重组人锰超氧化物歧化酶的胃蛋白酶酶解特性及其稳定性

目的探讨重组人锰超氧化物歧化酶(rhMn-SOD)的胃蛋白酶酶解特性及其稳定性。方法在37℃条件下,用模拟胃液(0.084 mol/L HCl,35 mmol/L NaCl,pH 2.0,4 000 U胃蛋白酶)处理rhMn-SOD不同时间,SDS-PAGE分析酶解效果;使用总SOD测定试剂盒检测rhMn-SOD在不同温度(25、37、45、55、65、75℃)下保存不同时间(10、20、30、40、50、60 min)、不同pH(4.0～10.0)、不同化合物(氯仿、甘油、DMSO、10%SDS、β-巯基乙醇、苯酚、30%H2O2)处理后及日光和紫外照射后的相对活力。结果 rhMn-SOD在模拟胃液中处理2 min即可完全酶解。在25～55℃范围内,rhMn-SOD具有较好的热稳定性,在65～75℃范围内,rhMn-SOD的热稳定性较差;在pH 6.0～8.0范围内,rhMn-SOD具有较好的稳定性;rhMn-SOD对甘油和氯仿不敏感,经DMSO、SDS处理后,活力大幅下降,经β-巯基乙醇、苯酚、30%H2O2处理后,基本失活;rhMn-SOD经日光和紫外照射3 h,活力基本不变。结论分析了rhMnSOD的胃蛋白酶酶解特性、热稳定性、pH稳定性、对不同化合物的敏感性以及光稳定性,为其广泛应用于化妆品、药品、食品添加剂等领域奠定了基础。     

Trp128Tyr mutation in the N-lobe of recombinant human serum transferrin: 1H- and 15N-NMR and metal binding studies

The conserved Trp residue within helix 5 of the N-lobe of human serum transferrin (hTF/2N, 40 kDa) has been mutated to Tyr. NMR and CD spectra and energy calculations show that the mutation causes little perturbation of the overall structure of hTF/2N although the chelating agent Tiron removed Fe3+ from the mutant protein about three times faster than from wild-type hTF/2N. 1H-NMR resonances of residues in the Leu122-Trp128-Ile132 hydrophobic patch are assigned both by ring current calculations and with the aid of the mutation. [1H, 15N]-NMR resonances for 11 of the 14 Tyr residues were observed in the spectra of 15N-Tyr-hTF/2N and a resonance for Tyr128 was assignable in spectra of the mutant. The 15N resonance of Y128 was sensitive to oxalate and Ga3+ binding, and Ga3+ binding perturbed 15N resonances for most of the Tyr residues. Since these are well distributed over the N-lobe, it can be concluded that metal-induced structural changes are not merely local to the binding site.      

NMR relaxation studies of the role of conformational entropy in protein stability and ligand binding

Recent advances in the measurement and analysis of protein NMR relaxation data have made it possible to characterize the dynamical properties of many backbone and side chain groups. With certain caveats, changes in flexibility that occur upon ligand binding, mutation, or changes in sample conditions can be interpreted in terms of contributions to conformational entropy. Backbone and side chain flexibility can either decrease or increase upon ligand binding. Decreases are often associated with "enthalpy-entropy compensation" and "induced fit" binding, whereas increases in conformational entropy can contribute to stabilization of complexes. In certain cases, conformational entropy appears to play a role in cooperative binding and enzyme catalysis. In addition, variations in conformational entropy and heat capacity may both be important in stabilizing the folded structures of proteins.     

Differential scanning calorimetric studies on phase transition of glucose and cellulose oligosaccharides

The phase transitions of D-glucose and an homologous series of cellulose oligosaccharides up to cellotetraose have been studied using a differential scanning calorimeter. A detailed comparison of our experimental results with those reported by other workers has been made in order to derive precise information on melting and glass transitions of carbohydrates. Particular attention has been given to the static values of melting temperature of D-glucose and cellobiose. It has also been found that cellulose oligosaccharides which have more than three anhydroglucose units decompose below melting temperature.     

