Microbial pectic transeliminases

Pectic transeliminases, also known as pectic lyases or pectinases, are involved in the degradation of pectic substances. They have a wide range of applications in food and textile processing. Although Aspergillus and Penicillium spp. produce pectin lyases, bacteria are the major producers of polygalacturonate lyase. The yields of pectic transeliminases are less than other pectinases. Since new applications for pectic transeliminases are emerging, an improved process for the production of these enzymes is necessary.     

WAKs: cell wall-associated kinases linking the cytoplasm to the extracellular matrix

There are only a few proteins identified at the cell surface that could directly regulate plant cell wall functions. The cell wall-associated kinases (WAKs) of angiosperms physically link the plasma membrane to the carbohydrate matrix and are unique in that they have the potential to directly signal cellular events through their cytoplasmic kinase domain. In Arabidopsis there are five WAKs and each has a cytoplasmic serine/threonine protein kinase domain, spans the plasma membrane, and extends a domain into the cell wall. The WAK extracellular domain is variable among the five isoforms, and collectively the family is expressed in most vegetative tissues. WAK1 and WAK2 are the most ubiquitously and abundantly expressed of the five tandemly arrayed genes, and their messages are present in vegetative meristems, junctions of organ types, and areas of cell expansion. They are also induced by pathogen infection and wounding. Recent experiments demonstrate that antisense WAK expression leads to a reduction in WAK protein levels and the loss of cell expansion. A large amount of WAK is covalently linked to pectin, and most WAK that is bound to pectin is also phosphorylated. In addition, one WAK isoform binds to a secreted glycine-rich protein (GRP). The data support a model where WAK is bound to GRP as a phosphorylated kinase, and also binds to pectin. How WAKs are involved in signaling from the pectin extracellular matrix in coordination with GRPs will be key to our understanding of the cell wall's role in cell growth.     

Effectiveness of pectin extracted from the eelgrass Zostera marina for alleviating lead-induced liver injury

The effects of pectin from the eelgrass Zostera marina on toxic liver injury induced by enteral administration of lead acetate are examined in experiments on rats. The results show that pectin helps to rapidly reduce lead concentration in liver, to decrease lipid peroxidation, and to normalize the levels of total cholesterol and triglycerides in blood serum and liver.     

Impact of pectin esterification on the antimicrobial activity of nisin‐loaded pectin particles

The relationship between pectin structure and the antimicrobial activity of nisin‐loaded pectin particles was examined. The antimicrobial activity of five different nisin‐loaded pectin particles, i.e., nisin‐loaded high methoxyl pectin, low methoxyl pectin, pectic acid, dodecyl pectin with 5.4 and 25% degree of substitution were tested in the pH range of 4.0–7.0 by agar‐diffusion assay and agar plate count methods. It was found that the degree of esterification of carboxyl group of galacturonic acid in pectin molecule is important for the antimicrobial activity of nisin‐loaded pectin particles. Nisin‐loaded particles prepared using pectic acid or the pectin with low degree of esterification exhibit higher antimicrobial activity than nisin‐loaded high methoxyl pectin particles. Pectins with free carboxyl groups or of low degree of esterification are the most suitable for particles preparation. Moreover, nisin‐loaded pectin particles were active at close to neutral or neutral pH values. Therefore, they could be effectively applied for food preservation. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:245–251, 2017     

The mercury binding activity of pectin isolated from the seagrass Zostera marina

The kinetics of mercury binding by pectin isolated from the seagrass Zostera marina was described, and its maximum mercury binding activity at pH from 2.0 up to 6.0 was determined. It was shown that mercury binding by pectin in in vitro conditions did not depend on concentration of hydrogen ions in the environment. The maximum mercury binding activity estimated from the Langmuir equation was 2.64 mmol/g of dry mass of the pectin.     

