Brutus Alexandre, Reca Ida Barbara, Herga Sameh, Mattei Benedetta, Puigserver Antoine, Chaix Jean-Claude, Juge Nathalie, Bellincampi Daniela, Giardina Thierry
Institut Méditerranéen de Recherche en Nutrition, Laboratoire de Biochimie et Biologie de la Nutrition, UMR Université Paul Cézanne Aix Marseille III, INRA 1111, service 342, Faculté des Sciences et Techniques Saint-Jérôme, Marseille Cedex, France.
Biochem Biophys Res Commun. 2005 Nov 11;337(1):160-6. doi: 10.1016/j.bbrc.2005.09.030.
The phytopathogen fungus Botrytis cinerea produces various glycosidases which are secreted during plant infection. In this study, the XynBc1 cDNA that encodes a xylanase from family 11 glycoside hydrolase from B. cinerea was identified by homology-based analysis, cloned by reverse transcription RT-PCR, fully sequenced, and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by chelating-affinity chromatography demonstrated high catalytic activity (180+/-23 U/mg) and efficiently degraded low viscosity xylan [K(m) = 10+/-3 g L(-1), V(max) = 0.50+/-0.04 micromol xylose min(-1), and k(cat) = 136+/-11.5 s(-1) at pH 4.5 and 25 degrees C]. XynBc1 was further tested for its ability to interact with wheat XIP and TAXI type xylanase inhibitors which have been implicated in plant defence. The xylanase activity of XynBc1 produced in P. pastoris was strongly inhibited by both XIP-I and TAXI-I in a competitive manner, with a K(i) of 2.1+/-0.1 and 6.0+/-0.2 nM, respectively, whereas no inhibition was detected with TAXI-II. We also showed that XynBc1 mRNAs accumulated during early stages of plant tissue infection.
植物病原菌灰葡萄孢会产生多种糖苷酶，这些酶在植物感染过程中分泌。在本研究中，通过基于同源性的分析鉴定了编码灰葡萄孢11家族糖苷水解酶木聚糖酶的XynBc1 cDNA，通过逆转录RT-PCR进行克隆、全序列测定，并在毕赤酵母X-33中进行异源表达。通过螯合亲和层析获得的纯化重组蛋白表现出高催化活性（180±23 U/mg），并能有效降解低粘度木聚糖[在pH 4.5和25℃时，K(m)=10±3 g L(-1)，V(max)=0.50±0.04 μmol木糖min(-1)，k(cat)=136±11.5 s(-1)]。进一步测试了XynBc1与小麦XIP和TAXI型木聚糖酶抑制剂相互作用的能力，这两种抑制剂与植物防御有关。毕赤酵母中产生的XynBc1的木聚糖酶活性受到XIP-I和TAXI-I的强烈竞争性抑制，K(i)分别为2.1±0.1和6.0±0.2 nM，而TAXI-II未检测到抑制作用。我们还表明，XynBc1 mRNA在植物组织感染的早期阶段积累。
