海栖热袍菌耐高温β-甘露糖苷酶基因的克隆及表达

Cloning and Expression of a β-Mannosidase Gene in Escherichia coli from the Extreme-thermophilic Thermotoga maritima

以海栖热袍菌(Thermotoga maritima)MSB8基因组DNA为模板,克隆得到β-甘露糖苷酶基因(man2).测序分析表明,该基因全序列为2358bp,编码785个氨基酸,分子量约为92×103.根据蛋白质氨基酸的同源性分析,该β-甘露糖苷酶与新阿波罗栖热袍菌(Thermotoga neapolitana)Man2(accession No.AAK52304.1)的同源性最大,达到80%.将此基因连接至表达载体pET-28a(+)并转化到大肠杆菌BL21细胞中,经IPTG诱导,β-甘露糖苷酶活力达5.96U/mL.粗酶的温度稳定性分析表明,该酶的热稳定性好,90℃处理10min,活力回收率65%,具有重要的工业应用前景.

The β-mannosidase gene （man2） was amplified by PCR from the genomic DNA of Thermotoga maritima MSB8, and was subsequently cloned into vector pET-28a（ ＋ ） and expressed in Escherichia coli BL21. The gene consisted of 2358 bp, and the translated protein （Man2） encoded 785 amino acids and its molecular mass was approximately 92 103. The mannosidase activity was up to 5.96 U/mL after the recombinant E. coli BL21 was induced by IPTG. The homology analysis of the deduced amino acid sequences showed that the Man2 shared 80% identity with the Man2 from Thermotoga neapolitana （ accession No. AAK52304.1）. The crude lysate remained 65% of residual activity after incubated at 90 22 for 10 min. This result indicates the Man2 is extremely thermostable and of great potential in application of various industries. Fig 3, Tab 1, Ref 17