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Factors affecting production of Dengue viral coat protein in Pichia pastoris

Name: ปณัฐฐา กีรติพัฒน์

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Pichia pastoris

; Recombinant

; Dengue viral coat protein

; Methanol concentration

; Methanol consumption rate

Abstract: The factors affecting production of dengue viral coat protein (D2E) thatexpressed in P, pastoris (GPEsl7) was investigated in this study. Firstly, during thegrowth phase, an influence of glycerol concentration on growth and biomassproduction was studied. A linear relationship between biomass concentration andsubstrate concentration was observed. There, was no effect of substrate inhibition ongrowth of P, pastoris GPEs17 and the Basal salt medium could provide sufficientnutrients for its growth. Thus, any level of biomass within the range could beobtained through adjusting the concentration of glycerol supplied. From these results,the 2%(w/v) glycerol was chosen for the rest of experiments because it gave thehighest level of biomass during fed-batch compared with that of the 3% and the 4%(w~v). he dissolved oxygen in the 2%(w/v) culture could be easily controlled whileoxygen limitation within the bioreactor was not occurred. Additionally, less pureoxygen was supplied compared with that at higher glyccrol concentrations. In theproduction phase, methanol concentration was one major factor affecting the D2Eproduction. Within the range of methanol concentration fiom 0.25, 0.4, 0.5, 1, 2, 3 to4%(v/v), the maximum specific growth rate (pin,,), biomass concentration and D2Eprotein increased with methanol concentration only up to OS%(v/v). Maximumspecific growth rate (pin,,), biomass concentration generated and D2E proteinconcentration at the 0.5% methanol were 0.0064 h-I, 8.42 g . ~ ' l and 115 pg.~'],respectively. However, using 1% and 2%(v/v) gave lower growth and D2Eproduction than O.S%(v/v). Both growth and D2E protein was not observed in 3%and 4%(v/v). One possible reason could be that those concentrations might becometoxic if the proportion or ratio between biomass and methanol concentrations was notsuitable. Therefore, the influelice of biomass concentration and methanol ratio on theD2E production was studied in the shake flasks culture by varying the biomassconcentration from 5, 15, 20, 30 to 40 g . ~ ' ' in 0.5%, 1% and 2%(v/v) methanolBasal salt media. The level of D2E production could be enhanced through increasingbiomass concentration. However, the level increased was not proportional to the cellconcentration and the delay of D2E production was observed with increasingmethanol concentration.The D2E protein was monitored in a fully controlled fermentor of which thedissolved oxygen was controlled above 20% saturation level at all time. Themethanol consumption rate was estimated to be approximately 0.754 g,,,thanol.l.lh-l.Then, .this methanol consumption rate was used for a pulsed feeding strategy in thefermentor culture. The growth was observed throughout the process and the biomasswas increased higher than the shalte flask culture approximately 6.9 times whereas,the D2E production was not. The level achieved was close to the shalte flask, withinthe level of 200-400 JL~.L-'. The pulse feeding strategy might not be the properfeeding approach for the D2E production. Low D2E production obtained in this studymight depend on other factors such as cell status represented by the specific growthrate of the cell or the degradation of D2E during high cell concentration culture.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

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

Name: Assoc.Prof.Dr.Morakot Tanticharoen

Role: Supervisor

Created: 2543

Issued: 2005-10-28

Modified: 2006-05-30

วิทยานิพนธ์/Thesis

ISBN: 9746281704

BIT322

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวภาพ

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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