In this study, the benzaldehyde lyase (BAL, EC 4.1.2.38) production in E. coli
BL21 (DE3) pLySs as intracellular and in Bacillus species as extracellular were
investigated, and comparison of the production capacity of the enzyme in the
developed recombinant microorganisms were compared. For this purpose, firstly,
PCR amplified bal gene was cloned into pRSETA vector which is under the control
of strong T7 promoter and expressed in E. coli BL21 (DE3) pLysS strain. With
developed recombinant E. coli BL21 (DE3) pLySs cells, the effect of bioprocess
parameters was systematically investigated. Among the investigated media, the
highest cell concentration and benzaldehyde lyase activity were obtained as 2.0
kg m-3 and 1060 U cm-3, respectively, in the medium containing 20.0 kg m-3
glucose, 11.8 kg m-3 (NH4)2HPO4 and the salt solution. Thereafter, oxygen
transfer effects on benzaldehyde lyase production were investigated at air inlet
v
rate of QO/VR = 0.5 vvm, and agitation rates of N=500 and 750 min-1 and at
QO/VR = 0.7 vvm, N=750 min-1 in pilot scale bioreactor and the highest cell
concentration and volumetric BAL activity were found as 1.7 kg m-3 and 990 U
cm-3, respectively, at 0.5 vvm, 750 min-1 condition. Next, the signal DNA
sequence of serine alkaline protease (SAP) from B. licheniformis DSM 1969
chromosomal DNA (pre-subC) was fused in front of the bal by using PCR-based
gene splicing by overlap extension (SOE) method. The fusion product of hybrid
gene first cloned into pUC19 plasmid, thereafter sub-cloned into pBR374 shuttle
vector and recombinant plasmid was transferred into various Bacillus species.
However, no extracellular production of benzaldehyde lyase was observed in
none of the developed recombinant Bacillus species, probably because of
ineffective secretion system, inefficient folding of heterologous protein,
degradation of enzyme due to proteolytic activity or high inactivation rate of the
enzyme.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/3/12607272/index.pdf |
| Date | 01 May 2006 |
| Creators | Kaya, Hande |
| Contributors | Calik, Pinar |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
Page generated in 0.0347 seconds