Production of a soluble hydrogenase from R. Eutropha H16

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2) and are generally classified by the structure of the catalytic site. From a biotechnology perspective, hydrogenases have been extensively studied with a view to their potential application in H2-based energy systems. Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni-Fe] uptake hydrogenase (SH) produced by Ralstonia eutropha H16. The research conducted in this project can be divided into two main parts. Firstly, bioprocess optimisation to produce and purify the SH from R. eutropha H16 was undertaken. Partially optimised batch-fermentations in controlled bioreactors were carried out using Fructose-Glycerol-Nitrogen (FGN) media to induce the production of SH in a diauxic growth process. Subsequently, further optimisations were performed on the cell ultrasonication and ammonium sulphate precipitation methods followed by two consecutive ion exchange steps and a final size exclusion chromatography step to recover functional SH. As a result of this purification scheme developed, a pure active SH preparation thatexhibits a specific activity of 2.86 U/mg (NAD+ reduction) was obtained with 18.7% yield and 13.1 fold purification. Subsequent electrochemical studies using cyclic voltammetry confirmed that the immobilised active enzyme on modified EPG electrodes was capable of oxidising H2 at the electrode surface. Secondly, molecular characterisation of the SH operon was investigated, involving two approaches: the analysis of the transcriptional regulation of the SH genes by quantifying the expressions of these genes using qRT-PCR, and development of a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. The expression of hoxF, hypF2 and hoxA genes were observed to be up-regulated by 4.6 + 0.94, 2.5 + 1.13 and 4.4 + 0.57 fold, respectively, under the derepressing growth condition. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by PSH promoter under derepressing conditions in FGN media was demonstrated in the recombinant R. eutropha.

Grantor dc:publisher

UNSW, Sydney

Year dc:date

2013

Author dc:creator

• Jugder, Bat-Erdene

• R. Eutropha
• Hydrogenase
• Hydrogen oxidation

Statement dc:rights

• open access
• CC BY-NC-ND 3.0
• free_to_read

Licence

• https://purl.org/coar/access_right/c_abf2
• https://creativecommons.org/licenses/by-nc-nd/3.0/au/

Language dc:language

EN

Identifier

https://doi.org/10.26190/unsworks/16257

OAI identifier oai:identifier

oai:unsworks.library.unsw.edu.au:1959.4/52792