重组人乳头瘤病毒33型L1病毒样颗粒的表达、纯化及免疫原性评价

目的利用毕赤酵母表达人乳头瘤病毒(human papillomavirus,HPV)33型L1病毒样颗粒(virus-like particles,VLPs),评价其在小鼠体内的免疫原性。方法构建HPV33 L1表达质粒,电转化毕赤酵母,筛选高表达菌株,15 L发酵罐发酵,离子交换和凝胶体积排阻层析分离纯化HPV33 L1 VLPs,纯化后样品免疫小鼠,采用假病毒中和试验检测小鼠免疫后血清中和抗体滴度。结果质粒HPV33 L1-pPIC3. 5K经双酶切及测序鉴定证明构建成功;毕赤酵母表达产物经Western blot分析,可见相对分子质量约56 000的目的条带;透射电镜下可见直径约60 nm的病毒样颗粒;纯化后的HPV33 L1 VLPs纯度可达95%以上;0. 4和0. 04μg HPV33 L1 VLPs免疫后小鼠血清中和抗体几何平均滴度(geometric mean titer,GMT)分别为3 200和566,差异有统计学意义(P 0. 05)。结论利用毕赤酵母成功表达了HPV33 L1 VLPs,并具有良好的免疫原性。     

Stability and calorimetric studies of silico‐ferrites of calcium aluminum and magnesium

In a systematic study on silico‐ferrites of calcium aluminum and magnesium (SFCA phases) this investigation focuses on synthesis of silicon‐free SFCA‐type compounds with low‐MgO contents (~1.00 apfu—atoms per formula unit). Previous studies revealed the existence of iron‐rich SFCA phases similar to the SFCA‐I structure with the chemical composition Ca₃MgAl₆Fe₁₀O₂₈ (Metall Mater Trans B. 2017;48:2207). The experimental results in the quaternary Fe₂O₃‐CaO‐Al₂O₃‐MgO system confirm large stability fields of 2 silicon‐free ferrites FCAM‐I and FCAM‐III, which are members of the homologous series M_14+6nO_20+8n (n = 1, 2). Starting with compositions corresponding to Ca₃MgAl_xFe_16‐xO₂₈ (with increasing aluminum content from x = 0‐12 apfu), it was possible to synthesize these phases with an x‐value ≥2 apfu, which corresponds to Al₂O₃ contents ≥7.14 wt%. Synthesis of pure silicon‐free ferrites with n = 1 (FCAM‐I) and 2 (FCAM‐III) and silicon‐bearing ferrites with n = 0 (SFCA) was possible. Samples were characterized by electron probe microanalysis, powder diffraction, and subsequently studied using relaxation calorimetry measurements in combination with differential scanning calorimetry for determination of the heat capacities and standard entropies S°(298). The corresponding values are S°(298) = 650.3 ± 4.6 J/mol·K for SFCA, S°(298) = 864.5 ± 6.1 J/mol·K for FCAM‐I, and S°(298) = 1206.2 ± 8.4 J/mol·K for FCAM‐III. These thermodynamic data are a step toward a rigorous quantitative thermodynamic modeling of the iron ore sintering process.     

Infrared and calorimetric studies of the adsorption of carbon monoxide on zeolite-supported iridium catalysts

The adsorption of carbon monoxide on the surface of small iridium particles embedded in NaY zeolites was investigated by combined infrared spectroscopy and calorimetry. At low surface coverage (< 0.5), carbon monoxide is readily adsorbed on the metallic surface to form mainly linear species characterized by a single carbon monoxide band around 2050 cm⁻¹. The heat of formation of these species (around 130 kJ mol⁻¹) is consistent with that reported for carbon monoxide adsorbed on supported iridium metal particles. Slight variations in the heat of adsorption are tentatively ascribed to differences in the average particle size. Further addition of carbon monoxide molecules induces the slow formation of monovalent iridium dicarbonyl species, the heat of formation of which is the same as that evolved for carbon monoxide linearly bonding to the metallic surface. A mechanism for the formation of monovalent dicarbonyl complexes from zeolite-supported iridium particles is discussed.     

