【作者】 封霞；

【导师】 袁静明；

【作者基本信息】 山西大学 ， 生物化学与分子生物学， 2004， 硕士

【副题名】D-海因酶基因的克隆、表达、纯化及其性质

【摘要】 海因酶（Hydantoinase，EC 3.5.2），是一类催化海因、5’-单替代海因及其衍生物环酰胺键断裂的酰胺水解酶。主要用于手性氨基酸的生物合成，如D-型氨基酸被广泛应用于医药及食品领域，是合成半合成抗生素、多肽激素、拟除虫菊脂、杀虫剂、甜味剂等的重要中间物。D-型氨基酸的生物合成经历两个反应历程，首先由D-海因酶催化5’-单替代海因及其衍生物水解生成N-氨甲酰基-D-型氨基酸，产物再在N-氨甲酰基-D-氨基酸酰胺水解酶的作用下生成相应的D-型氨基酸。目前，用微生物菌体转化生产D-型氨基酸的工艺路线已经产业化，但天然菌体转化工艺存在一些限制因素，阻碍了D-型氨基酸的扩大生产，导致产品短缺。因此，利用基因工程菌生物转化D-型氨基酸已成为世界氨基酸产业的新潮流。 本文根据文献报道的海因酶基因序列及大肠杆菌对密码子的偏爱性分别设计了正向及反向引物，以基因组DNA为模板，利用PCR技术扩增得到菌株Pseudomonas putida YZ-26和Sinorhizobium morelense SS-ori的D-海因酶基因。序列分析表明，来自YZ-26菌的D-海因酶基因全长1440 bp，编码479氨基酸，基因序列已被GenBank登录（AY387829），被确定为一个新基因。来自SS-ori菌的D-海因酶基因全长1374 bp，编码457氨基酸。 将YZ-26菌的D-海因酶基因分别与载体pET-3a和pGEX-4T-1连接，构建重组质粒EHD-YZ26和GHD-YZ26。重组质粒EHD-YZ26在E．coli BL21（DE3）中30℃生长10 hrs，在不需添加任何诱导剂的情况下可获得可溶性表达，目的蛋白的表达量约占总菌蛋白的20％，其酶活力为13 U／mL，约是原始菌株的D-海因酶表观活性的13倍。将工程菌株发酵所得酶液，经（NH₄）₂SO₄分级沉淀、Phenyl Sepharose疏水层析、Sephacryl S-200凝胶层析三步，得到D-海因酶纯制剂。其比活为16 U／mg，活力回收为55％，纯化倍数为3.4。通过对其生化性质的研究表明，其单体M_r为52042，天然M_r为102600，最适pH为pH 9.5，最适温度为45℃，不同的二价金属离子对酶活性有不同的影响。该酶是金属依赖型D-海因酶。重组质粒GHD-YZ26在E．coli BL21（DE3）中30℃诱导培养8 hrs，可获得可溶性表达，融合蛋白的表达量约占总菌蛋白的15％，其酶活力为5 U／mL，约是原始菌株的D-海因酶表观活性的8倍。将工程菌株发酵所得酶液，经GSTrap亲和层析，得到融合蛋白GST-HDT纯品。其比活为4 U／mg，活力回收为20％，纯化倍数为4.7。山西大学2004届硕士研究生学位论文 将55一ori菌的D一海因酶基因分别与5种载体连接，转入5种不同的宿主菌中，构建25株_「程菌，并各白在最适的条件下进行培养与诱导表达。除3株工程菌具有D一海因酶活性外，其它22株l一程菌表达均为无酶活性的聚合物。对其中产酶活性最高的一工程菌pExsec一HDT/云colj BLZ]（DE3）海因酶的表达条件进行了研究，目的蛋白的表达量约占总菌蛋白的20%，其酶活力为0.92u/mL，约是原始菌株的D一海因酶表观活性的4.6倍。对一「程菌pET- HDT，/E.。。11 BL21（0E3）表达的包涵体蛋白进行了变性、复性研究，部分获得有活性的D一海因酶，酶活力约为0.90U/mgo更多还原

