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Estabilización de Penicilina G acilasa por inmovilización covalente multipuntual dirigida. Mutagénesis de la superficie de la enzima para mejorar la complementariedad enzima-soporte activado
Author
Grazú Bonavia, Maria ValeriaEntity
UAM. Departamento de Biología MolecularDate
2006-06-09Subjects
Enzimas inmovilizadas - Tesis doctorales; Enzimas - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 09-06-2006Abstract
A new strategy to achieve a vety high stabilization of industrial enzymes is here proposed.
A vety intense ngidification of a given region of the enzyme surface via its improved multipoint
covalent immobilization on activated preexisting supports has been developed. The main objective
of this Ph.D. Thesis involves the development of five different relevant subobjedwes:
a.- Preparation of tailor made disulfideepoxy supports containing i)- a small concentration
of highly reactive disulfide groups able to selectively immobilize enzyme through inserted cysteine
groups, ii).- a very high concentration of epoxy groups able to perfom an intense multipoint
immobilization with nucleophilic groups of the enzyme surface placed in the vicinity of the inserted
cysteine group. Furthermore. the optimization of multipoint covalent immobilization of enzymes on
these new supports was also studied.
b.- Preparation of different mutated enzyme containing, each mutant, a reactive cysteine
group placed in regions that are naturally rich in lysine residues. The tridimensional structure of
PGA was studied and diierent mutations (insertion of cysteines) were proposed. The theoretical
structure of the proposed mutants was evaluated and those mutants (with very similar structure with
regards to the native one) were cloned. expressed and purified.
c.- Site-direct multipoint covalent attachment of each mutated enzyme in order to detect the
region of the enzyme surface that is more sensitive for stabilization (in this case a region close to
the active site and surrounding the amino acid 8380 of the enzyme)
d.- Enrichment of the selected region of the enzyme surface with new lysine residues (up to
8 new lysine groups were inserted close the amino acid 8380). In this way. a highly impmved
ngidification of this region of the enzyme by multipoint covalent irnmobilization couM be achieved.
e.- Evaluation of dfferent activated supports in order to get the most intense directed
multipoint covalent irnmobilization of the mutated enzyme and subsequently the best stabilization
factors. Glyoxyi agarose selectwely immobilues the enzyme via this mutated region of its surface
(enriched with adiiional lysine residues) and this support promotes the more intense stabilization ((around 20 lysine residues of the enzyme surface seem to be imrolved in this very interesting
multipoint covalent immobilization).
The best immobilized derivatives were more than 300.000 fold more stable than the
corresponding soluble (and one-point immobilized) enzyme in experiments of irreversible thermal
inactivarion. This dramatic stabilization was not found in scientific literature for any anzyme and any stabilzation strategy. Similar stabilization factors were found for inactivation of the enzyme
in the pregence of organic cowlvents. In addition to that, these highly r i g i i i enzymes exhibit
W r ca talytic properae9 for interesüng biotransfomiaoons (enant¡o&dh hydmlysm of chiral
esters, kimatically wntrolled synthesis c4 amide bonds).
Furlhermore, very intense mulüpoint immobiliit'mn of the best mutated enzyme was
performed under distorting experimental condtimns in order to " n g i d i i inconect enzyme stnictures
with altered functional propeftb. In ths way, soma derivatives with hiihly improved propeities for
kinetically controlled synthesis of antiobitcs were obtained.
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