酶法合成抗病毒药物阿糖腺苷

目的:为了开发一种生产阿糖腺苷的有效方法。方法:研究了以产气肠杆菌完整细胞为催化剂酶法合成阿糖腺苷,优化了菌体培养条件以及酶反应条件。结果:在培养基中添加0.5%葡萄糖,33℃下培养16h,既能得到较多菌体,又能使菌体的催化活性保持较高。酶反应在pH7.0、25mmol/L的磷酸钾缓冲液中进行,底物浓度为阿糖尿苷30mmol/L,腺嘌呤10mmol/L,加入10%湿菌体,在60℃下振荡反应48h,腺嘌呤转化率可达90%。结论:酶法合成阿糖腺苷可应用于大规模工业化生产。     

产阿糖腺苷菌株的筛选

据文献报道,阿糖腺苷与干扰素合并使用,治疗病毒性肝炎疗效可达70%左右。国外市场上提供的阿糖腺苷是以发酵法生产为主。1983年以来。我们从广东省微生物研究所以及美国农业部农业研究所等单位索取了32个菌株,从中筛选产阿糖腺苷的菌株。经过摇瓶发酵试验,通过纸层析、紫外光谱及高压液相色     

产气肠杆菌菌体内核苷磷酸化酶的诱导表达

目的:阿糖腺苷(Ara-A)是一种广谱抗病毒药物,临床上用于治疗多种病毒性疾病.同时也是合成阿糖腺苷单磷酸(Ara-AMP)的重要原料.本课题旨在寻找一种高效酶法生产嘌呤类阿糖核苷的方法.方法:以产气肠杆菌完整细胞为酶源,研究产气肠杆菌菌体培养条件对核苷磷酸化酶的影响及其诱导性.结果:胸苷磷酸化酶、尿苷磷酸化酶和嘌呤核苷磷酸化酶均可被多种核苷、核苷酸甚至碱基诱导.胞苷或胞苷酸的添加量为15-20mmol/L,诱导时间在0-8小时均可.经胞苷和胞苷酸诱导的菌体可使酶反应时间缩短6倍,大大提高了反应效率.经诱导的菌体,在反应后仍保持较高的核苷磷酸化酶活力;而未经诱导的菌体,一次反应后即丧失大量的酶活力.结论:核苷磷酸化酶的活性可以通过诱导而提高,以此优化阿糖腺苷的生产.     

工程菌E.coli MM2的固定化细胞制备

固定化细胞技术是七十年代兴起的一门应用技术。固定化细胞具有细胞密度高、易进行连续生产且稀释率高、不易被污染、产物易分离、可降低设备规模等优点,已成为生物合成、生物转化的有效工具,得到广泛应用。近十年来,人们把固定化细胞技术应用到工程菌的培养中取得了一些有价值的结果。用工程菌制得的固定化细胞裂解青霉素生产6-APA,已达工业化生产水平。据文献报道,工程菌固定化后可大大提高重组质粒的稳定性,并且外源基因能稳定地表达。本所马清钧研究员等构建的工程菌     

左旋麻黄素半生物合成的研究固定化酵母细胞生物合成L-PAC(L-苯基乙酰基甲醇)

本文对影响酵母菌生物合成L-PAC的主要因素进行了研究.结果表明酵母菌株Sc-5按2g鲜细胞/3.2%50ml海藻酸钠凝胶固定化后,所获固定化凝胶珠置于装有4倍体积反应液的容器中,在振荡频率220转/分、温度28-30℃、添加0.2%的Vc时,枇次生物合成L-PAC产量最高,达2.0g/L.     

国内应用单磷酸阿糖腺苷联合免疫调节剂治疗慢性乙型肝炎的现状

国内应用单磷酸阿糖腺苷联合免疫调节剂治疗慢性乙型肝炎的现状江国庆姜玲张平龚文军（安徽省立医院２３０００１）（安徽医科大学附属医院２３００６１对于慢性乙型肝炎（下简称慢乙肝）目前尚缺少特效的治疗方法,鉴于其疗效与病毒株的毒力,受感染细胞的数量和机体免疫...     

