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.     

Enzymatic modification of pectin to increase its calcium sensitivity while preserving its molecular weight

A commercial high-methoxy citrus pectin was treated with a purified salt-independent pectin methylesterase (PME) isozyme isolated from Valencia orange peel to prepare a series of deesterified pectins. A series of alkali-deesterified pectins was also prepared at pH 10 under conditions permitting beta-elimination. Analysis of these pectins using high-performance size exclusion chromatography (HPSEC) with on-line multiangle laser light-scattering, differential viscometer, and refractive index (RI) detectors revealed no reduction in weight-average molecular weight (M(w); 150000) in the PME-treated pectin series, whereas a 16% reduction in intrinsic viscosity (IV) occurred below a degree of esterification (DE) of 47%. In contrast, alkali deesterification rapidly reduced both M(w) and IV to less than half of that observed for untreated pectin. PME treatment of a non-calcium-sensitive citrus pectin introduced calcium sensitivity with only a 6% reduction in the DE. Triad blocks of unesterified galacturonic acid were observed in (1)H nuclear magnetic resonance spectra of this calcium-sensitive pectin (CSP). These results demonstrate that the orange salt-independent PME isozyme utilizes a blockwise mode of action. This is the first report of the preparation of a CSP by PME treatment without significant loss of the pectin's M(w) due to depolymerization.     

六种果皮原料果胶的理化及凝胶特性比较

为了解不同品种水果的果皮(柚子皮、西番莲皮、脐橙皮、石榴皮、榴莲皮)以及向日葵盘所提取果胶的理化和质构特性,研究了不同原料果胶的得率、色泽、果胶酸含量、甲氧基含量、酯化度、黏度及质构特性,特别是采用高效液相色谱准确测定了各类果胶的分子量。结果表明:柚子皮、向日葵盘和脐橙皮果胶质量分数较高,分别为18.06%、14.61%和14.43%;西番莲皮果胶质量分数为8.76%;而石榴皮及榴莲皮果胶质量分数较低(均<3%)。从分子量看,石榴皮、脐橙皮果胶分子量较大(>1000kDa),向日葵盘果胶分子量最小(483kDa)。此外,几种果胶的溶胶均属低黏度值果胶(<25厘泊),且在pH值为7.0时黏度最大、在pH值为5.0时黏度最小。结合凝胶质构分析表明:石榴皮果胶分子量最大,凝胶强度最大,但为高甲氧基果胶,且得率较低;而向日葵盘果胶分子量最小,但得率较高,且为低甲基果胶,在非糖及含糖体系中均可形成性能优良的凝胶,因此是生产果胶的良好原料。该文为果胶的生产及应用提供参考。     

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.     

Purification and glycosylation analysis of an acidic pectin methylesterase in jelly fig (Ficus awkeotsang) achenes

An acidic pectin methylesterase (PME) is responsible for the gelation of water extract from jelly fig (Ficus awkeotasang) achenes. A new, fast and efficient, method has been developed to purify this acidic PME. The method includes preparing jelly curd by traditional hand washing, extracting proteins from the curd, and separating PME by anion-exchanger. The purified PME exists as a monomer of 38 kDa determined by gel filtration, and exerts enzymatic activity over a broad pH range, particularly in acidic environments where most known PME enzymes from various species are inactivated. Chemical staining and enzymatic cleavage suggest that the jelly fig PME is an N-linked glycoprotein. Fluorophore-assisted carbohydrate electrophoresis reveals that the polysaccharide of this glycoprotein putatively consists of 22 hexoses including 16 mannose, 4 N-acetylglucosamine, and 2 galactose residues.     

Mode of de-esterification of alkaline and acidic pectin methyl esterases at different pH conditions

Highly esterified citrus pectin was de-esterified at pH 4.5 and 8.0 by a fungal pectin methyl esterase (PME) that was shown to have an acidic isoelectric pH (pI) and an acidic pH optimum and by a plant PME that was characterized by an alkaline pI and an alkaline pH optimum. Interchain and intrachain de-esterification patterns were studied by digestion of the pectin products with endo-polygalacturonase and subsequent analysis using size exclusion and anion-exchange chromatography. No effect of pH was observed on the de-esterification mode of either of the two enzymes. Acidic, fungal PME converted pectin according to a multiple-chain mechanism, with a limited degree of multiple attack at the intrachain level, both at pH 4.5 and at pH 8.0. A multiple-attack mechanism, with a high degree of multiple attack, was more appropriate to describe the action mode of alkaline, plant PME, both at pH 4.5 and at pH 8.0.     

