Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-beta-1,3-glucanase bound to the outer cell surface.
Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-beta-1,3-glucanase bound to the outer cell surface.
复制标题
热纤梭菌的 Lic16A，一种与细胞外表面结合的非纤维素体、高度复杂的内切 β-1,3-葡聚糖酶。
作者：
K. Fuchs;V. Zverlov;G. Velikodvorskaya;F. Lottspeich;W. Schwarz
热纤梭菌产生一种主要的β-1，3-葡聚糖酶。从两个菌株DSM 1237（T）（6848 bp）和F7（9766 bp）中克隆了含有该基因的基因组DNA片段。重叠序列有99.9%相同。核苷酸序列含有推定的转座酶、内切-β-1，3-1，4-葡聚糖酶CelC、推定的LacI型转录调节因子、β-1，3-葡聚糖酶Lic 16 A和推定的膜蛋白的阅读框。这两种菌株的licA基因编码一个相同的蛋白质的1324 aa的计算分子量为148 kDa。Lic 16 A是一种异常复杂的蛋白质，由前导肽、S层同源模块（SLH）的三重重复、糖基水解酶家族16的未知模块、催化模块和家族CBM 4a的碳水化合物结合模块的四重重复组成。重组Lic 16 A蛋白被表征为内切-1，3（4）-β-葡聚糖酶，其对大麦β-葡聚糖和海带多糖的比活性分别为2680和340 U mg（-1），K（m）分别为0.94和2.1 mg ml（-1）。它对含有β-1，3-键的β-葡聚糖具有特异性，最适温度为70 ℃，pH 6.0。在大肠杆菌和C.热纤维素，但仍然紧密结合到其余的蛋白质。Lic 16 A位于细胞表面，分级溶解后可从细胞表面纯化。它的诱导产生使C.热纤维素在β-1，3-或β-1，3-1，4-葡聚糖上生长。
Clostridium thermocellum produces one major beta-1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237(T) (6848 bp) and F7 (9766 bp). Overlapping sequences were 99.9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo-beta-1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, beta-1,3-glucanase Lic16A and a putative membrane protein. The licA genes of both strains encoded an identical protein of 1324 aa with a calculated molecular mass of 148 kDa. Lic16A is an unusually complex protein consisting of a leader peptide, a threefold repeat of an S-layer homologous module (SLH), an unknown module, a catalytic module of glycosyl hydrolase family 16 and a fourfold repeat of a carbohydrate-binding module of family CBM4a. The recombinant Lic16A protein was characterized as an endo-1,3(4)-beta-glucanase with a specific activity of 2680 and 340 U mg(-1) and a K(m) of 0.94 and 2.1 mg ml(-1) towards barley beta-glucan and laminarin, respectively. It was specific for beta-glucans containing beta-1,3-linkages with an optimum temperature of 70 degrees C at pH 6.0. The N-terminal SLH modules were cleaved from the protein as well in Escherichia coli as in C. thermocellum, but nevertheless bound tightly to the rest of the protein. Lic16A was located on the cell surface from which it could be purified after fractionated solubilization. Its inducible production allowed C. thermocellum to grow on beta-1,3- or beta-1,3-1,4-glucan.