激素和活性多肽

动物细胞,另二则为分泌人生长激素人原生质周围空间中的大肠杆菌系统。本文讨论了后一种系统,提出证实生物合成和天然22k人生长激素的证据。(戴顺志)8803了3使用生吻电化学系统生物合成幽类化合物〔俄〕z‘Yaroploov,A.I.…了Biokhi-miya(Moseow)一1586,51(9)一1442一1445〔译自CBA,198了,(4),1722〕 在有外源性电子受体甲硫酸吩嗓存在时,使用冻干的球型节细菌(Arfhrobacter夕Iobijorm‘s)(3一幽酮类1一脱氢酶)细胞,使氢化一可的松氧化为强的松龙。此固定化冻干细胞保留着20%的原活性。在固定化和非固定化细胞的稳定性上实际上并无差别…     

微乳液凝胶及其固定化脂肪酶研究进展

利用微乳液胶凝现象来固定化酶是90年代初建立起来的酶固定化新技术,该技术为胶束酶学在生物合成与转化领域的应用奠定了基础.就微乳液凝胶及其固定化脂肪酶的制备、性质、微观结构及其潜在应用作了带有知识介绍性质的综述.     

无载体固定化细胞的研究进展

以无载体固定化酵母细胞酒精连续发酵的成功工业化应用为实例,并与通常的载体固定化细胞技术比较,阐述了无载体固定化细胞技术的优缺点,系统提出了无载体固定化细胞技术的概念,进而对无载体固定化细胞技术在其它微生物发酵和动植物细胞培养过程的应用前景进行了展望。     

光交联聚氨酯作载体的固定化细胞产生a-淀粉酶的研究

将可光交联的聚氨酯预聚物与枯草杆菌细胞悬浮液混匀后经紫外光照射交联,制得固定化细胞,用于产生a-淀粉酶。探讨了交联和固定化条件对裁体结构和固定化细胞产酶性能的影响,研究了这种固定化细胞的使用特性。结果表明:光交联聚氨酯能有效地固定枯草杆菌细胞,进行正常增殖和产酶,改变交联和固定化条件能调节载体的孔容、孔径和比表面,从而调节固定化细胞的产酶性能和其它使用特性;枯草杆菌经光交联聚氮酯载体固定化后对温度和pH的适应性提高,在同样条件下,这种固定化细胞的产酶能力比游离细胞提高约30％,亦高于用k-角叉菜胶作载体的固定化细胞．     

The biochemical basis for resistance to adenine arabinoside in a mutant of Toxoplasma gondii.

We have previously reported the isolation and preliminary characterization of a mutant of Toxoplasma gondii that was resistant to adenine arabinoside. Fiftyfold higher concentrations of adenine arabinoside were required to inhibit the growth of the resistant parasite in human fibroblast cultures. To determine the enzymic basis for resistance, we measured the kinases and deaminases that act on adenosine or deoxyadenosine. All of these enzymic activities were found in uninfected human fibroblast cells. The mutant and wild type parasite proved to have similar activities of adenosine deaminase, deoxyadenosine deaminase, and deoxyadenosine kinase. However, the adenine arabinoside resistant mutant had less than 0.1% of the adenosine kinase activity observed in the wild type T. gondii. The mutant parasite is presumably resistant because without adenosine kinase to phosphorylate adenine arabinoside it cannot carry out the first step in the conversion of the analogue to adenine arabinoside triphosphate, the active form. A mutant of 3T6 (mouse) cells previously selected for a loss of adenosine kinase also proved to be resistant to adenine arabinoside.     

Apparent suicide inactivation of human lymphoblast S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine and adenine arabinoside. A basis for direct toxic effects of analogs of adenosine.

Adenosylhomocysteine hydrolase from human lymphoblasts binds 2'-deoxy[3H]adenosine tightly. Binding is associated with time-dependent, saturable, irreversible inactivation of catalytic activity which occurs with first order kinetics, suggesting "suicide" inactivation. Adenine arabinoside produces similar inactivation but is more potent. These results suggest a basis for a heretofore unrecognized mechanism of action for these and other analogs of adenosine, in which toxicity results from actions of the nucleosides themselves, rather than from nucleotides to which they may be converted.     

Production of adenine arabinoside by gel-entrapped cells of Enterobacter aerogenes in water-organic cosolvent system

Summary Gel-entrapped whole cells of Enterobacter aerogenes, which has a transglycosylation activity, were used to produce adenine arabinoside from uracil arabinoside and adenine, in an appropriate water-organic cosolvent system. Cells of E. aerogenes entrapped with a hydrophilic photo-crosslinkable resin prepolymer, ENT-4000, or a urethane prepolymer, PU-6, had a high and stable transglycosylation activity. To improve the poor solubility in water of the substrate (adenine) and product (adenine arabinoside), dimethyl sulfoxide was selected as the cosolvent based on the criteria of operational stability of the immobilized biocatalyst and solubility of both substrate and product. Addition of 40% dimethyl sulfoxide to the reaction mixture permitted use of a high substrate concentration range which gave high productivity under homogeneous reaction conditions. The immobilized cells of E. aerogenes exhibited a markedly improved operational stability, retaining their initial level of activity during repeated use for at least 35 days at 60°C in 40% dimethyl sulfoxide. When the reaction was carried out with 150 mM uracil arabinoside and 50 mM adenine as the substrates, the yield of adenine arabinoside was maintained at 100% based on the molar ratio of adenine, throughout the reaction.Abbreviations used AraA adenine arabinoside - AraU uracil arabinoside     

