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Type :thesis
Subject :QH Natural history
Main Author :Nurul Hazwani Shamsudin
Title :Characteristics of elastase strain K-Repeat-In-Toxin fusion protein overexpressed from newly constructed genetic tools
Place of Production :Tanjong Malim
Publisher :Fakulti Sains dan Matematik
Year of Publication :2021
Corporate Name :Universiti Pendidikan Sultan Idris
PDF Guest :Click to view PDF file

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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