Pectin methylesterase from kiwi and kaki fruits: purification, characterization, and role of pH in the enzyme regulation and interaction with the kiwi proteinaceous inhibitor

Pectin methylesterase was purified from kiwi (Actinidia chinensis) and kaki fruit (Diospyros kaki). The pH values of the fruit homogenates were 3.5 and 6.2, respectively. The kiwi enzyme is localized in the cell wall and has a neutral-alkaline pI, whereas the kaki enzyme is localized in the soluble fraction and has a neutral-acidic pI. The molecular weights of the kiwi and kaki enzymes were 50 and 37 kDa, respectively. The two enzymes showed a similar salt and pH dependence of activity, and a different pH dependence of the inhibition by the kiwi proteinaceous inhibitor.     

Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.     

Effect of mild-heat and high-pressure processing on banana pectin methylesterase: a kinetic study

Pectin methylesterase (PME) was extracted from bananas and purified by affinity chromatography. The thermal-high-pressure inactivation (at moderate temperature, 30-76 degrees C, in combination with high pressure, 0.1-900 MPa) of PME was investigated in a model system at pH 7.0. Under these conditions, the stable fraction was not inactivated and isobaric-isothermal inactivation followed a fractional-conversion model. At lower pressure (< or =300-400 MPa) and higher temperature (> or =64 degrees C), an antagonistic effect of pressure and heat was observed. Third-degree polynomial models (derived from the thermodynamic model) were successfully used to describe the heat-pressure dependence of the inactivation rate constants.     

Partial purification and characterization of pectin methylesterase from acerola (Malpighia glabra L.)

The enzyme pectin methylesterase (PME) is present in acerola fruit and was partially purified by gel filtration on Sephadex G-100. The results of gel filtration showed different PME isoforms. The total PME (precipitated by 70% salt saturation) and one of these isoforms (fraction from Sephadex G-100 elution) that showed a molecular mass of 15.5 +/- 1.0 kDa were studied. The optimum pH values of both forms were 9.0. The total and the partially purified PME showed that PME specific activity increases with temperature. The total acerola PME retained 13.5% of its specific activity after 90 min of incubation at 98 degrees C. The partially purified acerola (PME isoform) showed 125.5% of its specific activity after 90 min of incubation at 98 degrees C. The K(m) values of the total PME and the partially purified PME isoform were 0.081 and 0.12 mg/mL, respectively. The V(max) values of the total PME and the partially purified PME were 2.92 and 6.21 micromol/min/mL/mg of protein, respectively.     

Cloning and expression of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

Pectin methylesterase (PME) is the key enzyme responsible for the gelation of jelly curd in the water extract of jelly fig (Ficus awkeotasang) achenes. The jelly fig PME extracted from achenes was isoelectrofocused at pH 2.5 and subjected to N-terminal amino acid sequencing. A cDNA fragment encoding the mature protein of this acidic PME was obtained by PCR cloning using a poly(T) primer and a degenerate primer designed according to the N-terminal sequence of the purified PME. The complete cDNA sequence of its precursor protein was further obtained by PCR using the same strategy. The PME clone was overexpressed in Escherichia coli, and its expressed protein was immunologically recognized as strongly as the original antigen using antibodies against purified PME. Fractionation analysis revealed that the overexpressed PME was predominantly present in the pellet and thus presumably formed insoluble inclusion bodies in E. coli cells.     

Characterization of the temperature activation of pectin methylesterase in green beans and tomatoes

Low-temperature blanching of vegetables activates the enzyme pectin methylesterase (PME), which demethylates cell wall pectins and improves tissue firmness. This temperature activation of PME has been investigated by measuring the formation of methanol in intact tissue of green beans and tomatoes. Rates of methanol formation at temperatures of 35-65 degrees C were obtained by measuring the release of methanol from thin slices of tomato pericarp or green bean pod material. Activation energies of 112 and 97 kJ mol(-1) were calculated for PME activity in green beans and tomatoes, respectively. These activation energies indicate that the rate of pectin demethylation at 65 degrees C will be nearly 100 times that at 25 degrees C. PME activity was also determined titrimetrically using a solubilized form of the enzyme and purified pectin at temperatures from 30 to 60 degrees C. Under these conditions, much lower activation energies of 37 and 35 kJ mol(-1) were obtained for green beans and tomatoes, respectively. Methanol accumulation during heating of whole intact green beans was also determined and yielded an activation energy similar to that obtained with sliced beans. Whole green beans held at room temperature did not accumulate any methanol, but sliced or homogenized beans did. If whole beans were first heated to 45 degrees C and then cooled, methanol accumulation was observed at room temperature. These results indicate that two factors contribute to the observed high rate of pectin de-esterification during low-temperature blanching: (1) An irreversible change, causing PME to become active, occurs by heating to > or = 45 degrees C. (2) The high activation energy for pectin de-esterification means that the rate of de-esterification increases substantially with increasing temperature.     

