利用重组大肠杆菌产聚-4-羟基丁酸的研究

重组大肠杆菌 E.coli XL-1 Blue（pKSSE5.3）携带Ralstonia　eutropha　H16的 PHA聚合酶基因（phaC）和Clostridium kluyveri的4-羟基丁酸:CoA转移酶基因（orfZ）,可以利用葡萄糖和4-羟基丁酸为碳源合成均聚的聚-4-羟基丁酸[P（4HB）]。优化培养基和培养条件后,进行了补料分批培养。结果表明,经68h左右培养,E.coli XL-1 Blue（pKSSE5.3）的发酵液中菌体干重达13g/L,P（4HB）的密度达5g/L,P（4HB）百分含量为36％。从收获的冻干细胞中提纯得到40g均聚的P（4HB）,为进一步分析检测P（4HB）生物、理化、加工特性及其应用价值成为可能。     

烟草肉桂酸-4-羟化酶、4-香豆酸-辅酶A基因的

肉桂酸-4-羟化酶(C4H)、4-香豆酸-辅酶A(4CL)是烟草苯丙烷代谢途径的关键酶,其多酚类产物与烟草品质密切相关。本研究以酚类物质含量合成差异较大的2个烤烟品种红花大金元(HD)和K326为试验材料,利用同源克隆技术获得这2种烟草Ntc4h和Nt4cl基因的cDNA序列并进行表达特性分析。结果表明,在2个品种中Ntc4h和Nt4cl各有2个同源基因,Ntc4h1、Ntc4h2、Nt4cl1和Nt4cl2的ORF长度分别为1518 bp、1518 bp、1644 bp和1629 bp。Nt4cl1、Ntc4h1和Ntc4h2在编码序列上存在品种间差异。实时荧光定量PCR分析结果表明,该2种酶的基因在烟草中具有明显的时空表达特异性,2种酶基因在根、茎、叶、花和萼片中都有表达,在茎的木质部和韧皮部中的表达量均显著高于其他组织;在圆顶期和适熟期表达水平较高,在适熟期达到最高;且两品种中的表达模式存在差异。     

生物催化3-(4-氯苯基)-戊二腈去对称性水解合成光学纯巴氯芬的关键前体

研究了利用生物催化剂制备(S)-4-氰基-3-(4-氯苯基)-丁酸.以3-(4-氯苯基)-戊二腈为底物,采用苯酚-次氯酸钠法对实验室保藏的菌株进行筛选,得到一株产物立体选择性较高的菌株赤霉菌Gibberella intermedia WX12,并对其催化特性和发酵条件进行了初步研究.以30 g/L的乳糖和20 g/L的蛋白胨分别为碳、氮源,发酵培养96 h,收集的菌体在50 mmol/L磷酸缓冲液(pH 8.0)中30℃催化反应24 h,将3-(4-氯苯基)-戊二腈转化为4-氰基-3-(4-氯苯基)-丁酸,产率为90％.将产物化学转化为巴氯芬,手性HPLC分析表明水解产物构型是(S),其对映异构体过量值ee＞ 99％.该产物可以用来合成光学纯的(R)-和(S)-巴氯芬.     

4-羟基苯并恶唑-2-酮对四氯化碳致小鼠急性肝损伤保护作用

日的研究4一羟基苯并恶唑-2-酮（4-hydroxy-2-benzoxazolone,HBOA）对四氯化碳所致小鼠急性肝损伤的保护作用,并探讨其疗效机制。方法采用腹腔注射四氯化碳（carbonte trachloride,cch）制备小鼠急性肝损伤模型,HBOA灌胃给药,检测小鼠血清中的乳酸脱氢酶（LDH）活性以及肝组织中过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）含量,并用免疫组化法观察肿瘤坏死因子（TNF-a）的表达情况。结果HBOA能明显降低CCh致急性肝损伤小鼠血清LDH活性,同时升高肝组织中CAT、GSH-Px的活性并降低肝组织中TNF-a的表达。结论HBOA对CCh所致小鼠急性肝损伤有一定的保护作用。     

非天然氨基酸N-Fmoc-D-β-4-吡啶丙氨酸的合成研究

以4-氯甲基吡啶盐酸盐和乙酰氨基丙二酸二乙酯为原料,经缩合、水解、拆分制得α-氨基酸N-Fmoc-D-4-吡啶丙氨酸,经活化后生成重氮酮,失去氮气,重排而制得N-Fmoc-D-β-4-吡啶丙氨酸。中间体和目的产物经熔点、旋光度、红外、核磁以及元素分析,其结构得到证实。     

