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首页> 美国卫生研究院文献>SpringerPlus >Analysis of functional xylanases in xylan degradation by Aspergillus niger E-1 and characterization of the GH family 10 xylanase XynVII
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Analysis of functional xylanases in xylan degradation by Aspergillus niger E-1 and characterization of the GH family 10 xylanase XynVII

机译：黑曲霉E-1对木聚糖降解中功能性木聚糖酶的分析和GH家族10木聚糖酶XynVII的表征

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Xylanases produced by Aspergillus niger are industrially important and many types of xylanases have been reported. Individual xylanases have been well studied for their enzymatic properties, gene cloning, and heterologous expression. However, less attention has been paid to the relationship between xylanase genes carried on the A. niger genome and xylanases produced by A. niger strains. Therefore, we examined xylanase genes encoded on the genome of A. niger E-1 and xylanases produced in culture. Seven putative xylanase genes, xynI–VII (named in ascending order of the molecular masses of the deduced amino acid sequences), were amplified from the strain E-1 genome using primers designed from the genome sequence of A. niger CBS 513.88 by PCR and phylogenetically classified into three clusters. Additionally, culture supernatant analysis by DE52 anion–exchange column chromatography revealed that this strain produced three xylanases, XynII, XynIII, and XynVII, which were identified by N-terminal amino acid sequencing and MALDI-TOF-MS analyses, in culture when gown in 0.5% xylan medium supplemented with 50 mM succinate. Furthermore, XynVII, the only GH family 10 xylanase in A. niger E-1, was purified and characterized. The purified enzyme showed a single band with a molecular mass of 35 kDa by SDS-PAGE. The highest activity of purified XynVII was observed at 55°C and pH 5.5. The enzyme was stable in the broad pH range of 3–10 and up to 60°C and was resistant to most metal ions and modifying regents. XynVII showed high specificity against beechwood xylan with Km and Vmax values of 2.8 mg mL–1 and 127 μmol min–1mg–1, respectively. TLC and MALDI-TOF-MS analyses showed that the final hydrolyzed products of the enzyme from beechwood xylan were xylose, xylobiose, and xylotriose substituted with a 4-o-metylglucuronic acid residue.
机译：黑曲霉产生的木聚糖酶在工业上是重要的，并且已经报道了许多类型的木聚糖酶。各种木聚糖酶的酶学性质，基因克隆和异源表达已得到很好的研究。但是，对黑曲霉基因组携带的木聚糖酶基因与黑曲霉菌株产生的木聚糖酶之间关系的关注较少。因此，我们检查了黑曲霉E-1基因组中编码的木聚糖酶基因和培养物中产生的木聚糖酶。使用A.niger CBS 513.88基因组序列设计的引物，通过PCR和PCR扩增了七个推定的木聚糖酶基因xynI-VII（按推导的氨基酸序列的分子质量的升序命名）。在系统发育上分为三类。此外，通过DE52阴离子交换柱色谱对培养物上清液进行分析后发现，该菌株在培养时，会在培养物中产生三种木聚糖酶，即XynII，XynIII和XynVII，这是通过N端氨基酸测序和MALDI-TOF-MS分析确定的。 0.5％木聚糖培养基，添加50 mM琥珀酸酯。此外，纯化并鉴定了黑曲霉E-1中唯一的GH家族10木聚糖酶XynVII。通过SDS-PAGE，纯化的酶显示出分子量为35kDa的单条带。在55℃和pH 5.5下观察到纯化的XynVII的最高活性。该酶在3-10的广泛pH范围内以及高达60°C的温度下均稳定，并且对大多数金属离子和修饰剂具有抗性。 XynVII对山毛榉木聚糖具有高度特异性，Km和Vmax值分别为2.8 mg mL –1 和127μmolmin –1 mg –1 。 TLC和MALDI-TOF-MS分析显示，来自山毛榉木聚糖的酶的最终水解产物是被4-邻-甲基-葡糖醛酸残基取代的木糖，木糖和木三糖。

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