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Functional analysis of endoinulinase from arthrobacter sp. S37 using site-directed mutagenesis and N-terminal domain deletion
위치-특이적 돌연변이 유도와 N-말단 영역 제거를 통한 Arthrobacter sp. S37 유래의 endoinulinase의 기능 분석

DC Field Value Language
dc.contributor.advisor김수일-
dc.contributor.author김용범-
dc.date.accessioned2010-06-07T07:10:44Z-
dc.date.available2010-06-07T07:10:44Z-
dc.date.copyright2003.-
dc.date.issued2003-
dc.identifier.urihttp://dcollection.snu.ac.kr:80/jsp/common/DcLoOrgPer.jsp?sItemId=000000058982eng
dc.identifier.urihttp://hdl.handle.net/10371/67615-
dc.descriptionThesis (master`s)--서울대학교 대학원 :농생명공학부,2003.en
dc.description.abstractEndoinulinase from Arthrobacter sp. S37 (EnIAb), a member of
family 32 glycosidase, catalyzes hydrolysis of inulin using two
catalytic residues, a nucleophile and an acid/base catalyst, as indicated
in the bell-shaped pH dependence profile. Sequence alignment of
EnIAb with other members of the family followed by a site-directed
mutagenesis and the pH dependence studies of mutants proposed a
detailed catalytic mechanism. Complete loss of enzyme activity in
D460A mutant and noticeable changes in the alkaline limb from 8.8 to
7.0 of pH dependence in D460E mutant revealed that Asp460 residue,
a component of the fully conserved RDP (Asn-Asp-Pro) motif among
family 32, elevates the pKa of the acid/base catalyst of EnIAb with
electrostatic interaction. Together with observations that only a kcat not
a Km value was reduced more than 100-fold in E323A, E519A, D460N
and D460E mutants, the active site of the enzyme is proposed to be a
triad of carboxylate groups Glu323 (nucleophile), Glu519 (acid/base
catalyst) and Asp460. Various single residue and truncated mutants of
the unique N-terminal domain (residue 1 ~ 240) suggested that the
domain has dual roles as a carbohydrate-binding domain and a direct
involvement in catalysis. Significant reductions in enzyme efficiency
(kcat/Km) were observed in W75K, W141A, and W141K mutants,suggesting these highly conserved aromatic residues W75 and W141
play a key role in substrate binding. In addition, N-terminal truncated
mutants Δ15, Δ45, Δ70 as well as Δ240 were almost inefficient in
enzyme catalysis, necessitating the structural integrity of the Nterminal
domain for catalysis. These results provide a model of the
active site common to a family 32 and a unique functional role of the
N-terminal domain in EnIAb.
en
dc.format.extentvii, 69 leavesen
dc.language.isoenen
dc.publisher서울대학교 대학원en
dc.subjectEnIAben
dc.subjectFamily 32 glycosidaseen
dc.subjectRetainining mechanismen
dc.subjectpKaen
dc.subjectSite-directed mutagenesisen
dc.subjectCarbohydrate-binding domainen
dc.titleFunctional analysis of endoinulinase from arthrobacter sp. S37 using site-directed mutagenesis and N-terminal domain deletionen
dc.title.alternative위치-특이적 돌연변이 유도와 N-말단 영역 제거를 통한 Arthrobacter sp. S37 유래의 endoinulinase의 기능 분석-
dc.typeThesis-
dc.contributor.department농생명공학부-
dc.description.degreeMasteren
Appears in Collections:
College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Food and Animal Biotechnology (식품·동물생명공학부)Theses (Master's Degree_식품·동물생명공학부)
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