Investigation of enzymatic degradation of pectin polysaccharides under limiting conditions

The dynamics of changes in spectra of oligosaccharide fragments formed during enzymatic degradation of plant pectins at low enzyme/substrate ratio was studied. It is shown that degradation of deesterified pectin molecules is a discrete and determined process manifested in establishment of a stable polysaccharide spectrum. It is noted that introduction of chemical modifications into the polysaccharide substrate structure preserves the discreteness of the polymer molecule fragmentation but changes the spectrum of formed oligosaccharide fragments. It is supposed that degradation is defined by the spatial (three-dimensional) organization of the polysaccharide molecule.     

大豆蛋白、苹果果胶及维生素C 排铅效果的探究

通过对铅中毒小鼠灌胃三种不同剂量的食物材料,探究大豆蛋白、苹果果胶及维生素C的排铅效果。实验通过每日对铅中毒小鼠喂食高、中、低三种不同剂量的苹果果胶、大豆蛋白和维生素C,连续28 d。然后收集小鼠的血液、肝脏和股骨,用原子吸收光谱仪进行铅检测。结果表明重金属铅的摄入会影响小鼠的健康和生长状况;大豆蛋白、苹果果胶和维生素C均具有一定的排铅效果,在最佳的条件下,血铅含量降低了65%~70%,肝脏铅含量下降了63%~85%,股骨铅含量下降了18%~26%。本实验证明大豆蛋白、苹果果胶及维生素C三种食品材料具有较好的降血铅效果,维生素C被认为是最适合用于开发排铅功能食品的原料。本研究为排铅功能食品的研发提供了重要的理论依据。     

The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus

The pectin matrix of the cell wall, a complex and dynamic network, impacts on cell growth, cell shape and signaling processes. A hallmark of pectin structure is the methylesterification status of its major component, homogalacturonan (HGA), which affects the biophysical properties and enzymatic turnover of pectin. The pectin methylesterases (PMEs), responsible for de-esterification, encompass a protein family of more than 60 isoforms in the Arabidopsis genome. The pivotal role of PME in the regulation of pectin properties also requires tight control at the post-translational level. Type-I PMEs are characterized by an N-terminal pro region, which exhibits homology with pectin methylesterase inhibitors (PMEIs). Here, we demonstrate that the proteolytic removal of the N-terminal pro region depends on conserved basic tetrad motifs, occurs in the early secretory pathway, and is required for the subsequent export of the PME core domain to the cell wall. In addition, we demonstrate the involvement of AtS1P, a subtilisin-like protease, in Arabidopsis PME processing. Our results indicate that the pro region operates as an effective retention mechanism, keeping unprocessed PME in the Golgi apparatus. Consequently, pro-protein processing could constitute a post-translational mechanism regulating PME activity.     

Cell wall changes in white spruce (Picea glauca) needles subjected to repeated drought stress

White spruce [ Picea glauca (Moench) Voss] seedlings were preconditioned by subjecting them to 3 cycles of a mild drought stress. After 1 week of stress relief their water status, soluble carbohydrate content and cell wall composition in newly formed needles were examined and compared with those in control seedlings. Both preconditioned and control seedlings were subsequently subjected to a severe drought stress and again analyzed. Preconditioning treatment both before and during subsequent stress exposure lowered osmotic potentials at full hydration, and after the loss of turgor, decreased lignin content and increased hemicellulose content of the cell walls. Severe drought had similar but more drastic effects on seedling water relations, sugar accumulation and cell wall hemicellulose content; it also decreased cell wall pectin levels. The decrease in pectin levels was accompanied by a loss of galactose and glucose from pectic substances. Little change in cellulose content was observed as a result of preconditioning and severe drought.     

Substitution of crude cell wall for neutral detergent fibre in the equations of the Cornell Net Carbohydrate and Protein System that predict carbohydrate fractions: application to sunflower ( Helianthus annuus L.)

