Doctoral theses
Current Record: 254 of 337
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| Identifier |
uch.biology.phd//2002mavrommatis |
| Title |
Μελέτη της προσαρμογής ενζύμων σε χαμηλές θερμοκρασίες: βιοχημική μελέτη δύο χιτινασών από το ψυχρόφιλο βακτήριο Arthrobacter sp str. TAD20 |
| Alternative Title |
Study of enzymes cold adaption: biochemical �study of two chitinases from the psychrophilic bacterium Arthrobacter sp. str. TAD20 |
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Author
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Μαυρομμάτης, Κωνσταντίνος
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Thesis advisor
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Μπουριώτης, Βασίλειος
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| Abstract |
In order to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutations were designed based on multiple sequence alignment, assisted by a homology based model. The mutagenesis targets were residues Trp-260 and Ala-219 of the catalytic site and His-135 of the Mg2+ binding site. The replacement of Trp-260 by Lys in mutant W260K, resulted in a less active than the wild-type enzyme in the temperature range 5-25oC. The additional replacement of Ala-219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation which is reflected in a considerably decreased activity at temperatures 5-15oC and a significantly increased activity at 20-25oC. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored the low energy of activation. In addition, the His135 to Asp replacement in mutants H135D and w260K/A219N/H135D resulted in a considerable stabilization. Furthermore, we designed point mutations aiming to alter the distribution of glycine residues close to the active site of the psychrophilic alkaline phosphatase. The mutagenesis targets were residues Gly-261 and Gly-262. The replacement of Gly-262 by Ala resulted in an inactive enzyme. Substitution of Gly-261 by Ala resulted in an enzyme with lower stability and increased energy of activation. The double mutant G261A/Y269A designed on the basis of side-chain packing criteria from a modeled structure of the enzyme resulted in restoration of the energy of activation to the levels of the native enzyme and in an increased stability compared to the mutant G261A. It seems therefore, that the Gly cluster in combination with its structural environment plays a significant role in the cold adaptation of the enzyme. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.The gene encoding chitinase ArChiB from the Antarctic bacterium Arthrobacter sp. TAD20 has been expressed in E.coli and the recombinant enzyme purified to homogeneity. In our effort to engineer cold adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase at remote from the active site positions. The mutations were designed based on multiple sequence alignment, assisted by a homology based model, and included restoration of a salt bridge and replacement of selected Gly residues by either Pro or Gln. Restoration of a salt bridge in mutant N198K resulted to a more stable protein (?Tm=0.6 oC). The mutant G93P exhibits a ?Tm of 1.2 oC while mutants G254P, G406Q exhibited decreased stability, indicating that the effect of mutating Gly residues on enzyme stability is rather complex and can be only seen in combination with their structural environment. Further mutations aiming to eliminate a unique in this enzyme loop, and restore a disulfide bond did not produce an active enzyme. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified. The mode of action of ArChiA and ArChiB from the Antarctic Arthrobacter sp. strain TAD20 on both N-acetylchitooligomers and chitin polymers was examined using HPLC. ArChiB resulted to be an exochitinase cleaving N,N-diacetylchitobiose from the non reducing end while ArChiA an endochitinase cleaving chitin substrates randomly. At 100 μg/ml concentration ArChiB inhibited spore germination and hyphal elongation of the phytopathogenic fungus Botrytis cinerea strain 309 by 15 % and 30 % respectively. With ArChiA the effect was more pronounced. At enzyme concentration of 100 μg/ml and 200 μg/ml a 70 % and 90 % inhibition of conidial germination was observed as well as a 60 % and 90 % inhibition of germination tube elongation respectively. The combination of both enzymes at concentrations of 100 μg/ml produced a 76 % and 53.8 % inhibition of conidial germination and hyphal elongation respectively.
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| Language |
Greek |
| Subject |
Χιτινάση; Αλκαλική φωσφατάση; Ψυχροφιλικότητα; Κατευθυνόμενη μεταλλαξογένεση; Βακτήρια |
| Issue date |
2002-07-15 |
| Collection
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School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
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Type of Work--Doctoral theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/8/2/0/metadata-dlib-2002mavrommatis.tkl
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| Views |
241 |
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