Distinct roles of conventional non-covalent and cation-π interactions in protein stability

The different roles played by conventional non-covalent and cation-π interactions in stabilizing protein structures have been investigated using a dataset of 62 non-redundant DNA binding proteins. The stabilizing residues have been identified by a consensus approach using the concepts of hydrophobicity, long-range interactions and conservation of amino acid residues. The cation-π interactions have been delineated based on a geometric approach, such as, distance and energy criteria. I have identified 138 and 196 stabilizing residues, based on non-covalent and cation-π interactions, respectively. Interestingly, the stabilizing residues identified by consensus approach are not contributing to cation-π interactions. Further, 99% of the cation-π interactions forming residues are not identified as stabilizing ones. These results demonstrate that the roles of cation-π and non-covalent interactions are different from each other to the stability of protein structures. I have further evaluated the range of surrounding hydrophobicity, long-range order, stabilization center and conservation score for the residues that are contributing to cation-π interactions. The average surrounding hydrophobicity and long-range order of cation-π interaction forming residues are in the range of 7-15 kcal/mol and 0-0.03, respectively. I suggest that the inclusion of cation-π interaction along with conventional non-covalent interactions would provide deep insights for understanding the stability of protein structures.     

重组戊型肝炎疫苗菌种库的建立及其稳定性

目的建立重组戊型肝炎疫苗工程菌三级种子库,并分析其稳定性。方法将工程菌株HEV179/BL21(DE3)复苏活化,经全面检测合格后冻干保存即为原始种子库;原始种子库传代扩增,经全面检定合格后,冻干保存即为主种子库;主种子库经传代扩增和全面检定合格后,甘油冻存即为工作种子库。对三级种子库进行全面检定,并对工程菌进行冻干及甘油冻存稳定性、甘油冻存时间稳定性及质粒稳定性检测。结果建立的三级种子库经全面检定合格;菌种冻干稳定性、甘油冻存稳定性、甘油冻存时间稳定性及质粒稳定性指标均合格,菌种生长特性、遗传和表达稳定性均未发生变化。结论成功建立了HEV179/BL21(DE3)工程菌三级种子库,菌种稳定性良好,适用于重组戊型肝炎疫苗的工业化生产。     

Interaction of antipsychotic drug with novel surfactants: Micellization and binding studies

The interaction of cationic gemini surfactants(alkanediyl-α,ω-bis(alkyl dimethylammonium bromide)) with an antipsychotic drug(chlorpromazine hydrochloride(CPZ)) has been investigated. Various micellar and interfacial parameters have been deliberated by surface tension measurement to report the nature of interactions between drug and novel surfactant mixtures. The behavior of mixed systems, their compositions and activities of components have been analyzed in the light of Rubingh's theory. The results indicate synergism in the binary mixtures.The binding study between CPZ and surfactants has been done by spectroscopic techniques such as UV–visible and fluorescence. The results are discussed in the light of the use of gemini surfactants as promising drug delivery agents for phenothiazine drugs, and hence, improve their bioavailability.     

Thermal stability and degradation studies of polyester resins

Thermal behavior and degradation mechanisms of three polyester resins, namely bisphenol A based, isophthalic acid based, and general purpose were studied. These resins were cured with styrene in the presence of benzoyl peroxide (BPO) and methyl ethyl ketone peroxide (MEKP) as initiators. The cured resins were studied by thermogravimetric analysis and mass spectrometry. From the thermogravimetric data it was observed that the cured bisphenol-A-based polyester resin was thermally more stable than the cured isophthalic acid-based and general purpose polyester resins. It was also observed that polyester resins cured with MEKP are thermally more stable than benzoyl peroxide-cured products. Thermogravimetric analysis data shows that there are two first-order degradation reactions during thermal degradation in nitrogen atmosphere. The first degradation step involves the scission of cross-links/weak links with the liberation of free linear chains. The second step involves a random scission of the free linear chains into smaller fragments. The framentation patterns of the polyester resins were identified by mass spectrometery. Possible mechanisms for the degradation of the cured polyesters accounting for the experimental observations are discussed.     

