Dissertação de mest., Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2008The present work is referent to the optimization of the expression and production of a novel
thermostable enzyme, CGTase (cyclodextrin glucanotransferase), from Archaeal origin.
The origin of this gene is an important point because it is a group that is unusual to have CGTase
activity. Some of the Archaeal organisms are known as thermophiles. The enzymes of
thermophiles are characterized by extreme stability and activity, large potential in industries like
food and chemical industries and chemical synthesis. Also in biotechnological processes the
increased reaction temperatures leads to reduction of viscosity, higher reaction rates and also
reduced risk of contaminations. CGTase is a thermostable enzyme and is a member of the α-
amylase superfamily. It is used to produce cyclodextrins that are used in pharmaceutical
industries, it is an extracellular enzyme and degrades large starch molecules that can be used by
the organism.
Four different strains were used to express this gene, where two of them were used to try to
control the problem at the transcriptional or at the translational level.
After comparison between codons from Escherichia coli, codons from the gene (CGTase) and
codons from pRARE (plasmid with rare codons), a mutation was introduced in those with less
percentage of codons in E.coli and one of the rare codons in the sequence showed better
expression. The sequence of the mutation revealed that the construct was not under the control of
the T7 promoter, having relatively lower constitutive expression of the gene, not being
significantly affected with the optimizations when they were effectuated.
Future work could be to put the gene of interest in the right direction (turning the gene), using the
appropriate restriction enzymes and again try the right expression with BL21 Star (DE3)
