringaldazine. The transformants gave rise to green colour after 24 hours on the ABTS plates and to purple colour after 72 hours on the syringaldazine plates. Experimets with different chromogenic substrates indicated that ABTS and syringaldazine were best suited for screening of mutants. Remazol Brilliant Blue and Phenol Red are two substrates that after optimisation can serve as alternatives for the selection of laccase-secreting transformants.

Ort, förlag, år, upplaga, sidor
2008. , s. 36
Nyckelord [en]
Biotechnology
Nyckelord [sv]
Bioteknik
Nationell ämneskategori
Industriell bioteknik
Identifikatorer
URN: urn:nbn:se:kau:diva-1580OAI: oai:DiVA.org:kau-1580DiVA, id: diva2:5495
Presentation
2008-01-11
Uppsök
teknik
Handledare
Examinatorer
Tillgänglig från: 2008-04-09 Skapad: 2008-04-09

Open Access i DiVA

fulltext(880 kB)1988 nedladdningar
Filinformation
Filnamn FULLTEXT01.pdfFilstorlek 880 kBChecksumma MD5
1feeb187ee0674748d8df378b9f42a4836cbbabd90c0926d4c99dd0d71cfc46d21846919
Typ fulltextMimetyp application/pdf

Av organisationen
Institutionen för kemi
Industriell bioteknik

Sök vidare utanför DiVA

GoogleGoogle Scholar
Totalt: 1988 nedladdningar
Antalet nedladdningar är summan av nedladdningar för alla fulltexter. Det kan inkludera t.ex tidigare versioner som nu inte längre är tillgängliga.

urn-nbn

Altmetricpoäng

urn-nbn
Totalt: 572 träffar
RefereraExporteraLänk till posten
Permanent länk

Direktlänk
Referera
Referensformat
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annat format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annat språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf