南海深海微生物酯酶EST12-7在制备(R)-2-氯丙酸乙酯中的应用研究

利用来源南海深海的微生物酯酶EST12-7不对称水解反应拆分制备（R）-2-氯丙酸乙酯。并探寻了温度、pH、底物浓度、有机溶剂和反应时间等因素对酯酶EST12-7催化制备（R）-2-氯丙酸乙酯的影响。结果表明,深海微生物酯酶EST12-7催化制备（R）-2-氯丙酸乙酯的最佳反应条件为：13.8 μg/ml酯酶EST12-7,50 mmol/L（±）-2-氯丙酸乙酯,2%正癸醇,pH8.5,30℃,0.05mol/L Tris-HCl,反应60 min。在最佳反应条件下,（±）-2-氯丙酸乙酯的转化率可达49%,所制备的（R）-2-氯丙酸乙酯的光学纯度为98%。通过对酯酶EST12-7拆分制备（R）-2-氯丙酸甲酯和（R）-2-氯丙酸乙酯进行比较,2-氯丙酸酯中的链长对酯酶EST12-7拆分反应有极大的影响。     

Conformational studies on poly-L-tryptophan: circular dichroism and x-ray diffraction studies

Circular dichroism (CD) measurements were carried out on various copolymers of L -tryptophan and γ-ethyl L -glutamate in ethylene glycol monomethyl ether as the solvent. On increasing the L -tryptophan content of the copolymers a gradual change in the CD spectra was observed. The typical spectrum of the right-handedα-helix becomes more and more evident as the L -tryptophan content decreases. On the basis of these results we assumed that no conformational transition occurs on proceeding from pure poly (γ-ethyl L -glutamate) to pure poly-L -tryptophan in ethylene glycol monomethyl ether: therefore the conformation of poly-L -tryptophan should be that of a right-handed α-helix. Moreover we observed that the change in the CD spectra of the copolymers is gradual but not linear on increasing the tryptophan content. The deviations from linearity were attributed to interactions among side-chain chromophores whose contributions to the optical activity are not simply additive. An x-ray analysis carried out on oriented films of poly-L -tryptophan casted from solutions of the polymer in dimethylformamide shows conclusively that the solid-state conformation of the polymer is that, of an α-helix.     

Effects of temperature on the polymerization of γ-ethyl DL-glutamate N-carboxy anhydride in DMF initiated by tri-n-propylamine

Polymerization of γ-ethyl DL -glutamate N-carboxy anhydride (NCA) in DMF has been carried out at various temperatures and with the use of tri-n-propylamine as the initiator. It was found that on decreasing the polymerization temperature the reaction rate is lowered but the molecular weight and the helix content of the final polymer are enhanced. An overall activation energy of ～4 kcal/mole has been found for the polymerization process. Preliminary experiments carried out on the polymerization of γ-benzyl D -glutamate NCA in DMF and with tri-n-propylamine as the initiator showed a strong depressing effect of the CO₂ evolved during the polymerization, both on the reaction rate and on the molecular weight of the polymer. All data are interpreted in terms of the Bamford-Swarc mechanism.     

Far ultraviolet optical rotatory properties of poly-L-tryptophan. I

A block copolymer [γ-Et-DL -Glu]_m [L -Trp]_n was prepared using N-carboxy anhydrides (NCA) of L -tryptohan and γ-ethyl DL -glutamate. The block copolymer, dissolved in trifluoroethanol (TFE)–dichloroacetic acid (DCA) mixtures, exhibited a sharp change in the specific rotation at 546 mμ when the solvent composition reached 70–75% DCA content. Optical rotatory dispersion (ORD) and circular dichroism (CD) measurement were carried out in TFE solution in the spectral range 180–350 mμ. Indole side-chain chromophores were found to be optically active in the polymer. On the other hand, these groups exhibit very small optical activity in the model compound C₆H₃? CH₂? O? CO? (L -Trp)₂? O? CH₃. Indole groups therefore appear to be in a dissymmetric environment only in the polymer. From these data it was concluded that poly-L -Trp is in some type of helical conformation in TFE. Strong overlapping of CD bands from side-chain chromophores and peptides chromophores in the wavelength range 185–240 mμ does not allow definite conclusions to be drawn about the type of helical conformation which exists in poly-L -Trp in TFE solution.     

