Microbial enzymes involved in carbon dioxide fixation

This review focuses on the enzymes involved in two microbial carbon dioxide fixation pathways, the Calvin-Benson-Bassham cycle and the reductive tricarboxylic acid cycle. The function, structural features, and gene regulation of microbial ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of the Calvin-Benson-Bassham cycle, is described. Some recent findings on Rubisco from archaea and Rubisco-like proteins are also outlined. In the final section, biochemical features of the key enzymes in the reductive tricarboxylic acid cycle are reviewed.     

红葱中小分子化合物对透明质酸酶的分子对接研究

鸢尾科植物红葱是我国西南地区民间药用植物。由于红葱化学成分分离纯化及鉴定方面的原因,妨碍了红葱药理学研究的深入。本文基于文献收集到的已知化学结构的39种红葱化学成分,利用分子模拟对接软件AutoDock 4.2,以分子对接结合自由能为标准,评价红葱化学成分与透明质酸酶（HAase）间的相互作用,从中筛选出HAase抑制剂,同时采用Elson-Morgan改良法测定红葱不同极性提取物对HAase活性的影响。实验中筛选出10个与HAase结合较好的化合物,其中化合物37,33的对接结合能最低,分别为-7.73和-7.72 kcal/mol,提示其可能具有较强的HAase抑制活性。实验结果显示HAase分子中氨基酸残基TYR75、ASN37和GLU131是化合物与酶形成氢键的重要作用位点。酶活性测定实验显示红葱不同极性提取物对HAase有一定的抑制作用。本研究结果为进一步探讨红葱化学成分对HAase活性的影响提供了参考依据。     

Use of time-temperature integrators and predictive modeling to evaluate microbiological quality loss in poultry products

The purpose of this study was to characterize the kinetics of the spoilage process of chicken drumsticks in order to evaluate the application of an enzyme process-based time-temperature integrator (TTI) as a continuous quality monitor of poultry products. Shelf life studies were conducted at several temperatures (3 to 20 degrees C) to characterize (i) the poultry spoilage process as a function of total aerobic bacteria and Pseudomonas species populations and (ii) the TTI chroma response function. Two types of poultry products were examined: ice-packed and chill-packed drumsticks. An enzyme-based TTI with a color change response from green to yellow was used. Activation energies for each of the poultry products and each of the bacterial populations were as follows: 21.8 +/- 1.6 kcal/mol (ca. 91.2 +/- 6.7 kJ/mol) for ice-packed drumsticks and total aerobic population, 18.8 +/- 4.5 kcal/mol ca. 78.7 +/- 18.8 kJ/mol) for ice-packed drumsticks and Pseudomonas spp., 17.0 +/- 2.3 kcal/mol (ca. 71.1 +/- 9.6 kJ/mol) for chill-packed drumsticks and total aerobic population, and 14.1 +/- 3.6 kcal/mol (ca. 59.0 +/- 15.1 kJ/mol) for chill-packed drumsticks and Pseudomonas spp. The activation energy calculated for the TTI, 19.1 +/- 1.8 kcal/mol (ca. 79.9 +/- 7.5 kJ/mol), was determined to be adequately close to that of the poultry spoilage process to make effective quality predictions possible. Initial bacteria levels on the chicken drumsticks were uniform and not judged as important limiting factors in the application of TTIs to poultry products. Because the poultry spoilage process was reasonably characterized on the basis of Arrhenius kinetics, there is further need to conduct validation studies to determine the ability of TTIs to provide a continuous quality monitoring system.     

Potential Energy Surface for Anaerobic Oxidation of Methane via Fumarate Addition

Microbially mediated anaerobic oxidation of methane (AOM) is an important sink in the global methane cycle, but the mechanism and microorganisms responsible for this oxidation are not fully known. Using quantum chemical calculations, fumarate addition to methane was examined to determine if it could be an energetically feasible mechanism for AOM. A potential energy surface (PES) for the initial reaction was created and the results suggest the reaction is exothermic, with a calculated overall energy change between -9.8 and -11.2 kcal/mol. The addition of methane to fumarate is calculated to be the highest point on the surface, 25.0-25.3 kcal/mol above the reactants. Of the three possible molecular configurations of fumarate considered, the one that presents the least steric obstacles to the addition reaction with methane yields the greatest energy gain. While 11.2 kcal/mol may support growth under energy limited conditions it is unknown if enzymes can mediate an energetic barrier of 25 kcal/mol. These calculated energies provide values for what could be one of the least reactive substrates to undergo fumarate addition, making methane a model substrate in defining the limits of energy barriers and minimal energy requirements for growth in reactions activated by glycyl radical-containing enzymes.     