Prediction of carbohydrate fractions using equations from the Cornell Net Carbohydrate and Protein System (CNCPS) is a valuable tool to assess the nutritional value of forages. In this paper, these carbohydrate fractions were predicted using data from three sunflower (Helianthus annuus L.) cultivars, fresh or as silage. The CNCPS equations for fractions B2 and C include measurement of ash and protein-free neutral detergent fibre (NDF) as one of their components. However, NDF lacks pectin and other non-starch polysaccharides that are found in the cell wall (CW) matrix, so this work compared the use of a crude CW preparation instead of NDF in the CNCPS equations. There were no differences in the estimates of fractions B1 and C when CW replaced NDF; however, there were differences in fractions A and B2. Some of the CNCPS equations could be simplified when using CW instead of NDF. Notably, lignin could be expressed as a proportion of DM, rather than on the basis of ash and protein-free NDF, when predicting CNCPS fraction C. The CNCPS fraction B1 (starch + pectin) values were lower than pectin determined through wet chemistry. This finding, along with the results obtained by the substitution of CW for NDF in the CNCPS equations, suggests that pectin was not part of fraction B1 but present in fraction A. We suggest that pectin and other non-starch polysaccharides that are dissolved by the neutral detergent solution be allocated to a specific fraction (B2) and that another fraction (B3) be adopted for the digestible cell wall carbohydrates.     

植物果胶的生物合成与功能

果胶作为植物细胞壁多糖之一,其结构和功能非常复杂。果胶主要由同型半乳糖醛酸聚糖(HG)、鼠李半乳糖醛酸聚糖I (RGI)和鼠李半乳糖醛酸聚糖II (RGII)组成。果胶类成分在维持细胞壁结构的完整性以及细胞间黏附和信号转导等方面发挥重要作用。研究果胶类成分的结构、分布和功能,对理解细胞壁高级结构的构建和功能具有重要意义...     

CGR2 and CGR3 have critical overlapping roles in pectin methylesterification and plant growth in Arabidopsis thaliana

Pectins are critical polysaccharides of the cell wall that are involved in key aspects of a plant's life, including cell‐wall stiffness, cell‐to‐cell adhesion, and mechanical strength. Pectins undergo methylesterification, which affects their cellular roles. Pectin methyltransferases are believed to methylesterify pectins in the Golgi, but little is known about their identity. To date, there is only circumstantial evidence to support a role for QUASIMODO2 (QUA2)‐like proteins and an unrelated plant‐specific protein, cotton Golgi‐related 3 (CGR3), in pectin methylesterification. To add to the knowledge of pectin biosynthesis, here we characterized a close homolog of CGR3, named CGR2, and evaluated the effect of loss‐of‐function mutants and over‐expression lines of CGR2 and CGR3 in planta. Our results show that, similar to CGR3, CGR2 is a Golgi protein whose enzyme active site is located in the Golgi lumen where pectin methylesterification occurs. Through phenotypical analyses, we also established that simultaneous loss of CGR2 and CGR3 causes severe defects in plant growth and development, supporting critical but overlapping functional roles of these proteins. Qualitative and quantitative cell‐wall analytical assays of the double knockout mutant demonstrated reduced levels of pectin methylesterification, coupled with decreased microsomal pectin methyltransferase activity. Conversely, CGR2 and CGR3 over‐expression lines have markedly opposite phenotypes to the double knockout mutant, with increased cell‐wall methylesterification levels and microsomal pectin methyltransferase activity. Based on these findings, we propose that CGR2 and CGR3 are critical proteins in plant growth and development that act redundantly in pectin methylesterification in the Golgi apparatus.     

