Characterization and N-Terminal Amino Acid Sequences of β-(1-4)Endoxylanases from Streptomyces roseiscleroticus: Purification Incorporating a Bioprocessing Agent

Abstract

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.

Chromophore release from kraft pulp by purified streptomyces roseiscleroticus xylanases

“…(Bastawde et al 1991) and to Xln C from S. lividans (Kluepfel et al 1992). Xyl3 also possesses amino acid sequence homologies with these enzymes (Grabski et al 1993). The other three enzymes do not have obvious analogs.…”

“…Moreover, some appear to have a particular affinity for chromophores in pulp. In comparison to many other microbial xylanases with pH optima around 4.5 and temperature stabilities less than 40°C, the thermal stability (60 ° C) and the activity of S. roseiscleroticus xylanases at an alkaline pH (7-9) (Grabski et al 1993) enable them to be used under conditions more appropriate for the kraft-pulping process. Moreover, the relative specificity of the enzymes for chromophores could minimize xylan losses.…”

“…The other three enzymes do not have obvious analogs. Xyll is superficially similar to S. lividans Xln B in molecular mass and action pattern, but it differs significantly in amino acid composition (Grabski et al 1993). Xyl2 and Xyl4 are similar to one another in action pattern, but differ in molecular mass and amino acid sequence.…”

“…Xyl2 and Xyl4 are similar to one another in action pattern, but differ in molecular mass and amino acid sequence. Xyl4 is similar to Xln A in amino acid sequence (Grabski 1993) but differs in action pattern.…”

“…For enzyme production the strain was cultivated in shake flasks on complex medium using oat-spelt xylan as the carbon source, as described by Grabski and Jeffries (1991). Xylanase isozymes Xyll, Xyl2, Xyl3, Xyl4 were purified by successive ion exchange, hydrophobic interaction and molecular sieve chromatography (Grabski et al 1993).…”