Aspergillus sp136抗细菌耐药活性成分研究

基于广谱抗细菌耐药性这一思路，本研究中心建立了一套抗细菌耐药性化合物的筛选方法。由此从3000多种西南地区特殊生境的微生物和植物样品提取物中筛选获得17个抗细菌耐药性活性样品。对其中一株来自峨嵋山土样的微生物（Aspergillus sp136）进行了深入研究。通过TLC自显影等方法从其发酵产物中追踪分离得到抗耐药有效成分，并鉴定为烟曲霉酸。 采用多种方法对烟曲霉酸的体外抗细菌耐药活性进行评价。在平板扩散法中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）的协同抗耐药能力，其活性大约3倍于克拉维酸。在MIC的测试实验中，烟曲霉酸表现出对青霉素（β-内酰氨抗生素）以及非β-内酰氨抗生素如红霉素、四环素、氯霉素、链霉素、卡那霉素、庆大霉素的抗耐药能力。在棋盘格杀菌以及时间致死曲线的研究中，烟曲霉酸也表现出对青霉素、红霉素、四环素的协同抗细菌耐药活性。 在广泛的活性筛选中发现烟曲霉酸对LDLR基因具有上调活性，表明烟曲霉酸可能具有降血脂的活性。 在研究中发现，同空白对照相比，烟曲霉酸使耐药菌（Bacillus cereus NCPF63509）细胞外β-内酰胺酶酶活大幅度下降，而细胞内β-内酰胺酶酶活仅略有上升，这表明烟曲霉酸对β-内酰胺酶分泌过程具有抑制作用。 综述了β-内酰胺酶的研究进展。 A two-step agar diffusion method was established to screen wide spectrum synergistic antibacterial agents. By using this method, 17 active samples against antibiotic resistance were discovered from more than 3000 plants and microbes, which were collected from southwest china. One isolate Aspergillus sp136 collected from E-mei mountain area was selected for further studies. From the metabolites of this strain, a synergistic antibacterial compound was isolated by bioautographic TLC assay-guided fractionation and identified as helvolic acid. The synergistic effect of helvolic acid was confirmed by several methods in vitro. The synergistic effect of helvolic acid with penicillin (β-lactam antibiotics) was about 3 times as that of clavulanic acid with penicillin in agar diffusion assay. In MIC studies, helvolic acid exhibited synergistic effects with β-lactam antibiotics such as penicillin and non β-lactam antibiotics such as erythromycin, tetracycline, kanamycin, streptomycin and gentamycin. In checkerboard and time-kill studies, helvolic acid also exhibited synergistic effects with penicillin, erythromycin and tetracycline. In general screen of bioactivities, helvolic acid upregulate LDLR gene, which was indirectly determined by the activity of fluorescent enzyme. Therefore, helvolic acid might have the ability to lower lipid in blood. Compared with blank control, the extracellular β-lactamase activity decrease significantly and the intracellular β-lactamase activity increase slightly in Bacillus cereus NCPF63509 in the presence of helvolic acid, indicating that the secretion of β-lactamase was inhibited by helvolic acid. The research of β-lactamase was reviewed.

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5 similar to 6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

Difference in Enzyme Activity and Conformation of Glucose Oxidase Before and After Purification

EEnzyme activity of commercial glucose oxidase was enhanced after purification through a strong anionic exchange resin. In order to get a better insight into this phenomenon, surface pressure–area ( –A) isotherms and surface pressure–time ( –t) isotherms was used to study the interaction and the absorption at different pH values of the subphases between octadecylamine and glucose oxidase purified by a styrene system quaternary ammonium type strongly basic anionic exchange resin. Circular dichroism (CD), electrophoresis and enzyme activity measurements were conducted to study these phenomena. A preliminary hypothesis has been suggested to explain why the enzyme activity of purified glucose oxidase was higher than that of the commercial one. © 2002 Elsevier Science B.V. All rights reserved.

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

Trimeresurus stejnegeri venom, which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin. (C) 1998 Elsevier Science Ltd. All rights reserved.

Characterization of a thrombin-like enzyme from the venom of Trimeresurus jerdonii

From the venom of Trimeresurus jerdonii, a distinct thrombin-like enzyme, called jerdonobin. was purified by DEAF A-25 ion-exchange chromatography, Sephadex G-75 gel filtration, and fast protein liquid chromatography (FPLC). SDS-PAGE analysis of this enzyme shows that it consists of a single polypeptide chain with a molecular weight of 38,000. The NH2-terminal amino acid sequence of jerdonobin has great homology with venom thrombin-like enzymes documented. Jerdonobin is able to hydrolyze several chromogenic substrates. The enzyme directly clots fibrinogen with an activity of 217 NIH units/mg, The fibrinopeptides released, identified by HPLC consisted of fibrinopeptide A and a small amount of fibrinopepide B. The activities of the enzyme were inhibited by phenylmethylsulfonyl fluoride (PMSF) and p-nitrophenyl-p-guanidinobenzoate (NPGB). However, metal chelator (EDTA) had no effect on it. indicating it is venom serine protease. (C) 2000 Elsevier Science Ltd. All rights reserved.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity = 89%) to TSV-PA, a specific plasminogen activator from venom of T stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys(239) in TSV-PA to Gln(239) in jerdonobin-II might play an important role on their plasminogen activating activity difference. (C) 2005 Elsevier Ltd. All rights reserved.