Effects of thermal treatments and storage on pectin methylesterase and peroxidase activity in freshly squeezed orange juice

A specific indicator of freshness, allowing routine distinction between freshly squeezed orange juices (FSOJs) and FSOJ-like products, was to be identified. Using the Actijoule unit of a tubular heater at a flow rate of 60 L/h, FSOJs from Citrus sinensis (L.) Osbeck cv. Valencia Late were continuously heated on a pilot plant scale at six different temperatures (42-92 degrees C), followed by continuous cooling to ambient temperature and subsequent filling into sterilized glass jars. The cloud stability and residual activities of pectin methylesterase (PE) and peroxidase (POD) were monitored over the storage at 4 degrees C for up to 62 days, thus considering the storage conditions of FSOJs in retail markets. As shown by the viable microbial counts throughout storage, microbial activity was insignificant due to good sanitary practice, thus proving that the enzyme activities detected were of plant origin. The juices processed at temperatures > or =62 degrees C were characterized by minor residual activities. When exposed to temperatures <62 degrees C in the genuine acidic matrix of the juices, the heat stability of PE exceeded that of POD. Compared with the aforementioned samples, the juice processed at 52 degrees C with a residual PE activity of 33.8% was hardly inferior in terms of cloud stability within the first 14 days. After the juice was processed at 42 degrees C, rapid clarification occurred within the first 8 days, consistent with undetectable PE deactivation. Hence, only the range of approximately 50-60 degrees C is relevant in minimal heat-processing for the retention of cloud stability within the short turnover period of FSOJ-like products, with partial PE and POD deactivation being already sufficient to distinguish those juices from FSOJs. Irrespective of the previous thermal treatment, the total PE activity remained nearly constant during storage, whereas the POD activity rapidly declined to minor levels after 20 days. Consequently, as to the future analysis of samples with unknown processing history, PE was suggested as an indicator enzyme for the freshness of FSOJs, allowing their unambiguous distinction from minimally heat-processed juices.     

猕猴桃黄化病营养诊断与土壤养分相关性的研究

西北地区以石灰性土壤为主,土壤微量元素有效性较低,种植猕猴桃易出现黄化现象.采集正常和黄化猕猴桃叶片及其树冠下土壤样品进行营养诊断分析.结果表明,除Ca外,黄化叶片中N、P、K、Cu、Fe含量均较低,与正常叶片含量相比差异达极显著,其中Cu和Fe分别为正常叶片的66.85％与65.61％.铁是叶绿素合成的必需元素,表明黄化原因与缺铁有关.正常和黄化树体下土层各养分测定值差异不显著;说明植株叶片养分缺乏是植株根系吸收养分受阻,与土壤养分丰缺无关.需要通过改善土壤的其他条件(如pH,水分,通气状况等)或叶面喷肥来促进树体吸收.     

Isolation, characterization, and pectin-modifying properties of a thermally tolerant pectin methylesterase from Citrus sinensis var. Valencia

The thermally tolerant pectin methylesterase (TT-PME) was isolated as a monocomponent enzyme from sweet orange fruit (Citrus sinensis var. Valencia). It was also isolated from flower and vegetative tissue. The apparent molecular weight of fruit TT-PME was 40800 by SDS-PAGE and the isoelectric point estimated as pI 9.31 by IEF-PAGE. MALDI-TOF MS identified no tryptic-peptide ions from TT-PME characteristic of previously described citrus PMEs. TT-PME did not absolutely require supplemented salt for activity, but salt activation and pH-dependent activity patterns were intermediate to those of thermolabile PMEs. Treatment of non-calcium-sensitive pectin with TT-PME (reducing the degree of methylesterification by 6%) increased the calcium-sensitive pectin ratio from 0.01 to 0.90, indicating a blockwise mode of action. TT-PME produced a significantly lower end-point degree of methylesterification at pH 7.5 than at pH 4.5. Extensive de-esterification with TT-PME did not reduce the pectin molecular weight or z-average radius of gyration, as determined by HPSEC.     

