Fuchs Klaus-Peter, Zverlov Vladimir V, Velikodvorskaya Galina A, Lottspeich Friedrich, Schwarz Wolfgang H
Research Group Microbial Biotechnology, Technical University of Muenchen, Am Hochanger 4, D-85350 Freising, Germany.
Institute of Molecular Genetics, Russian Academy of Science, Kurchatov Sq., 123182 Moscow, Russia.
Microbiology (Reading). 2003 Apr;149(Pt 4):1021-1031. doi: 10.1099/mic.0.26153-0.
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.
嗜热栖热菌产生一种主要的β-1,3-葡聚糖酶。从DSM1237(T)（6848 bp）和F7（9766 bp）两个菌株中克隆了包含该基因的基因组DNA片段。重叠序列的同源性为99.9%。核苷酸序列包含一个推定转座酶、内切β-1,3-1,4-葡聚糖酶CelC、一个推定的LacI型转录调节因子、β-1,3-葡聚糖酶Lic16A和一个推定膜蛋白的阅读框。两个菌株的licA基因编码一种相同的1324个氨基酸的蛋白质，计算分子量为148 kDa。Lic16A是一种异常复杂的蛋白质，由一个前导肽、S层同源模块（SLH）的三重重复、一个未知模块、糖基水解酶家族16的催化模块和CBM4a家族碳水化合物结合模块的四重重复组成。重组Lic16A蛋白被鉴定为一种内切-1,3(4)-β-葡聚糖酶，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，Km分别为0.94和2.1 mg ml-(-1)。它对含有β-1,3-连接的β-葡聚糖具有特异性，在pH 6.0时的最适温度为70℃。N端的SLH模块在大肠杆菌和嗜热栖热菌中都能从蛋白质上切割下来，但仍与蛋白质其余部分紧密结合。Lic16A位于细胞表面，经分级溶解后可从该表面纯化。其诱导表达使嗜热栖热菌能够在β-1,3-或β-1,3-1,4-葡聚糖上生长。
