Masuda Sumiko, Endo Kimiko, Koizumi Naoya, Hayami Tokusuke, Fukazawa Tetsuya, Yatsunami Rie, Fukui Toshiaki, Nakamura Satoshi
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan.
Extremophiles. 2006 Jun;10(3):251-5. doi: 10.1007/s00792-006-0514-3. Epub 2006 Apr 7.
Alkaliphilic Nocardiopsis sp. strain F96 produced three beta-1,3-glucanase isozymes of different molecular masses (BglF1, BglF2 and BglF3). The N-terminal amino acid sequences of BglFs indicated that these isozymes were the products of a single gene. The beta-1,3-glucanase gene (bglF) was cloned from the chromosomal DNA of strain F96. The bglF gene encoded a polypeptide of 270 amino acids including a signal sequence. The deduced amino acid sequence of mature BglF exhibited the highest homology to those of glycoside hydrolase (GH) family 16 beta-1,3-glucanases, suggesting that the enzyme belonged to the GH family 16. The mature region of bglF gene was functionally expressed in Escherichia coli. The optimum pH and temperature of purified recombinant BglF were pH 9.0 and 70 degrees C, respectively. This enzyme efficiently hydrolyzed insoluble beta-1,3-glucans and showed the highest activity toward a beta-1,3-1,4-glucan rather than beta-1,3-glucans. These results suggested that BglF would be a novel beta-1,3-glucanse. Mutational analysis revealed that Glu123 and Glu128 should be the catalytic residues of BglF.
嗜碱诺卡氏菌属菌株F96产生了三种不同分子量的β-1,3-葡聚糖酶同工酶（BglF1、BglF2和BglF3）。BglFs的N端氨基酸序列表明这些同工酶是单个基因的产物。从菌株F96的染色体DNA中克隆了β-1,3-葡聚糖酶基因（bglF）。bglF基因编码一个包含信号序列的270个氨基酸的多肽。推导的成熟BglF氨基酸序列与糖苷水解酶（GH）家族16的β-1,3-葡聚糖酶的序列具有最高同源性，表明该酶属于GH家族16。bglF基因的成熟区域在大肠杆菌中实现了功能表达。纯化的重组BglF的最适pH和温度分别为pH 9.0和70℃。该酶能有效水解不溶性β-1,3-葡聚糖，对β-1,3-1,4-葡聚糖的活性最高，而不是对β-1,3-葡聚糖。这些结果表明BglF将是一种新型的β-1,3-葡聚糖酶。突变分析表明，Glu123和Glu128应该是BglF的催化残基。
