重组海栖热袍菌极耐热甘露聚糖酶的纯化和性质研究

Purification and Characterization of a Recombinant Thermostable β-mannanase from Thermotoga maritima

摘要:

研究了海栖热袍菌(Thermotoga maritima) MSB8甘露聚糖酶基因 (TM_1227)的克隆、重组酶的纯化和性质。该基因全序列2010 bp, 编码669个氨基酸, 分子量为76.827 kD。根据氨基酸同源性分析, 该β-甘露聚糖酶与Thermotoga sp. RQ2来源的β-甘露聚糖酶(GenBank登录号ACB09927.1)同源性最高, 为99％。重组转化子经IPTG诱导酶比活可达39.7 U/mg蛋白。粗酶液经金属亲和层析, 得到电泳纯甘露聚糖酶。以槐豆胶为底物时, 该酶的最适反应温度和pH分别为95°C和pH 8.0, 85°C处理30 min酶活保存50％以上, 很有潜力用于高温、偏碱性的造纸工业。对椰子甘露聚糖和槐豆胶的主要水解产物是不同聚合度的甘露寡糖, 几乎没有单糖生成, 适合生产低聚甘露糖。

Abstract:

A β-mannanase gene (TM_1227) from Thermotoga maritima MSB8 was cloned and expressed in E.coli. The recombinant β-mannanase was purified and characterized. The gene consists of 2010 bp, and the translated protein encodes 669 amino acids and its molecular mass is approximately 76.827 kD. Homology analysis of the deduced amino acid sequences showed that the enzyme shared 99% identity with β-mannanase from Thermotoga sp. RQ2. The mannanase activity was up to 39.7 U/mg after the recombinant E. coli BL21 was induced by IPTG. Crude enzyme solution was purified to homogeneity by Ni-NTA agarose. Its optimum temperature and pH was 95°C and pH 8.0 respectively for LBG. The enzyme remained over 50% activity after treated at 85°C for 30 min. The above properties showed great potential of its application in paper industry. The mannanase hydrolyzed copra mannan and LBG to give various sizes of oligosaccharides, and almost no mannose was detected by TLC, which was suitable for mannooligosaccharides production.