【作者】 刘咏梅；

【导师】 孙志浩；

【作者基本信息】 江南大学 ， 发酵工程， 2010， 博士

【摘要】 精氨酸脱亚胺酶(Arginine deiminase, ADI, EC 3.5.3.6),属于一类称为“胍基修饰酶”总科,催化精氨酸产生瓜氨酸和氨。该酶广泛存在于多种微生物中,但哺乳类动物细胞中没有该种酶。因其具有的成为精氨酸缺陷型肿瘤如肝细胞瘤、黑色素瘤抗癌药物的巨大潜力而被研究。肝细胞癌是现在世界上病发率最高的癌症之一,每年新增一百万病例。近年来,精氨酸脱亚胺酶(ADI)的抗癌活性在国际上受到关注。美国食品药品管理局(FDA),欧洲药品审评署(EMEA)分别于1999年及2005年通过ADI-PEG-20作为罕见病药物用于治疗黑素瘤,肝细胞癌,目前三期临床正在进行中。本研究室通过筛选鉴定ADI产生菌株,并对该酶进行定向改造以改良其在体内生理条件下的酶活和酶学性质,为开发具有自主知识产权的抗癌新药提供酶源。通过建立一种ADI产生菌的筛选方法,得到一株产ADI的菌株SWP1。对该菌进行形态、生理生化特征鉴定和16S rRNA基因序列分析,鉴定该菌为变形假单胞菌。该菌现保藏于中国北京中关村中国微生物菌种保藏管理委员会普通微生物保藏中心,保存号CGMCC No.2039,并已申请专利(一株产精氨酸脱亚胺酶菌株及其应用,专利号：200710107822.X)。构建ADI编码基因arc A的重组表达载体pET24a-ADI,在大肠杆菌BL21(DE3)中获得高效表达。通过对诱导表达条件的优化,确定在OD600达到1.0时加入0.2 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG),30℃诱导4 h时酶活最高,为2.04 U/mL发酵液。重组精氨酸脱亚胺酶(rADI)经离子交换层析和凝胶过滤层析后,得到电泳纯的rADI。比酶活为20.85 U/mg (pH 6.0,37℃), Km为2.88 mmol/L, Vmax为31.68 (μmol/min/mg,最适反应pH6.0。经凝胶过滤分析,该酶分子量为92.6kDa, SDS-PAGE电泳显示ADI条带大小为46kDa,说明重组精氨酸脱亚胺酶为同源二聚体。研究表明,来源于变形假单胞菌Pseudomonas plecoglossicida CGMCC2039的重组ADI的最适反应pH为6.0,在pH 7.4(人体内生理pH在7.4左右)时残余酶活降低至10%以下；其Km (2.88 mmol/L, pH6.0),高于人血清精氨酸浓度(100-120μM)20倍以上。因此,该精氨酸脱亚胺酶的高Km值及生理pH条件下酶活较低的性质成为该酶抗癌活性研究的限制性因素。定向进化技术是改造分子朝既定目标进化的有力手段。基于改良的二乙酰一肟硫胺脲(测定瓜氨酸)方法,建立了简便灵敏的96孔板高通量筛选模型,用于获得生理pH条件下具有高酶活和低Km值的精氨酸脱亚胺酶。通过一轮易错PCR,从650个突变株中筛选获得一株优良的突变株M314,其生理pH条件下比酶活(9.02U/mg)较出发菌株(0.44 U/mg)提高20倍以上,Km值下降至0.65 mmol/L (pH7.4),最适pH从6.0提高至6.5。M314携带的三个突变位点分别为A128T,H404R和I410L,通过蛋白质同源建模分析了各突变位点在蛋白质结构和活性中的作用。更多还原

【Abstract】 Arginine deiminase (ADI; EC 3.5.3.6) belongs to a guanidine group-modifying enzyme superfamily and catalyzes the irreversible hydrolysis of arginine to citrulline and ammonia, is expressed in microorganisms, but not in mammalian cells. It has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas (HCCs).HCC is one of the most common malignancies in the world, and its annual incidence is approximately 1 million cases. In 1999 and 2005, ADI-PEG-20, PEGylated recombinant ADI was designated the US orphan drug status for the treatment of HCC and invasive malignant melanomas by FDA and European Agency for the Evaluation of Medicinal Products (EMEA). Currently, clinical trials of ADI-PEG-20 for the treatment of HCCs (Phase III) and melanomas (Phase I/II) are being investigated.In this study, a strain SWP1 producing high activity of ADI was isolated from the Wuxi canal. Based on its morphological, biochemical characteristics and 16S rRNA gene sequence analysis, SWP1 was identified as Pseudomonas plecoglossicida and is now deposited at CGMCC (China General Microbiological Culture Collection Center) as P. plecoglossicida CGMCC2039. In addition, a Chinese patent (An arginine deiminase producing strain and its application, Application No.200710107822.X) was applied.Recombinant vector pET 24a-ADI was constructed for the over-expressed of ADI in E. coli BL21(DE3). The effects of IPTG concentrations, induction temperatures and induction time were investigated. Under the optimal expression conditions the enzyme activity reached to 2.04 U/mL broth. The rADI was purified using DEAE Fast Flow anion exchange and Superdex 200 gel filtration column chromatography. The rADI had a molecular mass of about 92.6 kDa, and comprised a homodimer of 46 kDa in the native condition. The specific activity of rADI was up to 20.85 U/mg. The Km and Vmax values for arginine were 2.88 mmol/L and 31.68μmol/min/mg, respectively, and the optimum temperature and pH for the production of rADI were 40℃and 6.0.It was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10%of the activity was retained at pH 7.4 (pH of human plasma is around 7.4). Additionally, the Km value for wild type ADI (WT-ADI) was 2.88 mmol/L (pH 6.0), which is over 20 times of the serum arginine level (100-120μM). These are two major limitations for PpADI as a potential anti-cancer drug. Directed evolution has proven to be a powerful tool to modify a biological molecule towards a desirable property. A highly sensitive and efficient high throughput screening strategy employing ep-PCR and a modified diacetylmonoxime-thiosemicarbazide (DAM-TSC) method was established to isolate ADI mutants with higher activity and lower Km under physiological pH. One excellent mutant 314 (M314, A128T, H404R, I410L) was selected among 650 variants after one round of ep-PCR and screening. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the Km value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties.更多还原