利用重组大肠杆菌产聚-4-羟基丁酸的研究*

重组大肠杆菌 E.coli XL-1 Blue（pKSSE5.3）携带Ralstonia　eutropha　H16的 PHA聚合酶基因（phaC）和Clostridium kluyveri的4-羟基丁酸:CoA转移酶基因（orfZ）,可以利用葡萄糖和4-羟基丁酸为碳源合成均聚的聚-4-羟基丁酸[P（4HB）]。优化培养基和培养条件后,进行了补料分批培养。结果表明,经68h左右培养,E.coli XL-1 Blue（pKSSE5.3）的发酵液中菌体干重     

Bacillus megaterium WZ009催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯

以外消旋4-氯-3-羟基丁酸乙酯为唯一C源的富集培养筛选得到一株菌株WZ009,经16S rDNA测序鉴定为巨大芽胞杆菌(Bacillus megaterium)。B.megaterium WZ009静息细胞可以立体选择性催化(S)-4-氯-3-羟基丁酸乙酯水解和脱氯反应得到光学纯的(R)-4-氯-3-羟基丁酸乙酯(e.e.≥99%)和(S)-3-羟基-γ-丁内酯(e.e.≥95%)。笔者对B.megaterium WZ009不对称催化反应影响因素(温度、pH、中和剂、底物浓度、时间进程以及细胞重复利用)进行优化研究,确定了该反应体系最优条件:底物浓度200 mmol/L,中和剂氨水,pH 7.2,40℃反应12 h,转化率达到50.6%,底物对映体过量值为99.6%。该生物催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯过程具有良好的工业化应用前景。     

L-脯氨酸-4-羟化酶的异源表达和合成反式-4-羟基-L-脯氨酸的条件优化

L-脯氨酸-4-羟化酶(L-Proline-4-hydroxylase,P4H)是依赖α-酮戊二酸(α-KG)和Fe2+的双加氧酶成员之一,在反式-4-羟基-L-脯氨酸(trans-4-hydroxy-L-proline,t-4Hyp)等重要手性化合物的生物合成中发挥关键作用。本研究构建了来源于Bradyrhizobium japonicum USDA 6的P4H重组大肠杆菌Escherichia coli BL21(DE3)/p ET-28b-p4h BJ,SDS-PAGE和酶活检测结果表明,该菌株具有表达可溶性P4H和催化合成t-4Hyp的能力。通过优化,确定了该重组菌全细胞催化合成t-4Hyp较优的反应体系和条件:10 m L p H 6.5 80 mmol/LMES缓冲液、9 mmol/L L-Pro,6 mmol/L L-抗坏血酸,6 mmol/Lα-KG,0.8 mmol/L Fe SO4·7H2O,反应温度为35℃;在20 g/L湿细胞的催化反应中,t-4Hyp的合成量达到34.86 mg/L,比优化前(17.53 mg/L)提高了98.86%。该工作为进一步利用P4H生物催化法合成t-4Hyp奠定了一定的技术基础。     

表达3-甾酮-△1-脱氢酶降解植物甾醇合成雄甾-1,4-二烯-3,17-二酮

构建分枝杆菌表达载体pMTac并在分枝杆菌Mycobacterium neoaurum JC-12中加强表达甾醇降解过程中的关键酶3-甾酮-△1-脱氢酶(KSDD)以提高雄甾-1,4-二烯-3,17-二铜(ADD)的产量。将p MF41的启动子pACE替换成tac启动子构建载体pMTac,在分枝杆菌中分别表达报告基因绿色荧光蛋白(GFP)和关键酶KSDD,通过GFP亮度和KSDD酶活验证tac启动子在M.neoaurum JC-12中的效果,并发酵验证加强表达KSDD对产物ADD的影响。荧光显微照片表明两个载体均能在M.neoaurum JC-12表达GFP,但tac启动子的效果比pACE强。酶活测定结果为重组菌M.neoaurum JC-12/pMTac-ksdd破碎细胞上清液中KSDD酶活比原始菌提高了6.53倍,比M.neoaurum JC-12/pMF41-ksdd提高了4.36倍。摇瓶发酵显示重组菌M.neoaurum JC-12/pMTac-ksdd ADD的产量比原始菌提高了22.2%,由4.86 g/L提高到5.94 g/L,而AD的产量由0.92 g/L减少到0.17 g/L,降低了81.5%;与M.neoaurum JC-12/p MF41-ksdd比,ADD产量提高了12.7%,AD降低了71.2%。以20 g/L植物甾醇为底物,5 L发酵罐中重组菌M.neoaurum JC-12/pMTac-ksdd的ADD产量达到10.28 g/L。结果表明,构建的新型表达载体pMTac适用于在M.neoaurum JC-12中加强表达关键酶KSDD,而且在M.neoaurum JC-12中过量表达KSDD有助于ADD产量的提高,为目前报道的发酵法利用新金色分枝杆菌降解植物甾醇合成ADD的最高水平。     

