Enhanced Polyhydroxybutyrate (PHB) production by arthrospira platensis, structural modeling, and characterization of PHA synthase in recombinant Escherichia cloni 10G

Name: Chanchanok Duangsri

LCSH: Kasetsart University -- Dissertations. Ph.D. (Animal Health Technology) 2021

Classification :.LCCS: QR82.E6

LCSH: Kasetsart University. -- Department of Veterinary Technology -- Dissertations

LCSH: Escherichia

LCSH: Biopolymers

LCSH: Cyanobacteria

LCSH: Polyhydroxybutyrate

LCSH: Gamma-hydroxybutyrate

Abstract: Polyhydroxybutyrate (PHB) is biopolymer synthesized under unbalanced growth conditions in microorganism. The biosynthesis of PHB is catalyzed by three enzymes consisting of β-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB) and PHA synthase (PhaC). The PhaC is the key enzyme for PHB production. Arthrospira platensis is one of cyanobacteria representing the capacity for PHB biosynthesis. In the present study, the A. platensis was subjected to nitrate deprivation supplemented with sodium acetate, sodium butyrate, sodium propionate or glucose. The results showed that biomass and chlorophyll-a contents of A. platensis were reduced in the absence of nitrate. However, the addition of carbon sources to culture, could alleviate the effects of nitrate deprivation. PHB content was observed by Nile-red staining (orange red fluorescent granule) and HPLC. Noticeably, increase of PHB was observed in cells grown photoheterotrophically for 3 days under nitrogen-deprived condition with 0.50%(w/v) acetate supplementation showing the maximum PHB contents of 19.2 ± 0.5% (w/cdw). Furthermore, the representative FTIR spectra of the extracted polymer from A. platensis gave the prominent peaks at 1721 cm−1 and 1279 cm−1 for the carbonyl (C=O) stretching of the ester group and asymmetric C–O–C stretching vibration, respectively. Moreover, this study successively constructed the recombinant E. cloni ®10G cells harboring A. platensis phaC (His-tagged PhaCAp). The purified His-tagged PhaCAp had a predicted molecular mass of 69 kDa, which had Vmax and Km values of 0.0234 mmol/min/mg protein and 0.0195 mM, respectively. Moreover, the activity and kcat of the purified His-tagged PhaCAp were 0.6 mol/min/mg protein and 412.7 s-1 . The sequence identity between A. platensis phaC (PhaCAp) and Chromobacterium sp. USM2 (PhaCCs) was only 24.06%, but the overall folding and the active site were found to be well conserved. Based on the three-dimensional structural model, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrates 3HB-CoA.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Wuttinun Raksajit

Role: Thesis Advisor

Name: Sarawan Kaewmongkol

Role: Thesis CO-Advisor

Created: 2021

Modified: 2025-07-26

Issued: 2025-07-26

วิทยานิพนธ์/Thesis

application/pdf

URL: https://www.lib.ku.ac.th/KUthesis/2564/chanchanok-dua-all.pdf

CallNumber: QR82.E6 .C43

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degraee

Descipline: Animal Health Technology

Grantor: Kasetsart University

©copyrights Kasetsart University

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	chanchanok-dua-all.pdf	3.53 MB

ใช้เวลา

0.038998 วินาที