定点突变提高青霉素G酰化酶的稳定性

以大肠杆菌青霉素G酰化酶的晶体结构为模板 ,用软件PMODELING同源模建巨大芽孢杆菌青霉素G酰化酶的三维结构。在此基础上 ,将 β亚基 4 2 7位 (突变A)和 4 3 0位 (突变B)赖氨酸残基突变为丙氨酸 ,降低了该酶的等电点 ,增加了疏水性 ,从而提高其在酸性和有机溶剂环境中的稳定性。两个突变体与亲本相比 ,比活力和Km相近 ,最适pH减少了 0 .5个单位 ,突变B在 pH 5 .2的溶液中的稳定性明显提高。突变A和B在 15 %DMF中的半衰期分别比亲本酶提高了 60 %和 166%     

定点突变提高青梅素G酰化酶的稳定性

以大肠杆菌青霉素Ｇ酰化酶的晶体结构为模板,用软件ＰＭＯＤＥＬＩＮＧ同源模建巨大芽孢杆菌青霉素Ｇ酰化酶的三维结构。在此基础上,将β亚基４２７位（突变Ａ）和４３０位（突变Ｂ）赖氨酸残基突变为丙氨酸,降低了该酶的等电点,增加了疏水性,从而提高其在酸性和有机溶剂环境中的稳定性。两个突变与亲本相比,比活力和Ｋｍ相近,最适ｐＨ减少了０．５个单位,突变Ｂ在ｐＨ５．２的溶液中的稳定性明显提高。突变Ａ和Ｂ在１５％和     

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。     

利用DNA家族重排提高青霉素G酰化酶合成活力

NA家族重排技术是酶定向进化的有力工具 ,已在实际应用中获得了巨大成功。来源于Providenciarettgeri、Escherichiacoli和Kluyveracitrophila的青霉素酰化酶基因序列同源性为 6 2 .5 %～ 96 .9%。在Providenciarettgeri青霉素酰化酶基因克隆和表达的基础上 ,利用DNA家族重排技术构建了上述基因的嵌合体突变库。通过平板初筛获得有活力的阳性克隆 ,表达提取突变酶测定其合成水解活力比。对突变酶进行随机测序的结果表明多基因嵌合体突变库显示出明显的多样性。通过一轮重排及筛选 ,获得了合成活力提高 4 0 %的突变酶 ,并且发现α亚基的重排对酶合成活力的提高更加有效。上述方法的应用有望获得合成活力进一步提高的青霉素G酰化酶。     

GL—7ACA酰化酶（C130）β亚基N端氨基酸残基的突变及功能

戊二酰 7 氨基头孢烷酸酰化酶 (即GL 7ACA酰化酶 ,EC .3.5 .1.11)的催化中心通常在 β亚基N端的第一个氨基酸 ,底物亲和标记的研究亦显示N端存在着结合靶点 ,因而该区域的结构可能与酶的功能密切相关。对C130 β亚基N端的 2～ 8位氨基酸残基分别进行了肽段置换和定点突变研究。将N端前 8位肽段置换为来源于Arthrobacterviscosus的青霉素G酰化酶 (PAC)的对应序列后 ,C130酰化酶活力丧失 ;而置换为来源于E .coli的青霉素G酰化酶 (PGA)的对应序列后 ,酰化酶活力仍然保留 ,但Km 值从 0 .44× 10 -3 mol·L-1增大为 0 .5 5× 10 -3mol·L-1,kcat值由 4.92s-1降低为 1.6 4s-1。另对C130 β亚基N端 2～ 4位氨基酸残基作了单点突变 :第 4位的Trp为可能的底物类似物结合位点 ,被变为Tyr后 ,它对底物GL 7ACA的结合能力略为减弱 ,kcat则降低为 2 .2 9s-1;而变为Leu后 ,Km 为 0 .34× 10 -3 mol·L-1,kcat为 3.15s-1;第 3位的Ser变为Met、Ala及Cys后 ,随着Km值逐渐降低 ,kcat也有所降低 ,而S3 M、S3 A突变体的kcat/Km 值比野生型的分别增加了 2 2 .3%和 39.3% ;将活性中心Ser(β1)邻位的Asn(β2 )变为Gln后 ,C130酶活大幅度下降 ,kcat减为 0 .47s-1。上述结果表明 ,C130 β亚基N端的前几个氨基酸残基均可对酶的功能     

