Cooperativity and substrate specificity of an alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1

The saccharifying alkaline amylase and neopullulanase complex of Micrococcus halobius OR-1 hydrolyzes both α-(1,4)- and α-(1,6)-glycosidic linkages of different linear and branched polysaccharides. The following observations were made concerning the analysis of the coexpressed amylase and neopullulanase enzymes. Even though the enzymes were subjected to a rigorous purification protocol, the activities could not be separated, because both the enzymes were found to migrate in a single peak. By contrast, two independent bands of amylolytic activity at 70 kDa and pullulanolytic activity at 53 kDa were evident by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reducing and nonreducing PAGE, and zymographic analysis on different polysaccharides. Preferential chemical modification of the enzyme and concomitant high-performance thin-layer chromatographic analyses of the saccharides liberated showed that amylase is sensitive to 1-(dimethylamino-propyl)-3-ethyl carbodiimide-HCl and cleaved α-(1,4) linkages of starch, amylose, and amylopectin producing predominantly maltotriose. On the other hand, formalin-sensitive neopullulanase acts on both α-(1,4) and α-(1,6) linkages of pullulan and starch with maltotriose and panose as major products. It is understood that neopullulanase exhibits dual activity and acts in synergy with amylase toward the hydrolysis of α-(1,4) linkages, thereby increasing the overall reaction rate; however, such a synergism is not seen in zymograms, in which the enzymes are physically separated during electrophoresis. It is presumed that SDS-protein intercalation dissociated the enzyme complex, without altering the individual active site architecture, with only the synergism lost. The optimum temperature and pH of amylase and neopullulanase were 60°C and 8.0, respectively. The enzymes were found stable in high alkaline pH for 24 h. Therefore, the saccharifying alkaline amylase and neopullulanase of M. halobius OR-1 evolved from tapioca cultivar shows a highly active and unique enzyme complex with several valuable biochemical features.     

A computational tool to optimize ligand selectivity between two similar biomacromolecular targets

Summary Algorithms for a new computer program designed to increase ligand--receptor selectivity between two proteins are described. In this program ligand--receptor selectivity is increased by functional modifications to the ligand so as to increase the calculated binding affinity of it to one protein and/or decrease the calculated binding affinity of it to the other protein. The structure of the ligand is modified by selective replacement of atoms and/or functional groups in silico based on a specific set of steric and/or hydropathic complementarity rules involving atoms and functional groups. Relative binding scores are calculated with simple grid-based steric penalty, hydrogen bond complementarity, and with the HINT score model. Two examples are shown. First, modifying the structure of the ligand CB3717 is illustrated in a number of ways such that the binding selectivity to wild type L. casei thymidylate synthase or its E60Q mutant may be improved. Second, starting with a non-selective lead compound that had been co-crystallized with both plant and mammalian 4-hydroxyphenylpyruvate dioxygenases, new compounds (similar to selective ligands discovered by screening) to improve the selectivity of (herbicidal) inhibitors for the plant enzyme were designed by the program.     

Do Intrinsically Disordered Proteins Possess High Specificity in Protein–Protein Interactions?

Specific protein–protein interactions are critical to cellular function. Structural flexibility and disorder‐to‐order transitions upon binding enable intrinsically disordered proteins (IDPs) to overcome steric restrictions and form complementary binding interfaces, and thus, IDPs are widely considered to have high specificity and low affinity for molecular recognition. However, flexibility may also enable IDPs to form complementary binding interfaces with misbinding partners, resulting in a great number of nonspecific interactions. Consequently, it is questionable whether IDPs really possess high specificity. In this work, we investigated this question from a thermodynamic viewpoint. We collected mutant thermodynamic data for 35 ordered protein complexes and 43 disordered protein complexes. We found that the enthalpy–entropy compensation for disordered protein complexes was more complete than that for ordered protein complexes. We further simulated the binding processes of ordered and disordered protein complexes under mutations. Simulation data confirmed the observation of experimental data analyses and further revealed that disordered protein complexes possessed smaller changes in binding free energy than ordered protein complexes under the same mutation perturbations. Therefore, interactions of IDPs are more malleable than those of ordered proteins due to their structural flexibility in the complex. Our results provide new clues for exploring the relationship between protein flexibility, adaptability, and specificity.     

Decision threshold adjustment in class prediction

Standard classification algorithms are generally designed to maximize the number of correct predictions (concordance). The criterion of maximizing the concordance may not be appropriate in certain applications. In practice, some applications may emphasize high sensitivity (e.g., clinical diagnostic tests) and others may emphasize high specificity (e.g., epidemiology screening studies). This paper considers effects of the decision threshold on sensitivity, specificity, and concordance for four classification methods: logistic regression, classification tree, Fisher's linear discriminant analysis, and a weighted k-nearest neighbor. We investigated the use of decision threshold adjustment to improve performance of either sensitivity or specificity of a classifier under specific conditions. We conducted a Monte Carlo simulation showing that as the decision threshold increases, the sensitivity decreases and the specificity increases; but, the concordance values in an interval around the maximum concordance are similar. For specified sensitivity and specificity levels, an optimal decision threshold might be determined in an interval around the maximum concordance that meets the specified requirement. Three example data sets were analyzed for illustrations.     