Thermal Destruction of Pteroylglutamic Acid in Buffer and Model Food Systems

First order rate constants (k) and activation energies (Ea) of pteroylglutamic acid (PteGlu) were determined between 100 and 140°C in citrate buffer (pH 3–6) and model food systems. The rate constants (hr^-1) in citrate buffer ranged from 0.145–2.36 at pH 3; 0.074–0.840 at pH 4.0; 0.021–0.207 at pH 5.0, and 0.015–0.133 at pH 6.0. Corresponding Ea values were 22.6, 19.5, 17.8, and 16.8 kcal/mole at pH 3, 4,5, and 6, respectively. In an apple juice model (pH 3.4), the rate constants ranged from 0.063–0.822 and in tomato juice model (pH 4.3), from 0.056–0.723. Ea values in apple juice an tomato juice were 20.0 and 19.7 kcal/mole, respectively.     

烤烟营养生殖转换期叶片生长标志蛋白的筛选

通过双向电泳的方法对烤烟叶片营养、生殖生长转换时期叶片的蛋白质表达谱进行比较研究,结果分析出两时期叶片中差异表达的8个标志蛋白质。其中有6个蛋白在营养生长时期表达量较高,分别是与光合相关的叶绿体放氧复合蛋白（chloroplast oxygen-evolving complex/thylakoid luminal 25.6kDa protein）;光呼吸过程中的关键酶丝氨酸羟甲基转移酶1（SHM1,Serine transhydroxymethyltransferase 1）;与蛋白质合成及植物生长发育和细胞分裂有关的核糖体60S与30S蛋白、谷胱甘肽硫转移酶（GST,glutathione S-transferase）和葡聚糖内糖基转移酶（XET,xyloglucan endotransglycosylase ）。另外2个碳同化过程中的关键蛋白1,5-二磷酸核酮糖羧化酶/加氧酶Rubisco大亚基（Ribulose-1,5-biphosphate carboxylase/ oxygenase large subunit）和小亚基（Small subunit ribulose 1,5-bisphosphate carboxylase）则在生殖生长时期表达量较高。结果表明烤烟在营养向生殖生长转换过程中叶片通过调节关键途径的蛋白表达以降低叶片的呼吸作用、蛋白质合成过程及抗氧化能力,同时增强叶片的碳同化过程。      

Partial Separation and Characterization of Papaya Endo- and Exo-Polygalacturonase

Exo- and endo-polygalacturonases (EC 3.2.1.40 and EC 3.2.1.15) in papayas were extracted, purified 20- and 90-fold, respectively, and characterized. Both enzymes functioned optimally at pH 4.6 and 45°C. Heat inactivation of the papaya PGases was biphasic and both phases followed first order kinetics. Decreasing the pH from 4.6 to 3.6 decreased the time required for their heat inactivation. The activation energies for the thermal inactivation at pH 3.6 and 4.6 were 85 and 92 kcal/mol, respectively, for endo-PGase (PG I) and 140 and 102 kcal/mol, respectively, for exo-PGase (PG II). The apparent molecular weight was determined by gel filtration to be 164,000 daltons for PG I and 34,000 daltons for PG II.     

肾素与抑制性地肤子皂苷相互作用的分子机制

地肤子中的木鳖子皂苷Ic和2’-O-β-D-吡喃葡萄糖木鳖子皂苷Ic有较强抑制肾素体外活性的功能。分子对接证实两皂苷与肾素结合较好,分别形成9 个和4 个氢键,氨基酸Ser230与Tyr231是氢键作用的关键残基,而Ala229、Met303、His301、Asp38、Arg82、Tyr83与Ile137则对疏水结合起重要作用。分子动力学模拟约1 000 ps后,两复合物平衡,均方根偏差分别为0.224 nm和0.219 nm,两皂苷降低了肾素链开始约160 个氨基酸的均方根波动。分子力学泊松-波尔兹曼表面积法获得的结合自由能分别为－44.36 kcal/mol与－62.46 kcal/mol,其中主要驱动力是静电和范德华作用,而极性溶剂化能则强烈阻碍结合。3 种方法综合揭示了两种地肤子皂苷抑制肾素的分子机制。     