Engineered disulfide bonds in recombinant human interferon-{gamma}: the impact of the N-terminal helix A and the AB-loop on protein stability

Insertion sites for cysteines with optimal stereochemistry forthe formation of unstrained disulfide bridges were identifiedin recombinant human interferon- (rhu-IFN-) by computer modelling.We have engineered two different disulfide cross-linked mutants,containing a pair of symmetry-related disulfide bonds, whichstabilize the N-termini of both monomers of the homodimenc protein.Mutations E7C and S69C allow the formation of an intramonomerdisuffide bond between helices A and D. In contrast, the A17Cand H111C mutations lead to a covalent cross-link between bothmonomers. The AB-loop is linked to helix F. The fluorescenceproperties of native and disulfide cross-linked proteins werestudied as a function of guanidine hydrochloride concentration.Melting temperatures (T_m) were calculated from the decreasein CD ellipticity at 220 nm. The induction of the antiviraleffect was measured using A549 fibroblast cells infected withencephalomyocarditis virus. The ability to induce the expressionof the HLA-DR antigen in Colo 205 cells was determined by fluorescence-activatedcell scanning analysis. The stability of both mutants was stronglyenhanced against temperature- and cosolvent-induced unfolding.The T_m of mutant IFN- E7C/S69C was 15°C. All measured biologicalactivities of this mutant were equal to wild type. In the caseof the other mutant IFN- A17C/H111C, the T_m value was 25°C.This mutation abolishes nearly the entire biological activity(<1%) with no detectable changes of secondary structure inthe CD spectrum. Our results illustrate the importance of theN-terminal helix A and the AB-loop for the unfolding pathwayand thermodynamic stability of rhu-IFN-.     

重组H21G蛋白衍生物的制备及其免疫原性

目的分析重组H21G蛋白的化学修饰方法及化学修饰与免疫原性之间的相关性。方法应用琥珀酸酐和己二酸二酰肼(adipic acid dihydrazide,ADH)分别修饰重组H21G蛋白制备衍生物,赖氨酸单位减少率法测定重组H21G蛋白琥珀酰化物的琥珀酰化率;2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenesulfonic acid,TNBS)法测定重组H21G蛋白衍生物的-AH衍生率;SDS-PAGE及HPLC法分析重组H21G蛋白各衍生物的纯度及分子量分布。用各衍生物分别经皮下免疫小鼠,免疫浓度为25μg/ml,共免疫3次,于末次免疫后2周,经小鼠眼眶采血,分离血清,采用间接ELISA法检测血清IgG含量。采用非还原型SDS-PAGE分析重组H21G蛋白衍生物在制备初期、制备后4℃储存2周及6个月的稳定性。结果随着重组H21G蛋白与琥珀酸酐质量比(10∶1、10∶3、10∶4和10∶5)的提高,琥珀酰化率也逐渐升高,但质量比为10∶4和10∶5时无明显差异。重组H21G蛋白ADH衍生1和2 h的衍生率无明显差异。质量比为10∶4及10∶5的琥珀酰化物与原蛋白分子量分布差异较大;ADH衍生后,原蛋白的分子量分布也发生改变。重组H21G蛋白与琥珀酸酐质量比高于10∶3时,对蛋白的降解作用明显,当质量比达到10∶5时,重组H21G蛋白几乎不再以单体形式存在;而ADH衍生1和2 h对蛋白聚合作用无明显差异,均产生了二聚体和多聚体。重组H21G蛋白及其衍生物免疫小鼠后表现明显的剂次加强效应,且ADH衍生的产物的免疫原性高于琥珀酰化产物。琥珀酰化产物的稳定性较差,目标蛋白均明显降解,而ADH衍生的产物在4℃储存6个月后未见明显聚合或降解。结论重组H21G蛋白经ADH衍生的产物免疫原性及稳定性均优于琥珀酰化产物。     

Studies on fermentation with recombinant yeast cells: plasmid stability and kinetics of biomass and protein production

Plasmid stability of the recombinant Saccharomyces cerevisiae YPB‐G strain harbouring a YEp plasmid with α‐amylase and glucoamylase genes as a fusion has been investigated in shake flasks and in a bioreactor using various compositions of media containing glucose or starch as the main carbon source. The medium composition affected both the growth characteristics and the stability of the plasmid. Superior plasmid stability was obtained in yeast minimal medium and in complex medium with glucose. Plasmid stability was substantially increased at high growth rates. Additional data were collected in the same system to investigate the kinetic characteristics of biomass and protein production, and unstructured kinetic models were used to interpret the results. At high initial glucose concentrations, where the biomass and protein production rates were similar, the kinetic models displayed good fits associated with high degrees of correlation. © 2000 Society of Chemical Industry     

