Download 150715215111Abstract_Shweta_IITKanpur

SusChemE 2015
International Conference on Sustainable Chemistry & Engineering
October 8-9, 2015, Hotel Lalit, Mumbai
Cloning, Sequencing and Expression of the Gene Encoding Azoreductase from Klebsiella sp.
Shweta Dixit1, Sanjeev Garg1
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Department of Chemical Engineering, IIT Kanpur, Kanpur UP 208016, India
[email protected], [email protected]
1. Introduction:
Azo dyes are the largest and most versatile class of dyes used in different industries including textile production, food, printing
and pharmaceuticals [1]. Azo dyes are toxic (carcinogenic) and are not completely degraded by conventional treatment
processes due to its recalcitrant nature. Bioremediation is most attractive and effective method to enhance the color removal
from wastewater due to its low cost and ecofriendly nature [2]. In this study, isolation, identification, cloning, sequencing and
expression of azoreductase gene from Klebsiella sp. is reported.
2. Material and Methods:
Klebsiella sp. was isolated from the sludge of a local dying industry. Methyl Orange was purchased from SD fine chemical,
India. pGEM-T cloning vector and T4 DNA Ligase were purchased from Promega, India. E. coli (DH5 alpha) and E. coli
BL21 (DE3) competent cells used for cloning and expression respectively were purchased from NEB, India.
The azoreductase gene was amplified from chromosomal DNA of Klebsiella sp. by polymerase chain reaction (PCR). PCR
products amplified were extracted from gel using Qiagen gel extraction kit. The purified DNA was directly cloned into the
pGEM-T cloning vector. The cloned gene was transformed into competent E. coli BL21 cells for expression.
3. Significant Results and Discussion
3.1 Isolation and identification of bacterial strain Klebsiella sp.
Bacterial strain was isolated in the laboratory from the sludge of a local dying industry by repeated enrichment culture [3]. The
bacterial strain was identified as Klebsiella sp. by 16S rDNA gene sequence analysis. Methyl Orange (Model dye, 100mg/l)
decolorization was achieved under microaerophilic (or anaerobic) condition in the presence of organic carbon source.
Decolorizations of other azo dyes are under progress.
3.2 Cloning, sequencing and expression of the gene encoding the azoreductase from Klebsiella sp.
A gene that encoded a protein with azoreductase activity was cloned by polymerase chain reaction amplification from the
genomic DNA of Klebsiella sp. The azo gene encoded a protein of 201 amino acids which showed azoreductase activity. The
azoreductase enzyme was heterologously expressed in E. coli with a strong band of 23 kDa on sodium dodecyl sulfate
polyacrylamide gel electrophoresis [Fig.1]. The integrity of the cloned gene was verified by sequencing.
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SusChemE 2015
International Conference on Sustainable Chemistry & Engineering
October 8-9, 2015, Hotel Lalit, Mumbai
Figure 1: Molecular mass determination of recombinant azo gene using SDS-PAGE
Lane M: Molecular mass marker; Lanes 1and 2: Crude extract of transformed cells;
Lane 3: Control (Transformed with pET only)
4. Conclusions:
It is concluded that the isolated Klebsiella sp. is highly efficient in decolorization of azo dyes e.g. Methyl Orange. The
recombinant azoreductase (after expression) decolorized MO (in the presence of NADH) more efficiently as compared to wild
type strain. The results confer that Klebsiella sp. has a great potential for the treatment of azo dyes containing waste water.
References
[1] J. T. Chacko and K. Subramaniam, Int. J. of Environ. Sci., 1(6) 2011, 1250-1260.
[2] I.M. Banat, P. Nigam, D. Singh and R. Marchant, Bioresour. Technol., 58 (3) 1996, 217-227.
[3] T. Joshi, L. Iyengar, K. Singh and S. Garg, Bioresour. Technol., 99 (15) 2008, 7115-7121.
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