Glucose-induced secretion of Trichoderma reesei xylanases
Glucose-induced secretion of Trichoderma reesei xylanases
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DOI：
10.1128/aem.62.8.2859-2865.1996
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发表时间：
1996-08-01
影响因子：
4.4
通讯作者：
Kubicek, CP
中科院分区：
文献类型：
--
作者：
Kurzatkowski, W;Torronen, A;Kubicek, CP
为了用在葡萄糖上生长的里氏木霉产生两种木聚糖酶，构建了在同源pki 1（丙酮酸激酶编码）基因的表达信号下携带xyn 1或xyn 2（木聚糖酶I和II [XYN I和XYN II]编码）结构基因的重组菌株。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫染色结果表明，两种转化子在葡萄糖上分别分泌XYN Ⅰ和XYN Ⅱ。最佳转化子在葡萄糖上的相应木聚糖酶比活力分别为76和145 U/mg蛋白质，与亲本菌株相比，然而，当与形成的生物量相关时，它们仅产生约4至5 U/g，与高得多的活性相反，（10至12 U/g）在木聚糖上生长期间，XYN II在产生最高组成型XYN II形成的菌株（ATX 2 -12）中的超微结构位置通过免疫电镜观察，并与木聚糖上生长的野生型菌株进行比较，两种类型的转化子的细胞提取物在葡萄糖上生长时均表现出比在木聚糖上生长的亲本菌株更高的胞内木聚糖酶活性。通过电镜和免疫金标记，在内质网中检测到XYN II，高尔基体样囊泡，分泌囊泡，空泡，和细胞壁，空泡中的免疫标记被检测到优先在亚顶端细胞，当一个重组菌株表达xyn 2从pki 1启动子与亲本菌株相比，在木聚糖生长过程中，前者表现出较少增殖的内质网和分泌囊泡的数量较少，但是，观察到较高的标记密度。这些研究结果的效力，蛋白质分泌生长过程中对葡萄糖的关系进行了讨论。
To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining, The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein), When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan, The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan, Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan, By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls, The immunolabel in the vacuoles was detected preferentially in subapical cells, When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.