枝链霉菌L2001的鉴定及其木聚糖酶性质的研究
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摘要
从土壤中筛出一株木聚糖酶产生菌株Streptomyces sp. L2001,经鉴定为枝链霉菌。该菌株的最适产酶条件为:碳源是浓度为2.5%、粒度为80目的玉米芯水不溶性木聚糖,氮源为酵母浸膏(0.5%)+蛋白胨(1.0%),培养基初始pH值为6.0,培养温度为40℃,转速为140r/min。表面活性剂吐温80(0.8%)的添加对菌株产酶有促进作用。在最优条件下,枝链霉菌L2001木聚糖酶的产量高达1810.9U/ml。
枝链霉菌L2001所产的木聚糖酶经硫酸铵分级沉淀、DEAE-52柱层析和CM Sepharose Fast Flow柱层析,最终两种酶在SDS-PAGE中都显示为单一条带。其中,Xyn A是该菌株所产的最主要木聚糖酶,回收率为21.8%,纯化倍数为13.3,分子量为21.1kDa。Xyn B回收率为2.6%,纯化倍数为2.4,分子量为39.4kDa。
木聚糖酶Xyn A的最适反应pH值为5.3;50℃时pH值稳定范围是2.2~(-1)1.3,70℃时pH值稳定范围是4.3-6.7。该酶的最适反应温度为70℃;70℃时处理30min依然保留60%以上的相对酶活力。在各种金属离子和螯合剂中,Co~(2+)对酶促反应的促进作用最强。该酶具有很高的底物特异性,仅对木聚糖底物表现出特异性,对其它种类的多糖和糖苷底物均无活性。该酶对桦木木聚糖和榉木木聚糖的K_m值分别为5.8mg·ml~(-1)和5.3mg·ml~(-1)。在以桦木木聚糖和榉木木聚糖为底物时,水解产物主要为木二糖和木三糖,基本无木糖产生。该酶N端15个氨基酸残基序列为ATVVTTNQTGTDNGF。远紫外CD光谱显示,Xyn A的二级结构中大约含有37%的α-螺旋,26%的β-折叠和37%的无规则卷曲结构。
木聚糖酶Xyn B的最适反应pH值为6.7;50℃时pH值稳定范围是4.2-9.15。Xyn B的最适反应温度为80℃;60℃时处理30min依然保留90%的相对酶活力。金属离子对Xyn B的作用同木聚糖酶Xyn A相似,金属离子Co~(2+)对酶促反应有较强的促进作用。Xyn B对多种底物都表现出底物特异性,对各种木聚糖、羧甲基纤维素、pNP-β- D-纤维二糖苷、pNP-β-D-吡喃木糖苷、pNP-α-L-呋喃阿拉伯糖苷都有活性。
木聚糖酶Xyn A助漂麦草浆的实验结果显示,当酶用量为15U/g绝干浆时,麦草浆的白度与对照浆相比提高了2.8%ISO。在纸浆达到相同白度的情况下,酶助漂麦草浆可节约15%左右的用氯量。
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~(-1)1.3 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 Co~(2+). 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 Co~(2+). 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.
枝链霉菌L2001所产的木聚糖酶经硫酸铵分级沉淀、DEAE-52柱层析和CM Sepharose Fast Flow柱层析,最终两种酶在SDS-PAGE中都显示为单一条带。其中,Xyn A是该菌株所产的最主要木聚糖酶,回收率为21.8%,纯化倍数为13.3,分子量为21.1kDa。Xyn B回收率为2.6%,纯化倍数为2.4,分子量为39.4kDa。
木聚糖酶Xyn A的最适反应pH值为5.3;50℃时pH值稳定范围是2.2~(-1)1.3,70℃时pH值稳定范围是4.3-6.7。该酶的最适反应温度为70℃;70℃时处理30min依然保留60%以上的相对酶活力。在各种金属离子和螯合剂中,Co~(2+)对酶促反应的促进作用最强。该酶具有很高的底物特异性,仅对木聚糖底物表现出特异性,对其它种类的多糖和糖苷底物均无活性。该酶对桦木木聚糖和榉木木聚糖的K_m值分别为5.8mg·ml~(-1)和5.3mg·ml~(-1)。在以桦木木聚糖和榉木木聚糖为底物时,水解产物主要为木二糖和木三糖,基本无木糖产生。该酶N端15个氨基酸残基序列为ATVVTTNQTGTDNGF。远紫外CD光谱显示,Xyn A的二级结构中大约含有37%的α-螺旋,26%的β-折叠和37%的无规则卷曲结构。
木聚糖酶Xyn B的最适反应pH值为6.7;50℃时pH值稳定范围是4.2-9.15。Xyn B的最适反应温度为80℃;60℃时处理30min依然保留90%的相对酶活力。金属离子对Xyn B的作用同木聚糖酶Xyn A相似,金属离子Co~(2+)对酶促反应有较强的促进作用。Xyn B对多种底物都表现出底物特异性,对各种木聚糖、羧甲基纤维素、pNP-β- D-纤维二糖苷、pNP-β-D-吡喃木糖苷、pNP-α-L-呋喃阿拉伯糖苷都有活性。
木聚糖酶Xyn A助漂麦草浆的实验结果显示,当酶用量为15U/g绝干浆时,麦草浆的白度与对照浆相比提高了2.8%ISO。在纸浆达到相同白度的情况下,酶助漂麦草浆可节约15%左右的用氯量。
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~(-1)1.3 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 Co~(2+). 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 Co~(2+). 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.
引文
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