Okai N, Fukasaku M, Kaneko J, Tomita T, Muramoto K, Kamio Y
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohuku University, Sendai, Japan.
Biosci Biotechnol Biochem. 1998 Aug;62(8):1560-7. doi: 10.1271/bbb.62.1560.
Aeromonas caviae W-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993) and Appl. Environ. Microbiol., 57, 445-449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviae in solution at 4 degrees C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from beta-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.
豚鼠气单胞菌W-61可产生5种木聚糖酶，即木聚糖酶1、2、3、4和5[Nguyen, V.D.等人，《生物科学、生物技术与生物化学》，56, 1708 - 1712 (1993)以及《应用与环境微生物学》，57, 445 - 449 (1991)]。在4℃将豚鼠气单胞菌纯化的木聚糖酶3制剂保存在溶液中时，发现木聚糖酶3被蛋白酶解，产生了一种分子量比完整形式小的截短形式。在这个特定的纯化实验中，截短形式的木聚糖酶3似乎是由污染的蛋白酶作用产生的。我们分离出了木聚糖酶3的截短形式（Xyn3tr），其缺失了C端的102个氨基酸残基片段。通过对Xyn3tr的N端和C端氨基酸残基进行化学分析以及对木聚糖酶3基因（xyn3）进行DNA测序分析，发现木聚糖酶3的N端第398个脯氨酸残基是Xyn3tr的C端。Xyn3tr具有将燕麦 spelto 木聚糖水解形成木三糖（X3）、木四糖（X4）、木五糖（X5）和木六糖（X6）作为主要终产物的活性。相比之下，完整的木聚糖酶3释放出X6和更高的木寡糖作为主要产物。木聚糖酶3可水解X4至X6。然而，Xyn3tr对X4和X5没有活性。从分别包含xyn3和xyn3tr基因的质粒pXYN3和pXYN3tr的大肠杆菌周质空间中均匀纯化出重组Xyn3tr和重组木聚糖酶3（XYN3），并测定了它们的酶活性。XYN3tr对燕麦spelto木聚糖和木寡糖的切割模式与完整的Xyn3tr相同。因此，我们得出结论，木聚糖酶3中包含一个由102个氨基酸残基组成的C端区域，该区域参与控制该酶从β-1,4-木聚糖上切割下来的木寡糖的分子大小以及对X4和X5的水解活性。
