固定化在多孔玻璃上的青霉素酰化酶性质

从大肠杆菌As 1．76提取青霉素酰化酶,经纯化,用戊二醛圈定在氯烷基硅烷化多孔玻璃上。初步摸索了固定化条件。固定化酶的米氏常数为7．7 x 10^-4M,比自然酶大10倍,但竞争性抑制剂苯乙酸对固定化酶和自然酶的抑制常数基本相同,固定化酶的最大反应速度为5．8×10^-2M／min,比自然酶大2．5倍,水解NIPAB的最适pH为7．O,比自然酶低1个pH单位,最适反应温度比自然酶低i0℃,固定化酶在pH 5．8—8．0之间较稳定,较自然酶的范围略窄;固定化酶在40℃以下稳定,而自然酶在45℃以下稳定。     

在甜椒花药培养中马来酰胼对小孢子发育的抑制作用

选用甜椒的小孢子单核期花药,用100、300、500、2000ppm的马来酰肼溶液浸泡处理,并设对照,进行无激素MS固体培养基培养。分别取样用各种组化方法对花药内部多糖、蛋白质、核酸及ATP酶进行组化反应和形态学上的观察。与对照组相比,处理组花药外部形态和内部结构出现许多变异。小孢子内多糖,蛋白质含量减少;绒毡层无明显变化;两组中,核酸的含量均无明显变化;ATP酶的活性低于对照组。可能,马来酰肼对于花药中ATP酶等产生抑制作用,致使花药败育。     

氨基酰化酶中金属锌离子的功能作用

 氨基酰化酶是含锌金属酶。该酶每摩尔蛋白中含2摩尔Zn(Ⅱ)离子。金属鳌合剂与酶作用,通过竞争螯合Zn(Ⅱ)离子使酶活力下降。残余活力与残留金属含量呈正相关。竞争螯合的结果,生成不含金属的脱辅基酶蛋白,并导致酶活力的丧失。脱辅基酶由于加入Zn(Ⅱ)离子而恢复其活力。实验表明金属锌离子是氨基酰化酶催化活力所必需。与Zn(Ⅱ)离子相似,Co(Ⅱ)离子也可与脱辅基酶相结合并使之复活。 在190—240nm区域内对比了天然酶、脱辅基酶蛋白与Co(Ⅱ)置换氨基酰化酶的圆二色谱。远紫外圆二色谱表明,与天然酶相比,在脱辅基酶中由于金属离子的丧失导致主链构象发生变化,其中α螺旋增加约7％。因而锌离子(钴离子)对蛋白主链的反应最适构象有一定的稳定作用。脱辅基酶与Co(Ⅱ)离子结合,酶的主链构象恢复至与天然酶几近相同。可认为这是促使酶复活的内在因素。     

青霉素G酰化酶α亚基Ser177的突变对酶活性的影响

用盒式突变和定点突变对大肠杆菌青霉素G酰化酶α亚基177位ser进行了突变研究,结果发现所挑选的突变体均无酶的活力,这一结果可能可以用来解释Ser 177附近肽段和一些青霉素结合蛋白青霉素结合区在一级结构上保持同源性的原因。     

体外培养周期型马来丝虫感染期幼虫

关于体外培养周期型马来丝虫(periodic Brugia malayi)感染期幼虫(L_3),近年来国内外有一些报道,系应用含哺乳动物组织细胞系或原代细胞的培养系统培养彭亨丝虫和马来丝虫L_3,可使其幼虫发育至Ⅳ期蚴(L-4)或早期成虫(Chen and Howells,1979; Wong et al.,1982;Mak et al.,1983;郑惠君等,1984)。但未见应用含人体组织细胞系的体外培养系统培养布鲁属丝虫L_3的报道。本研究     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

固定化尖镰孢菌细胞的某些酶学特性

尖镰孢菌(Fusariun oxysporum)33—11是一株青霉素V酰化酶的高产菌株。用二醋酸纤维素固定的该菌细胞裂解青霉素V最适的Ph范围为7．4至7.6;最适的反应温度为45℃至48℃;其酶活性受8-羟基喹啉和EDTA的可逆抑制,Mg²⁺、Zn^2-和Mn^2-离子则有激活作用。采用问歇式搅拌裂解青霉素V,测得0．6rnm颗粒度固定化细胞的表观km值为11．7mmol／L。裂解青霉素V的反应活化能为33kJ／mol;底物抑制常数Ks为1950mmol/L;苯氧乙酸和6-APA的抑制常数分别为220mmol／L和270mm0I／L,在裂解反应中．前者是竞争性抑制剂,后者是非竞争性抑制剂。     

固定化青霉素酰化酶的研究

将巨大芽孢杆菌胞外青霉素酰化酶通过共价键连接到醋酸纤维素载体上,制成的固定化青霉素酰化酶的表观活力达2000 u／g左右(PDAB法)。水解lO％(w／v)的青霉素G钾盐落液,使用30批,保留活力70％以上。6-氨基青毒烷酸(6-APA)总收率平均达88．37％。固定化青霉素酰化酶水解青霉素G的最适pH为9．95,最适温度为55℃,表观米氏常数为1.093×10-2mol／L,在pH 5．8-10．7,温度45℃以下酶的活力稳定。     

氨基酰化酶在LDS溶液中的失活与去折叠的比较研究

氨基酰化酶在阴离子去污剂十二烷基硫酸溶液中的失活与去折叠的研究结果表明,在低浓度的ＬＤＳ溶液中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化,而酶的活力已经大部分或几乎全部丧失。当ＬＤＳ浓度达１．６ｍｍｏｌ／Ｌ时,此时酶分子的构象变化才达到最大程度。在实验使用的ＬＤＳ的浓度范围内,用远紫外ＣＤ光谱监测的二级结构没有发生明显的变化。     

锌离子对氨基酰化酶构象及其稳定性的影响

天然氨基酰化酶和脱谷氨基酰化酶无论在二级结构（用ＣＤ和ＦＴＩＲ监测）还是三级结构上（以荧光发射光谱监测）都有明显的差异,表明了脱锌后酶的有序度降低;当比较天然和脱锌氨基酸化酶对去圬剂的稳定性时,结果表明脱锌后酶的构象的稳定性明显降低．因此可以认为锌离子对维持酶分子活性部位的特定构象以及构象的稳定性具有重要的作用．