Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6.
Xylanase T-6 is a thermostable alkaline-tolerant enzyme that is produced by Bacillus stearothermophilus T-6. Xylanase T-6 was found to bleach pulp effectively at pH 9 and 65 degrees C and was used successfully on an industrial-scale mill trial. To facilitate the future characterization of the protein via X-ray analysis and protein engineering, it was necessary to overexpress the enzyme in Escherichia coli. The xylanase gene was cloned into T-7 polymerase expression vectors and its expression was optimized. The enzyme was found to constitute over 70% of the cell protein and it was efficiently purified from the host proteins by a single heating step. Over 2 g soluble and active enzyme per 1 culture were achieved.
Lapidot
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A
Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa, Israel.
Mechaly
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A
Shoham
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Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000235:genetics* / Q000302:isolation & purification*
D017931:DNA Primers; D011994:Recombinant Proteins; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase