Evolution of a new function in an esterase: simple amino acid substitutions enable the activity present in the larger paralog, BioH

Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual, involving simple amino acid residue substitutions, or drastic, such that larger structural elements are inserted, deleted or rearranged. Vast protein sequence comparisons, supported by some experimental evidence, argue that large structural modifications have been necessary for certain catalytic activities to evolve. However, it is not clear whether these activities could not have been attained by gradual changes. Interestingly, catalytic promiscuity could play a fundamental evolutionary role: a preexistent secondary activity could be increased by simple amino acid residue substitutions that do not affect the enzyme's primary activity. The promiscuous profile of the enzyme may be modified gradually by genetic drift, making a pool of potentially useful activities that can be selected before duplication. In this work, we used random mutagenesis and in vivo selection to evolve the Pseudomonas aeruginosa PAO1 carboxylesterase PA3859, a small protein, to attain the function of BioH, a much larger paralog involved in biotin biosynthesis. BioH was chosen as a target activity because it provides a highly sensitive selection for evolved enzymatic activities by auxotrophy complementation. After only two cycles of directed evolution, mutants with the ability to efficiently complement biotin auxotrophy were selected. The in vivo and in vitro characterization showed that the activity of one of our mutant proteins was similar to that of the wild-type BioH enzyme. Our results demonstrate that it is possible to evolve enzymatic activities present in larger proteins by discrete amino acid substitutions.     

Mutational analyses of restriction endonuclease--HindIII mutant E86K with higher activity and altered specificity

We have performed mutational analyses of restriction endonucleaseHindIII in order to identify the amino acid residues responsiblefor enzyme activity. Four of the seven HindIII mutants, whichhad His-tag sequences at the N-termini, were expressed in Escherichiacoli, and purified to homogeneity. The His-tag sequence didnot affect enzyme activity, whereas it hindered binding of theDNA probe in gel retardation assays. A mutant E86K in whichLys was substituted for Glu at residue 86 exhibited high endonucleaseactivity. Gel retardation assays showed high affinity of thismutant to the DNA probe. Surprisingly, in the presence of atransition metal, Mo²⁺ or Mn²⁺, the E86K mutant cleaved substrateDNA at a site other than HindIII. Substitution of Glu for Valat residue 106 (V106E), and Asn for Lys at residue 125 (K125N)resulted in a decrease in both endonucleolytic and DNA bindingactivities of the enzyme. Furthermore, substitution of Leu forAsp at residue 108 (D108L) abolished both HindIII endonucleaseand DNA binding activities. CD spectra of the wild type andthe two mutants, E86K and D108L, were similar to each other,suggesting that there was little change in conformation as aresult of the mutations. These results account for the notionthat Asp108 could be directly involved in HindIII catalyticfunction, and that the substitution at residue 86 may bringabout new interactions between DNA and cations.     

Synthesis and characterization of novel amphiphiles containing amino acid and carbohydrate

Amphiphiles composed of two different natural renewable raw materials, amino acid and carbohydrate (chitin), were synthesized, and their colloidal properties were studied. N-Higher acylated glutamic and aspartic acid were used as amino acid raw materials. Chitin monomer (N-acetyl glucosamine) and dimer (N,N′-diacetylchitobiose) were employed as the carbohydrate hydrophilic moiety in the amphiphiles. These surfactants showed a surface tension in the range of 30–36 mN/m at their critical micelle concentration. In the study of colloidal properties, such as surface tension, emulsification, and foaming, the surfactants containing the monosaccharide hydrophilic group showed much better results than those with disaccharide. Biodegradation measurements showed that the tested surfactants are biodegraded by environmental microorganisms to 57–73% of the initial levels in 14 d. Their biodegradation extent depended on neither the saccharide structure nor the kind of amino acid.     

Synthesis, characterization and high in vitro antitumour activity of novel triphenyltin carboxylates

The synthesis and spectral characterization of six novel triphenyltin compounds are described. The in vitro antitumour activity of three of these compounds against two human tumour cell lines, MCF-7, a mammary tumour, and WiDr, a colon carcinoma, was determined. All three compounds are more active than cis-platin, etoposide and doxorubicin against both tumour cell lines. They are as active as mitomycin C against WiDr, but less active against MCF-7.     

