东方肉座菌GH10家族木聚糖酶基因的克隆及异源表达

摘要

Endo-1,4-xylanase (E.C.3.2.1.8) is the major enzyme to the conversion of hemicelluloses into xylo-oligosaccharide.In this research, a novel GH10 xylanaseⅢ ( xynⅢ) gene was cloned from Hypocer a orientalis EU7 -22 by chromosome walking and PCR.The results showed that the DNA fragment (1283 bp) encoding xynⅢ (gxynⅢ) contained three introns. The CDS of xynⅢ( cyx nⅢ) encoded 331 amino acids of putative mature protein and a 16 aa signal in N terminator.The amino acid sequence of yx nⅢis highly homologous with the endoxylanase of Trichoderma pseudokno ingii.The bioinformatics analysis showed that the theoretical isoelec-tric point and the molecular weight of putative mature protein of XYNⅢwere 6.14 and 36.55 ku, respectively.It is a soluble hydro-philic protein containing 18 N-glycosylation sites.The 3D structure predicted with SWISS-Model showed that XYNⅢprotein contained 11 alpha helices and 8 extended strands.A recombinant plasmid pPIC9K-xynⅢwas constructed and then transformed into Pichia pas-toris.The transformant identified by PCR was induced to produce XYNⅢenzyme with 1%methanol.And after 168 hours induced ex-pression, the produced crude enzyme was detected to reach a high enzymatic activity of 127.5 IU/mL.%木聚糖酶是降解半纤维素最主要的酶，对于开发可再生生物能源具有重要的应用价值。分别以东方肉座菌（ Hypocrea orientalis） EU7－22的基因组DNA和cDNA为模板，利用染色体步移和PCR技术首次克隆获得该菌GH10家族木聚糖酶Ⅲ的基因（ xynⅢ），并对其进行生物信息学分析。结果表明：该基因全长1283 bp （ gxynⅢ），含有3个内含子；CDS序列为1044 bp（ cxynⅢ），编码347个氨基酸，N端含有一个16 aa的信号肽序列；XYNⅢ氨基酸序列与Trichoderma pseudokoningii的endoxylanase具有较高的同源性。经生物信息学分析，XYNⅢ成熟蛋白可能含有18个N－糖基化位点，其理论等电点（pI）为6．14，蛋白质分子质量为36．55 ku，属于亲水性蛋白；SWISS－Model 建模预测，XYNⅢ成熟蛋白中含有11个α螺旋，其核心结构为8个β折叠片围成一个柱状结构。同时将编码成熟蛋白的基因片段mxynⅢ与pPIC9K质粒连接构建表达载体后转化毕赤酵母，对重组子表达产物进行酶活检测显示该基因能在毕赤酵母中表达有生物活性的XYNⅢ并分泌到胞外，发酵液中的木聚糖酶活在诱导培养168 h后可达到127．5 IU／mL。