การศึกษาบทบาทหน้าที่ Histidine Cluster ของเอนไซม์ desaturase ใน Spirulina platensis strain C1

Study of Functional Histidine Clusters of 6-desaturase in Spirulina platensis strain C1Miss Pavinee Kurdrit

Name: ภาวิณี เกิดฤทธิ์

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Spirulina platensis

; desD Gene

LCSH: ∆6-desaturase

; Histidine Cluster

; γ-linolenic acid (GLA)

Abstract: Spirulina platensis is a high potential cyanobacterium and thus used as a source of high value chemicals production. This cyanobacterium contains high value chemical constituents especially ?-linolenic acid (GLA). GLA is a polyunsaturated fatty acid (PUFA) used as a precursor in the synthesis of prostaglandin series and thus has got its importance in pharmaceuticals. Desaturation process in S. platensis is carried out by three desaturase genes desC, desA and desD encoding ?9-, ?12- and ?6-acyl-lipid desaturases respectively. The desD gene encoded ?6-desaturase is a key enzyme that catalyzes desaturation process of linoleic acid to GLA. Like other ?6-desaturases, ?6-desaturase of S. platensis contains three conserved histidine clusters, comprising seven conserved histidine residues (H89, H93, H124, H128, H129, H305 and H306). In order to understand the functional role of these conserved histidine residues, site directedmutagenesis was carried out to change three individual histidine residues in the second and the third conserved histidine motifs to glycine. Then, the desD gene and the mutated desD gene were expresssed in a heterologous host, Saccharomyces cerevisiae. Analysis of the enzyme activity in the presence of exogenously added substrate, linoleic acid, showed a significant GLA production when wild type S. platensis-?6-desaturase was being used. In contrast, the substitution of histidine by glycine (H129G, H305G and H306G) causes a complete elimination of ?6-desaturase activity. Interestingly, the activity of H129G, H305G and H306G could be partially restored by the addition of exogenous imidazole at the concentration of 150 mM. This study reveals that the histidine residues in the conserved histidine clusters play critical role in ?6-desaturase activity possibly by providing a diiron catalytic center.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร

Email: info.lib@mail.kmutt.ac.th

Name: Asst.Prof.Dr. Marasri Ruengjitchatchawalya

Role: Supervisor

Created: 2546

Issued: 2005-10-12

Modified: 2019-06-24

วิทยานิพนธ์/Thesis

ISBN: 9744565063

CallNumber: BIT466

tha

DegreeName: Master of Science

Level: Master's Degree

Descipline: Biotechnology

Grantor: King Mongkut's University of Technology Thonburi

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