新型高活性聚醚多元醇的制备及应用

详细介绍了用三(五氟苯基)硼烷为代表的新型Lewis酸催化剂制备高活性聚醚多元醇的工艺,并介绍了该高活性聚醚多元醇在聚氨酯(PU)泡沫及弹性体中的应用情况,指出用该新型聚醚多元醇制备的PU树脂具有优异的固化特性、力学性能、耐水性和湿热稳定性。     

Development and analysis of a novel collagen composite encapsulating a natural pharmic amino acid,dencichine

In this work, we have conducted a blend of type I collagen and dencichine under acid conditions, followed by investigating the intermolecular interactions between the two components. Fourier transform infrared spectroscopy (FTIR), molecular modeling methods, the circular dichrorism (CD) measurement, and X‐ray photoelectron spectroscopy (XPS) studies inducate that the structure integrity of collagen is still maintained obviously after introducing dencichine. Furthermore, we also analyze the mechanism of the combination, which is just more than hydrogen bonding. Moreover, differential scanning calorimetry (DSC) reveals that compared to pure collagen, dencichine could promote the thermalstability of collagen‐dencichine composite sponge (CDCS) with the increasing content of dencichine. Atomic force microscopy (AFM) is utilized to determine the morphology changes of collagen by inducing dencichine. The fibril aggregation occurs due to the crosslinking with dencichine and the fibrous structure of collagen after inducing dencichine still remains compact and clear, which is the foundation of its biological activity. The present study, reveals that intermolecular interactions exist in the CDCS indeed, which is benefit for the development of new biological material and fabrication of novel functionalized hemostatic biomaterials. POLYM. COMPOS., 37:2027–2036, 2016. © 2015 Society of Plastics Engineers     

以高产量、高产率、高生产强度为目标的发酵过程优化技术

以高产量、高底物转化率和高生产强度为目标,综合运用微生物反应计量学、生化反应和传递动力学、生物反应器工程及代谢工程理论,开发了:(1)基于微生物反应计量学的培养环境优化技术;(2)基于微生物代谢特性的分阶段培养技术;(3)基于反应动力学模型的优化技术;(4)基于代谢通量分析的优化技术;(5)基于系统观点的生物反应系统优化技术。将这些技术广泛应用于多种产品的发酵过程优化研究中并取得了成功。在此基础上总结出“简化、定量化、模型化和阶段化”的发酵过程优化基本原理,这一基本原理对提高我国发酵工业技术水平、促进生物食品产业的健康发展将起重要作用。     

Dioxomolybdenum(VI) complexes with amino acid functionalized N-salicylidene hydrazides: Synthesis,structure and catalytic activity

A cis-dioxomolybdenum(VI) complex with an amino acid functionalized hydrazone ligand derived from salicylaldehyde and Boc-protected β-alanine hydrazide (Boc = t-butyloxycarbonyl) has been synthesized. The X-ray crystal structure reveals a distorted octahedral geometry at the molybdenum atom with an O₅N donor set and vast hydrogen bonding interactions including the amino acid side chain of the ligand. The new cis-dioxomolybdenum(VI) complex is an efficient catalyst for the peroxidic oxidation of sulfides, but does not show any activity towards the oxidation of bromide.     

Synthesis and pro-apoptotic activity of novel glycyrrhetinic acid derivatives

Triterpenoids are used for medicinal purposes in many countries. Some, such as oleanolic and glycyrrhetinic acids, are known to be anti-inflammatory and anticarcinogenic. However, the biological activities of these naturally occurring molecules against their particular targets are weak, so the synthesis of new synthetic analogues with enhanced potency is needed. By combining modifications to both the A and C rings of 18βH-glycyrrhetinic acid, the novel synthetic derivative methyl 2-cyano-3,12-dioxo-18βH-olean-9(11),1(2)-dien-30-oate was obtained. This derivative displays high antiproliferative activity in cancer cells, including a cell line with a multidrug-resistance phenotype. It causes cell death by inducing the intrinsic caspase-dependent apoptotic pathway.     