Inhibitory alkaloids from Dictamnus dasycarpus root barks on lipopolysaccharide-induced nitric oxide production in BV2 cells

The methanolic extract of Dictamnus dasycarpus root barks afforded one new glycosidic quinoline alkaloid, 3-[1β-hydroxy-2-(β-D-glucopyranosyloxy)-ethyl)-4-methoxy-2(1H)-quinolinone (1), together with nine known compounds, preskimmianine (2), 8-methoxy-N-methylflindersine (3), dictamine (4), γ-fagarine (5), halopine (6), skimmianine (7), dictangustine-A (8), iso-γ-fagarine (9), isomaculosidine (10). The isolated alkaloids significantly inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV2 cells. Among them, compounds 3 and 7 showed the most potent inhibitory activities on LPS-induced NO production.     

Structural Differences in the Hinge Region of Human Gamma A Myeloma Proteins of Different Subclasses

THE two antigenic subclasses of human γA myeloma proteins that have been identified are γA₁ and γA₂ (refs. 1-3). H—H interchain disulphide bonds are present in molecules of both subclasses, but H—L interchain disulphide bonds are present only in γA₁ proteins and the minor allotype of γA₂, Am 2–(ref. 4). Light chains of Am 2+ γA proteins occur as disulphide bonded dimers non-covalently linked to the heavy chains⁵.     

谷氨酸棒杆菌一步法发酵糖质原料生产γ-聚谷氨酸

γ-聚谷氨酸在食品、化妆品、生物医药等领域具有广泛的应用,目前主要的生产菌株是谷氨酸依赖型菌株,在生产过程中需要添加谷氨酸作为前体,因而生产γ-聚谷氨酸的成本较高。文中主要研究从糖质原料一步法发酵合成γ-聚谷氨酸的生产工艺。首先,从产γ-聚谷氨酸的菌株枯草芽孢杆菌中克隆γ-聚谷氨酸合成酶的基因簇pgs BCA,在谷氨酸棒杆菌模式菌株ATCC13032中进行诱导型和组成型表达,结果显示,仅诱导型表达菌株可以积累γ-聚谷氨酸,产量为1.43 g/L。进一步对诱导条件进行优化,确定诱导时间为2 h,IPTG浓度为0.8 mmol/L,γ-聚谷氨酸产量为1.98g/L。在此基础上,在一株高产谷氨酸的谷氨酸棒杆菌F343中外源表达pgs BCA,对重组菌进行发酵,结果表明,在摇瓶发酵中γ-聚谷氨酸产量达到10.23g/L,在5L发酵罐中产量达到20.08g/L;继而对γ-聚谷氨酸进行分子量测定,结果显示,产自F343重组菌的γ-聚谷氨酸的重均分子量比产自枯草芽孢杆菌的提高34.77%。文中构建了一步法发酵糖质原料生产γ-聚谷氨酸的新途径,同时为开发其潜在应用奠定了基础。     

小克银汉霉γ-亚麻酸高产菌株的快速筛选和发酵条件研究

以小克银汉霉C0为出发菌株,经过5-氟尿嘧啶和紫外线复合诱变,采用抗失水苹果酰肼与抗低温(15℃)相结合的筛选方法,获得一株生产性能比出发菌株显著提高的突变株C23。采用5L全自动发酵罐对小克银汉霉C23发酵生产γ-亚麻酸的pH值控制和补料工艺进行研究,发现将发酵液pH值维持在5.5,当发酵进行到60h、84h、108h时,分别补糖15g/L,发酵192h后收获,结果生物量、油脂产量和γ-亚麻酸产量分别达到49.88g/L、21.93g/L、2.69g/L。     

