St John Franz J, Rice John D, Preston James F
Department of Microbiology and Cell Science, University of Florida, Box 110700, Bldg. 981, Museum Rd., Gainesville, FL 32611, USA.
J Bacteriol. 2006 Dec;188(24):8617-26. doi: 10.1128/JB.01283-06. Epub 2006 Oct 6.
Secretion of xylanase activities by Bacillus subtilis 168 supports the development of this well-defined genetic system for conversion of methylglucuronoxylan (MeGAXn [where n represents the number of xylose residues]) in the hemicellulose component of lignocellulosics to biobased products. In addition to the characterized glycosyl hydrolase family 11 (GH 11) endoxylanase designated XynA, B. subtilis 168 secretes a second endoxylanase as the translated product of the ynfF gene. This sequence shows remarkable homology to the GH 5 endoxylanase secreted by strains of Erwinia chrysanthemi. To determine its properties and potential role in the depolymerization of MeGAXn, the ynfF gene was cloned and overexpressed to provide an endoxylanase, designated XynC, which was characterized with respect to substrate preference, kinetic properties, and product formation. With different sources of MeGAXn as the substrate, the specific activity increased with increasing methylglucuronosyl substitutions on the beta-1,4-xylan chain. With MeGAXn from sweetgum as a preferred substrate, XynC exhibited a Vmax of 59.9 units/mg XynC, a Km of 1.63 mg MeGAXn/ml, and a k(cat) of 2,635/minute at pH 6.0 and 37 degrees C. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and 1H nuclear magnetic resonance data revealed that each hydrolysis product has a single glucuronosyl substitution penultimate to the reducing terminal xylose. This detailed analysis of XynC from B. subtilis 168 defines the unique depolymerization process catalyzed by the GH 5 endoxylanases. Based upon product analysis, B. subtilis 168 secretes both XynA and XynC. Expression of xynA was subject to MeGAXn induction; xynC expression was constitutive with growth on different substrates. Translation and secretion of both GH 11 and GH 5 endoxylanases by the fully sequenced and genetically malleable B. subtilis 168 recommends this bacterium for the introduction of genes required for the complete utilization of products of the enzyme-catalyzed depolymerization of MeGAXn. B. subtilis may serve as a model platform for development of gram-positive biocatalysts for conversion of lignocellulosic materials to renewable fuels and chemicals.
枯草芽孢杆菌168分泌木聚糖酶活性，这有助于开发这个定义明确的遗传系统，用于将木质纤维素半纤维素成分中的甲基葡糖醛酸木聚糖（MeGAXn [其中n代表木糖残基的数量]）转化为生物基产品。除了已鉴定的糖基水解酶家族11（GH 11）内切木聚糖酶XynA外，枯草芽孢杆菌168还分泌第二种内切木聚糖酶，即ynfF基因的翻译产物。该序列与菊欧文氏菌菌株分泌的GH 5内切木聚糖酶具有显著同源性。为了确定其性质以及在MeGAXn解聚中的潜在作用，克隆并过表达了ynfF基因以提供一种内切木聚糖酶，命名为XynC，并对其底物偏好、动力学性质和产物形成进行了表征。以不同来源的MeGAXn为底物，比活性随着β-1,4-木聚糖链上甲基葡糖醛酸基取代的增加而增加。以枫香树的MeGAXn作为优选底物时，XynC在pH 6.0和37℃下表现出的Vmax为59.9单位/mg XynC，Km为1.63 mg MeGAXn/ml，k(cat)为2635/分钟。基质辅助激光解吸电离飞行时间质谱和1H核磁共振数据表明，每个水解产物在还原端木糖的倒数第二个位置有一个单一的葡糖醛酸基取代。对枯草芽孢杆菌168的XynC的详细分析定义了GH 5内切木聚糖酶催化的独特解聚过程。基于产物分析，枯草芽孢杆菌168分泌XynA和XynC。xynA的表达受MeGAXn诱导；xynC的表达在不同底物上生长时是组成型的。完全测序且具有遗传可塑性的枯草芽孢杆菌168对GH 11和GH 5内切木聚糖酶的翻译和分泌表明，这种细菌适合引入完全利用MeGAXn酶催化解聚产物所需的基因。枯草芽孢杆菌可作为开发用于将木质纤维素材料转化为可再生燃料和化学品的革兰氏阳性生物催化剂的模型平台。
