Kester H C, Kusters-van Someren M A, Müller Y, Visser J
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, The Netherlands.
Eur J Biochem. 1996 Sep 15;240(3):738-46. doi: 10.1111/j.1432-1033.1996.0738h.x.
From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH-range 3.7-4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly(1,4-alpha-D-galacturonide)galacturonohydrolase] that cleaves monomer units from the non-reducing end of the substrate molecule. K(m) and Vmax for polygalacturonic acid hydrolysis were 3.2 mg ml-1 and 3.1 mg ml-1 and 255 U mg-1 and 262 U mg-1 for the wild-type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2-7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2-7), the Michaelis constant (K(m)) decreased with increasing chain length (n). The Vmax value increased with chain length up to n = 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid (Ki = 0.3 mM) as well as by reduced digalacturonate (Ki = 0.4 mM). The exopolygalacturonase gene (pgaX) was cloned by reverse genetics and shows only 13% overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pgaX-coding region was used to achieve high level production of exopolygalacturonase under conditions where no endopolygalacturonases were produced.
从丝状真菌土曲霉的培养液中纯化出一种胞外多聚半乳糖醛酸酶，其分子量为78 kDa，等电点在pH 3.7 - 4.4范围内，最适pH为4.2。该酶已被鉴定为一种胞外多聚半乳糖醛酸酶[聚(1,4-α-D-半乳糖醛酸)半乳糖醛酸水解酶]，可从底物分子的非还原端切割单体单元。野生型和重组酶催化多聚半乳糖醛酸水解的米氏常数（K(m)）分别为3.2 mg ml-1和3.1 mg ml-1，Vmax分别为255 U mg-1和262 U mg-1。根据亚位点模型解释了胞外多聚半乳糖醛酸酶对不同聚合度（2 - 7）的低聚半乳糖醛酸的动力学数据，以更深入了解催化作用和底物结合。在不同聚合度（2 - 7）的低聚半乳糖醛酸上，米氏常数（K(m)）随链长（n）增加而降低。Vmax值随链长增加至n = 4，然后达到平稳值。该酶受到半乳糖醛酸（Ki = 0.3 mM）以及还原二半乳糖醛酸（Ki = 0.4 mM）的竞争性抑制。通过反向遗传学克隆了胞外多聚半乳糖醛酸酶基因（pgaX），其与黑曲霉内切多聚半乳糖醛酸酶的总体氨基酸序列同一性仅为13%。该胞外多聚半乳糖醛酸酶与植物多聚半乳糖醛酸酶关系最为密切。在所有已知的内切半乳糖醛酸酶和胞外多聚半乳糖醛酸酶之间，仅四个小的氨基酸片段是保守的。pgaX基因的表达可被半乳糖醛酸诱导，并受到分解代谢物阻遏。使用黑曲霉编码丙酮酸激酶的糖酵解基因的启动子与pgaX编码区之间的融合，在不产生内切多聚半乳糖醛酸酶的条件下实现胞外多聚半乳糖醛酸酶的高水平生产。
