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

作　　者：张敏[1] 江正强[1] 李里特[1] 

机构地区：[1]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《微生物学通报》2008年第10期1565-1571,共7页Microbiology China

基　　金：国家"863计划"(No.2006AA10Z337)部分内容;教育部"新世纪优秀人才支持计划"(No.NCET-05-0130)部分内容

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2010bp,编码669个氨基酸,分子量为76.827kD。根据氨基酸同源性分析,该β-甘露聚糖酶与Thermotogasp.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39.7U/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pH8.0,85°C处理30min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。A β-mannanase gene （TM_1227） from Thermotoga rnaritima 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℃ and pH 8.0 respectively for LBG. The enzyme remained over 50% activity after treated at 85℃ 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 oligosaccha- rides, and almost no mannose was detected by TLC, which was suitable for mannooligosaccharides production.

关 键 词：海栖热袍菌 甘露聚糖酶 表达 高温酶 

分 类 号：Q814[生物学—生物工程]