Comparative study on foaming and foam stability of multiple mixed systems of fluorocarbon,hydrocarbon, and amino acid surfactants

This study focuses on the effect of amino acid surfactant (sodium lauroyl glutamate [SLG]) on foam properties of a mixture of fluorocarbon (FS-50) and hydrocarbon (APG0810) surfactants. The molecular interactions, foamability, and foam stability of the surfactant solutions are systematically studied. The results show that SLG has stronger ability to lower surface tension of water compared to APG0810, but worse that than FS-50, and to adsorb onto gas/liquid interface in mixed solutions with FS-50 compared to APG0810. The addition of SLG increased foamability of APG0810/FS-50 mixed solution, and decelerated foam drainage and coarsening of individual APG0810, individual FS-50, and mixture of APG0810/FS-50 solutions. The mixture of SLG/FS-50 exhibited the higher foamability and foam stability than the frequently used critical component of the new developed AFFF, the mixture of APG0810/FS-50. This study can provide guidance for the scientific application of amino acid surfactants in the development of environmentally friendly firefighting foams.     

7,10-Dihydroxy-8(E)-Octadecenoic acid: Stereochemistry and a novel derivative, 7,10-Dihydroxyoctadecanoic acid

7,10-Dihydroxy-8(E)-octadecenoic acid (obtained by bioconversion of oleic acid with the bacterial strain PR3) was hydrogenated with hydrazine hydrate under air in ethanolic solution to give a novel compound, 7,10-dihydroxy-octadecanoic acid. The absolute configuration of both compounds was determined with the aid of circular dichroism to be R,R. A different purification method for 7,10-dihydroxy-8(E)-octadecenoic acid is described. Presented in part at the 82nd AOCS Annual Meeting, Chicago, IL, May 1991.     

Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala

The 3-D structure of subtilisin YaB was computer modelled using thestructures of subtilisin BPN', subtilisin Carlsberg and thermitase astemplates. Gly124 and Gly151 located on both sides of the waist of the S1pocket were selected for site-directed mutagenesis based on the modelledstructure. The mutated ale genes coding for the mutant subtilisin YaB wereexpressed in Bacillus subtilis DB104. All of the G124 and G151 series ofmutants exhibited an increase of relative catalytic activity forelastin-orcein against casein and myofibrillar proteins. The S1 substratespecificity of G124A, G124V and G151A mutants were assessed using variouscarbobenzoxy-amino acid-nitrophenyl esters and succinyl-Ala-Ala-(Pro orVal)-(Ala, Phe or Leu)-p- nitroanilide [AA(P/V) (A/F/L)]. While G124A andG124V mutants hydrolyzed only Ala and Gly esters, G151A mutant hydrolyzedAla, Leu and Gly esters. The G124A and G124V mutants did not hydrolyze AAPFand AAPL. However, these two mutants hydrolyzed AAPA and AAVA with kcat/Kmvalues approximately 3-10-fold higher than those of the wild-type enzyme.The G151A mutant did not hydrolyze AAPF, but hydrolyzed AAPL, AAPA and AAVAwith kcat/Km values approximately 1-4-fold higher than those of thewild-type enzyme. These results clearly indicate that the S1 substratespecificity of G124A and G124V mutants was restricted to Ala and Gly, andG151A mutant to Ala, Gly and Leu.     