微波辅助提取桔皮果胶的理化及凝胶特性比较

为了增加桔皮利用率,提高桔皮中果胶得率和品质,以5种新鲜桔皮(丑橘、砂糖橘、芦柑、金橘、贡桔)为原料,采用微波辅助法提取桔皮中果胶,对果胶理化特性和凝胶特性进行研究。分析果胶理化及凝胶特性的相关性,并采用主成分分析法确定影响果胶凝胶特性的主要因素,优化微波辅助法提取工艺参数。结果表明:5种桔皮果胶都是高酯果胶,5种果胶理化及凝胶特性存在显著差异(P0.05),其中芦柑果胶得率和半乳糖醛酸质量分数最高,分别为12.36%、78.61%;且5种果胶理化及凝胶特性呈显著相关性(P0.05),主成分分析法确定甲氧基质量分数和果胶得率分别为主成分1和2中主要影响因子,甲氧基质量分数决定果胶种类,因此果胶种类和得率是桔皮果胶提取的主要因素。微波提取优化参数:以芦柑桔皮为原料,料液比为1:26 g/g,p H值1.30,微波功率440 W,微波时间62 s,果胶得率为16.00%。研究结果为桔皮果胶加工和应用提供理论指导。     

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.     

Characterization of a salt-independent pectin methylesterase purified from valencia orange peel

The pectin methylesterase (PME; EC 3.1.1.11) present in a commercial orange peel enzyme preparation was characterized to establish its identity among the multiple PME isozymes present in Valencia orange (Citrus sinensis L.) peel. We show the commercial enzyme corresponds to the major peak 2 PME previously separated by heparin-Sepharose chromatography (Cameron et al., J. Food Sci. 1998, 63, 253). Both PMEs have comparable elution profiles on cation-exchange and hydrophobic-interaction perfusion chromatography columns, molecular weights (ca. 34 kDa) and pI (pH 9.2), and biochemical properties, including a broad pH activity range and activity in the absence of added cations. An identical partial amino terminal peptide sequence was also obtained for the PMEs, which further demonstrated a structural identity with other plant PMEs. The biochemical and structural properties readily distinguish this Valencia orange PME from salt-dependent isozymes and further suggest that it is an ortholog to the salt-independent fruit-specific isozyme of tomato. This work provides a well-defined, enzymatically homogeneous, salt-independent (type 1) plant PME isozyme that is suitable for studying details of the enzyme's mode of action and for use in modifying methylester patterns for studying the structure-functional property relationships in pectin.     

Extraction of green labeled pectins and pectic oligosaccharides from plant byproducts

Green labeled pectins were extracted by an environmentally friendly way using proteases and cellulases being able to act on proteins and cellulose present in cell walls. Pectins were isolated from different plant byproducts, i.e., chicory roots, citrus peel, cauliflower florets and leaves, endive, and sugar beet pulps. Enzymatic extraction was performed at 50 degrees C for 4 h, in order to fulfill the conditions required for microbiological safety of extracted products. High methoxy (HM) pectins of high molar mass were extracted with three different enzyme mixtures. These pectins were subsequently demethylated with two pectin methyl esterases (PMEs), either the fungal PME from Aspergillus aculeatus or the orange PME. It was further demonstrated that high molar mass low methoxy (LM) pectins could also be extracted directly from cell walls by adding the fungal PME to the mixture of protease and cellulase. Moreover, health benefit pectic oligosaccharides, the so-called modified hairy regions, were obtained after enzymatic treatment of the residue recovered after pectin extraction. The enzymatic method demonstrates that it is possible to convert vegetable byproducts into high-added value compounds, such as pectins and pectic oligosaccharides, and thus considerably reduce the amount of these residues generated by food industries.     

