基因工程菌发酵表达核苷磷酸化酶条件的优化

目的 优化苷磷酸化酶（包括嘌呤和嘧啶核苷磷酸化酶）基因工程菌的发酵表达条件。方法通过工程菌摇瓶培养,测定吸光度D值,考马斯亮蓝（Bradford）法测定蛋白,SDS—PAGE电泳和凝胶成像扫描分析表达量,优化表达条件;通过正交试验优化50L发酵罐发酵条件。结果摇瓶培养起始pH为7．0～7．2,于30℃培养4h,加入终浓度为0．4mmol／L的异丙基硫代半乳糖苷（IPTG）诱导8h后收获菌体,可得到较高的生物量和重组酶蛋白表达量。50L发酵罐的最佳条件为起始pH为7．0～7．2,于32oC培养4h,加入终浓度为0．4mm01／L的IPTG诱导9h后收获菌体,每升发酵液可得2g以上的酶蛋白。结论基因工程菌发酵表达核苷磷酸化酶产量较高,可工业化生产．为酶法合成核苷酸类似物的研究奠定了基础。     

利用嘧啶核苷磷酸化酶基因工程菌酶法合成5-氟尿苷的转化率影响因素的研究

研究酶法合成5-氟尿苷(5-FUR)的反应条件.利用嘧啶核苷磷酸化酶(Pyrimidine-nudeoside phosphorylase,PyPNase)基因工程菌大肠杆菌BL21(DE3)/pET28a 合成5-氟尿苷(5-FUR),考察不同反应条件对转化率的影响;尝试不同反应体系对转化率的影响.结果:(1)最佳转化条件:菌体:0.5 WCG/L;UR:10 mmol/L;5-FU:45mmol/L;PBS:40 mmol/L;T:50℃;pH 7.4;t:60 min,转化率可达91.27%;(2)以Na2SO4溶液为溶剂,比以PBS溶液为溶剂的转化率略有提高.微生物酶法合成5-氟尿苷(5-FUR)的转化率较高,可考虑用于工业生产.     

酶法合成5-氟尿苷

采用产气肠杆菌ＣＰＵ７８５６变异株的游离细胞,利用其中的嘧啶核苷磷酸化酶通过碱基交换进行酶法合成目的物。试验以３０ｍｍｏｌ／Ｌ尿苷（ＵＲ）,３０ｍｍｏｌ／Ｌ５－氟尿嘧啶（ＦＵ）,３０ｍｍｏｌ／Ｌ磷酸盐缓冲液ＰＨ７．８）的混合物加入１０％的产气肠杆菌湿菌体反应３ｈ,底物ＵＲ的转化率可达５０％。     

酶法合成2’—脱氧—5—氟尿苷

本文采用大肠杆菌核苷磷酸化酶，以5-氟尿嘧啶（FU）、2’-脱氧尿苷（dU)和磷酸盐为底物酶法合成2’-脱氧-5-氟尿苷，实验结果表明，含10mmol/L dU、30mmol/L FU、10-30mmol/L磷酸盐的混合物加入5%培养24h的大肠杆菌湿菌体于pH7.0反应3h，底物dU的转化率可达45.9%。     

嘌呤核苷磷酸化酶检测体系优化及芒果苷对其活性的影响

目的优化体外嘌呤核苷磷酸化酶(purine nucleoside phosphorylase,PNP)活性检测体系,研究芒果苷及芒果苷元对PNP活性的影响。方法采用紫外分光光度法,通过考察酶浓度、底物浓度、二硫苏糖醇(DTT)浓度对酶促反应的影响,确定检测酶活性的最适反应条件,以此为基础研究芒果苷和芒果苷元对PNP活性的影响。结果成功建立并优化了体外PNP活性检测体系,反应温度为25℃时,在磷酸钾盐缓冲液中,当PNP浓度为250 U·L-1、底物鸟苷为400μmol·L-1、DTT为0.5 mmol·L-1的反应条件下于波长258nm处动态监测15 min,芒果苷及其代谢产物苷元在浓度高达100μmol·L-1时,对PNP活性无明显抑制作用。结论以优化的PNP活性检测体系研究发现芒果苷及芒果苷元体外对PNP活性无影响。     

发酵条件对产酶的影响

产凝乳酶活力及C/P(clotting activities/proteolytic activities)值不仅受菌株本身性质的影响,还和发酵条件有关。由于液态发酵的发酵条件可控,在工业化生产凝乳酶方面有较大优势。由于丝状真菌适合在固态培养条件下生长,本章首先将固态发酵培养与液态发酵培养进行对比实验,选择优势较大的液态发酵培养基;然后对液态培养不同孢子培养基、接种量、发酵时间及温度进行优化试验。     