新型无毒可生物降解氨基酸衍生环氧树脂的合成与表征

用天然氨基酸为起始材料,合成了一种具有双酚羟基官能团的环肽,并以此环肽为联酚合成了主链包含酰胺键的可生物降解环氧树脂。用FT-IR与NMR表征了合成氨基酸衍生环肽与对应环氧树脂的化学结构,结果表明合成了预期结构。用盐酸丙酮法测定了合成环氧树脂的环氧值。     

Comparative modelling and analysis of amino acid substitutions suggests that the family of pregnancy-associated glycoproteins includes both active and inactive aspartic proteinases

The pregnancy-associated glycoproteins (PAGs) are secretoryproducts synthesized by the outer epithelial cell layer (chorion)of the placentas of various ungulate species. The amino acidsequences of eight PAGs have been inferred from cloned cDNAof cattle and sheep, as well as of the non-ruminant pig andhorse. We compare the PAG sequences and present results of thethree-dimensional models of boPAG-1 and ovPAG-1 that were constructedon the basis of the crystal structures of homologous porcinepepsin and bovine chymosin using a rule-based comparative modellingapproach. Further, we compare peptide binding subsites definedby interactions with pepstatin and a decapeptide inhibitor (CH-66)modelled on the basis of crystal structures of other asparticproteinases. We have extended our analysis of the peptide bindingsubsites to the other PAG molecules of known sequence by aligningthe PAG sequences to the structural template derived from thepepsin family and by making use of the three-dimensional modelsof the boPAG-1 and ovPAG-1. The residues that are likely toaffect peptide binding in the boPAG-1, ovPAG-1 and other PAGmolecules have been identified. Sequence comparisons revealthat all PAG molecules may have evolved from a pepsin-like progenitormolecule with the equine PAG most closely related to the pepsins.The presence of substitutions at the S₁ and other subsites relativeto pepsin make it unlikely that either bovine, ovine or theporcine PAG-1 have catalytic activity. Only two of the eightPAGs examined (porcine PAG-2 and equine PAG-1) retain featuresof active aspartic proteinases with pepsin-like activity. Ourresults indicate that in the PAGs so far characterized the peptidebinding specificities differ significantly from each other andfrom pepsin, despite their high sequence identities. Analysisof the various peptide binding subsites demonstrates why bothbovine and ovine PAG-1 are capable of binding pepstatin. Thestrong negative charge in the binding cleft of boPAG-1 and ovPAG-1indicates a preference for lysine- or arginine-rich peptides.PAGs represent a family where the possible peptide binding functionmay be retained through their binding specificities, but wherethe catalytic activity may be lost in some cases, such as theboPAG-1, ovPAG-1 and the poPAG-1.     

Modeling and prediction of pH and water activity in aqueous amino acid solutions

Water activity and pH of amino acids (alanine, glicine, arginine and proline) in aqueous and buffer solutions were measured at various concentrations and 25°C. The partial dissociation phenomenon was considered and the UNTFAC model, combined with solvation equations, was used for the water activity and pH predictions. The validity of the model was tested for the aminoacids in water and two buffer solutions. Mean deviations of 0.64% for the water activity and 2.5% for the pH were obtained.     

Isolation, amino acid sequence and biological activities of novel long-chain polyamine-associated peptide toxins from the sponge Axinyssa aculeata

A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N-terminal residues that are modified by the addition of long-chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU-A and B, which were purified in this study, share a common polypeptide chain but differ in their N-terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU-A and B was deduced from 3' and 5' RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides.     

Modified macroporous adsorption resins with amino and acetyl groups through a novel method and adsorption behaviors for alizarin yellow GG

Under bubble with air compressor, macroporous adsorption resin was functionalized with amino and acetyl groups. The method avoided the fragmentation of the resin during modification. Alizarin yellow GG (AYGG) was used as an adsorbate to investigate adsorption kinetics of the modified resins. It showed that pseudofirst‐order and pseudosecond‐order kinetics cannot reasonably express the adsorption process. A new kinetic model, multi‐layer adsorption model, showed much better fit to the adsorption kinetic data and corresponding kinetic parameters could predict adsorption mechanism. Meantime, AYGG can be easily recovered, and the resins can be regenerated. Due to π – π , electrostatic force and hydrogen bond interaction between the resin and carboxyl, phenolic hydroxyl, and azo groups of AYGG, the resin with amino group showed higher adsorption capacity than the other resins used in this study. Steric hindrance and decrease in electrostatic force were unfavorable for the enrichment of AYGG by the resin with acetyl group. Response surface model combining with central composite design was used to determine effects of pH and initial concentration on adsorption. It showed that a second‐order polynomial regression model could reasonably express the experimental data and optimum adsorption conditions were obtained. The design provided an effective methodology to optimize an adsorption process. POLYM. ENG. SCI., 54:1960–1968, 2014. © 2013 Society of Plastics Engineers     