Subsite affinities of beta-glucosidase from Aspergillus sojae on various xylooligosaccharides

The subsite affinities of beta-glucosidase (EC 3.2.1.21) with high beta-xylosidase activity from Aspergillus sojae on various xylooligosaccharides (degree of polymerization: n = 2-6) were investigated by steady-state kinetic analysis. The molecular activity (k0) value of the enzyme for xylobiose was not markedly different from those of other substrates (n = 3-6). The arrangement of the subsite affinities (A(i), i = 1-6) was evaluated; A1 = 2.93 kcal/mol, A2 = 3.67 kcal/mol, A3 = 0.64 kcal/mol, A4 = 0.12 kcal/mol, A5 = -0.07 kcal/mol, A6 = -0.05 kcal/mol, and the intrinsic rate constant (K(int)) was 7.6 s(-1). The subsite structure was similar to those of beta-glucosidase from A. niger and alpha-glucosidases from A. niger and Mucor javanicus, where the values for A1 were much larger than those for A3.     

Thermal Inactivation of Asparagus Lipoxygenase and Peroxidase

Thermal stability of lipoxygenase (LOX) and peroxidase (POD) in fresh asparagus tips and partially purified asparagus LOX and POD were compared. In all cases, heating at 50, 60 and 70°C resulted in higher percentages of residual LOX activity than POD activity. Inactivation of LOX followed first order kinetics while inactivation of POD followed a biphasic curve. Activation energies for thermal denaturation of the partially purified enzymes were 47.5 kcal/mol for LOX and 41.9 kcal/mol for POD.     

Kinetics of Ascorbic Acid Loss and Nonenzymatic Browning in Orange Juice Serum: Experimental Rate Constants

The kinetics of ascorbic acid loss and nonenzymatic browning in clarified orange juice (serum) were investigated in an anaerobic environment from 70.3 to 97.6°C and from 11.7 to 80.6Brix. Data were fitted to firstorder kinetic models. Rate constants of ascorbic acid degradation in serum were not different from rate constants in whole juice. Activation energies were ±30 kcal/mol and largely independent of solids concentration. Rate constants of browning pigment formation were 30–50% greater in serum. Activation energies were 19–25 kcal/mol and increased slightly with solids concentration.     

Secretagogue and bacteriostatic active fractions derived from a peptic hydro‐ lysate of alfalfa RuBisCO small purified subunit

For the first time a purification process for small RuBisCO (ribulose‐1,5‐bisphosphate carboxylase/oxygenase) subunit (SRS) was developed from an industrial by‐product of alfalfa, taking advantage of its solubility at low pH. Only one protein strip (14 kDa) was clearly detected in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) profile of the supernatant at pH 2. The recovery of SRS was 48% by this method, with a purity estimated as 98% by densitometry and reverse phase high‐performance liquid chromatography (RP‐HPLC). Moreover, most polyphenolic compounds were discarded, as confirmed by spectrophotometry and RP‐HPLC. SRS hydrolysis was performed for 20 h at 37 °C using pepsin in ammonia/formic acid buffer at pH 3. The hydrolysate was fractionated on a Sephadex G25 column equilibrated with ethanolamine/HCl buffer. Biological activities were found in two fractions. The first fraction showed slight bacteriostatic properties against two pathogenic bacteria, Salmonella arizonae and Shigella sonnei. The second fraction, tested by radioimmunoassay (RIA), presented a secretagogue activity comparable to that of gastrin. Copyright © 2006 Society of Chemical Industry     

Reduction in carbon dioxide emission, and enhancement of cell yield by control of light intensity in photomixotrophic batch culture of Marchantia polymorpha

Photomixotrophic cultures of Marchantia polymorpha were examined under light irradiation using glucose as an organic carbon source. The activity of ribulose 1,5-bisphosphate carboxylase/oxygenase in the cells was found to be maximum at an absorbed light energy of E(c) = 8.7 x 10(2)W/kg and the respiration rate of the cells remained at a low level in the range of E(c) = 1.7 x 10(2) to 1.5 x 10(3) W/kg. Batch culture of M. polymorpha was carried out in a bioreactor while keeping the E(c) value at about 3.5 x 10(2) W/kg by regulating incident light intensity between 19 and 220 W/m2. During the culture conducted under controlled light, CO2 evolution from the reactor was effectively suppressed and the obtained cell yield was 0.88 kg dry cells/kg glucose, whereas the yield was 0.62 kg dry cells/kg glucose throughout the culture conducted at the constant incident light intensity of 50 W/m2.     

