北冰洋海单胞菌β-D-半乳糖苷酶的异源表达及酶学特性研究  

Heterologous expression and its enzymatic properties of β-galactosidase from a Marinomonas strain isolated from the Arctic Ocean

作　　者：孙茜 廖丽[2] 丁海涛[2] 刘双[1] 陈波[1,2] 

机构地区：[1]华东理工大学生物工程学院,上海200237 [2]中国极地研究中心国家海洋局极地科学重点实验室,上海200136

出　　处：《海洋学报》2015年第11期165-177,共13页

基　　金：国家"863"计划(2012AA092105);海洋公益性行业科研专项经费项目(201005032-4);上海市自然科学基金项目(13ZR1462700);南北极环境综合考察与评估专项(CHINARE04-03/04-05/01-06)

摘　　要：初筛表明,一株分离自北极加拿大海盆海冰心芯样品的海单胞菌（Marinomonas sp.BSi20584）具有较高的β-D-半乳糖苷酶活性,为了研究清楚其酶学性质,将经hiTAIL-PCR扩增得到的β-D-半乳糖苷酶基因（galt）与pET-28a（＋）原核表达载体结合,转入大肠杆菌BL21（DE3）。经IPTG诱导后对重组β-D-半乳糖苷酶（GALT）的表达条件进行了优化,采用金属螯合亲和层析技术制备纯酶,并对重组GALT的酶学性质进行了研究。结果显示,重组酶的最适诱导温度为20℃,在IPTG浓度为0.07mmol/L时诱导22h后,酶活和产酶量达到最大值。GALT单体分子量约为6.6×104 g/mol,天然酶为同源三聚体。GALT最适作用温度为35℃,其热稳定性较好,在60℃处理5h后,仍可保持50%以上的相对活性。GALT的最适作用pH为9.0,在pH为6.0~11.0范围内比较稳定。GALT的最适NaCl浓度为0.5mol/L,对盐度具有较高的耐受性。Mg2＋、K＋、DTT和EDTA对酶活不具有显著影响,而Mn2＋、Fe2＋对酶活有促进作用,Zn2＋和L-谷胱甘肽对酶活有抑制作用。GALT对Galβ1-4GlcNAc具有水解作用,而对Galβ1-3GalNAc和Galβ1-3GlcNAc糖苷键型没有水解能力。本研究实现了海单胞菌属菌株的β-D-半乳糖苷酶基因在大肠杆菌系统中的高效表达,并系统研究了重组酶的酶学特性,为后续开展该酶的代谢适应性和潜在应用研究提供详细的酶学数据基础。A Marinornonas strain isolated from an ice core sample collected from Canada Basin, Arctic Ocean, dis- played high β-galactosidase activity in the preliminary screening process. To get its overall understanding towards its enzymatic properties,the galt gene obtained by hiTAIL-PCR amplification was inserted into plasmid pET-28a （＋） ,then transformed into E. coli BL21 （DE3）. The recombinant enzyme was purified to electrophoretic homoge- neity by one step Ni2＋ affinity chromatography. The highest yield was achieved with 0.07mM IPTG when cultivat- ed in 20 ℃ for 22 h. The molecular weight of the monomeric GALT was estimated as about 6. 6 ;〈 104 g/mol and the native GALT was confirmed as homologous trimer through native-PAGE. The optimum temperature of GALT was 35 ℃ and showed good thermal stability at the temperature of 60℃ below. The optimum pH of GALT was 9. 0 and was stable between pH 6.0 and 11.0. GALT showed high tolerance to salinity and the optimum NaC1 concentration was 0.5 mol/L. Mineral ions Fe2＋ and Mn2＋ were identified as enzyme activators, Mg2＋ ,K＋ ,DTT and EDTA showed no significant influence towards the enzyme activity, while Zn2＋ and L-glutathione inhibited the activity strongly. GALT was able to hydrolyse Gal β1-4 GlcNAc but unable to hydrolyze Gal β1-3 GalNAc and Gal β1-3 GIcNAc. In this study,the β-galactosidase gene from Marinomonas sp. BSi20584 was successfully expressed in E. coli system, and a systematic understanding of the enzymatic properties of the recombinant GALT was acquired, which will provide the detailed enzymatic data for further studies on its metabolic adaptation and potential applications.

关 键 词：北冰洋 海单胞菌 Β-D-半乳糖苷酶 异源表达 酶学特性 

分 类 号：TQ925[轻工技术与工程—发酵工程]