Kawai Rie, Igarashi Kiyohiko, Yoshida Makoto, Kitaoka Motomitsu, Samejima Masahiro
Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
Appl Microbiol Biotechnol. 2006 Aug;71(6):898-906. doi: 10.1007/s00253-005-0214-4. Epub 2005 Dec 23.
When Phanerochaete chrysosporium was grown with laminarin (a beta-1,3/1,6-glucan) as the sole carbon source, a beta-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear beta-1,3-glucan, branched beta-1,3/1,6-glucan, and beta-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-beta-glucanase (EC 3.2.1.6) with broad substrate specificity for beta-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (beta-D-Glcp-(1->6)-beta-D-Glcp-(1->3)-beta-D-Glcp-(1->3)-D-Glc) and 4-O-glucosyl-laminaribiose (beta-D-Glcp-(1->4)-beta-D-Glcp-(1->3)-D-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes beta-D-Glcp-(1->3)-D-Glcp at subsites -2 and -1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a beta-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched beta-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular beta-1,3-glucanases.
当黄孢原毛平革菌以海带多糖（一种β-1,3/1,6-葡聚糖）作为唯一碳源生长时，会产生一种分子量为36 kDa的β-1,3-葡聚糖酶作为主要的细胞外蛋白。编码该酶的cDNA被克隆出来，推导的氨基酸序列表明该酶属于糖苷水解酶家族16；它被命名为Lam16A。在甲基营养型酵母毕赤酵母中表达的重组Lam16A能随机水解线性β-1,3-葡聚糖、分支的β-1,3/1,6-葡聚糖和β-1,3-1,4-葡聚糖，这表明该酶是一种典型的具有广泛β-1,3-葡聚糖底物特异性的内切-1,3(4)-β-葡聚糖酶（EC 3.2.1.6）。当以海带多糖和地衣多糖作为底物时，Lam16A分别产生6-O-葡糖基-海带三糖（β-D-葡萄糖吡喃糖基-(1->6)-β-D-葡萄糖吡喃糖基-(1->3)-β-D-葡萄糖吡喃糖基-(1->3)-D-葡萄糖）和4-O-葡糖基-海带二糖（β-D-葡萄糖吡喃糖基-(1->4)-β-D-葡萄糖吡喃糖基-(1->3)-D-葡萄糖）作为主要产物之一。这些结果表明，该酶在亚位点-2和-1处严格识别β-D-葡萄糖吡喃糖基-(1->3)-D-葡萄糖吡喃糖基，而在亚位点+1处允许6-O-葡糖基取代，并且在催化位点允许β-1,4-糖苷键连接。因此，Lam16A能从分支的β-1,3/1,6-葡聚糖产生非分支的寡糖，从而可能与其他细胞外β-1,3-葡聚糖酶一起有助于此类分子的有效降解。