紫穗槐-4,11-二烯合酶基因在酵母工程菌中具有协同作用

为了构建高产的紫穗槐-4,11-二烯酵母工程菌,主要探究了含紫穗槐-4,11-二烯合酶基因的不同表达载体在酵母工程菌中是否存在协同效应。首先构建了含紫穗槐-4,11-二烯合酶基因的酵母表达载体pGADADS,分别将pGADADS和pYeDP60/G/ADS转入酿酒酵母W303-1B和WK1中,获得6种能产生紫穗槐-4,11-二烯的酵母工程菌：W303B[pGADADS]、W303B[pYGADS]、W303B[pYGADS+pGADADS]、WK1[pGADADS]、WK1[pYGADS]和WK1[pYG     

Acquirement of fetal properties in hepatoma on glutathione metabolism

Glutathione peroxidase/glutathionè reductase activity ratio was determined in the high-speed supernatant fraction of the rat livers. The ratio was dependent on age and the ratio increase gradually with the increase in age. The fetal liver showed a ratio of 1.5–2.0, which was almost the same value to those of the 4-dimethylaminoazobenzene-induced primary hepatoma and some transplantable hepatomas originating from the azodye-induced hepatoma. Four cell lines of transplantable ascites hepatoma examined in this study showed the value of 1.2–1.8 for the activity ratio, however, the values of two strains were found to be 2.8–3.0, even though these cell lines were also originated from the azodye-induced hepatoma.Glutathione contents of azodye-induced hepatoma and ascites hepatomas were also similar with those of fetal rat livers.The acquirement of the fetal properties in hepatoma was discussed in relation to glutathione metabolism.     

Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.     

Synthesis of metabolism-resistant substrates for the transport system for cationic amino acids; their stimulation of the release of insulin and glucagon, and of the urinary loss of amino acids related to cystinuria

Several amino acids have been synthesized as model transport substrates building on the piperidine and cyclohexane rings. Only when the distal N atom is part of an unambiguously cationic structure are these compounds transported predominantly by the cationic amino acid system. These amino acids in labeled form are excreted rather slowly in unmodified state, very little ¹⁴CO₂ being released. Those which are unambiguously cationic (including also homoarginine) led to a greatly increased excretion of arginine, lysine, ornithine and citrulline. Those which might be expected to act as lysine analogs had little effect on the excretion of the basic amino acids, although the excretion of citrulline and the sum of glutamine plus asparagine was accelerated. Certain of the analogs intensified the excretion of citrulline in dissociation from effects on resorption of the basic amino acids, also in dissociation from effects on cystine resorption. These results indicate citrulline resorption does not occur principally by the same agency serving for the basic amino acids, nor by the agency serving for cystine, despite the observed interactions for resorption. The injection of either of three transport analogs for arginine into the rat leads to early increases in the circulating levels of immunologically reactive insulin and glucagon.     

The effect of 4-isothiocyanatobenzoic acid on the oxygen-linked chloride binding sites in human hemoglobin: Influence on the alkaline Bohr effect

Human oxyhemoglobin reacted with 4-isothiocyanatobenzoic acid shows a decreased oxygen affinity that does not change with increasing chloride concentration indicating that all of the oxygen-linked chloride binding sites are blocked in the modified protein. By contrast, reaction of oxyhemoglobin with 4-isothiocyanatobenzenesulfonamide produces a modified protein with increased oxygen affinity below pH 7.3 that shows the expected decrease in oxygen affinity with increasing chloride concentration. The latter result demonstrates the importance of the negatively charged moiety in producing both the decrease in oxygen affinity and the effect on the oxygen-linked chloride binding sites produced by 4-isothiocyanatobenzoic acid. Reduction in the alkaline Bohr effect by 50% in the protein modified by 4-isothiocyanatobenzoic acid indicates that contribution to the alkaline Bohr effect is evenly divided between chloride dependent and chloride independent groups.     

