Construction of a preconstruct for recombinant thermostable Carboxy methyl cellulase from the strain of Bacillus pumilus Z14

Akrami, Ania and Zahri, Saber and Latifi-Navid, Saeid (2014) Construction of a preconstruct for recombinant thermostable Carboxy methyl cellulase from the strain of Bacillus pumilus Z14. Masters thesis, University of Mohaghegh Ardabili.

Text (ساخت پیش سازواره کربوکسی متیل سلولاز نوترکیب مقاوم به حرارت حاصل از سویه Bacillus pumilus Z14) Ania Akrami.pdf Download (858kB)

Abstract

The endoglucanase are industrial enzymes which their recombinantly productions are economically important. The laboratory findings showed that the enzymes from Bacillus pumilus is thermostable, active in wide pH scale range, has significant stability in presence of the ions and detergents and can hydrolyse native lignocellulosic materials of fodder. In order to heterologous production of the enzyme, the pET21-b(+) expression vector was selected. The vector has important features for heterologous expression including: strong and regulative promoter, suitable multiple cloning site and histidine tag in downstream of cloning site. The two specific tailed primers was designed to amplify the ORF of the gene from B.pumilus Z14 which was included two restriction sites of BamHI-5ˊ and XhoI-3ˊ. In order to increment of restriction efficiency and future modifications of the gene, an expression preconstruct was prepared by cloning of the PCR fragment in vector pTG19-T. The recombinant molcules were transformed in E.coliDH5α using heat shock/CaCl2 method. The correct colonies were selected using blue/white screening and toothpick methods. The recombinant molecule was confirmed by appropriate restriction endoglucanase digestion. The insert was cloned in pTG19-T between two digestion sites of BamHI and digestion with the restriction enzyme produced two linear fragment. The lower band was matched to the size of the full length gene. Keywords: Bacillus pumilus, Cloning, Endoglucanase, Expression vector, Heterologous expression, Preconstruct.

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