Cobtorin target analysis reveals that pectin functions in the deposition of cellulose microfibrils in parallel with cortical microtubules

Cellulose and pectin are major components of primary cell walls in plants, and it is believed that their mechanical properties are important for cell morphogenesis. It has been hypothesized that cortical microtubules guide the movement of cellulose microfibril synthase in a direction parallel with the microtubules, but the mechanism by which this alignment occurs remains unclear. We have previously identified cobtorin as an inhibitor that perturbs the parallel relationship between cortical microtubules and nascent cellulose microfibrils. In this study, we searched for the protein target of cobtorin, and we found that overexpression of pectin methylesterase and polygalacturonase suppressed the cobtorin-induced cell-swelling phenotype. Furthermore, treatment with polygalacturonase restored the deposition of cellulose microfibrils in the direction parallel with cortical microtubules, and cobtorin perturbed the distribution of methylated pectin. These results suggest that control over the properties of pectin is important for the deposition of cellulose microfibrils and/or the maintenance of their orientation parallel with the cortical microtubules.     

Atomic force microscopy of tomato and sugar beet pectin molecules

Atomic force microscopy has been used to image the structure of pectin molecules isolated from unripe tomato and sugar beet tissue. The tomato pectin molecules were found to be extended stiff chains with a weight average contour length of L_W = 174 nm and a number average contour length of L_N = 132 nm (L_W/L_N = 1.32). A proportion of the pectin molecules (30%) were found to be branched structures. Chemical analysis of the sugar beet pectin extracts showed that the samples contained protein (8.6%). This protein proved difficult to remove and is believed to be covalently attached to the polysaccharide. Imaging of the extracted pectin revealed largely un-aggregated chains: a small fraction (33%) of which were extended stiff polysaccharide chains and a major fraction (67%) of which were of polysaccharide–protein complexes containing a single protein molecule attached to one end of the polysaccharide chains (‘tadpoles’). In addition the sample contained a small number of aggregated structures. The un-aggregated pectin molecules were found to be predominately linear structures with a small fraction (17%) of branched structures. The branched structures were all in the free polysaccharide fraction and no branched pectin chains were observed in the protein–polysaccharide complexes. Alkali treatment was found to remove the protein. For the alkali-treated, un-aggregated structures the average contour lengths were found to be L_W = 137 nm, L_N = 108 nm with L_W/L_N = 1.27. It is proposed that the ‘tadpole’ structures contribute to the unusual emulsifying properties of sugar beet pectin.     

Polygalacturonase, Pectate Lyase and Pectin Methylesterase Activity in Pathogenic Strains of Phytophthora capsici Incubated under Different Conditions

Pectolytic enzymes are found mainly in fungi and bacteria. The most widely occurring enzymes are polygalacturonase (PGs), pectin methylesterase (PMEs) and pectate lyase (PLs) produced during the infection process and during culturing. The secretion of these enzymes results in the disorganization of the plant cell walls, which is responsible for the pathogenicity of the pathogens. These enzymes degrade the pectin of plants causing maceration of plant tissues and the enzyme activity increases under favourable environmental conditions. We have found that Phytophthora capsici , a pathogenic oomycete, produces levels of these three enzymes equal to those produced by soft-rotting Erwinia chrysanthemi . The activity of PGs, PLs and PMEs was investigated at the optimum temperature, pH and ionic strength in highly pathogenic P. capsici strains cultivated in two kinds of liquid medium containing either crude pepper extracts plus pectin or pectin as the carbon source. Virulence tests and enzymes activity showed that there was a high correlation between the enzyme activity and the pathogenicity of P. capsici . The effects of different carbon sources on the enzyme activity showed that pepper extract plus pectin was the best source for the carbon source.     