Extent and stability of the alpha-helix in gamma-benzyl L-glutamate-glycine copolymers

Block and random copolymers of γ-benzyl L -glutamate and glycine were studied by optical rotatory dispersion methods in order to ascertain the extent and stability of the α-helix formed by these polymers in appropriate solvent mixtures. Results indicate that when in solution such polymers do contain sections of α-helix. However, it appears that glycine does not readily fit into the α-helix which is largely due to the γ-benzyl L -glutamate segments. Further, there are indications that the helical sections in the random copolymers are due to the nonrandomness of the polymerization.     

A newly isolated Bacillus siamensis SB1001 for mass production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a retaining agent; it has applications in the food, medicine, agriculture, cosmetics and wastewater treatment industries. Most of the γ-PGA producing strains belong to the genus Bacillus. This study reports on a novel γ-PGA producing species. Bacillus siamensis SB1001 was screened and isolated from organically cultivated soybeans exhibiting a high γ-PGA producing ability. The fermentation medium and culture parameters for γ-PGA production by Bacillus siamensis SB1001 were optimized by statistical methods. The sucrose, l-glutamic acid and dipotassium phosphate in the medium were shown to be the significant factors of the γ-PGA production, and the optimum medium obtained consisted of the following: 106.86 g/L sucrose, 69.84 g/L l-glutamic acid and 2.39 g/L dipotassium phosphate. Using the optimized medium, 25.22 g/L γ-PGA were produced with a productivity of 1.05 g/L/h. The γ-PGA obtained had a molecular weight of 7.9 × 10⁵ Da and a polydispersity index of 2.34, and the ratio of d-/L-glutamic acid was 89.71%:10.29%. To the best of our knowledge, this is the first report of γ-PGA production by B. siamensis strain. B. siamensis SB1001 has great potential as an industrial γ-PGA producer.     

N-(n-Butylamide) of (S)-2-(phenylcarbamoyloxy)propionic acid: A new chiral solvating agent,derived from L-lactic acid,for the enantiomeric purity determination of derivatized amino acids

The N-(n-butylamide) of (S)-2-(phenylcarbamoyloxy)propionic acid, easily prepared starting from the inexpensive L -ethyl lactate, can be used as convenient chiral solvating agent (CSA) to determine the enantiomeric composition of N-(3,5-dinitrobenzoyl)amino acid methyl esters.     

Optimization of γ-polyglutamic acid synthesis using response surface methodology of a newly isolated glutamate dependent <Emphasis Type="Italic">Bacillus velezensis</Emphasis> Z3

A new glutamate-dependent γ-polyglutamic acid (γ-PGA) producer Z3 isolated from soil samples in Daxinganling forest region of China was identified, and its optimal medium components were investigated using response surface methodology. Strain Z3 was identified as Bacillus velezensis by physiology and biochemistry and 16S rDNA sequence analysis. This is the first report of glutamate-dependent B. velezensis with the ability to synthesize γ-PGA. Then, the optimum γ-PGA yield (5.58 g/L) was achieved with glutamate 86 g/L, glucose 36 g/L, yeast extract powder 5.5 g/L, and NaH₂PO₄ 7.5 g/L. Furthermore, activities of enzymes participating in glutamate synthesis were assessed, and the results showed that lower ketoglutaric dehydrogenase activity (KGDH) and higher glutamate dehydrogenase activity (GDH) resulted in higher γ-PGA yield. Identification of glutamate-dependent γ-PGA producer named B. velezensis Z3 enriches microbiological resources with γ-PGA-producing capacity. B. velezensis optimization of nutrients and analysis of enzymes activities will not only help to increase γ-PGA productivity but also to understand the γ-PGA synthesis mechanism in B. velezensis Z3.     