螺旋型抗菌肽的疏水性对抗细菌与抗真菌活性的影响比较

α-螺旋型多肽HPRP-A1由15个氨基酸残基组成,来源于幽门螺杆菌核糖体蛋白L1的N端.本研究以HPRP-A1为模板,在其非极性面中心通过单个氨基酸定点取代的方法,形成一系列疏水性不同的多肽类似物,系统地研究疏水性对α-螺旋型多肽生物活性的影响.结果显示,多肽疏水性及所带净电荷对多肽生物活性起着重要的作用;HPRP-A1及疏水性相对较高的多肽类似物具有较好的广谱抗菌活性（包括革兰氏阳性菌、革兰氏阴性菌及真菌）,但也有相对较高的溶血活性;多肽的疏水性与所带净电荷的变化对多肽抗细菌活性与抗真菌活性所产生的影响有着相似的变化趋势和程度.这意味着多肽与细菌的作用机制和多肽与真菌的作用机制存在一定的相关性.多肽对细菌和真菌的抗菌活性存在特异性,为设计出具有临床应用前景的抗菌肽药物奠定了基础.     

Understanding the role of ion interactions in soluble salt flotation with alkylammonium and alkylsulfate collectors

There is anecdotal evidence for the significant effects of salt ions on the flotation separation of minerals using process water of high salt content. Examples include flotation of soluble salt minerals such as potash, trona and borax in brine solutions using alkylammonium and alkylsulfate collectors such as dodecylamine hydrochloride and sodium dodecylsulfate. Although some of the effects are expected, some do not seem to be encompassed by classical theories of colloid science. Several experimental and modeling techniques for determining solution viscosity, surface tension, bubble-particle attachment time, contact angle, and molecular dynamics simulation have been used to provide further information on air–solution and solid–solution interfacial phenomena, especially with respect to the interfacial water structure due to the presence of dissolved ions. In addition atomic force microscopy, and sum frequency generation vibrational spectroscopy have been used to provide further information on surface states. These studies indicate that the ion specificity effect is the most significant factor influencing flotation in brine solutions.     

Switching Demethylation Activities between AlkB Family RNA/DNA Demethylases through Exchange of Active‐Site Residues

The AlkB family demethylases AlkB, FTO, and ALKBH5 recognize differentially methylated RNA/DNA substrates, which results in their distinct biological roles. Here we identify key active‐site residues that contribute to their substrate specificity. Swapping such active‐site residues between the demethylases leads to partially switched demethylation activities. Combined evidence from X‐ray structures and enzyme kinetics suggests a role of the active‐site residues in substrate recognition. Such a divergent active‐site sequence may aid the design of selective inhibitors that can discriminate these homologue RNA/DNA demethylases.     

甲氧基有机磷杀虫剂广谱特异性抗体的制备

以通用结构O,O-二甲基硫代磷酸酯为目标检测基团,制备针对甲氧基有机磷杀虫剂的广谱特异性抗体。利用O,O-二甲基硫代磷酸钠和氯乙酸合成半抗原S-羧甲基-O,O-二甲基二硫代磷酸酯(CMP),通过混合酸酐法(MA)和活性酯法(AE)分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联。CMP-MA-BSA、CMP-AE-BSA作为免疫原均获得了免疫应答。其中,CMP-AE-BSA所获得的抗血清效价最高,为256000。研究了有机溶剂种类及含量、pH因素对ELISA曲线的影响,确定CMP酶联免疫分析方法(ELISA)的最佳工作条件,CMP的最低检测浓度为0.076μg/L,IC50为93.97μg/L。以14种常见有机磷杀虫剂为对象,检测抗体对其交叉反应,测定结果表明:抗体对马拉硫磷、稻丰散、乐果、亚胺硫磷、倍硫磷、甲基嘧啶磷、甲基对硫磷、杀螟硫磷及杀扑磷等均有识别作用。IC50分别为69.92、136.90、230.39、416.84、508.57、510.38、607.21、835.30和850.21μg/L。该技术可用于多种甲氧基有机磷杀虫剂的快速定性或半定量检测。     

磺胺间甲嘧啶单克隆抗体的制备

研究了磺胺间甲嘧啶单克隆抗体的制备。结果表明,得到三株单抗,其中3F7株抗体效价最高,细胞培养上清为1∶5.2×102,腹水为1∶5.5×105。通过交叉反应试验可知,单抗具有较好的特异性。