Partial purification,characterization, and thermal and high-pressure inactivation of pectin methylesterase from carrots (Daucus carrota L.)

Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model.     

Effect of different water supply on plant growth and fruit quality of Lycopersicon esculentum

It is well known from earlier work that water stress and salinity results in depressed plant growth and high fruit quality of tomato (e.g. increased sugar and acid levels), but generally is associated with a low marketable fruit yield. In the present work we investigated whether even a small reduction in water supply (without visible symptoms of water stress) also results in a high fruit quality together with high marketable fruit proportions. To characterize fruit quality sugars (glucose and fructose), titratable acids, odour-active aroma volatiles and vitamin C were investigated. Tomato plants (Lycopersicon esculentum Mill. cv Vanessa) were grown in soil and with the onset of fruit development water supply was varied (70% and 50% water capacity). In the treatment with lower water supply plant growth, and in particular the number of fruit settings were depressed and the sugar and vitamin C concentrations in the fruits were significantly increased, especially during fruit ripening. Furthermore, with lower water supply the concentrations of titratable acids and of C₆ aldehydes (hexanal, (Z)-3-hexenal and (E)-2-hexenal) were significantly increased in the red fruits. Fruit growth was identical in both treatments. The higher levels of sugars, titratable acids, aroma volatiles and vitamin C are responsible for the higher fruit quality under conditions of lower water supply. Since not all fruits of the well watered plants became mature, the marketable yield in both treatments was rather similar and hence, together with a higher fruit quality in the treatment with lower water supply, high proportions of marketable fruits can be harvested.     

微波干燥预处理对苹果渣提取果胶的影响

利用微波干燥技术对湿苹果渣进行预处理,研究了微波干燥预处理对苹果渣提取果胶的影响.以探索一种新的干燥方法和提高果胶品质.结果表明:微波能量越大,苹果渣组织结构破碎越严重,其复水能力越弱;但对苹果渣颜色无显著影响;微波干燥预处理能显著提高果胶得率,微波预处理条件为300 W、15 min、7.5×10-2kW·h或500 W、10 min、8.3×10-2kW·h时微波预处理苹果皮渣的果胶提取得率比对照提高了50%左右,是较为适宜的微波处理条件,但微波能量大于8.3×10-2kW·h时,对果胶得率有不利影响;微波干燥预处理对果胶半乳糖醛酸含量无显著性影响,但显著提高了果胶的酯化度、固有黏度和黏均分子质量.     

柑橘果实发育中果胶酸钙、草酸钙和果胶动态的研究

以单性结实的龟井蜜柑和自花结实的鄂柑1号橘为试材,对整个果实发育期的子房(幼果)、果皮和果肉的果胶酸钙、草酸钙和果胶含量变化进行了测定。结果表明,1)两品种子房(幼果)果胶酸钙含量呈类似的下降趋势;草酸钙则相反,龟井花后趋下降,而鄂柑1号却明显上升;而且鄂柑1号子房(幼果)果胶酸钙、草酸钙和果胶含量均相对较高。2)在果实增大期内,两品种果皮和果肉的果胶酸钙含量均出现显著上升,对应果皮草酸钙含量虽有波动但居相对较高水平,而果肉草酸钙则趋明显下降。3)两品种果皮和果肉水溶性果胶含量均在增大期内呈显著上升,对应原果胶含量均相对较高,进入增大后期均明显下降。     

Application of wood ash compared with fertigation for improving the nutritional status and fruit production of kiwi vines