Stabilization of soluble and immobilized horse liver alcohol dehydrogenase by adenosine 5'-monophosphate

Horse liver alcohol dehydrogenase, which catalyzes oxidoreductions for a broad spectrum of substrates of organic chemical interest, was immobilized on CNBr-activated Sepharose and on decylamine-substituted agarose. The specific activities of the immobilized enzyme preparations were compared with the free enzyme, and the apparent K(m) values of the preparations were determined for a selection of substrates. At pH 9 and 60 degrees C, soluble liver alcohol dehydrogenase was rapidly inactivated, while the enzyme immobilized on CNBr-activated Sepharose was more stable. Adenosine monophosphate (AMP), adenosine diphosphate, and adenosine diphosphoribose protected the free and immobilized alcohol dehydrogenase against heat inactivation. On storage under a variety of conditions, AMP effectively stabilized free horse liver alcohol dehydrogenase and the immobilized preparations.     

Continuous production of NADP by immobilized Achromobacter aceris cells.

Several microorganisms having higher nicotinamide adenine dinucleotide kinase (NAD kinase, EC 2.7.1.23) activity were immobilized into polyacrylamide gel lattices. The enzyme activity field by immobilization was highest in Achromobacter aceris AKU 0120. By the incubation of the immobilized A. aceris cells at pH 4.0, the NAD kinase activity increased and the adenosine triphosphate (ATP)-degradation activity disappeared completely. Enzymatic properties of the immobilized A. aceris cells were investigated and compared with those of intact cells. The optimal pH and the optimal temperature of immobilized cells were the same as those of intact cells. Immobilized cell NAD kinase was more stable than that of intact cells. The operational half-life of immobilized cells was 20 days when the substrate solution was passed through a column packed with immobilized cells at a flow rate which gives a space velocity (SV) of 0.1 hr-1 at 37 degrees C. On the other hand, the half-life of the intact cells was only 6 hr.     

Substrate specificity, kinetics, and stoichiometry of sodium-dependent adenosine transport in L1210/AM mouse leukemia cells

Two equilibrative (facilitated diffusion) nucleoside transport processes and a concentrative Na(+)-dependent co-transport process contribute to zero-trans inward fluxes of nucleosides in L1210 mouse leukemia cells. Na(+)-linked inward adenosine fluxes in L1210/AM cells (a clone deficient in adenosine, deoxyadenosine, and deoxycytidine kinase activities) were measured as initial rates of [3H]adenosine influx in medium containing Na+ salts and 10 microM dipyridamole. The Na(+)-linked transporter distinguished between the D- and L-enantiomers of adenosine, the latter being a virtual nonpermeant in the initial-rate assay. Adenine arabinoside, inosine, 2'-deoxyadenosine and 2'-deoxyadenosine derivatives with halogen atoms at the purine C-2 position were recognized as substrates of the Na(+)-linked system because of their inhibition of adenosine (10 microM) fluxes under the condition of Na(+)-dependence with IC50 values ranging between 25 and 183 microM; uridine, deoxycytidine, and cytosine arabinoside (each at 400 microM) inhibited adenosine fluxes by 10-40%. Inward Na(+)-linked adenosine fluxes were saturable with respect to extracellular adenosine and Na+ concentrations [( Na+]o); Km and Vmax values for adenosine influx were 9.4 +/- 2.6 microM and 1.67 +/- 0.2 pmol/microliter cell water/s when [Na+]o was 100 mM. The stoichiometry of Na+:adenosine co-transport, determined by Hill analysis of the dependence of adenosine fluxes on [Na+]o, was 1:1. The thiol-reactive agents, N-ethylmaleimide (NEM), showdomycin and p-chloromercuriphenylsulphonate (pCMPS), inhibited Na(+)-linked adenosine fluxes with IC50 values of 40, 10, and 2 microM, respectively. This inhibition was partially reversed by the presence of adenosine in incubation media containing pCMPS, but not NEM. Thiol groups accessible to pCMPS may be involved in substrate recognition by the transporter and in the permeation step.     

