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真养产碱杆菌112R4酰亚胺酶基因的克隆、序列分析及其在大肠杆菌中的表达
引用本文:王宇,张英姿,丁久元,刘阳剑,王绛,余志华.真养产碱杆菌112R4酰亚胺酶基因的克隆、序列分析及其在大肠杆菌中的表达[J].微生物学报,2002,42(2):153-162.
作者姓名:王宇  张英姿  丁久元  刘阳剑  王绛  余志华
作者单位:中国科学院微生物研究所,北京,100080
基金项目:国家自然科学基金资助项目 ( 395 70 0 1 5 )~~
摘    要:筛选到一株具海因水解活力的微生物,经鉴定后命名为真养产碱杆菌112R4。该菌能水解海因、二氢尿嘧啶和琥珀酰亚胺,且对琥珀酰亚胺活力最高,但不水解5单取代海因和5,5’双取代海因,因而被确定为含有酰亚胺酶。真养产碱杆菌112R4能在以琥珀酰亚胺为唯一碳、氮源的培养基上生长,表明该菌中存在琥珀酰亚胺完整的转化途径。从112R4基因组DNA出发,用鸟枪法克隆了一个6kb的与环酰亚胺水解相关的DNA片段;进一步亚克隆得到了带酰亚胺酶基因的2kb的DNA片段,并进行了序列测定。缺失分析确定了一个876bp的ORF为真养产碱杆菌112R4的酰亚胺酶基因,推测编码一个291个氨基酸的多肽,这是第一次报道微生物酰亚胺酶的核酸和蛋白序列。推测的氨基酸序列在蛋白数据库中进行了比较,结果表明,酰亚胺酶与已知的环酰胺酶没有明显的同源性,也不属于氨酰水解酶蛋白超家族,因而被分类为一种新的环酰胺酶。真养产碱杆菌112R4的酰亚胺酶与芽生杆菌A17p4的酰亚胺酶N端的20个氨基酸有较高的同源性,一致性为60%,与多糖脱乙酰酶保守序列也部分同源,一致性为14%。带有酰亚胺酶基因的重组质粒在大肠杆菌中得到表达,在lac启动子控制下,使用1mmol/L IPTG诱导5h,酰亚胺酶活力达到3200U/L,为供体菌真养产碱杆菌112R4的7倍。

关 键 词:酰亚胺酶,  基因克隆,  序列分析,  基因表达,  真养产碱杆菌
文章编号:0001-6209(2002)02-0153-10

Cloming, Sequence Analysis of Imidase Gene from Alcaligenes eatrophus and Its Expression in E. coli
Yu Wang,Yingzi Zhang,Jiuyuan Ding,Yangjian Liu,Jiang Wang,Zhihua Yu.Cloming, Sequence Analysis of Imidase Gene from Alcaligenes eatrophus and Its Expression in E. coli[J].Acta Microbiologica Sinica,2002,42(2):153-162.
Authors:Yu Wang  Yingzi Zhang  Jiuyuan Ding  Yangjian Liu  Jiang Wang  Zhihua Yu
Institution:Institute of Microbiology, The Chinese Academy of Science, Beijing 100080, China.
Abstract:A hydantoin-cleaving microorganism 112R 4 is screened and identified to be Alcaligenes eutrophus. The resting cell of Alcaligenes eutrophus 112R 4 can catalyze the hydrolysis of hydantoin, dihydropyrimidine and succinimide effectively, but not function to 5-monosubstituted hydantoins or 5,5'-disubstituted hydantoins. The microorganism can utilize succinimide as a sole carbon source and nitrogen source, which indicates the presence of a complete transformation pathway of succinimide, and a hydantoin-cleaving enzyme, imidase, is suggested to be contained in this metabolic pathway. A 6kb EcoRI-EcoRI fragment isolated from the genome DNA of Alcaligenes eutrophus 112R 4 is shown to be correlative with the transformation of succinimide. A 2kb DNA fragment containing the gene of imidase is subcloned and sequenced. Deletion analysis verifies that one open reading frame of 876 nucleotides, which encodes a peptide of 291 amino acids, with a calculated molecular weight of 33688, is responsible for the encoding of imidase. This is the first report of the nucleotide and amino acid sequences of imidase (GenBank accession number: AF373287). A homology search performed in protein database reveals an identity of 14% with polysaccharide deacetylase conserved domain, an identity of 60% with N-terminal 20 amino acids of Blastobacter sp. A17p-4, but no apparent similarity with all known cyclic-amide-cleaving enzymes. This result suggested that the imidase should be classified as a new member of cyclic amidases. Under the control of lac promoter and IPTG induction, the imidase activity of transformed E.coli reached 3200U/L, which is about 7-fold higher than that of gene donor strain.
Keywords:Imidase  Gene cloning  Sequence analysis  Gene expression  Alcaligenes eutrophus  
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