Indentification Of Streptomyces Rameus L2001 And Characterization Of Its Xylanase

Streptomyces sp. L2001 as the most promising strain was isolated and identified as Streptomyces rameus. 2.5%, 80# corncob xylan (water-insoluble) was the best carbon source. 0.5% yeast extract and 1.0% peptone was best nitrogen source. The optimal temperature, the initial pH value and the rotation rate of the cultures were 40℃, pH 6.0 and 140r/min, repectively. The maximum increase in xylanase production was observed in the presence of Tween 80 (0.8%). Under the optimized conditions, the highest xylanase acitiviy of 1810.9U/ml was achieved in the 6th day.Two extracellular xylanases were purified by ammonium sulfate precipitation, DEAE-52 chromatography and CM Sepharose Fast Flow chromatography. Xyn A as the most important xylanase, the molecular mass of approximately 21.1kD, was purified 13.3-fold and 21.8% activity recovery. Xyn B, the molecular mass of approximately 39.4kD,was purified 2.4-fold and 2.6% activity recovery.Xyn A had an optimum pH of 5.3, it was stable over pH 2.2^-11.3 at 50℃and stable over pH 4.3-6.7 at 70℃. The optimal temperature was 70℃and retained 60% of its activity at 70℃after 30min. The influence of metal ions and other reagent on xylanase activity was also studied, and the xylanase was strongly activated by Co²⁺. The xylanase was highly specific towards different xylans and did not act towards other polysacchrides. Apparent K_m value of the xylanase for birchwood and beechwood were 5.8mg·ml^-1 and 5.3mg·ml^-1, respectively. Xyn A released mainly xylobiose and xylotriose from birchwood xylan or beechwood xylan. Its N-terminal seguence of 15 amino acid residues was determined as ATVVTTNQTGTDNGF. Circular dichroism studies indicated that the protein contains about 37% ofα-helix, 26% ofβ-sheet and 37% of radom coil.Xyn B had an optimum pH of 6.7 and was stable over pH 4.2-9.15 at 50℃. The optimal temperature was 80℃and retained 90% of its activity at 60℃after 30min. As the same as Xyn A, the xylanase was strongly activated by Co²⁺. The xylanase was highly specific towards different xylans, but had a little activity towards CMC, pNP-β-D- cellobioside, pNP-β-D-xylopyranosideand pNP-α-L-arabinofuranoside.The potential application of Xyn A was investigated in wheat straw pulp. Enzymatic treatment at a charge of 15U/g dry pulp, the wheat straw pulp revealed an increase in brightness index by 2.8%ISO. Chlorine dioxide consumption was reduced by 15% in xylanase-pretreated pulp with the pulp still maintaining brightness as the control level.