重组干酪乳杆菌贮存期内质粒稳定性及活菌数的检测

目的检测产肠毒素性大肠杆菌(Enterotoxigenic E.coli,ETEC)基因重组干酪乳杆菌(Lactobacillus casei,L.casei)贮存过程中质粒的稳定性及活菌数的变化。方法将ETEC K88重组干酪乳杆菌(pLA-K88/L.casei)接种于MRS培养基,转种WPC培养基,收集菌液,置4、15、37℃贮存。通过抗生素MRS(+)和MRS(-)平板计数菌落数的方法及PCR法检测重组质粒在4℃贮存1、15、30、60、90和120 d的稳定性;Dot ELISA法检测菌液外源蛋白免疫学活性的变化;平板计数法检测4、15、37℃贮存1、2、3、7、15、30、60、90、120、210和240 d的活菌数。结果在4℃贮存120 d内,重组质粒稳定性良好,目的基因片段无缺失,外源蛋白活性稳定,乳清蛋白对工程菌的稳定性及外源蛋白的免疫原性均无影响;工程菌在4℃贮存效果最好,存活时间最长,第240 d活菌数仍保持在9×106cfu/ml以上,15和37℃贮存期仅为90和15 d。结论 ETEC基因重组干酪乳杆菌于4℃贮存条件下,质粒及外源基因表达稳定。     

Combustion of fly-ash carbon: Part II: Thermodynamic aspects and calorimetric experiment

Thermodynamic aspects of carbon combustion are discussed. At lower temperatures (<400°C), carbon dioxide is the dominant species in the reacting system. Thermodynamic calculations indicate that at low temperatures methane can be produced. Based on literature data, the reaction rate of methane generation is low and therefore the methane combustion represents probably only a marginal contribution to the overall energy balance. Calorimetric experiments proved that impregnation of pelletized carbon fly-ash samples by sodium and/or potassium carbonates have beneficial effect on conversion of CO to CO₂. The value of the effective heat of combustion, calculated from CO and CO₂ outlet concentrations, increased for sample modified by Na₂CO₃ by 5% and for sample modified with K₂CO₃ almost by 20%.     

Spectroscopic studies of copper and silver binding to metallothioneins

MAMMALIAN METALLOTHIONEIN IS REMARKABLE IN ITS METAL BINDING PROPERTIES: well-characterized species exist for metal to sulfur ratios of M(7)S(20), M(12)S(20), and M(18)S(20), where M = Cd(ll), Zn(ll), Hg(ll), Ag(I), Au(I), and Cu(I). Circular dichroism and luminescence spectra provide rich details of a complicated metal binding chemistry when metals are added directly to the metal free- or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. Qualitative and quantitative interpretations show that the final structure adopted by Ag-MT is not the same as that formed by Cu(I) ions in Cu-MT. XAFS structural data are reported for a number of metallothioneins, including Ag(12)-MT and Ag(17)-MT. Electrospray ionization mass spectrometry provides details on the species formed when Ag(I) binds to metallothionein. Mass spectral data are reported for metal-free MT 2A and Ag(n)-MT (n = 14-18).     

Effect of H2 addition during PECVD on the moisture barrier property and environmental stability of H:SiNx film

A hydrogenated silicon nitride (H:SiN_x) film with enhanced moisture barrier property and environmental stability was developed using plasma-enhanced chemical vapor deposition (PECVD) with the addition of H₂ gas at 100°C. The moisture barrier property and film density of the 100-nm-thick H:SiN_x film were ameliorated by increasing the H₂ gas flow rate during PECVD. X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy studies demonstrated that the improved performance was a result of an increase in the amount of Si–N bonds compared to hydrogen-terminated bonds with an increase in the H₂ gas flow rate. It is believed that H₂ gas assisted the formation of aminosilane, which contributed to the condensation of silicon nitride by lowering the activation energy for radicalization reactions of silane and ammonia. After the 85°C/85% RH test, the optimized H:SiN_x film maintained a water vapor transmission rate lower than 5 × 10⁻⁵ g/m²/day owing to the suppression of oxidation. The optimized H:SiN_x film was rarely oxidized owing to the decrease in hydrogen-terminated bonds and increase in the film density. The results indicated that the introduction of H₂ gas during the PECVD process strengthened the environmental stability of the H:SiN_x film.