新型海洋微生物β-葡萄糖苷酶基因的克隆、表达及重组酶性质  

Cloning and characterization of a β-glucosidase from marine metagenome

作　　者：房伟[1] 方泽民[1] 刘娟娟[1] 洪宇植[1] 彭惠[1] 张学成[1] 孙宝林[2] 肖亚中[1] 

机构地区：[1]安徽大学生命科学学院生物工程研究中心,合肥230039 [2]中国科技大学生命科学学院,合肥230026

出　　处：《生物工程学报》2009年第12期1914-1920,共7页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划(863计划)(No.2007AA09Z421);安徽省优秀青年科技基金(No.08040106908)资助~~

摘　　要：通过功能筛选方法,从中国南海海洋表层海水微生物元基因组文库筛选得到了6个β-葡萄糖苷酶阳性克隆。对其中的一个阳性克隆pSB47B2进一步亚克隆和序列分析,获得一新型β-葡萄糖苷酶基因(命名为bgl1B)开放阅读框。以pET22b(+)为载体、Escherichia coli BL21(DE3)为宿主菌,bgl1B被高效活性重组表达。通过Ni-NTA亲和层析柱纯化了重组Bgl1B(rBgl1B)。纯化的rBgl1B催化pNPG水解反应的最适pH为6.5,最适温度为40oC。在最适反应条件下,rBgl1B水解pNPG的活性达到39.7U/mg,Km和Vmax分别为0.288mmol/L、36.9μmol/min。纤维二糖是rBgl1B的有效作用底物,其Km和Vmax分别为0.173mmol/L、35μmol/min。但rBgl1B不能催化转化蔗糖、乳糖、麦芽糖以及CMC。rBgl1B催化pNPG的水解反应对高浓度的Na+有较好的耐受性,而低浓度的Ca2+、Mn2+对该酶活有一定促进作用。不同于许多来源于真菌的酸性β-葡萄糖苷酶,rBgl1B在pH7.0～9.0范围内具有比较高的酶活力并具有较好的稳定性。In the present study,through a functional strategy,a metagenome library of the marine microbes from the surface water of the South China Sea was screened for β-glucosidase and six positive clones were obtained.One of these clones,pSB47B2,was subcloned and further analysed in sequence.The result showed that there was an open reading frame for a novel β-glucosidase,which was nominated as bgl1B.Using pET22b（＋） as vector and Escherichia coli BL21（DE3） as host,Bgl1B was overexpressed recombinantly with high yield obtained and substantial enzymatic activity detected. The recombinant protein （rBgllB） was purified by Ni-NTA affinity chromatography and further biochemically characterized. The results indicated that, with pNPG as substrate, the optimum pH and temperature for the hydrolytic activity of rBgllB were about 6.5 and 40℃ respectively. Under the optimumconditions, rBgllB hydrolyzed pNPG with an activity up to 39.7 U/mg, Km and Vmax being 0.288 mmol/L and 36.9 lamol/min respectively. In addition, rBgllB could also hydrolyze cellobiose, with a Km of 0.173 mmol/L and a Vmax of 35 μmol/min. However, we did not detect evident hydrolytic activity of rBgllB to lactose, maltose, sucrose, and CMC. The enzymatic activity of rBgllB to pNPG was stimulated to certain degrees by low concentration of Ca^2＋ or Mn^2＋, whereas it exhibited significant tolerance against high Na＋. Distinguished from most of the β-glucosidases derived from fungi, which display the highest activities under acidic conditions, rBgllB exhibited relatively higher activity and stability at pH between 7.0 and 9.0.

关 键 词：海洋微生物 元基因组 Β-葡萄糖苷酶 葡萄糖苷水解酶Ⅰ 

分 类 号：Q78[生物学—分子生物学]