【Abstract】 Hydantoinase (EC 3.5.2) hydrolyzes its substrate 5’-monosubstantial hydantoin to enantiomerical N-carbamyl-amino acids and in turn, the resulting product can be chemically or enzymatically converted into the corresponding optically active amino acids. A number of bacterial D-hydantoinases with the different stereoselectivity and substrate specificity have been used in industrial bioconversion of optically active D-amino acids. D-Amino acids as unnatural chiral products are important intermediates in the synthesis of products, such as β-lactam semisynthetic antibiotics, antiviral agents, artificial sweeteners, pesticide, peptide hormones, and pyrethroids, etc. Though bioconversion of D-amino acids using microbial cells has been realized in industrialization for decades, some limited factors hinder the development of D-amino acid production, and ultimately result in shortage of the raw material in the related industrial fields. By the technology of gene cloning, bioconversion of D-amino acids with engineered cells containing D-hydantoinase and D-carbamoylase would be expected to overcome the drawbacks presented by using the original strains described above.According to the reported amino acid sequence of D-hydantoinases, two primers were designed and synthesized. Two DNA fragments encoding D-hydantoinase gene were amplified by PCR from chromosome DNA of Pseudomonas putida YZ-26 and Sinorhizobium morelense SS-ori, respectively, and confirmed by DNA sequence analysis. D-Hydantoinase from strain YZ-26 contains an open reading frame of 1440 bp corresponding 479 amino acids, which is a new gene as accepted by GenBank (AY387829). Whilst D-hydantoinase from strain SS-ori contains an open reading frame of 1374 bp encoding 457 amino acids.D-Hydantoinase gene from strain YZ-26 was cloned to pET-3a and pGEX-4T-1 vectors, respectively. The engineered strain EHD-YZ26/E.coli BL21(DE3) was incubated at 30 ℃ for 10 hrs without adding any inducers. The results show that the target product is soluble and active, which account for about 20 % compared with the total proteins in cells, and the enzymatic activity reaches 13 U/mL that is about 13 folds compared with the activity of strain YZ-26. The D-hydantoinase was purified by three steps, ammonium sulfate fractionation, Phenyl Sepharose hydrophobic chromatography and Sephacryl S-200 gel-filtration. With the procedures, the overall recovery of enzymatic activity reached 55 % and the specific activity for substrate hydantoin was about 16 U/mg protein. The purification factor was 3.4 folds with the purity about more than 95 % as estimated by SDS-PAGE analysis. The enzyme is homologous dimmer with molecular mass 102600 as determined by gel filtration on HPLC and subunit mass 52042 as determined by MALDI-TOF mass spectrometry. The optimal pH of D-hydantoinase is at near 9.5 and the optimal temperature is at 45 ℃. Based on the effect of divalent metal ions and chelator EDTA on the activity, the enzyme is metal-dependent only. The engineered strain GHD-YZ26/E.coli BL21(DE3) was incubated and induced at 30 ℃ for 8 hrs. The results show that the target fusion protein is soluble and active, which is about 15 % compared with the total proteins in cells, and the enzymatic activity reaches 5 U/mL that is about 8 folds compared with the activity of strain YZ-26. Fusion protein GST-HDT was purified by GSTrap affinity chromatography. With the procedures, the overall recovery of enzymatic activity reached 20 % and the specific activity for substrate hydantoin was about 4 U/mg protein. The purification factor was about 4.7 folds with the purity about more than 95 % as estimated by SDS-PAGE analysis.D-Hydantoinase gene from strain SS-ori was cloned to five different vectors to be five recombinant plasmids and in turn to transfer into five different E.coli strains, respectively. Then the total 25 engineered strains were cultured and induced in the respective conditions. The results show that the target products expressed in 3 strains of them are partly soluble and active, whils更多还原