乙型脑炎减毒活疫苗E蛋白基因及氨基酸序列稳定性分析

目的分析2003²011年连续9年出口韩国的23批乙型脑炎减毒活疫苗E蛋白基因及氨基酸序列的稳定性。方法提取出口韩国的23批乙型脑炎减毒活疫苗及其生产该疫苗的主种子和工作种子病毒RNA,RT-PCR法扩增乙型脑炎减毒活疫苗病毒E蛋白基因片段后进行测序;将23批乙型脑炎减毒活疫苗E蛋白基因序列中8个关键位点氨基酸序列与GenBank中登录的乙型脑炎病毒弱毒株SA14-14-2应用AlignX比对软件进行分析;检测23批乙型脑炎减毒活疫苗及生产该疫苗的主种子和工作种子病毒滴度。结果 23批疫苗与生产该疫苗的主种子、工作种子的病毒E蛋白基因序列与GenBank中登录的乙型脑炎病毒减毒株SA14-14-2 E蛋白基因序列完全一致,均由1 500个核苷酸组成,并编码500个氨基酸,同源性100%;23批疫苗E蛋白基因中与弱毒力密切相关的8个关键位点的氨基酸均未发生改变,与生产该疫苗的主种子、工作种子以及GenBank中登录的乙型脑炎病毒减毒株SA14-14-2(D90195)E蛋白相应位点一致性为100%;生产23批疫苗的主种子和工作种子病毒滴度变化幅度较小,为6.722011年连续9年出口韩国的23批乙型脑炎减毒活疫苗E蛋白基因及氨基酸序列的稳定性。方法提取出口韩国的23批乙型脑炎减毒活疫苗及其生产该疫苗的主种子和工作种子病毒RNA,RT-PCR法扩增乙型脑炎减毒活疫苗病毒E蛋白基因片段后进行测序;将23批乙型脑炎减毒活疫苗E蛋白基因序列中8个关键位点氨基酸序列与GenBank中登录的乙型脑炎病毒弱毒株SA14-14-2应用AlignX比对软件进行分析;检测23批乙型脑炎减毒活疫苗及生产该疫苗的主种子和工作种子病毒滴度。结果 23批疫苗与生产该疫苗的主种子、工作种子的病毒E蛋白基因序列与GenBank中登录的乙型脑炎病毒减毒株SA14-14-2 E蛋白基因序列完全一致,均由1 500个核苷酸组成,并编码500个氨基酸,同源性100%;23批疫苗E蛋白基因中与弱毒力密切相关的8个关键位点的氨基酸均未发生改变,与生产该疫苗的主种子、工作种子以及GenBank中登录的乙型脑炎病毒减毒株SA14-14-2(D90195)E蛋白相应位点一致性为100%;生产23批疫苗的主种子和工作种子病毒滴度变化幅度较小,为6.72⁷.17 lgPFU/ml,而23批疫苗的病毒滴度变化幅度也较小,为7.027.17 lgPFU/ml,而23批疫苗的病毒滴度变化幅度也较小,为7.02⁷.61 lgPFU/ml。结论连续9年出口韩国的23批乙型脑炎减毒活疫苗病毒E蛋白基因稳定,一致性良好,该疫苗质量稳定,安全可靠。     

氨基酸表面活性剂的合成、性质及工业应用（续完）

正7毒性传统表面活性剂,特别是阳离子表面活性剂对水生生物具有很强的毒性。其急性毒性是由于表面活性剂在细胞-水界面处的吸附-离子相互作用现象。降低表面活性剂的cmc通常导致表面活性剂更强的界面吸附,通常导致其急性毒性升高。表面活性剂疏水链的长度增加也导致表面活性剂急性毒性增大。大部分AAS为对人体和环境(特别是对海洋生物)低毒或无毒,使其适合做为食品成分、药物