1株产α-环糊精葡萄糖基转移酶菌株的产酶条件优化

目的提高菌株Y112产α-环糊精葡萄糖基转移酶（α-CGTase）的能力。方法采用单因素法和响应面法,对菌株Y112产酶的培养基成分和培养条件进行了优化。结果最终确定了菌株Y112产酶最佳培养基成分为：玉米淀粉 7.5 g/L,蛋白胨17.8 g/L,酵母浸粉 9.1 g/L,Mn2+ 0.6 mmol/L,NaCl 50 g/L,Na2CO3 15 g/L(pH 9.6);最佳产酶的培养条件为：27 ℃下发酵50 h。结论优化后的酶活为优化前的2.8倍。     

利用产气肠杆菌EAM-Z1转化合成5-氟尿苷的转化率影响因素的研究

利用产气肠杆菌EAM Z1转化合成5 氟尿苷,从筛选中间体稳定剂、底物流加、底物诱导、菌体冻融和石英砂研磨等方面研究转化率的影响因素,结果:(1)转化过程中加入40 mmol/L的硼砂可以将转化率达到75.56%,提高30.28%;加入10 mmol/L的甘氨酸可以使转化率达到61.92%,提高6.76%;(2)流加5 氟尿嘧啶可以将转化率提高到87.78%;(3)培养过程中加入尿苷诱导效果并不明显,尿苷浓度为 0.001 g/L时效果最佳,提高3.63%;(4)菌体冻融和石英砂研磨使转化率下降26%。     

来源于产气肠杆菌的嘧啶核苷磷酸化酶基因在大肠杆菌BL21中的表达

实现产气肠杆菌（Enterobacter aerogenes）嘧啶核苷磷酸化酶（Pyrimidine-nucleoside phosphorylase，PyPNase）基因在大肠杆菌中的高效表达。用来源于产气肠杆菌的核苷磷酸化酶N末端序列与NCBI中公布的已知序列进行比对，设计一对引物，以该菌株基因组DNA为模板扩增出PyPNase基因。定向克隆到PET-28a（＋）载体后转化到表达宿主大肠杆菌BL21中，筛选阳性克隆并测序，然后在LB培养基中优化发酵条件。结果：目的基因片断长度为1302bp。表达产物经SDS-PAGE和酶活测定表明PyPNase基因在大肠杆菌中获得活性表达，43ku处有表达蛋白。37℃培养2h至对数生长中期，降温到31℃，0．1mmol／LIPTG诱导5h获得最大酶活力为512．3U／mg，为出发菌株的17．2倍。重组PyPNase的成功表达为今后大规模生产5-FUR打下了基础。     

青霉素酰化酶产生菌的分子育种及其产酶条件的研究

用EcoR1分别酶切质粒pACYC184和Ec108染色体,经连接、转化、筛选,得到7株克隆了青霉素酰化酶基因的转化体-C600(pPAHD1-7)。重组质粒pPAHD1在不同受体细胞内的酶活表达水平相差20倍。据此构建的工程菌其青霉素酰化酶活比出发菌株提高4～5倍。本文还对工程菌的产酶条件进行了研究。     

由鸟苷经酶法生产利巴韦林

利用乙酰短杆菌ＴＱ－９５２细胞为酶源，酶法生产利巴韦林。当在２５ｍｍｏｌ／Ｌ（ｐＨ７．０）磷酸钾缓冲液中鸟苷和１，２，４－三唑－３－甲酰胺浓度分别为２００ｍｍｏｌ／Ｌ，酶用量为５０ｍｇ／ｍｌ（湿重），最适反应温度为６５℃，２４ｈ达到反应终点，收率为７８％。该菌种酶活力高，稳定性好，本法高效低耗，有较高实用价值     

抗真菌药萘替芬的合成

抗真菌药萘替芬的合成ＳＹＮＴＨＥＳＩＳＯＦＡＮＴＩＭＹＣＯＴＩＣＡＧＥＮＴＮＡＦＴＩＦＩＮＥ左淑芳＊杨凤艳（河北省药物研究所，石家庄０５００６１）ＺＵＯＳｈｕ－Ｆａｎｇ＊，ＹＡＮＧＦｅｎｇ－Ｙａｎ（ＨｅｂｅｉＰｈａｒｍａｃｅｕｔｉｃａｌＩｎｓｔｉｔｕ...     

