Department of Chemistry, Biochemistry, & Physics, Marist College, Poughkeepsie, NY, USA.
J Appl Microbiol. 2019 Aug;127(2):508-519. doi: 10.1111/jam.14306. Epub 2019 Jun 18.
This work aims to determine the tolerance of xylanase towards enzyme-generated oxidative conditions, such as those produced by the peroxidase or laccase mediator systems (LMS).
The activity of Thermomyces lanuginosus xylanase was measured after incubation with lignin peroxidase, manganese peroxidase or laccase plus various mediators. The laccase system, using mediators such as 1-hydroxybenzotriazole and violuric acid, resulted in complete loss of xylanase activity, accompanied by an increase in the solution potential. However, an increase in solution potential alone was not sufficient to inactivate xylanase, nor was loss of xylanase activity always accompanied by a significant increase in solution potential, as observed with N-hydroxyphthalimide as the mediator. Neither lignin peroxidase nor manganese peroxidase impacted xylanase activity; only extended treatment with elevated hydrogen peroxide concentration promoted modest xylanase activity loss. The mechanism of inactivation as determined by the tryptophan-modifying reagent N-bromosuccinimide (NBS) indicated that oxidation of just one of the eight tryptophan residues of T. lanuginosus xylanase would be sufficient to result in complete loss of xylanase activity, since xylanase is completely inactivated at 1 : 1 molar ratio of NBS to xylanase.
While showing tolerance to peroxidase-based enzyme systems, T. lanuginosus xylanase is readily inactivated in the presence of the LMS. Based upon treatment with NBS as the oxidant, inactivation can be attributed to modification of a single tryptophan residue.
The simultaneous application of mixed hydrolytic and oxidative enzyme systems is of importance to biomass processing industries. Understanding the tolerance of xylanase to oxidative conditions will facilitate the design of reaction conditions or enzyme variants to maximize the impact of mixed enzyme systems.
本研究旨在确定木聚糖酶对酶促氧化条件的耐受性，例如过氧化物酶或漆酶介体系统（LMS）产生的氧化条件。
在与木质素过氧化物酶、锰过氧化物酶或漆酶加各种介体孵育后，测定嗜热真菌木聚糖酶的活性。使用 1-羟基苯并三唑和尿囊酸等介体的漆酶系统导致木聚糖酶活性完全丧失，同时溶液电势增加。然而，单独增加溶液电势不足以使木聚糖酶失活，并且木聚糖酶活性的丧失并不总是伴随着溶液电势的显著增加，如使用 N-羟基邻苯二甲酰亚胺作为介体时观察到的那样。木质素过氧化物酶和锰过氧化物酶均不影响木聚糖酶活性；只有延长处理时间并提高过氧化氢浓度才会导致适度的木聚糖酶活性丧失。通过色氨酸修饰试剂 N-溴代丁二酰亚胺（NBS）确定的失活动力学表明，只需氧化嗜热真菌木聚糖酶的 8 个色氨酸残基之一，就足以导致木聚糖酶活性完全丧失，因为木聚糖酶在 NBS 与木聚糖酶摩尔比为 1 ：1 时完全失活。
尽管嗜热真菌木聚糖酶对基于过氧化物酶的酶系统具有耐受性，但在 LMS 存在下，木聚糖酶很容易失活。根据 NBS 作为氧化剂的处理，失活可以归因于单个色氨酸残基的修饰。
混合水解和氧化酶系统的同时应用对生物质加工行业很重要。了解木聚糖酶对氧化条件的耐受性将有助于设计反应条件或酶变体，以最大限度地提高混合酶系统的效果。
