Purification and biochemical characteristics of β‐D‐xylanase from a thermophilic fungus, Thermomyces lanuginosus‐SSBP
Purification and biochemical characteristics of β‐D‐xylanase from a thermophilic fungus, Thermomyces lanuginosus‐SSBP
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DOI：
10.1111/j.1470-8744.1999.tb01162.x
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发表时间：
1999-08
影响因子：
2.8
通讯作者：
Johnson Lin;L. Ndlovu;Suren Singh;B. Pillay
中科院分区：
文献类型：
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作者：
Johnson Lin;L. Ndlovu;Suren Singh;B. Pillay
从嗜热真菌Thermomomorphilanossus-SSBP的培养滤液中纯化得到一种胞外木聚糖酶，并对其生化特性进行了研究。通过80%饱和度的程序实现了70-80%的产率。硫酸铵沉淀、DEAE-Sephadex A25和季氨乙基（QAE）-Sephadex A25柱层析。经SDS/PAGE分析，纯化的木聚糖酶的分子量为23.6 kDa，pI值为3.8。280 nm处吸光度的摩尔吸光系数为6.8×104 M−1·cm−1。使用二硝基水杨酸（DNS）方法计算的比活度为3500单位/mg。酶反应遵循Michaelis-Menten动力学，Kappm和Vmax值分别为3.26 mg/ml和6300单位/ml/mg蛋白质，如从Lineweaver-Burk图获得的。该木聚糖酶除水解木聚糖底物外不含其他酶活性（纤维素酶、β-葡萄糖苷酶、β-甘露糖苷酶、α-阿拉伯呋喃糖苷酶或β-木糖苷酶）。酶法的最适温度为70-75 °C。该酶在60 °C温育3小时后保持完全活性。木聚糖酶的最适pH为6.5，在较宽的pH范围内（pH 5-12）酶活性稳定。除了Pb 2+（中等抑制剂）和Hg 2+（强抑制剂）外，大多数测试的金属离子对酶活性没有影响。结果表明，每个酶分子中只要有1 ~ 2个色氨酸残基被N-溴代琥珀酰胺氧化，就足以完全抑制酶的活性，表明色氨酸残基在酶的催化过程中起着重要的作用。由于纯化的SSBP木聚糖酶的优异性质，该木聚糖酶在生物制浆过程和其他工业应用中具有潜在的用途。
An extracellular xylanase was purified to homogeneity from the culture filtrate of a thermophilic fungus, Thermomyces lanuginosus‐SSBP, and its biochemical characteristics were studied. A yield of 70–80% was achieved through the procedures of 80%‐satd. ammonium sulphate precipitation, DEAE‐Sephadex A25 and quaternary aminoethyl (QAE)‐Sephadex A25 column chromatography. The molecular mass of the purified xylanase was 23.6 kDa, as analysed by SDS/PAGE, with a pI value of 3.8. The molar absorption coefficient of the absorbance at 280 nm was 6.8×104 M−1·cm−1. The specific activity, calculated using the dinitrosalicylic acid (DNS) method, was 3500 units/mg. The enzyme reactions followed Michaelis–Menten kinetics with Kappm and Vmax values of 3.26 mg/ml and 6300 units/ml per mg of protein respectively, as obtained from a Lineweaver–Burk plot. The xylanase contained no other enzyme activity (cellulase, β‐glucosidase, β‐mannosidase, α‐arabinofuranosidase, or β‐xylosidase) except for the hydrolysis of xylan substrate. The optimal temperature of the enzyme assay was 70–75 °C. The enzyme retained full activity after a 60 °C incubation for 3 h. The optimal pH of xylanase activity was 6.5 and the enzyme appeared to be stable over a broad pH range (pH 5–12) under the assay conditions. The majority of the metal ions tested had no effect on the enzyme activity, with the exception of Pb2+ (modest inhibitor) and Hg2+ (strong inhibitor). The results showed that one or two tryptophan residues oxidized by N‐bromosuccinamide per enzyme molecule was sufficient to inhibit the enzyme activity completely, thus indicating that the tryptophan residues play an important role in the catalytical processes of the enzyme reaction. Because of the outstanding properties of the purified xylanase from the SSBP strain, this xylanase has a potential use in biopulping processes and other industrial applications.