Identification of 4-acetoxyaminoquinoline from the hydrolysis of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline,in vitro and in vivo properties

4-Acetoxyaminoquinoline (Ac-4-HAQ) (1) was identified as a hydrolysis product of 1-acetoxy-4-acetoxyimino-1,4-dihydroquinoline (diAc-4-HAQO). The reaction allowing the obtention of (1) obeys to a reduction mechanism implying the N₁-O cleavage. The carcinogenic properties of (1) observed by Sato et al. (Japan J. Exp. Med., 40 (1970) 475) in mice were studied in rats with the in vivo system we used previously with 4-nitroquinoline-1-oxide (4-NQO) and 4-hydroxyaminoquinoline-1-oxide (4-HAQO). In rats (1) does not covalently bind DNA. It was, therefore, possible to propose an interpretation of the results obtained by Enomoto et al. (Proc. Soc. Exp. Biol. Med., 136 (1971) 1206) who injected diAc-4-HAQO s.c. to mice and rats. Compound 1 could be responsible for the carcinogenic effects observed through the following pathway: (1) should be formed by hydrolysis of diAc-4-HAQO and reactivated by an enzymatic system to N-oxide derivative, the 4-acetoxyaminoquinoline-1-oxide (Ac-4-HAQO), which constitutes an ultimate carcinogen model of 4-NQO.     

Phytosterols accumulation in the seeds of Linum usitatissimum L.

A comparative study was performed to determine the free sterols content and composition during the development of three varieties of linseed (H52, O116 and P129). Seed samples were collected at regular intervals from 7 to 60 days after flowering (DAF). Ten compounds were identified: cholesterol, campesterol, brassicasterol, stigmasterol, β-sitosterol, Δ5-avenasterol, cycloartenol; 24-methylene cycloartanol, obtusifoliol, citrostadienol. The maximum level of 4-desmethylsterols (1515 mg/100 g oil) was reached at 7 DAF in P129 variety. H52 had the highest level of 4-4 dimethylsterols (355 mg/100 g oil) at 28 DAF. The greatest amount of 4-monomethylsterols (35 mg/100 g oil) was detected in H52 at 14 DAF. During linseed development, β sitosterol (830 mg/100 g oil) was the major 4-desmethylsterols, followed by campesterol (564 mg/100 g oil) and stigmasterol (265 mg/100 g oil). Some of these compounds followed nearly the same accumulation pattern during linseed maturation.     

Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.     

Studies on the mechanism of action of the aromatase inhibitor, 4-hydroxyandrostenedione

[1 beta-3H], [1 alpha,2 alpha-3H] and [1 beta,2 beta-3H] 4-Hydroxyandrostenedione (4-OH-A) were synthesized to study the mechanism of inhibition of aromatase by 4-OH-A. Incubations of [1 beta-3H] and [1 beta,2 beta-3H] 4-OH-A with placental microsomes in the presence of NADPH showed very little loss of tritium, with aromatization of 4-OH-A ranging from 0.3 to 0.6 percent. No loss of tritium was observed in the absence of NADPH. The extent of covalent binding of 4-OH-A to microsomal proteins was higher with incubations in the absence of NADPH than with those in the presence of NADPH. These results are discussed in light of what has been proposed for the mechanism of androgen aromatization.     

Determination of 4-(4-chlorophenyl)-4-hydroxypiperidine, a metabolite of haloperidol, by gas chromatography with electron-capture detection

An electron-capture gas chromatographic procedure was developed for the analysis of 4-(4-chlorophenyl)-4-hydroxypiperidine (CPHP), a metabolite of haloperidol. The assay involved basic extraction of this metabolite from the biological samples, followed by back-extraction with HCl. After basification of the acid phase, extractive derivatization with pentafluorobenzoyl chloride in toluene was conducted. The pentafluorobenzoyl derivative was quantified on a gas chromatograph equipped with a fused-silica capillary column, an electron-capture detector and a printer-integrator. N-(3-Trifluoromethylphenyl)piperazine was carried through the procedure as an internal standard and calibration curves were determined for each assay run. The procedure was demonstrated to be linear and reproducible and was utilized to detect and quantify CPHP in urine, plasma, brain and liver samples from rats treated with haloperidol. The structure of the derivatized metabolite was confirmed by gas chromatography-mass spectrometry.