Proteinové inženýrství halogenalkandehalogenasy LinB: změna reakčního mechanismu

Bc. Peter Holub, Ph.D.

Diplomová práce

Protein engineering of haloalkane dehalogenase LinB: Towards the change of reaction mechanism

Abstract:

Haloalkane dehalogenases are microbial hydrolases catalyzing removal of halogens from aliphatic hydrocarbons. Based on structural and sequence similarities between haloalkane dehalogenase LinB and luciferase from Renilla reniformis, we propose that it could be possible to construct a mutant of LinB with luminescence activity or to obtain an enzyme with both luminescence and dehalogenase activities. This Diploma Thesis is focused on selection of the LinB variant suitable for directed evolution towards luminescence activity from previously prepared mutants LinB53, LinB54, LinB55 and LinB56. Based on the level of expression, solubility, folding, and activity of mutants, the variants LinB54 and LinB55 were chosen for fine-tuning by directed evolution. Sensitive high-throughput screening, which is an important prerequisite for directed evolution experiments, was developed. The screening uses the multi-well plates format and can be applied to the cell lysates. The optimization of the testing conditions resulted in 22-fold increase of luminescence enabling detection of low activities.

Keywords

Enzyme, reaction mechanism, dehalogenase, luciferase, luminescence, directed evolution, high-throughput screening

Jazyk práce: angličtina

Datum vytvoření / odevzdání či podání práce: 30. 4. 2008

Obhajoba závěrečné práce

• Obhajoba proběhla 9. 6. 2008
• Vedoucí: prof. Mgr. Jiří Damborský, Dr.
• Oponent: doc. Mgr. Štěpánka Vaňáčová, Ph.D.

Masarykova univerzita

Přírodovědecká fakulta

Magisterský studijní program / obor:
Biochemie / Biochemie