Ribulose-1,5-bisphosphate carboxylase/oxygenase from an ammonia-oxidizing bacterium, Nitrosomonas sp. K1: purification and properties

The ammonia-oxidizing chemoautotrophic Nitrosomonas sp. strain K1 exhibited marked ribulose-1,5-bisphosphate carboxylase (RubisCO) activity. The RubisCO [EC 4.1.1.39] was purified as an electrophoretically homogeneous protein. The molecular mass of the enzyme was estimated to be about 460 kDa by gel filtration, and it consists of two subunits [large (L): 52.2 kDa; small (S): 13.3 kDa] as demonstrated by SDS-PAGE. This confirmed that the enzyme has an L(8)S(8) structure. The K(m) values of the enzyme for RuBP, NaHCO3, and Mg2+ were estimated to be 0.112, 0.415, and 1.063 mM, respectively. The optimum pH and temperature for its activity were approximately 7.0 and 45 degrees C. The enzyme was stable up to 45 degrees C and in a pH range from 7.0-9.0 (4 degrees C, 48 h). The enzyme activity was inhibited by Cu2+, Hg2+, N-ethylmaleimide, p-chloromercuribenzoate, and SDS (0.1 mM). The activity was also inhibited by ammonium sulfate at high concentrations (38-303 mM) but the stability of the enzyme showed no inhibition at the same ammonium sulfate concentrations. The N-terminal amino acid sequences of the large and small subunits are AIKTYQAGVKEYRQTYW QPDYVPL and AIQAYHLTKKYETFSYLPQM, respectively.     

Supramolecular structure modification of RuBisCO from alfalfa during removal of chloroplastic materials

The use of alfalfa as a dietary protein source is of great interest. Membrane filtration shows potential for removal of unwanted compounds in the purification of ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO), but fouling remains a challenge. It was hypothesized that treatments, namely centrifugation, ultracentrifugation, and heat clearing, prior to membrane processing modify the supramolecular assembly of the proteins, facilitating its filtration. The pre-treatments led to different aggregate structures. In particular, heating at 50 °C decreased the aggregate sizes by ∼500 folds relative to the raw juice, as measured by light scattering. The molar mass of RuBisCO decreased by ∼180 kDa after treatment with heat. Similarly, supernatants after ultracentrifugation also showed smaller structures. Unlike the other treatments, heating led to an efficient transmission of RuBisCO during microfiltration. These results demonstrate for the first time, the importance of controlling the supramolecular structure of RuBisCO when processing proteins from alfalfa juice.Industrial relevanceThe demand for more sustainable protein sources is continuously growing. RuBisCO is of particular interest because of its favorable amino acid composition. However, improved purification strategies are needed to extract it from green biomass. The results of this study serve as a proof-of-concept for the application of an industrially relevant process, mild heat treatment in combination with microfiltration, for the purification of RuBisCO from alfalfa green biomass.     

油菜短柄叶光合衰退及其对产量的影响

以中双9号、中油杂11号和华油杂14号油菜为材料,测定花后短柄叶光合速率及生理指标,探讨短柄叶光合衰退与叶绿素含量等因素之间的关系及其对油菜产量的影响。结果表明,油菜短柄叶光合速率随叶片衰老而逐渐降低,1,5-二磷酸羧化酶（RuBPCase）、可溶性蛋白和叶绿素含量,以及超氧化物歧化酶（SOD）和过氧化氢酶（CAT）活性均呈现出下降趋势,膜脂过氧化作用产物丙二醛（MDA）在叶片光合功能衰退过程中含量逐渐上升。油菜叶片光合衰退与RuBPCase含量、可溶性蛋白含量、叶绿素含量和SOD活性存在显著相关关系,表明光合速率的下降可能与油菜叶片衰老过程中N含量、叶绿素降解及蛋白质合成受抑制相关;SOD和CAT水平逐渐降低及MDA含量升高也表明,叶片衰老过程中氧自由基胁迫抑制光合作用。叶片光合作用与油菜单株产量的相关关系分析结果表明,油菜短柄叶光合速率,特别是短柄叶衰退的中后期光合速率是影响油菜产量的重要因素。     

