真养产碱杆菌112R_4酰亚胺酶基因的克隆、序列分析及其在大肠杆菌中的表达

Cloming, Sequence Analysis of Imidase Gene from Alcaligenes eatrophus and Its Expression in E.coli

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 1 1 2R4 。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 1 1 2R4 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 1 1 2R4 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 1 1 2R4 的酰亚胺酶基因 ,推测编码一个 2 91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 1 1 2R4 的酰亚胺酶与芽生杆菌A1 7p 4的酰亚胺酶N端的 2 0个氨基酸有较高的同源性 ,一致性为 6 0 % ,与多糖脱乙酰酶保守序列也部分同源 ,一致性为 1 4%。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 LIPTG诱导 5h 。

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.