de Vries R P, Poulsen C H, Madrid S, Visser J
Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
J Bacteriol. 1998 Jan;180(2):243-9. doi: 10.1128/JB.180.2.243-249.1998.
An extracellular alpha-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70 degrees C. The alpha-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and beta-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this alpha-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and alpha-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.
从一种市售曲霉菌制剂以及泡盛曲霉的培养滤液中纯化并鉴定了一种胞外α-葡萄糖醛酸酶。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，该酶的分子量为107 kDa，通过质谱测定为112 kDa，测定的pI略低于5.2，并且在pH 6.0下长时间稳定。该酶的最适pH在4.5至6.0之间，最适温度为70℃。α-葡萄糖醛酸酶主要作用于小的取代木寡糖，但也能够从桦木木聚糖中释放少量的4-O-甲基葡萄糖醛酸。该酶在木聚糖水解中与内切木聚糖酶和β-木糖苷酶协同作用。该酶进行N-糖基化，含有14个推定的N-糖基化位点。从泡盛曲霉中克隆了编码这种α-葡萄糖醛酸酶（aguA）的基因。它由一个2523 bp的开放阅读框组成，不含内含子。该基因编码一个841个氨基酸的蛋白质，含有一个20个氨基酸的真核信号序列。成熟蛋白的预测分子量为91,790 Da，计算的pI为5.13。将该基因的多个拷贝导入泡盛曲霉，并在一个高产转化体中研究其表达。aguA基因在木糖、木二糖和木聚糖上表达，类似于编码内切木聚糖酶的基因，表明木聚糖酶和α-葡萄糖醛酸酶的表达存在协同调控。葡萄糖醛酸不诱导aguA的表达，也不调节木糖上的表达。添加葡萄糖可阻止aguA在木聚糖上的表达，但仅降低木糖上的表达。