胆固醇致人血管内皮细胞DNA损伤

本文研究胆固醇是否通过氧化应激机制损伤人内皮细胞DNA.实验用125 mg/L, 25 mg/L, 50 mg/L胆固醇作用于人内皮细胞12 h,用免疫荧光法观察到不同浓度的胆固醇均可诱导细胞内γH2AX形成焦点,随着胆固醇浓度的增加,γH2AX焦点的荧光强度也逐渐增强.利用Western印迹及流式细胞术检测到细胞内γH2AX的量和ROS水平也随胆固醇浓度增大而增加.彗星电泳实验检测到细胞内拖尾DNA量随胆固醇浓度逐渐增加,DNA损伤加重.用抗氧化剂10 mmol/L N 乙酰半胱氨酸(NAC)预作用细胞1 h后,再用50 mg/L胆固醇作用细胞12 h,细胞内γH2AX焦点的荧光强度明显减弱,γH2AX量减少,ROS的水平下降.结果表明,胆固醇可诱导人脐静脉内皮细胞中ROS升高,导致DNA损伤.     

γ-Glutamylcysteinylserine — A New Homologue of Glutathione in Plants of the Family Poaceae*

In addition to glutathione (γ-GluCysGly), many species of the family Poaceae have another tripeptide which has the amino acid sequence γ-GluCysSer. This thiol was isolated from etiolated leaves of wheat (Triticum aestivum L. cv. Star). Its structure was elucidated by quantitative amino acid analysis after total hydrolysis and by partial hydrolysis with carboxypeptidase A and γ-glutamyltranspeptidase. The content of γ-GluCysSer in the leaves of T. aestivum is increased by incubation with sulfate and is severely diminished by incubation with buthionine sulfoximine, a specific inhibitor of γ-glutamylcysteine synthetase. Oxidized γ-GluCysSer is reduced by yeast glutathione reductase with a rate somewhat lower than for glutathione, but the new tripeptide is not a substrate of glutathione-S-transferase from equine liver. Besides homoglutathione (γ-GluCysßAla), a tripeptide found in plants of the order Fabales, the tripeptide γ-GluCysSer is the second homologue of glutathione detected in plants.     

Polymerization of N-carboxyanhydrides of various α-amino acids using secondary amines as initiator

In the polymerizations of alanine, γ-ethyl glutamate, and leucine N-carboxyanhydrides (NCA's) initiated by tertiary amines and some secondary amines such as N-methyl-L -alanine dialkylamide, a stereoselectivity was observed: the polymerization rates of L - and D -NCA's were identical to each other and larger than that of DL -NCA. However, this selectivity was not observed in the polymerizations of valine and isoleucine NCA's initiated by N-methyl-L -alanine dialkylamide. The stereoselective polymerizations of valine and isoleucine NCA's were induced only with tetriary amines such as tri-n-butylamine. N-Methyl-L -alanine di-alkylamide has been shown to initiate the polymerization of usual α-amino acid NCA according to the activated-NCA mechanism, but it initiated the polymerizations of valine and isoleucine NCA's according to the primary amine-type mechanism. This is because in the latter NCA's the N–H group is masked by the adjacent C^β-branched alkyl substituent against the approach of the secondary amine. Poly(DL -alanine)s produced in the stereoselective polymerization had higher viscosities and were more stereoblock-like than those produced without the stereoselectivity. These experimental results indicate that the stereoselective polymerization is possible only when the polymerization proceeds through the activated-NCA mechanism.     

不同D/L单体比γ-聚谷氨酸的合成与调控

γ-聚谷氨酸(γ-PGA)是由L-谷氨酸和/或D-谷氨酸聚合而成的一种聚氨基酸,广泛应用于化妆品、医药等领域.高聚物单体的立体构型会影响产品性质和应用,因此调控γ-PGA中D-谷氨酸/L-谷氨酸单体比(D/L单体比)具有重要意义.前期以谷氨酸棒杆菌为底盘,表达来自于地衣芽孢杆菌的γ-PGA合成酶,合成以L-Glu(97...     

