Penicillin acylase-catalyzed synthesis of β-lactam antibiotics in water-methanol mixtures: effect of cosolvent content and chemical nature of substrate on reaction rates and yields

The synthesis of four β-lactam antibiotics (penicillin G, pivaloyloxymethyl ester of penicillin G, ampicillin and pivampicillin) catalyzed byEscherichia coli penicillin acylase has been investigated in water-methanol mixtures. The enzyme reactions were either thermodynamically or kinetically controlled at the same conditions using phenylacetic acid andd--phenylglycine methyl ester as acyl donors and 6-aminopenicillanic acid and pivaloyloxymethyl 6-aminopenicillanic acid as acyl acceptors. It has been found that the influences of the cosolvent content on the reaction rates and synthetic yields are significantly different depending on the substrates used in the experiments. On the other hand, within certain ranges of the methanol content (up to ca. 40% (v/v) the residual activities of the enzymes in water-methanol mixtures were only slightly lower than those in aqueous media. To analyze the factors that determine the reaction rate in water-cosolvent mixtures, the effect of methanol on the apparent pK values of the substrates has been investigated, and a mathematical model has been developed on the basis of the assumption that the enzyme binds non-ionized substrates. Model simulation results indicate that the solvent effect on reaction rates is mainly attributed to the kinetic effects of changes in apparent pK values.     

Ucon-benzoyl dextran aqueous two-phase systems: protein purification with phase component recycling

Benzoyl dextran with a degree of substitution of 0.18 was synthesized by reacting dextran T500 with benzoyl chloride. A new type of aqueous two-phase system composed of benzoyl dextran as bottom phase polymer and the random copolymer of ethylene oxide and propylene oxide (Ucon 50-HB-5100) as top phase polymer has been formed. The phase diagram for the system Ucon 50-HB-5100-benzoyl dextran with a degree of substitution of 0.18 was determined at room temperature. This two-phase system has been used to purify 3-phosphoglycerate kinase from bakers' yeast. The top-phase polymer (Ucon) can be separated from target enzyme by increasing the temperature. The bottom-phase polymer (benzyol dextran) could be recovered by addition of salt. Yeast homogenate was partitioned in a primary Ucon 50-HB-5100-benzoyl dextran aqueous two-phase system. After phase separation the top phase was removed and temperature-induced phase separation was used for formation of a water phase and a Ucon-rich phase. The benzoyl dextran-enriched bottom phase from the primary system was diluted, and the polymer was separated from water by addition of Na₂SO₄.     

玉米根微粒体ABA结合蛋白的性质及逆境胁迫的影响

以玉米根微粒体为材料进行的微量放射配体结合（ＭＲＬＢ）实验表明玉米根微粒体膜上存在着ＡＢＡ结合位点,ＡＢＡ与ＡＢＡ结合蛋白（ＡＢＡ－ＢＰ）结合的最适ｐＨ为６．５,结合反应对温度（０℃和２５℃）不太敏感,ＡＢＡ与ＡＢＡ－ＢＰ的结合反应是一个动态平衡过程,５ｍｉｎ即可达最大结合（Ｂｍａｘ）的５０％,３０ｍｉｎ达到最大结合,１ｈ内基本保持不变。胰蛋白酶处理表明此结合位点为蛋白质,冻融实验则表明此蛋白与ＡＢＡ的结合不仅要求其自身具有特定构象而且需要有一定的膜脂环境,ＤＴＴ处理实验结果显示ＡＢＡ－ＢＰ中可能存在着二硫键。逆境处理可以提高玉米根微粒体膜对ＡＢＡ的结合活性,盐胁迫、渗透胁迫、干旱胁迫和热冲激处理分别使结合活性上升３４．９％、１７．８％、２３．１％和１３．３％。     

一例智力低下患者7q~ 标记染色体的来源鉴定

以人类染色体显微切割、PCR技术构建的现有人类染色体特异性和染色体区带特异性探针池作为绘画探针,采用正向染色体绘画技术,结合染色体筛查方法,查明了一例7q~ 标记染色体患者的染色体附加片段来源于3q26→3qter。确定该患者的核型为46,XX,-7, der(7)t(7;3)(7pter→7q32::3q26→3qter)。应用这个策略,能够快速有效地鉴定标记染色体的来源。     

野生猕猴声行为的研究

本文通过在华南四省区对四个野生种群猕猴声行为的观察和现场录音,对它们的叫声声学特性,行为模式和其它个体对其相应的行为反应进行了定量的分析研究,并得出１１种不同的声行为,这些声行为有相互不同的声学特性,不同的行为模式并引起其它个体相应的行为反应,从而起着通讯的作用。叫声在猕猴的声行为通讯中,一些借助视觉提示,至少以不同的刺激度来“表示”某一类事物,并得到个体间的“共识”有些甚至无需视觉提示而单独传递     

Flow cytometric analysis of antibody producing cells using double immunofluorescent staining

Summary Double fluorescent labeling, with fluorescein isothiocyanate (FITC)-labeled F(ab)₂ specific for the heavy chain and R-phycoerythrin (R-PE)-labeled F(ab)₂ specific for the light chain, was demonstrated as a convenient means for the accurate evaluation of a heterogeneous non-antibody-producing population. Furthermore, it could be used for monitoring the changes in each immunoglobulin (Ig) chain content of the cells during the batch culture, which will facilitate the study on antibody synthesis, assembly and secretion.     

人细胞差异表达基因cDNA消减杂交体系的建立

为克隆肺腺癌分化相关基因, 采用诱导分化与消减杂交相结合的策略, 建立了全反式维甲酸(RA)诱导前后人肺腺癌细胞系的cDNA消减文库, 得到124个cDNA消减克隆. 经加减法杂交差异筛选、DNA和RNA印迹、cDNA全序列测定和生物学功能分析, 分离到3个在人肺腺癌细胞系分化过程中由RA激活而特异表达的新的cDNA序列这一策略和技术路线适用于分离细胞中呈过量表达或表达抑制基因的cDNA克隆, 并具有反映细胞分化过程中基因表达动态变化特征和相对简便适用的特点.