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Degradation of polychlorinated biphe...

Fortin, Pascal.

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Degradation of polychlorinated biphenyl (PCB) metabolites: Directed evolution and enzymatic fortuity.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Degradation of polychlorinated biphenyl (PCB) metabolites: Directed evolution and enzymatic fortuity./
Author:	Fortin, Pascal.
Description:	120 p.
Notes:	Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6411.
Contained By:	Dissertation Abstracts International66-12B.
Subject:	Biology, Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=NR10492
ISBN:	9780494104927

Degradation of polychlorinated biphenyl (PCB) metabolites: Directed evolution and enzymatic fortuity.
Fortin, Pascal.

Degradation of polychlorinated biphenyl (PCB) metabolites: Directed evolution and enzymatic fortuity. - 120 p.

Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6411.

Thesis (Ph.D.)--The University of British Columbia (Canada), 2005.

A multi-faceted approach was used to investigate and overcome the PCB-degrading limitations of the Bph enzymes, responsible for the catabolism of biphenyl. First, the reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di- and trichlorinated 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia xenovorans sp. LB400 (DHBDLB400), DHBDP6-I and DHBDP6-III from Rhodococcus globerulus P6 and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 (THBDRW1). The specificity of each enzyme for particular DHBs differed by up to 3 orders of magnitude. Moreover, each enzyme cleaved at least one of the tested chlorinated DHBs better than DHB. However, no enzyme was able to cleave 2',6'-diCl DHB or 3,4-DHB, two recalcitrant PCB metabolites.

ISBN: 9780494104927Subjects--Topical Terms:

1017734
Biology, Microbiology.

Degradation of polychlorinated biphenyl (PCB) metabolites: Directed evolution and enzymatic fortuity.
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100 1 $a Fortin, Pascal. $3 1912050
245 1 0 $a Degradation of polychlorinated biphenyl (PCB) metabolites: Directed evolution and enzymatic fortuity.
300 $a 120 p.
500 $a Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6411.
502 $a Thesis (Ph.D.)--The University of British Columbia (Canada), 2005.
520 $a A multi-faceted approach was used to investigate and overcome the PCB-degrading limitations of the Bph enzymes, responsible for the catabolism of biphenyl. First, the reactivities of four evolutionarily divergent extradiol dioxygenases towards mono-, di- and trichlorinated 2,3-dihydroxybiphenyls (DHBs) were investigated: 2,3-dihydroxybiphenyl dioxygenase (EC 1.13.11.39) from Burkholderia xenovorans sp. LB400 (DHBDLB400), DHBDP6-I and DHBDP6-III from Rhodococcus globerulus P6 and 2,2',3-trihydroxybiphenyl dioxygenase from Sphingomonas sp. RW1 (THBDRW1). The specificity of each enzyme for particular DHBs differed by up to 3 orders of magnitude. Moreover, each enzyme cleaved at least one of the tested chlorinated DHBs better than DHB. However, no enzyme was able to cleave 2',6'-diCl DHB or 3,4-DHB, two recalcitrant PCB metabolites.
520 $a In the second facet of the study, biological selections were designed to facilitate the engineering of PCB-degrading enzymes via directed evolution. Although these selections failed to link the desired activities to host cell viability, they showed promise for other biocatalysts. In addition, a broadly useful high-throughput colorimetric screen was developed. The latter was applied to increase the specificity of DoxG, an extradiol dioxygenase from Pseudomonas sp. C18, for 3,4-DHB. A single round of directed evolution yielded DoxGSMA2, a variant that cleaved 3,4-DHB &sim;1000-fold more specifically than the wild-type enzyme. DoxGSMA2 contained three substituted residues: L190M, S191W and L242S. The crystal structure of the DoxG:3,4-DHB binary complex indicates that residues at position 190 and 242 occur on opposite sides of the DHB-binding pocket and may interact directly with the distal ring of the substrate. Kinetic analyses revealed that the substitutions are anti-cooperative.
520 $a Finally, characterization of BphK, the glutathione S-transferase from the bph pathway of B. xenovorans sp. LB400, revealed that the enzyme catalyzes the glutathione-dependant dehalogenation of certain inhibitory Cl HOPDAs. BphK catalyzed the dechlorination of 3-Cl HOPDA with a specificity constant of &sim;104 M -1s-1 in a reaction that utilized a ternary complex mechanism and 2 equivalents of GSH. The identified product of the reaction, HOPDA, is the substrate of the hydrolase from the bph pathway. The relatively low specificity constant of BphK for 3-Cl HOPDAs corroborate genetic evidence that the enzyme was recently recruited to the bph pathway to facilitate PCB degradation.
590 $a School code: 2500.
650 4 $a Biology, Microbiology. $3 1017734
650 4 $a Engineering, Environmental. $3 783782
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710 2 0 $a The University of British Columbia (Canada). $3 626643
773 0 $t Dissertation Abstracts International $g 66-12B.
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791 $a Ph.D.
792 $a 2005
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