Rheological behaviour and microstructure of microfibrillated cellulose suspensions/low-methoxyl pectin mixed systems. Effect of calcium ions

In this study, the influence of the presence of low-methoxyl pectin (LM pectin) on the rheological and microstructural properties of microfibrillated cellulose suspensions was elucidated in order to create new structures with new and interesting textures. For that purpose, the rheological properties of the cellulose/LM pectin mixtures in variable proportions were compared with those of the individual biopolymers. The influence of the presence of calcium and/or sodium ions on the properties of the mixed systems was studied. The microstructure of the resulting system was studied by transmission electron microscopy and confocal laser scanning microscopy. It was found that, in the presence of LM pectin, a synergistic effect was observed when calcium ions were also present, leading to increased rheological properties of the composites. Indeed, addition of calcium to the mixtures induced LM pectin gelation, which was favoured in the presence of sodium, the pectin network contributing to the formation of a stronger cellulose/LM pectin composite gel. The presence of LM pectin alone in the microfibrillated cellulose suspensions does not significantly modify the viscoelastic and microstructural properties of microfibrillated cellulose suspensions. Whether calcium was added to the mixtures or not in water, the viscoelastic properties of the mixtures are mainly controlled by cellulose. The same behaviour was observed for the mixtures in NaCl without added calcium. Contrary to this observation, it was noticed that in presence of both sodium and calcium ions, the viscoelastic properties of the mixtures are largely governed by LM pectin. On the other hand, it was showed that the flow behaviour of microfibrillated cellulose suspensions is modified in the presence of LM pectin with an increase in thixotropic character shear-thinning behaviour, which was more pronounced in the presence of NaCl. It was also shown, from TEM observations, that an interpenetrating network formed in cellulose/LM pectin composites gel in the presence of calcium ions. In the same way, the CLSM observations allowed the separate localization of cellulose and LM pectin within the composite systems to be highlighted. The results obtained suggests that it is possible to thus create new structures with new interesting textures, by mixing microfibrillated cellulose suspensions and LM pectin in suitable proportions in the simultaneous presence of both sodium and calcium ions.     

Pectin microspheres for oral colon delivery: Preparation using spray drying method and<Emphasis Type="Italic">in vitro</Emphasis> release of indomethacin

Drug delivery systems that are based on pectin have been studied for colon specific delivery using the specific activity of colon microflora. The aim of this study was to design a novel method of manufacturing pectin microspheres without oils and surfactants and to investigate the potential use of the pectin microsphere as an oral colon-specific drug carrier. The pectin microspheres were successfully formed using the spray drying method and crosslinking with calcium chloride. From the crosslinked pectin microspheres, indomethancin (IND) release was more supressed than its release from non-crosslinked microspheres. In a low pH (pH 1.4) environment, the pectin microspheres released IND at an amount of about 18±2% of the total loaded weight for 24h while the release rate of IND was stimulated at neutral pH (pH 7.4). IND release from the pectin microspheres was increased by the addition of pectinase. The results clearly demonstrate that the pectin microspheres that were prepared by the spray drying and crosslinking methods are potential carriers for colon-specific drug deliveries.     

豆腐柴果胶的纯化及其理化性质研究

豆腐柴为一种民间用作制作传统观音豆腐的小型灌木。通过对所采集的鲜豆腐柴叶经pH值3．5的稀酸性溶液保温,70℃杀菌15min,使叶中果胶酶等酶失活后,于60℃烘干至恒重;干叶用料液比为1：20的盐酸进行酸解,滤液用2．5倍体积的食用酒精进行沉淀、纯化果胶。结果显示每千克干叶可得到纯化果胶0．27kg,果胶得率为27％,酯化度为53％,符合国家对果胶规定的标准,表明该提取方法可行。     

Enzymatic degradation of cell walls of apples and characterization of solubilized products

Partially depectinated apple cell walls were digested by pectin lyase or endoglucanase or a combination. By combining these commercial enzymes, a higher yield of 22.2% of carbohydrate material was obtained compared with only 13.9% and 5.7%, respectively, when using them singly. Only small amounts of carbohydrates were extracted by buffer (0.8%). The solubilized extracts were fractionated using a combination of ion-exchange chromatography and gel filtration. The indidividual subfractions were analysed for neutral sugar and uronic acid content. The results indicated the existence of a synergistic effect between pectin lyase and endoglucanase based on the percentage of material extracted.