UPSI Digital Repository (UDRep)
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Abstract : Universiti Pendidikan Sultan Idris |
This study was intended to construct a new Escherichia coli-Pseudomonas shuttle
vector for overexpression of elastase strain K in both E. coli and Pseudomonas as well
as for rapid purification using new RTX-tag. A 6.5 kb novel shuttle vector, designated
as pSIT/RTX, was constructed from pCon2(3) as to improvise the expression of
pCon2(3). pSIT/RTX was employed with tightly regulated promoter PT7(A1/O4/O3) for
controlling gene expression, stabilizing fragment (SF) for replication and maintenance
of plasmid in E. coli and P. aeruginosa, attB gene for genome integration, elastase
strain K as passenger enzyme and RTX-tag which is located at C-terminal for rapid
purification. E. coli TOP10/pSIT/RTX was chosen to proceed with purification as the
highest amount of proteolytic activity was detected at 12 h after incooperation with 0.6
mM IPTG (Isopropyl β- d-1-thiogalactopyranoside). Elastase strain K-RTX fusion
protein was purified using Ca2+ as novel ligand in immobilized-metal affinity
chromatography (IMAC) with 28 % recovery and 3.8 fold. The estimated molecular
weight as observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was 73 kDA, with optimal temperature and pH were 40oC and pH6,
respectively. The proteolytic activity was significantly enhanced by increasing the
concentration of Na+ and Cu2+ ions and more stable in phenylmethylsulfonyl fluoride
(PMSF), Tween20 and Triton-X-100. On the downside, Ni2+, Zn2+, n-dodecane, ntetradocane,
dithiothreitol (DTT) and SDS showed strong inhibition on the proteolytic
activity. Elastase strain K exhibited preference towards 25% (v/v) of Dimethyl
sulfoxide (DMSO), methanol and pyridine as their uniqueness as an organic solvent
tolerant enzyme. Congo-red as the most specified substrate for elastase recorded the
lowest release of its product. As a concluding remark, experimental work conducted in
this study had indeed highlighted several achievements, novelties and findings
including construction of vectors which had led to the overexpression of elastase strain
K by constructed vectors, the using of RTX-tag for purification via IMAC and most
importantly, the remarkable stability of elastase strain K in hydrophilic organic
solvents. |
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