Glutamic acid independent production of poly-γ-glutamic acid by Bacillus amyloliquefaciens LL3 and cloning of pgsBCA genes

A new glutamic acid independent poly-γ-glutamic acid (γ-PGA) producing strain, which was identified as Bacillusamyloliquefaciens LL3 by analysis of 16S rDNA and gyrase subunit A gene (gyrA), was isolated from fermented food. The product had a molecular weight of 470, 801 and l-glutamate monomer content of 98.47%. The pre-optimal medium, based on single-factor tests and orthogonal design, contained 50 g/L sucrose, 2 g/L (NH₄)₂SO₄, 0.6 g/L MgSO₄, and provided well-balanced changes in processing parameters and a γ-PGA yield of 4.36 g/L in 200 L system. The γ-PGA synthetase genes pgsBCA were cloned from LL3, and successfully expressed by pTrcLpgs vector in Escherichia coli JM109, resulting the synthesis of γ-PGA without glutamate. This study demonstrates the designedly improved yield of γ-PGA in 200 L system and the first report of pgsBCA from glutamic acid independent strain, which will benefit the metabolized mechanism investigation and the wide-ranging application of γ-PGA.     

New Synthesis of Optically Active O-Aryl O-Ethyl Phenylphosphonothionates

The mixed anhydride method was applied to synthesize O-aryl O-ethyl phenylphosphonothionate. The reaction of O, O-diethyl phosphorochloridate with O-ethyl phenylphosphonothioic acid afforded O, O-diethyl phosphoric O-ethyl phenylphosphonothioic anhydride in a good yield. This anhydride was converted to O-aryl O-ethyl phenylphosphonothionates by reacting with the appropriate sodium phenoxide. This esterification occurred without racemization and produced optically pure O-ethyl O-(4-nitrophenyl) phenylphosphonothionate (EPN) and O-(4-cyanophenyl) O-ethyl phenylphosphonothionate (cyanofenphos).     

Crystal Structure of γ-Hexachlorocyclohexane Dehydrochlorinase LinA from Sphingobium japonicum UT26

LinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from γ hexachlorocyclohexane (HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene via γ-pentachlorocyclohexene. We determined the crystal structure of LinA at 2.25 Å by single anomalous dispersion. LinA exists as a homotrimer, and each protomer forms a cone-shaped α + β barrel fold. The C-terminal region of LinA is extended to the neighboring subunit, unlike that of scytalone dehydratase from Magnaporthe grisea, which is one of the most structurally similar proteins identified by the DALI server. The structure we obtained in this study is in open form, in which γ-HCH can enter the active site. There is a hydrophobic cavity inside the barrel fold, and the active site is largely surrounded by the side chains of K20, L21, V24, D25, W42, L64, F68, C71, H73, V94, L96, I109, F113, and R129. H73 was considered to function as a base that abstracts the proton of γ-HCH through its interaction with D25. Docking simulations with γ-HCH and γ-pentachlorocyclohexene suggest that 11 residues (K20, I44, L64, V94, L96, I109, A111, F113, A131, C132, and T133) are involved in the binding of these compounds and support the degradation mechanism.     

一株γ-聚谷氨酸合成菌的筛选与鉴定

从土壤中筛选分离获得一株γ-聚谷氨酸合成菌PGS-1,经鉴定为枯草芽孢杆菌(Bacillus subtilis),在富含谷氨酸和葡萄糖的培养基中可大量合成γ-聚谷氨酸,摇瓶发酵产量达26 g/L,不同于大多文献报道的微生物合成的γ-聚谷氨酸具有较高的分子量,该菌株合成的γ-聚谷氨酸分子量较低(3×105-4×105 kD),分子量分布较窄,可适用于低分子量要求的应用领域,如作为药物的控缓释载体,值得深入开发研究。