Ike Masakazu, Nagamatsu Kazuhisa, Shioya Akiko, Nogawa Masahiro, Ogasawara Wataru, Okada Hirofumi, Morikawa Yasushi
Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
Appl Microbiol Biotechnol. 2006 Jul;71(3):294-303. doi: 10.1007/s00253-005-0171-y. Epub 2005 Dec 10.
We purified a chitinase (named Chi46), with a molecular mass of 46 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from the culture filtrate of Trichoderma reesei PC-3-7 grown on N-acetylglucosamine (GlcNAc). The relative activity of this enzyme reduced when the degrees of acetylation (DA) of chitosan decreased. Furthermore, the enzyme was able to hydrolyze colloidal chitin and ethylene glycol chitin. The gene chi46 was cloned and sequenced. chi46 encodes a protein of 424 amino acid residues containing a 35-amino acid prepro-type secretion signal peptide. The molecular mass of mature Chi46 calculated from deduced amino acid sequence was 42,265 Da. The chi46 transcript was biphasic when the mycelia were grown on GlcNAc, suggesting that the multiple regulatory proteins are involved in the chi46 expression. The chi46 cDNA was expressed in Escherichia coli (ca. 0.23 mg/ml culture). To determine substrate cleavage fashion of Chi46 in more detail, we carried out high-performance liquid chromatography analysis and viscosimetric assay using recombinant Chi46 (rChi46). Chi46 was shown to release mainly (GlcNAc)(2) from colloidal chitin (insoluble chitin) as an exo-type manner and to act on chitosan 7B (DA ca. 30%) and N-acetylchitooligosaccharides (soluble chitins) in an endo-type one.
我们从在 N-乙酰葡糖胺（GlcNAc）上生长的里氏木霉 PC-3-7 的培养滤液中纯化出一种几丁质酶（命名为 Chi46），通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳估计其分子量为 46 kDa。当壳聚糖的乙酰化度（DA）降低时，该酶的相对活性降低。此外，该酶能够水解胶体几丁质和乙二醇几丁质。chi46 基因被克隆并测序。chi46 编码一个由 424 个氨基酸残基组成的蛋白质，含有一个 35 个氨基酸的前原类型分泌信号肽。根据推导的氨基酸序列计算出的成熟 Chi46 的分子量为 42,265 Da。当菌丝体在 GlcNAc 上生长时，chi46 转录本呈双相性，这表明多种调节蛋白参与了 chi46 的表达。chi46 cDNA 在大肠杆菌中表达（约 0.23 mg/ml 培养物）。为了更详细地确定 Chi46 的底物切割方式，我们使用重组 Chi46（rChi46）进行了高效液相色谱分析和粘度测定。结果表明，Chi46 主要以外切型方式从胶体几丁质（不溶性几丁质）中释放出 (GlcNAc)₂，并以内切型方式作用于壳聚糖 7B（DA 约 30%）和 N-乙酰壳寡糖（可溶性几丁质）。
