Kester H C, Benen J A, Visser J
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.
Biotechnol Appl Biochem. 1999 Aug;30(1):53-7.
Apple-pectin hairy regions were prepared from apple pectin by combined action of the recombinant Aspergillus niger enzymes endopolygalacturonase II and pectin methylesterase and the A. tubigensis exopolygalacturonase. Using this enzymically prepared pectin fraction, an additional activity of the A. tubigensis exopolygalacturonase was discovered only when the substrate was chemically saponified and when D-galacturonate, a potent inhibitor of the enzyme, was removed from the incubation mixture. The new reaction product was purified and could be hydrolysed by A. niger beta-xylosidase into D-galacturonate and beta-D-xylose in a 1:1 ratio, which identified it as xylogalacturonate. The results demonstrate that exopolygalacturonase is not only active on galacturonan but also on xylogalacturonan. The enzyme thus accomodates a substrate in which the terminal galacturonic acid residue carries a single xylose substitution. The well-defined substrate specificity of exopolygalacturonase opens the possibility for use of this enzyme in biotechnological applications, such as preparing pectins that are methylated at the non-reducing end, and for studying the fine structure of xylogalacturonan in pectin.
通过重组黑曲霉内切多聚半乳糖醛酸酶II和果胶甲酯酶以及管囊曲霉外切多聚半乳糖醛酸酶的联合作用，从苹果果胶中制备了苹果果胶毛状区域。使用这种酶法制备的果胶组分，仅当底物进行化学皂化且从孵育混合物中去除该酶的强效抑制剂D-半乳糖醛酸时，才发现管囊曲霉外切多聚半乳糖醛酸酶具有额外活性。新的反应产物被纯化，并且可以被黑曲霉β-木糖苷酶以1:1的比例水解为D-半乳糖醛酸和β-D-木糖，这将其鉴定为木糖半乳糖醛酸。结果表明，外切多聚半乳糖醛酸酶不仅对半乳糖醛酸聚糖有活性，而且对木糖半乳糖醛酸聚糖也有活性。因此，该酶能够作用于末端半乳糖醛酸残基带有单个木糖取代的底物。外切多聚半乳糖醛酸酶明确的底物特异性为该酶在生物技术应用中的使用提供了可能性，例如制备在非还原端甲基化的果胶，以及用于研究果胶中木糖半乳糖醛酸聚糖的精细结构。
