用于阿糖鸟苷合成的腺苷脱氨酶在大肠埃希菌中的表达

Expression of Adenosine Deaminase in Escherichia coli Used for Synthesis of Guanine Arabinoside

铜绿假单胞菌(Pseudomonas aeruginosa TX16)具有腺苷脱氨酶活性,可以催化2,6-二氨基嘌呤核苷脱氨合成阿糖鸟苷,但铜绿假单胞菌TX16是一种条件致病菌,直接用于工业生产存在一定风险。将铜绿假单胞菌TX16菌株来源的腺苷脱氨酶基因(add)克隆至载体p ET-28a中,然后转化至大肠埃希菌Escherichia coli BL21(DE3),经过筛选获得工程菌E.coli(add)。经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导和聚丙烯酰胺凝胶电泳(SDS-PAGE)检测及酶活力测定,表明E.coli(add)能够表达大量腺苷脱氨酶,其产酶能力是含空载质粒的BL21(DE3)菌株E.coli(p ET-28a)的16.2倍。以E.coli(add)制备的粗酶液为催化剂,催化阿糖2,6-二氨基嘌呤核苷发生脱氨反应得到产物阿糖鸟苷,转化率可达96.8%。

Guanine arabinoside （Ara-G） could be synthesized from 2,6-diaminopurine arabinoside （Ara- DA） by Pseudomonas aeruginosa TX16 producing adenosine deaminase. However, as a conditional pathogen, there are potential risks to use the enzyme from Pseudomonas aeruginosa directly for Ara-G production. The adenosine deaminase gene （add） was amplified from P aeruginosa TX16 genome and cloned into the expression plasmid pET- 28a. The recombinant plasmid was transformed into E. coli BL21 （DE3） and add gene was successfully expressed after induction. The enzyme activity of engineering strain was 16.2 times of E. coli BL21 （DE3）, and was 16.0% higher than that ofP aeruginosa TX16. The crude enzyme prepared from E. coli BL21 （add） were used for the deamination of 2,6-diaminopurine arabinoside to produce guanine arabinoside with a 96.8 % conversion ratio.