核苷类药物的酶法合成

综述了酶法合成核苷类药物的最新研究进展 ,内容包括酶法合成核苷类药物的优点 ,利用含核苷磷酸化酶的菌体一步合成核苷类药物或药物前体 ,利用中间体进行转化制备和通过基因工程菌转化制备 ,并指出工业化应用过程中几个需要解决的关键步骤。     

The cytostatic activity of pyrimidine nucleosides is strongly modulated by Mycoplasma hyorhinis infection: Implications for cancer therapy

Nucleoside analogues are widely used as chemotherapeutic agents in the treatment of cancer. Several cancers are reported to be associated with mycoplasmas (i.e. Mycoplasma hyorhinis), which contain a number of nucleoside-metabolizing enzymes. Pyrimidine nucleoside analogues, such as 5-fluoro-2'-deoxyuridine (FdUrd), 5-trifluorothymidine (TFT) and 5-halogenated 2'-deoxyuridines can be degraded by thymidine phosphorylase (TP) to their inactive bases. We found in M. hyorhinis-infected MCF-7 breast carcinoma cells (MCF-7/HYOR) a mycoplasma-encoded TP that dramatically (20-150-fold) reduces the cytostatic activity of these compounds. The reduction in cytostatic activity could be fully restored in the presence of TPI (5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride), a known inhibitor of human TP. This observation is in agreement with the markedly decreased formation of active metabolite (i.e. FdUMP for FdUrd) or diminished drug incorporation into nucleic acids (i.e. for TFT and 5-bromo-2'-deoxyuridine) in MCF-7/HYOR cells compared with uninfected MCF-7 cells. Antimetabolite formation is fully restored in the presence of TPI. In contrast, 5-fluoro-5'-deoxyuridine (5'DFUR), an intermediate metabolite of capecitabine, was markedly more cytostatic in MCF-7/HYOR cells than in uninfected cells, due to the activation of this prodrug by the mycoplasma-encoded TP. Thus, our data reveal that M. hyorhinis expresses a TP that activates 5'DFUR but inactivates FdUrd, TFT and 5-halogenated 2'-deoxyuridines, and that is highly sensitive to the inhibitory effect of the TP inhibitor TPI. Given the association of M. hyorhinis with several human cancers, our findings suggest that pyrimidine nucleoside-based but not 5FU-based anti-cancer therapy might be more effective when combined with a mycoplasmal TP inhibitor.     

The cytostatic activity of NUC-3073, a phosphoramidate prodrug of 5-fluoro-2'-deoxyuridine, is independent of activation by thymidine kinase and insensitive to degradation by phosphorolytic enzymes

A novel phosphoramidate nucleotide prodrug of the anticancer nucleoside analogue 5-fluoro-2′-deoxyuridine (5-FdUrd) was synthesized and evaluated for its cytostatic activity. Whereas 5-FdUrd substantially lost its cytostatic potential in thymidine kinase (TK)-deficient murine leukaemia L1210 and human lymphocyte CEM cell cultures, NUC-3073 markedly kept its antiproliferative activity in TK-deficient tumour cells, and thus is largely independent of intracellular TK activity to exert its cytostatic action. NUC-3073 was found to inhibit thymidylate synthase (TS) in the TK-deficient and wild-type cell lines at drug concentrations that correlated well with its cytostatic activity in these cells. NUC-3073 does not seem to be susceptible to inactivation by catabolic enzymes such as thymidine phosphorylase (TP) and uridine phosphorylase (UP). These findings are in line with our observations that 5-FdUrd, but not NUC-3073, substantially loses its cytostatic potential in the presence of TP-expressing mycoplasmas in the tumour cell cultures. Therefore, we propose NUC-3073 as a novel 5-FdUrd phosphoramidate prodrug that (i) may circumvent potential resistance mechanisms of tumour cells (e.g. decreased TK activity) and (ii) is not degraded by catabolic enzymes such as TP which is often upregulated in tumour cells or expressed in mycoplasma-infected tumour tissue.     