Purification and characterization of a novel nucleoside phosphorylase from a Klebsiella sp. and its use in the enzymatic production of adenine arabinoside

An adenosine-assimilating bacterium, Klebsiella sp. strain LF1202, inducibly formed a novel nucleoside phosphorylase which acted on both purine and pyrimidine nucleosides when the cells were cultured in medium containing adenosine as a sole source of carbon and nitrogen. The enzyme was purified (approximately 83-fold, with a 17% activity yield) to the homogeneous state by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was calculated to be 125,000 by gel filtration of Sephadex G-200 column chromatography, although the enzyme migrated as a single protein band with a molecular weight of 25,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; thus, it was thought to consist of five identical subunits. Besides purine nucleosides (adenosine, inosine, and guanosine), the purified enzyme also acted on pyrimidine nucleosides such as uridine, 2'-deoxyuridine, and thymidine. The purified enzyme catalyzed the synthesis of adenine arabinoside, a selective antiviral pharmaceutic agent, from uridine arabinoside and adenine.     

Purification and characterization of a novel nucleoside phosphorylase from a Klebsiella sp. and its use in the enzymatic production of adenine arabinoside.

An adenosine-assimilating bacterium, Klebsiella sp. strain LF1202, inducibly formed a novel nucleoside phosphorylase which acted on both purine and pyrimidine nucleosides when the cells were cultured in medium containing adenosine as a sole source of carbon and nitrogen. The enzyme was purified (approximately 83-fold, with a 17% activity yield) to the homogeneous state by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was calculated to be 125,000 by gel filtration of Sephadex G-200 column chromatography, although the enzyme migrated as a single protein band with a molecular weight of 25,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; thus, it was thought to consist of five identical subunits. Besides purine nucleosides (adenosine, inosine, and guanosine), the purified enzyme also acted on pyrimidine nucleosides such as uridine, 2'-deoxyuridine, and thymidine. The purified enzyme catalyzed the synthesis of adenine arabinoside, a selective antiviral pharmaceutic agent, from uridine arabinoside and adenine.     

Adenosine kinase mediates high affinity adenosine salvage in Trypanosoma brucei

African sleeping sickness is caused by Trypanosoma brucei. This extracellular parasite lacks de novo purine biosynthesis, and it is therefore dependent on exogenous purines such as adenosine that is taken up from the blood and other body fluids by high affinity transporters. The general belief is that adenosine needs to be cleaved to adenine inside the parasites in order to be used for purine nucleotide synthesis. We have found that T. brucei also can salvage this nucleoside by adenosine kinase (AK), which has a higher affinity to adenosine than the cleavage-dependent pathway. The recombinant T. brucei AK (TbAK) preferably used ATP or GTP to phosphorylate both natural and synthetic nucleosides in the following order of catalytic efficiencies: adenosine > cordycepin > deoxyadenosine > adenine arabinoside (Ara-A) > inosine > fludarabine (F-Ara-A). TbAK differed from the AK of the related intracellular parasite Leishmania donovani by having a high affinity to adenosine (K m = 0.04-0.08 microm depending on [phosphate]) and by being negatively regulated by adenosine (K i = 8-14 microm). These properties make the enzyme functionally related to the mammalian AKs, although a phylogenetic analysis grouped it together with the L. donovani enzyme. The combination of a high affinity AK and efficient adenosine transporters yields a strong salvage system in T. brucei, a potential Achilles' heel making the parasites more sensitive than mammalian cells to adenosine analogs such as Ara-A. Studies of wild-type and AK knockdown trypanosomes showed that Ara-A inhibited parasite proliferation and survival in an AK-dependent manner by affecting nucleotide levels and by inhibiting nucleic acid biosynthesis.     

Flow system for fish freshness determination based on double multi-enzyme reactor electrodes

A double reactor system for the determination of fish and shellfish freshness using the freshness indicator, K-value (K=[(HxR+Hx)/(ATP+ADP+AMP+IMP+HxR+Hx)]x100), was developed, where ATP, ADP, AMP, IMP, HxR and Hx are adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, inosine monophosphate, inosine and hypoxanthine, respectively. The system consisted of a pair of enzyme reactors with an oxygen electrode positioned close to the respective reactor. The enzyme reactor (I) was packed with nucleoside phosphorylase and xanthine oxidase immobilized simultaneously on chitosan beads (immobilized enzyme A). Similarly, the enzyme reactor (II) was packed with immobilized enzyme A and immobilized enzyme B (co-immobilized alkaline phosphatase and adenosine deaminase). Moreover, this reactor consisted of two layers, the enzyme A and enzyme B (1:1). A good correlation was obtained between K values, which were determination by the proposed system and by the HPLC method. One assay could be completed within 5 min. The signal for the determination of K value of fish and shellfish was reproducible within 2.3%. The long-term stability of the enzyme reactors was evaluated at 30 degrees C for 28 days.