Application of wood ash can potentially improve the fertility of acid soils and the nutritional status of crops. However, there is limited information about the effectiveness of this practice with fruit trees. The application of wood ash as a fertilizer in a kiwifruit plantation—both with and without fertigation/irrigation—was compared with that of a conventional fertigation program in a completely randomized field experiment on an acid soil in northwest (NW) Spain. The effects on plant nutritional status and on fruit yield, as well as environmental effects, were evaluated over a period of 2 y. The application of wood ash led to modest increases in soil pH and extractable nutrients (phosphorus, P; calcium, Ca; magnesium, Mg; potassium, K; boron, B). However, no consistent effects in foliar nutrient concentrations were found. Ash application led to an increase of up to 45% in the number of fruits produced, which was mainly attributed to the inputs of Ca and Mg. Although moderate increases in soil available manganese (Mn) and nickel (Ni) after ash application were recorded, there were no changes in heavy‐metal concentrations in leaves or fruits. From the results of the study it can be concluded that wood ash can be used to improve the growth conditions of kiwi vines on acidic soils. Wood ash should be applied at rates adapted to the liming needs of the soil, while also taking into account the chemical composition of the ash.     

Enzymatic modification of a model homogalacturonan with the thermally tolerant pectin methylesterase from Citrus: 1. Nanostructural characterization, enzyme mode of action, and effect of pH

Methyl ester distribution in pectin homogalacturonan has a major influence on functionality. Enzymatic engineering of the pectin nanostructure for tailoring functionality can expand the role of pectin as a food-formulating agent and the use of in situ modification in prepared foods. We report on the mode of action of a unique citrus thermally tolerant pectin methylesterase (TT-PME) and the nanostructural modifications that it produces. The enzyme was used to produce a controlled demethylesterification series from a model homogalacturonan. Oligogalacturonides released from the resulting demethylesterified blocks introduced by TT-PME using a limited endopolygalacturonase digestion were separated and quantified by high-pressure anion-exchange chromatography (HPAEC) coupled to an evaporative light-scattering detector (ELSD). The results were consistent with the predictions of a numerical simulation, which assumed a multiple-attack mechanism and a degree of processivity ～10, at both pH 4.5 and 7.5. The average demethylesterified block size (0.6-2.8 nm) and number of average-sized blocks per molecule (0.8-1.9) differed, depending upon pH of the enzyme treatment. The mode of action of this enzyme and consequent nanostructural modifications of pectin differ from a previously characterized citrus salt-independent pectin methylesterase (SI-PME).     

Antcin B and its ester derivative from Antrodia camphorata induce apoptosis in hepatocellular carcinoma cells involves enhancing oxidative stress coincident with activation of intrinsic and extrinsic apoptotic pathway

The triterpenoids methylantcinate B (MAB) and antcin B (AB), isolated from the medicinal mushroom Antrodia camphorata , have been identified as strong cytotoxic agents against various type of cancer cells; however, the mechanisms of MAB- and AB-induced cytotoxicity have not been adequately explored. This study investigated the roles of caspase cascades, reactive oxygen species (ROS), DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in MAB- and AB-induced apoptosis of hepatocellular carcinoma (HCC) HepG2 cells. Here, we showed that MAB and AB induced apoptosis in HepG2 cells, as characterized by increased DNA fragmentation, cleavage of PARP, sub-G1 population, chromatin condensation, loss of mitochondrial membrane potential, and release of cytochrome c. Increasing the levels of caspase-2, -3, -8, and -9 activities was involved in MAB- and AB-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that MAB and AB triggered the caspase-dependent apoptotic pathway. Additionally, the enhanced apoptotic effect correlates with high expression of Fas, Fas ligand, as well as Bax and decreased protein levels of Bcl-(XL) and Bcl-2, suggesting that both the extrinsic and intrinsic apoptosis pathways were involved in the apoptotic processes. Incubation of HepG2 cells with antioxidant enzymes superoxide dismutase and catalase and antioxidants N-acetylcysteine and ascorbic acid attenuated the ROS generation and apoptosis induced by MAB and AB, which indicate that ROS plays a pivotal role in cell death. NADPH oxidase activation was observed in MAB- and AB-stimulated HepG2 cells; however, inhibition of such activation by diphenylamine significantly blocked MAB- and AB-induced ROS production and increased cell viability. Taken together, our results provide the first evidence that triterpenoids MAB and AB induced a NADPH oxidase-provoked oxidative stress and extrinsic and intrinsic apoptosis as a critical mechanism of cause cell death in HCC cells.     

