To better understand the roles of genes involved in mannan degradation in filamentous fungi, in this study we searched, identified, and characterized one putative GH5 endo-β-mannanase (GH5-7) and two putative GH2 mannan-degrading enzymes (GH2-1 and GH2-4) in Neurospora crassa. Real-time RT-PCR analyses showed that the expression levels of these genes were significantly up-regulated when the cells were grown in mannan-containing media where the induction level of gh5-7 was the highest. All three proteins were heterologously expressed and purified. GH5-7 displayed a substrate preference toward galactomannan by showing 10-times higher catalytic efficiency than to linear β-mannan. In contrast, GH2-1 preferred short manno-oligosaccharides or β-mannan as substrates. Compared to the wild type strain, the growth of Δgh5-7 and Δgh5-7Δgh2-4 mutants, but not Δgh2-1, Δgh2-4, and Δgh2-1Δgh2-4 mutants, was poor in the cultures containing glucomannan or galactomannan as the sole carbon source, suggesting that GH5-7 plays a critical role in the utilization of heteromannans in vivo. On the other hand, all the mutants showed significantly slow growth when grown in the medium containing linear β-mannan. Collectively, these results indicate that N. crassa can utilize glucomannan and galactomannan without GH2-1 and GH2-4, but efficient degradation of β-mannan requires a concerted action of three enzymes, GH5-7, GH2-1, and GH2-4.

为了更好地了解参与丝状真菌中甘露聚糖降解的基因的作用，在本研究中，我们搜索、鉴定并表征了一种推定的 GH5 内切β-甘露聚糖酶 (GH5-7) 和两种推定的 GH2 甘露聚糖降解酶 (GH2-1和 GH2-4) 在粗糙脉孢菌中。实时 RT-PCR 分析表明，当细胞在含甘露聚糖的培养基中生长时，这些基因的表达水平显着上调，其中gh5-7的诱导水平是最高的。所有三种蛋白质均异源表达和纯化。GH5-7 显示出对半乳甘露聚糖的底物偏好，其催化效率是线性 β-甘露聚糖的 10 倍。相比之下，GH2-1 首选短甘露寡糖或 β-甘露聚糖作为底物。相比于野生型菌株，Δ的生长gh5-7和Δ gh5- 7Δ gh2-4突变体，但不Δ gh2-1，Δ gh2-4，和Δ gh2- 1Δ gh2-4突变体，是在差含有葡甘露聚糖或半乳甘露聚糖作为唯一碳源的培养物，表明 GH5-7在体内异甘露聚糖的利用中起关键作用. 另一方面，当在含有线性β-甘露聚糖的培养基中生长时，所有突变体都显示出显着缓慢的生长。总的来说，这些结果表明粗糙脉孢菌可以在没有 GH2-1 和 GH2-4 的情况下利用葡甘露聚糖和半乳甘露聚糖，但 β-甘露聚糖的有效降解需要三种酶 GH5-7、GH2-1 和 GH2-4 的协同作用.