Watanabe Taisuke, Nasukawa Masashi, Yoshida Yuki, Kogo Takashi, Ogihara Jun, Kasumi Takafumi
1 Applied Microbiology and Biotechnology Laboratory, College of Bioresource Sciences, Nihon University.
2 Biotechnology Center, Chitose Laboratory Incorporated.
J Appl Glycosci (1999). 2019 May 21;66(2):59-64. doi: 10.5458/jag.jag.JAG-2018_0004. eCollection 2019.
In the current study, we attempted to enhance the xylanase activity of ATCC66589 by using disparity mutagenesis, wherein a plasmid harboring proofreading-impaired DNA polymerase δ was inserted. Following selection on xylan-rich media and successive plasmid curing, a mutant showing conidiospores strikingly different from those of the parent strain, with many small humped-surface spheres, was generated. Xylanase and β-xylosidase activities of the mutant XM1, cultivated in xylan medium, were 15.8- and 11.0-fold higher than those of the parent strain, respectively. Furthermore, xylanase activity was generated approximately 24 h in advance compared to that in the parent. In contrast, when cultivated in Avicel medium, its xylanase and β-xylosidase activities were 0.14- and 0.33-fold, respectively, compared to those in the parent. Among the xylan component sugars and related polyols, D-xylose and xylobiose exerted a distinct inductive effect on the xylanase activity in Avicel media, while xylitol and L-arabinose did not. Mutagenesis involved in xylose catabolism is suggestive of changes at the gene transcription level. Although the induction mechanism remains unclear in details, disparity mutagenesis may be useful for obtaining mutants with high xylanase activity.
在本研究中，我们尝试通过差异诱变提高ATCC66589的木聚糖酶活性，即将携带校对功能受损的DNA聚合酶δ的质粒插入其中。在富含木聚糖的培养基上进行筛选并连续去除质粒后，获得了一个突变体，其分生孢子与亲本菌株的分生孢子显著不同，有许多小的驼峰状表面球体。在木聚糖培养基中培养的突变体XM1的木聚糖酶和β-木糖苷酶活性分别比亲本菌株高15.8倍和11.0倍。此外，与亲本相比，木聚糖酶活性提前约24小时产生。相反，在微晶纤维素培养基中培养时，其木聚糖酶和β-木糖苷酶活性分别为亲本的0.14倍和0.33倍。在木聚糖组成糖和相关多元醇中，D-木糖和木二糖对微晶纤维素培养基中的木聚糖酶活性有明显的诱导作用，而木糖醇和L-阿拉伯糖则没有。参与木糖分解代谢的诱变表明基因转录水平发生了变化。虽然诱导机制的细节尚不清楚，但差异诱变可能有助于获得具有高木聚糖酶活性的突变体。