植物化感物质对种子萌发的影响及其生态学意义

化感现象作为植物之间的一种相互作用方式,在农林业生产中广泛存在。合理利用植物之间的化感作用,对于生产实践具有重要的指导意义。研究表明,化感物质可促进或抑制不同物种种子的萌发过程,这对于植物的生长发育、植物群落的组成与分布以及生态系统的平衡有着重要影响。本文从化感物质对种子萌发的影响与其生态学意义两个方面进行了综述。一方面,在阐述化感作用影响种子萌发的基础上,进一步总结了化感物质抑制植物种子萌发的生理生化机制。包括:化感物质通过抑制胚根和胚轴的伸长,破坏亚细胞结构,干扰植物激素及活性氧的合成与代谢,造成细胞损伤,从而阻碍种子萌发;抑制种子中储存物质的代谢,阻碍种子萌发过程中的物质以及能量转换,导致种子萌发受阻等。另一方面,本文从化感作用在抑制农田杂草及影响生态系统稳定性两个方面,阐述了化感物质调控种子萌发的生态学意义。讨论了农作物的化感抑草作用,农林业生产中的化感自毒作用以及化感作用造成的生物入侵等,以期为农林业生产提供借鉴。最后,根据目前研究进展,对本领域未来研究方向进行了展望和讨论。     

Effect of pepper lipoxygenase activity and its linked reactions on pigments of the pepper fruit.

The products formed during the enzymatic reaction catalyzed by the lipoxygenase of pepper (variety Agridulce) have in vitro a strong destructive action on the carotenoid pigments of the fruit. When conditions and proportions of enzyme and pigments are similar to those found in the fruit, and at a reaction temperature of 20 degrees C, almost 30% of the pigments are destroyed after 24 h of reaction. Of this amount, 2.5% is due to autoxidation of pigments, 4. 5% to oxidation induced by the presence of linoleic under saturating conditions, and the remaining 22% to the presence in the medium of reaction products of the lipoxygenase-catalyzed reaction. When the enzyme acts under substrate-saturating conditions, the rate of pigment destruction by lipoxygenase can be considered maximal at the experimental temperature. The fact that in vitro pepper lipoxygenase induces a heavy destruction of pigments and that, in vivo, its activity remains almost constant during over-ripening could explain why up to 40% of the pigment content in some varieties is lost during the postharvest period.     

微波辅助提取中pH值与脱色对苹果果胶的影响

为了得到高得率、高品质的苹果果胶,该文利用不同pH值的盐酸溶液对苹果渣中果胶进行微波辅助提取（Microwave-assisted extraction, MAE）,之后对果胶提取液进行大孔树脂XAD-16HP脱色,研究了pH值与脱色对果胶得率和品质的影响。结果表明：微波辅助提取工艺中随着pH值的升高,果胶得率、半乳糖醛酸质量分数和总离子含量减少,酯化度、黏均分子量和总多酚增加,褐变度无显著变化;大孔树脂吸附脱色后果胶褐变度、总多酚、彩度C*值显著下降,色调角H°值显著增加,半乳糖醛酸质量分数和酯化度没有     

生态因子对冬枣果实品质的影响

以滨州市博兴、泰安市新泰冬枣示范基地的冬枣园为试验对象, 研究了两地冬枣园生育期主要气象因素、土壤肥力因子以及冬枣果实不同成熟度品质指标之间的关系.结果表明: 土壤因素对枣果可溶性糖、可溶性蛋白质的影响大于气象因子, 增施钾肥、有机肥有助于改善两者含量; 雨量大, 偏施磷、氮肥均可提高枣果可滴定酸、游离氨基酸含量, 而偏施钾肥可降低两者含量; 枣果Vc含量的高低受土壤、气候两因素的共同作用, 与土壤中有效铜含量呈显著正相关.果实含水量的高低受土壤质地、肥力的影响较大; 降雨量大, 土壤富含Fe、P、Zn等元素可增大果形指数, 使果实近长圆形, 反之则可使果实近卵圆形.此外, 土壤增施Ca、Mg等微肥有利于提高果实含糖量、Vc和可溶性蛋白质含量.