Characterization and N-terminal amino acid sequences of beta-(1-4)endoxylanases from Streptomyces roseiscleroticus: purification incorporating a bioprocessing agent.
Streptomyces roseiscleroticus produces extracellular xylanases when cultured on a liquid xylan medium. Purified xylanases are used to facilitate bleaching of kraft pulps in the pulp and paper industry. Downstream processing and purification of xylanases from S. roseiscleroticus is difficult unless red pigments produced by the bacterium are removed. We report that the bioprocessing agent, Biocryl BPA-1000, removes these pigments allowing purification of four xylanases by HPLC employing cation exchange, hydrophobic interaction, and gel filtration. The xylanases have been named Xyl1, Xyl2, Xyl3, and Xyl4 according to their order of elution from the cation exchange column. The purified xylanases have been characterized according to their molecular weights, pH and temperature stabilities, N-terminal amino acid sequences, and hydrolysis action patterns on oat spelt xylan. The molecular weights by mass spectroscopy for Xyl1-Xyl4 are 33,647, 33,655, 21,070, and 46,855, respectively. All four xylanases exhibit pH optima between 5.0 and 7.0 and temperature optima between 50 and 60 degrees C. The N-terminal amino acid sequences are compared to sequences from Streptomyces lividans, Streptomyces 36A, and a Chainia sp. The N-terminal amino acid sequence of Xyl1 appears to be unique, but sequences from Xyl2, 3, and 4 bear strong homology to xylanases cloned from S. lividans. Xyl3 is also homologous to xylanases from Streptomyces 36A, and a Chainia sp. Predominant products of arabinoxylan hydrolysis by the purified xylanases included xylotriose, tetraose, and pentaose. None of the xylanases purified from S. roseiscleroticus produced xylose.
Grabski
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Protein Purification Facility, University of Wisconsin Biotechnology Center, Madison 53705.
Forrester
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Patel
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Jeffries
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TW
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D043364:Endo-1,4-beta Xylanases; D005374:Filtration; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*
D006026:Glycoside Hydrolases; D043364:Endo-1,4-beta Xylanases