D-海因酶基因重组子的构建和酶的表达活性

以中华根瘤菌(Sinorhizobium morelense)SS—ori总DNA为模板，用PCR法扩增D-海因酶基因，分别克隆到5种不同的载体，并转入5种不同的Escherichia coli菌株，获得25株工程菌，进行培养与诱导表达．细胞经超声处理后，通过SDS—PAGE和酶活性两种指标比较表达产物．结果表明，除3株工程菌具有D-海因酶活性外，其它均为无酶活性的不溶性包涵体，包涵体经变性、复性后，可部分获得有活性的D-海因酶，其比活为0．90U／mg，复性率约为20％．此外，对包涵体产生的原因及可能解决办法也进行了讨论．

Construction Strategy of Recombinant Harboring D-hydantoinase Gene and Expression Activity of D-Hydantoinase

A DNA fragment coding D-hydantoinase gene was amplified by PCR from the chromosome DNA of Sinorhizobium morelense SS-ori and confirmed by DNA sequence analysis.The gene was cloned to five different vectors to be five recombinant plasmids and in ture to transfer into five different Escherichia coli strains,respectively.Then the total 25 engineered strains were cultured and induced in LB medium supplemented with antibiotics in the respective conditions.The results show that the target products expressed in 3 strains of them,pExSec-HDT/ E.coli BL21(DE3),pJLA-HDT/ E.coli M15 and pMAL-HDT/ E.coli BL21(DE3) are partly soluble and active,whilst that of the other 22 strains are all precipitates named inclusion bodies by the analysis both SDS-PAGE and enzymatic activity.It is also pointed out from a test of denaturation and renaturation of the inclusion body from the strain pET-HDT/ E.coli BL21(DE3) that the solubility of the enzyme after renaturation of inclusion body is about 20 % compared with the precipitate and the apparent enzymatic activity reaches 0.9 U/mg.In addition,the possible reasons and resolutions of inclusion bodies formed were also discussed.