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GH3家族新型海洋微生物β-葡萄糖苷酶基因的克隆、表达及重组酶性质研究

发表时间:2011-03-15  浏览量:1359  下载量:485
全部作者: 刘娟娟,方泽民,房伟,洪宇植,彭惠,张学成,肖亚中
作者单位: 安徽大学生命科学学院,生物工程研究中心
摘 要: 从中国南海表层海水微生物宏基因组文库筛选到一β-葡萄糖苷酶阳性克隆pSB16D15,经亚克隆测序分析鉴定,其含一新型GH3家族β-葡萄糖苷酶基因(bgl3)开放阅读框(open reading frame, ORF)。bgl3由2 535个核苷酸组成,G+C含量为43.03%,编码蛋白Bgl3由844个氨基酸组成,理论分子量为90 807 u,与来自Glaciecola sp. HTCC2999以及Alteromonas macleodii ATCC 27126的β-葡萄糖苷酶分别具有98%和61%的序列一致性。以pET22b(+)为载体,成功实现bgl3在E. coli BL21(DE3)细胞异源表达,并获得性质稳定的重组蛋白产物。理化性质分析表明:重组蛋白rBgl3氧化pNPG的最适pH值为6.5~7.0,最适温度为45℃. rBgl3在pH值为7.0~9.0范围内具有较高的酶活及稳定性:35℃下保存1 h能保持至少90%的活力。在pH值为7.0、45℃条件下,rBgl3催化pNPG的比酶活为17.07 IU·mg-1,Km,Vmax分别为1.80 mmol/L和0.81 μmol·min-1·mg-1. 当溶液中有10 mmol/L Cu2+,Fe2+和Fe3+存在时,rBgl3的活性完全被抑制;葡萄糖、乙醇和EDTA对酶活性也有一定抑制作用。
关 键 词: 分子生物学;β-葡萄糖苷酶;GH3家族;宏基因组文库
Title: Cloning, expression and characterization of a novel β-glucosidase of GH3 family from marine microbial metagenome
Author: LIU Juanjuan, FANG Zemin, FANG Wei, HONG Yuzhi, PENG Hui, ZHANG Xuecheng, XIAO Yazhong
Organization: Biotechnology Center, School of Life Science, Anhui University
Abstract: In the present study, a metagenome library of the marine microbes from the surface water of the South China Sea was screened for β-glucosidase. One positive clone named as pSB16D15 was obtained. It was subcloned and further analyzed in sequence. The results showed that there was an open reading frame (ORF) coding for a novel β-glucosidase of GH3 family member, which was nominated as bgl3. Further analysis showed that the 2 535-nucleotide-long ORF had a G+C content of 43.03%. The deduced product of bgl3 consisted of 844 amino acids (aa) with the predicted molecular masses of about 90, 807 u, sharing the highest homology with β-glucosidases from Glaciecola sp. HTCC2999 and Alteromonas macleodii with 98% and 61% identity, respectively. Using pET22b(+) as vector and E.coli BL21(DE3) as host, Bgl3 was overexpressed recombinantly, with substantial enzymic activity obtained. The recombinant protein (rBgl3) was purified with Ni-NTA affinity chromatography and further biochemically characterized. With pNPG as substrate, the optimum pH and temperature for the hydrolytic activity of rBgl3 were determined as 6.5~7.0 and 45℃, respectively. The recombinant protein maintained the highest activity at pH range of 7.0~9.0. Distinguished from most other β-glucosidase, rBgl3 remained 90% of the initial activity after heat treatment at 35℃ for 1 h. Under the optimum conditions, rBgl3 hydrolyzed pNPG with an activity of 17.07 IU·mg-1, a Km of 1.80 mmol/L and a Vmax of 0.81 μmol·min-1·mg-1. The activity of rBgl3 was completely inhibited by Fe3+, Cu2+, Fe2+ of 10 mmol/L, as well as EDTA, while 10 mmol/L K+ had a little effect on it. Glucose and ethanol suppressed the activity of rBgl3, with 80% of the original activity lost in 10 mmol/L glucose and 50% lost in 5% ethanol.
Key words: molecular biology; β-glucosidase; GH3 family; metagenome
发表期数: 2011年3月第5期
引用格式: 刘娟娟,方泽民,房伟,等. GH3家族新型海洋微生物β-葡萄糖苷酶基因的克隆、表达及重组酶性质研究[J]. 中国科技论文在线精品论文,2011,4(5):445-452.
 
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