Masson J Y, Boucher I, Neugebauer W A, Ramotar D, Brzezinski R
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, QC, Canada.
Microbiology (Reading). 1995 Oct;141 ( Pt 10):2629-35. doi: 10.1099/13500872-141-10-2629.
Strain N106, a newly isolated soil actinomycete classified in the genus Nocardioides on the basis of its chemotaxonomy, produced an extracellular chitosanase and was highly active in chitosan degradation. A gene library of Nocardioides sp. N106 was constructed in the shuttle vector pFD666 and recombinant plasmids carrying the chitosanase gene (csnN106) were identified using the 5'-terminal portion of the chitosanase gene from Streptomyces sp. N174 as a hybridization probe. One plasmid, pCSN106-2, was used to transform Streptomyces lividans TK24. The chitosanase produced by S. lividans (pCSN106-2) is a protein of 29.5 kDa, with a pI 8.1, and hydrolyses chitosan by an endo-mechanism giving a mixture of dimers and trimers as end-products. N-terminal sequencing revealed that the mature chitosanase is a mixture of two enzyme forms differing by one N-terminal amino acid. The csnN106 gene is 79.5% homologous to the csn gene from Streptomyces sp. N174. At the amino acid level, both chitosanases are homologous at 74.4% and hydrophobic cluster analysis revealed a strict conservation of structural features. This chitosanase is the third known member of family 46 of glycosyl hydrolases.
菌株N106是一种新分离的土壤放线菌，根据其化学分类法被归类为诺卡氏菌属，它能产生一种细胞外壳聚糖酶，且在壳聚糖降解方面具有高活性。构建了诺卡氏菌属N106的基因文库，该文库使用穿梭载体pFD666构建，并且使用来自链霉菌属N174的壳聚糖酶基因的5'-末端部分作为杂交探针，鉴定携带壳聚糖酶基因（csnN106）的重组质粒。一个质粒pCSN106 - 2被用于转化变铅青链霉菌TK24。变铅青链霉菌（pCSN106 - 2）产生的壳聚糖酶是一种29.5 kDa的蛋白质，其pI为8.1，通过内切机制水解壳聚糖，产生二聚体和三聚体的混合物作为终产物。N端测序表明，成熟的壳聚糖酶是两种酶形式的混合物，它们在一个N端氨基酸上有所不同。csnN106基因与来自链霉菌属N174的csn基因具有79.5%的同源性。在氨基酸水平上，两种壳聚糖酶的同源性为74.4%，疏水簇分析显示结构特征严格保守。这种壳聚糖酶是糖基水解酶家族46的第三个已知成员。
