Mattéotti Christel, Bauwens Julien, Brasseur Catherine, Tarayre Cédric, Thonart Philippe, Destain Jacqueline, Francis Frédéric, Haubruge Eric, De Pauw Edwin, Portetelle Daniel, Vandenbol Micheline
Unité de Biologie Animale et Microbienne, Gembloux Agro-Bio Tech, Université de Liège, B_5030 Gembloux, Belgium.
Protein Expr Purif. 2012 Jun;83(2):117-27. doi: 10.1016/j.pep.2012.03.009. Epub 2012 Apr 2.
Termites are world champions at digesting lignocellulosic compounds, thanks to cooperation between their own enzymes and exogenous enzymes from microorganisms. Prokaryotic cells are responsible for a large part of this lignocellulolytic activity. Bacterial enzyme activities have been demonstrated in the higher and the lower termite gut. From five clones of Gram-positive bacteria isolated and identified in a previous work, we constructed a genomic DNA library and performed functional screening for alpha-amylase, beta-glucosidase, and xylanase activities. One candidate, Xyl8B8, showed xylanase activity. Sequence analysis of the genomic insert revealed five complete ORFs on the cloned DNA (5746bp). Among the encoded proteins were a putative endo-1,4-beta-xylanase (XylB8) belonging to glycoside hydrolase family 11 (GH11). On the basis of sequence analyses, genomic DNA organization, and phylogenetic analysis, the insert was shown to come from an actinobacterium. The mature xylanase (mXylB8) was expressed in Escherichia coli and purified by affinity chromatography and detected by zymogram analysis after renaturing. It showed maximal xylanase activity in sodium acetate buffer, pH 5.0 at 55 °C. Its activity was increased by reducing agents and decreased by Cu(2+), some detergents, and chelating agents. Its substrate specificity appeared limited to xylan.
白蚁是消化木质纤维素化合物的世界冠军，这得益于它们自身的酶与微生物来源的外源酶之间的协同作用。原核细胞在这种木质纤维素分解活性中发挥了很大作用。在高等和低等白蚁肠道中均已证实存在细菌酶活性。从先前工作中分离并鉴定出的五个革兰氏阳性细菌克隆中，我们构建了一个基因组DNA文库，并对α-淀粉酶、β-葡萄糖苷酶和木聚糖酶活性进行了功能筛选。一个候选克隆Xyl8B8表现出木聚糖酶活性。对基因组插入片段的序列分析显示，克隆的DNA（5746bp）上有五个完整的开放阅读框（ORF）。编码的蛋白质中有一种推定的内切-1,4-β-木聚糖酶（XylB8），属于糖苷水解酶家族11（GH11）。基于序列分析、基因组DNA组织分析和系统发育分析，该插入片段显示来自一种放线菌。成熟的木聚糖酶（mXylB8）在大肠杆菌中表达，通过亲和层析纯化，并在复性后通过酶谱分析进行检测。它在pH 5.0的醋酸钠缓冲液中、55℃时表现出最大木聚糖酶活性。其活性因还原剂而增加，因Cu(2+)、一些去污剂和螯合剂而降低。其底物特异性似乎仅限于木聚糖。
