CAJ | 학술논문

通过对前期筛选到的一株嗜热真菌Talaromyces thermophilus WYN9进行产木聚糖酶的诱导和非诱导培养。诱导培养的上清液中可以检测到木聚糖酶酶活。在非变性胶和变性胶SDS-PAGE中，诱导和非诱导的酶谱条带差异明显。以非诱导酶谱为对照，将诱导酶谱中与其有显著差异的条带切下，回收蛋白，测定酶活。将有木聚糖酶酶活的蛋白通过SDS-PAGE进一步分离纯化，切胶后使用胶内酶切法进行预处理，使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）进行分析。结果表明该蛋白与拟青霉属Paecilomyces sp. J18的内切木聚糖酶高度同源。根据质谱分析结果设计兼并引物，通过交错式热不对称PCR（thermal asymmetric interlaced PCR，TAIL-PCR）扩增，获得了TtXynA基因全长。根据Kimura双参数模型用临近相邻法构建了TtXynA及其同源蛋白的进化树。
통과대전기사선도적일주기열진균Talaromyces thermophilus WYN9진행산목취당매적유도화비유도배양。유도배양적상청액중가이검측도목취당매매활。재비변성효화변성효SDS-PAGE중，유도화비유도적매보조대차이명현。이비유도매보위대조，장유도매보중여기유현저차이적조대절하，회수단백，측정매활。장유목취당매매활적단백통과SDS-PAGE진일보분리순화，절효후사용효내매절법진행예처리，사용기질보조격광해흡전리비행시간질보（MALDI-TOF）진행분석。결과표명해단백여의청매속Paecilomyces sp. J18적내절목취당매고도동원。근거질보분석결과설계겸병인물，통과교착식열불대칭PCR（thermal asymmetric interlaced PCR，TAIL-PCR）확증，획득료TtXynA기인전장。근거Kimura쌍삼수모형용림근상린법구건료TtXynA급기동원단백적진화수。
A thermophilic fungus with xylanase activity, Talaromyces thermophilus WYN9, isolated in our previous study was cultured by the induced and non-induced mode. The xylanase activity was detected in liquid supernatant of induced culture model. The significant differences were observed between induced and non-induced zymogram bands both in non-denaturing gel and denaturing gel SDS-PAGE. The different bands were cut and the xylanase activities were detected. The recovered protein with xylanase activity was analyzed by MALDI-TOF. The result showed that the identified protein has high homology with an endo-xylanse from Paecilomyces sp. J18. Degenerate primers were designed according to the analysis results of mass spectrum and the full-length of TtXynA was obtained by TAIL-PCR amplification. NJ tree of TtXynA homologous proteins was constructed based on Kimura two-parameter distance.