Thermal Destruction of 5-Methyltetrahydrofolic Acid in Buffer and Model Food Systems

Thermal kinetic data (rate constants, k, and activation energies, Ea) for 5-Methyltetrahydrofolic acid (5-CH₃-H₄PteGlu) were determined in citrate buffers (pH 3-6) and in model food systems between 100° and 140°C. As pH increased from 3.0 to 6.0, the rate constants decreased as the temperature increased from 100° to 130°C, the rate constants increased. Ea values were 19.0, 17.0, 19.7 and 19.8 kcal/mole at pH 3, 4, 5 and 6, respectively. In the model food systems, the Ea values (kcal/mole) were 7.85 in apple juice and 10.6 in tomato juice. When dissolved oxygen content was reduced to 5.3 ppm, the stability of the 5-CH₃H₄PteGlu was increased substantially.     

豆粕血管紧张素转化酶抑制肽的结构鉴定及作用机制解析

以大豆粕为原材料,利用超声辅助酶解技术、超滤-?KTA层析相结合的方法分离纯化获取豆粕酶解产物中血管紧张素转化酶（angiotensin-converting enzyme,ACE）抑制肽,对其分子质量分布进行研究,后通过质谱分析与分子对接技术鉴定并筛选出ACE抑制活性肽的氨基酸序列,经固相合成肽序列,检测其ACE抑制肽的活性并基于分子对接技术探索其抑制机制。结果表明：经超声辅助酶解提取获得的豆粕肽分子质量主要分布在6 000 Da以下;根据分子对接的最低预测自由能筛选出的GVRP（－8.44 kcal/mol）和IIVTP（－9.04 kcal/mol）可以抑制ACE活性,半抑制浓度（50% inhibitory concentration,IC50）分别为（84±0.06）、（77±0.08）μmol/L;分子对接结果表明：GVRP、IIVTP能够与ACE的活性口袋S1、S1′、S2形成氢键相互作用,共有的过近接触（3.5 ?范围内）ACE氨基酸残基为His513、Ala354和Glu384。本研究基于串联质谱与分子对接技术,建立从混合多肽中快速鉴定、筛选活性多肽的方法,探究活性多肽与ACE稳定结合并体现其ACE活性的抑制机制,为后续的深入研究提供一定参考。     

Effect of Oxygen on Thermal Degradation of 5-Methyl-5,6,7,8-Tetrahydrofolic Acid

Previous studies of thermal degradation of folic acid have not given consistent rate expressions or activation energies. The present study employed a model system which eliminated oxygen mass transfer limitations to examine degradation as a result of heating. A pseudo first-order reaction rate constant (k_365.K) and an activation energy (E_a) for thermal degradation due to an oxidative mechanism were estimated to be 0.154 ± 0.044 min^?1 and 16.3 kcal/mol, respectively. In the presence of N₂, these parameters were estimated to be 0.094 ± 0.009 min^?1 and 23.3 kcal/mol, respectively. While elimination of oxygen will not ensure folic acid retention, the overall degradation rate will be reduced significantly.     

Water-insoluble complexes of ribulose-1,5-bisphosphate carboxylase of alfalfa with pectin

The formation of water-insoluble complexes of ribulose-1,5-bisphosphate carboxylase with pectins was studied using gel-permeation chromatography and phase analysis. The decrease in the pectin charge density accompanying an increase of its esterification degree caused both the lowering of its content in the complex precipitate and the shift of pH value, corresponding to the maximum of ribulose-1,5-bisphosphate carboxylase subunit precipitation, towards the isoelectric point of the protein. The increase in ionic strength decreases the amount of pectin in the complex precipitate. Pectins with an exterification degree of 98% presumably form the soluble protein-pectin complexes. The evidence of fractionation of protein subunits accompanying the formation of soluble and insoluble complexes with pectin has been given. The results obtained suggest a principal role of the electrostatic factor in the formation of water-insoluble complexes of ribulose-1,5-bisphosphate carboxylase with pectins. The formation of water-soluble complexes of ribulose-1,5-bisphosphate carboxylase with pectin of a high esterification degree presumably occurs due to the forces of non-electrostatic origin.