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芽孢杆菌同源脱氧核糖醛缩酶的克隆及序列分析

发表时间:2010-09-15  浏览量:1499  下载量:719
全部作者: 陈丽伟,杨立荣,陈雄,徐刚,吴坚平
作者单位: 浙江大学化学工程与生物工程学系
摘 要: 采用基因采矿方法从Genbank中选取4株芽孢杆菌,以聚合酶链反应(Polymerase chain reaction,PCR)方法扩增了脱氧核糖醛缩酶(deoxyribose aldolases, DERA)基因,并在E.coli中实现过量表达。与E.coli K12 DERA(EcoDERA)相比,各种来源于芽孢杆菌的DERA催化天然底物2-脱氧-D-核糖-5-磷酸(2-deoxy-D-ribose-5-phosphate, DRP)进行裂解反应的活性接近,但对非磷酸化底物2-脱氧-D-核糖(2-deoxyribose, DR)的催化能力要远大于EcoDERA,且各种酶对2种底物的专一性常数之比([Kcat/Km(DR)]/[Kcat/Km(DRP)])均比EcoDERA高2~3个数量级,在热稳定性上也有明显优势,在70℃仍能保持40%~65%的酶活。经活性位点比较发现4种酶底物结合位点具有相似的结构特征,EcoDERA中Lys172,Phe200,Val206和Ser239分别在BsuDERA,BliDERA,BamDERA和BthDERA中被Phe,Val,Ile和Ala全部或部分取代。分析表明,这种替换可能影响底物结合位点静电环境变化和2个关键活性位点Lys残基架构周围的疏水相互作用,因此影响底物专一性。
关 键 词: 生物化学工程;脱氧核糖醛缩酶;基因采矿;底物专一性
Title: Cloning and sequence analysis of Deoxyribose aldolases from Bacillus strains
Author: CHEN Liwei, YANG Lirong, CHEN Xiong, XU Gang, WU Jianping
Organization: Department of Chemical and Biological Engineering, Zhejiang University
Abstract: Four Bacillus strains were chosen via genome mining, and the putative deoxyribose aldolases (DERA) origined from the chromosome of these microorganisms were individually cloned via Polymerase chain reaction (PCR), and over-expressed in E.coli. It was found that these Bacillus DERAs had similar cleavage activity for their natural substrate 2-deoxy-D-ribose-5-phosphate (DRP), compared with E.coli DERA(EcoDERA), while they exhibited higher catalysis ability over EcoDERA in the retroaldol reaction of nonphosphorylated substrate 2-deoxyribose (DR). The ratio of specifcity constants for DR compared to DRP ([Kcat/Km(DR)]/[Kcat/Km(DRP)]) of four Bacillus strains DERA were all two or three orders of magnitude higher than EcoDERA. On the other hand, they had high tolerance towards heat, considering 40%~65% of the activity could be retained when they were heated at 70℃. It showed similar structure characters in the alignment of five DERA amino acid sequences, that Lys172, Phe200, Val206, and Ser239 in EcoDERA were all or partly replaced by Phe, Val, Ile and Ala in BsuDERA, BliDERA and BamDERA, and BthDERA, respectively. The differences might affect the electrostatic environment in substrate-binding pocket and the hydrophobic interaction around the scaffold of the two key Lys residues, herein were responsible for the differences in substrate specificities.
Key words: biochemical engineering; deoxyriboaldolase; genome mining; substrate specificity
发表期数: 2010年9月第17期
引用格式: 陈丽伟,杨立荣,陈雄,等. 芽孢杆菌同源脱氧核糖醛缩酶的克隆及序列分析[J]. 中国科技论文在线精品论文,2010,3(17):1782-1789.
 
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