Organ Selective Delivery Using a Tissue-Directed Sreptavidin-Biotin System: Targeting 5-Fluorouridine via TNP-Streptavidin

Abstract

Trinitrophenyl (TNP) modification of streptavidin (St) resulted in high and prolonged accumulation in mouse liver following intravenous administration of radioiodinated TNP streptavidin (TNP-St). Uptake, which is correlated with increased TNP substitution, was first observed at 2-3 h, increased to 40-50% of injected dose/gram tissue (%/g) at 24 h and slowly declined later on. A low degree of accumulation (10%/g) was observed in the spleen. TNP substitution of other proteins such as bovine serum albumin (BSA) or ovalbumin (Ova) led to a transient short-term liver uptake. The enzyme-resistance property of streptavidin and its biotin binding sites render TNP-modified streptavidin a potential targeting vehicle to the liver. 5-Fluorouridine (FUR) was attached to high molecular weight carrier carboxymethyldextran (CMdex, derived from 40 kDa dextran) and the dextran-FUR conjugate was charged with 2-4 biotinyl groups (in the form of biotinyl-diaminopropionyl-tyrosine, BDT) for complexing to TNP-St. Biodistribution monitoring of the BDT-CMdex-FUR ligand, radiolabeled at the tyrosyl residue of BDT and targeted via non-radiolabeled TNP-St, showed that ligand accumulation in the liver was similar to TNP-St itself. Liver targeting of FUR was demonstrated by trace-labeling FUR with its structural analog 5,6-[³H]uridine prior to conjugation to dextran hydrazide. Specific liver accumulation of [³H] radioactivity occurred following administration of the conjugate only when complexed to TNP-St. Hepatic levels of [³H] radioactivity were in the range of 25%/g or 35% per whole liver during a period of at least 8h, as compared to the rapid elimination of free FUR + [³H]uridine (4%/g at 20 min). [³H]-drug radioactivity disappeared at a faster rate as compared to ¹²⁵I-dextran radioactivity, suggesting that metabolic processes required to generate the 5,6-[³H]uracil-containing active metabolites took place.     

肿节风颗粒特定条件下最优制粒区间研究

目的:初步探索获得中药制剂最优制粒区间的可行性。方法:以肿节风颗粒成型工艺为模型,采用析因设计进行试验,以颗粒得率、颗粒成型率加权后的综合评分值为考察指标,采用Origin 8.0软件分别绘制不同含水量混合粉(浸膏粉-可溶性淀粉)综合评分值与润湿剂浓度、液固比关系效果图,并用Origin 8.0软件中的Screen Reader功能进行坐标提取,综合得到肿节风颗粒在特定条件下的最优制粒区间;在该制粒区间下,选择不同批次浸膏粉进行实验室规模及处方放大试验进行验证。结果 :当浸膏粉-可溶性淀粉配比为1∶2时,肿节风颗粒存在一个最适制粒区间,即当物料含水量在[2.0%,6.0%]、润湿剂浓度在[50%,80%]及液固比在[15.3 mL/g,18.6 mL/g]范围内时,所制肿节风颗粒均符合要求;验证试验的综合评分值均大于85%,证实所制颗粒合格。结论:本研究建立的考察中药浸膏在特定条件下的最优制粒区间的方法,对解决中药制粒工业生产批间差异大、重现性差等问题,具有一定的参考作用。     

L-缬氨酸高产菌选育及营养需求研究

以黄色短杆菌 (Brevibacteriumflavum)L 缬氨酸产生菌ZQ -2为出发菌株 ,经硫酸二乙酯(DES)和亚硝基胍 (NTG)诱变处理 ,氨基酸结构类似物定向筛选 ,获得一株L 缬氨酸高产菌XQ -6 (LeulAHVrα ABhr2 TAhr)。在以 1 4 %葡萄糖为碳源、5 %硫酸铵为氮源的培养基中直接发酵72h,产酸达 5 8～ 6 2g/L。同时添加Fe2 + 和Mn2 + 比单独添加Fe2 + 更有效。还进行了氨基酸、核酸、维生素和其它营养对L 缬氨酸发酵影响的试验。     

基因重组卡介苗高效电转化条件的实验研究

目的探讨卡介苗(BCG)电转化中影响转化效率的因素。方法利用电转化仪将穿梭质粒pYL-hB7-2转入BCG中,考察其影响因素并优化转化条件。转化子利用卡那霉素抗性筛选和聚合酶链反应(PCR)及酶链免疫吸附试验(ELISA)鉴定。结果重组BCG获得较高的转化效率,最高可达8/9。外加电场强度和质粒浓度是决定电转化效率的关键因素,感受态细胞的状态,如生长期和冰浴时间均可影响转化效率。结论成功解决了基因重组BCG构建过程中电转化的问题。