高产青霉素酰化酶巨大芽胞杆菌的诱变选育及产酶条件优化

【目的】选育高产青霉素G酰化酶(PGA)工业菌株。【方法】采用LiCl-紫外线复合诱变以及常压室温等离子体(ARTP)诱变技术对巨大芽胞杆菌(Bacillus megaterium) ATCC 14945进行处理。处理菌体涂平板后,将长出的菌落接种到液体培养基中,向培养6 h后的二代菌液中添加终浓度为0.1%的苯乙酸,28 °C、250 r/min条件下诱导培养40 h。对离心后获得的上清(粗酶液)采用NIPAB法测定PGA酶活力。以PGA酶活力最高的菌株为材料,对苯乙酸最佳添加量和最佳诱导时间进行优化,采用NIPAB法测定PGA酶活力。采用SDS-PAGE检测诱变前后巨大芽胞杆菌粗酶液中PGA的蛋白特性。【结果】从诱变菌落中筛选到PGA酶活力为39.60 U/mL的菌株12-4,酶活力比出发菌株提高了8.5倍。该菌株在液体培养6 h后添加终浓度为0.2%的苯乙酸,继续培养50 h后,PGA酶活力可达78.45 U/mL,比出发菌株提高了16.8倍。诱变前后菌株培养液中的PGA蛋白均具α、β亚基;诱变后菌株PGA α亚基的量没有明显变化,β亚基的量明显增多;α、β亚基之间的蛋白条带明显增多。【结论】采用诱变技术可提高巨大芽胞杆菌PGA活性,获得的诱变菌株12-4及培养条件对PGA工业化生产具有重要价值。     

巨大芽孢杆菌青霉素G酰化酶在新型环氧载体ZH-HA上的固定化

巨大芽孢杆菌青霉素G酰化酶共价结合在新型环氧-氨基型载体ZH-HA 上,通过对酶浓度、固定化时间、pH以及缓冲液浓度等条件的考察,确定了最优固定化条件:50 mg比活力6000 U/g的巨大芽孢杆菌青霉素G酰化酶蛋白和1g ZH-HA悬浮于pH 9.01 mol/L磷酸缓冲液,室温搅拌6 h,制得固定化巨大芽孢杆菌青霉素G酰化酶,活力2126 U/g湿载体,活力回收率7.67%.比较研究了固定化酶与原酶性质,原酶最适温度45℃,最适pH为8.0.固定化酶则分别是50℃和9.0,分别比溶液酶偏移5℃、1.0个pH单位.经过40批连续水解青霉素G钾盐,固定化巨大芽孢杆菌青霉素酰化酶仍保持80%的活力,显示出良好的工作稳定性.     

青霉素G酰化酶β亚基Ser~(579)、Arg~(580)的定点突变研究

比较青霉素酰化酶(PGA)和青霉素结合蛋白的一级结构,我们推测PGAβ亚基中565～595肽段可能和酶的底物结合功能有关。为此我们将2.6 kb 长的完整的PGA 基因克隆到pTz 18 U 中构建成质粒pTZGA,并用定点突变的技术对Ser~(579)。和Arg~(580)两个氨基酸残基进行了突变研究。在所得到的四种突变子中·Ser~(579)→Gly~(579),Arg~(580)→Gly~(580),Arg~(580)→Glu~(580),Arg~(580)→Lys~(580))Glu~(580)和Gly~(580)没有酶活力,Lys~(580)约有30%的酶活力,Gly~(579)约有70%的酶活力。用ELISA 方法检测了四种突变子和野生型酶蛋白表达量,没有显著差异,这表明Arg~(580)对酶活性有重要意义,是酶活力中心的重要组成部分,可能与催化活性有关。     

大肠杆菌AE109青霉素G酰化酶的分离纯化及性质研究

 由发酵培养液所得大肠杆菌AE109菌体,先经高渗休克处理,继经D-苯甘氨酸-Sepharose 4B和DEAE-纤维素柱层析分离纯化得到青霉素G酰化酶,酶制品在非变性条件下的聚丙烯酰胺凝胶电泳上呈一条区带,而且可以结晶。在SDS变性条件下解离为α和β两个亚基。 酶性质的研究结果表明,由大肠杆菌工程菌AE109菌株所得青霉素G酰化酶与其亲本大肠杆菌AS1.76菌株所得青霉素G酰化酶性质相同。     

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

我们由E.coli AS1.76克隆了青霉素G酰化酶的基因,并且测定了其全部核苷酸序列。青霉素G酰化酶结构基因是由下述功能片段组成的:(1)编码信号肽(26个氨基酸残基)的78个碱基对;(2)编码α-亚基(209个氨基酸残基)的627个碱基对;(3)编码间隔肽(54个氨基酸残基)的162个碱基对;(4)编码β亚基(557个氨基酸残基)的1671个碱基对。此外,我们还发现起始密码子(ATG)前有个核糖体结合位点和启动子序列以及在终止密码子(TAA)之后有个转录终止信号。与最近发表的青霉素G酰化酶基因的DNA序列比较,同源性达99.7%。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.