摘要
采用PCR从海栖热袍菌 (Thermotogamaritima)克隆出编码极耐热稳定性阿拉伯糖苷酶基因 ,以pET 2 0b为表达质粒 ,与其C末端 6个组氨酸标签序列融合 ,在大肠杆菌中得到高效表达。基因表达产物通过热处理和亲和层析柱纯化后 ,酶纯度达电泳均一。纯化重组酶稳定性检测表明 ,阿拉伯糖苷酶活性最适作用温度和最适作用pH分别为 90～ 95℃和pH 5 .0～ 5 .5 ,在pH 4 .2～ 8.2之间酶活力稳定 ,95℃的半衰期为 4h ;SDS PAGE测得酶的分子量为 5 6 .5 7kD ,与理论推算值相吻合。在所测定的底物中 ,阿拉伯糖苷酶仅对对硝基苯 阿拉伯呋喃糖苷 (pNPAF)有专一性水解作用 ,其动力学参数Km 值为 0 18mmol L ,Vmax为 139μmol min·mg。
The gene of arabinofuranosidase from Thermotoga maritima was amplified by PCR, and inserted into the plasmid pET-20b with the 6-His tag, and expressed in E.coli JM109(DE3). The recombinant protein was purified by the heat treatment and immobilized metal affinity chromatography, purified enzyme presented as a single protein band on SDS-PAGE with a molecular weight of 56.57kD. The optimum activity of arabinofuranosidase was found to be at pH 5.0～5.5 and 90～95℃ and at the pH range of 4.2～8.2, the enzyme was stable with an half-life of 4h at 95℃. Among all tested substrates, the enzyme exhibited the specific activity towards p-nitrophenyl α-L-arabinofuranoside (pNPAF). The apparent Michaelis constant of the α-L-arabinofuranosidase was 0.18 mmol/L for pNPAF, and V max was 139μmol/min·mg protein.
出处
《微生物学报》
CAS
CSCD
北大核心
2004年第2期220-225,共6页
Acta Microbiologica Sinica
基金
国家轻工总局 2 11专项基金资助~~