镉安全水稻材料根系细胞壁果胶对镉的响应特征

【目的】研究镉(Cd)处理下水稻根系细胞壁果胶对Cd胁迫的响应,进一步深化Cd安全水稻材料根系细胞壁Cd的固持机制。【方法】以Cd安全水稻材料D62B为研究对象,普通材料Luhui17为对照进行水培试验。设4个Cd质量浓度处理：0 mg/L (CK)、0.5 mg/L (Cd0.5)、1.0 mg/L (Cd1)、2.0 mg/L (Cd2)。在水稻分蘖期采集根系样品,分析细胞壁多糖中果胶、半纤维1、半纤维2以及残渣部分的Cd含量,测定果胶糖醛酸含量、果胶酯化度、果胶甲酯酶(PME)活性、根系过氧化氢(H₂O₂)含量以及细胞壁过氧化物酶(POD)活性,进而分析根系细胞壁果胶对Cd的响应特征。【结果】1) Cd胁迫下,D62B和Luhui17根系细胞壁果胶合成增加,根系细胞壁低酯化和高酯化果胶糖醛酸含量均表现为D62B高于Luhui17。Cd处理下D62B根系细胞壁低酯化和高酯化果胶糖醛酸含量较对照分别增加了13.21%～71.82%和22.10%～64.27%,Luhui17分别增加了24.14%～137.86%和13.12%～41.26%。...     

高密度CO2处理虾肌球蛋白形成凝胶的临界浓度与凝胶强度

为了探讨高密度CO_2(dense phase carbon dioxide,DPCD)诱导蛋白质形成凝胶的机制,以凡纳滨对虾肌球蛋白为研究对象,研究了DPCD处理压强、温度和时间对虾肌球蛋白形成凝胶的临界浓度和对虾肉糜凝胶强度的影响。研究结果表明:DPCD处理压强和温度对虾肌球蛋白溶液形成凝胶的临界浓度有显著影响,处理时间对肌球蛋白溶液形成凝胶的临界浓度无显著影响,但增加处理时间,可以形成更加紧实的凝胶。在40℃和5~30 MPa时虾肌球蛋白溶液形成凝胶的临界质量浓度为14 mg/mL,在50℃和5、10 MPa时虾肌球蛋白溶液形成凝胶的临界质量浓度为12 mg/mL,在50℃和15~30 MPa时虾肌球蛋白溶液形成凝胶的临界质量浓度为11 mg/mL,在60℃和5~30 MPa时虾肌球蛋白溶液形成凝胶的临界质量浓度为10 mg/mL。DPCD处理压强和温度对虾肉糜的凝胶强度也具有显著影响(P0.05),且随着压强增加和温度升高,虾肉糜凝胶强度呈增加趋势(P0.05);在50℃和25 MPa下处理虾肉糜20 min,形成的凝胶强度较好,达到了(14.28±0.57)N·mm。DPCD处理温度越高,虾肌球蛋白形成凝胶的临界浓度就越低,而虾肉糜形成凝胶的强度越高;DPCD处理压强越高,虽然对虾肌球蛋白形成凝胶的临界浓度影响较小,但能使虾肌球蛋白和虾肉糜形成凝胶的强度增加。从分析中还可以推断,DPCD低压(5~10 MPa)诱导虾肉糜形成凝胶主要是热效应的作用,DPCD较高压强(10 MPa)诱导虾肉糜形成凝胶是热和CO_2分子效应的共同作用。研究结果为进一步阐明DPCD诱导蛋白质形成凝胶的机制提供了基础数据。     

Effect of intrinsic and extrinsic factors on the interaction of plant pectin methylesterase and its proteinaceous inhibitor from kiwi fruit

A proteinaceous pectin methylesterase inhibitor (PMEI) was isolated from kiwi fruit (Actinidia chinensiscv. Hayward) and purified by affinity chromatography on a cyanogen bromide (CNBr) Sepharose 4B-orange PME column. The optimal pH of banana PME activity was 7.0, whereas that for carrot and strawberry PME activity was 9.0. The optimal pH for the binding between kiwi fruit PMEI and these PMEs was 7.0. The kiwi fruit PMEI has a different affinity for PME depending on the plant source. The inhibition kinetics of kiwi fruit PMEI to banana and strawberry PME followed a noncompetitive type, whereas that to carrot PME followed a competitive type. The kiwi fruit PMEI was mixed with banana, carrot, and strawberry PME to obtain PMEI-PME complexes, which were then subjected to thermal (40-80 degrees C, atmospheric pressure) or high-pressure (10 degrees C, 100-600 MPa) treatment. Experimental data showed that the PMEI-PME complexes were easily dissociated by both thermal and high-pressure treatments.     

Activity and process stability of purified green pepper (Capsicum annuum) pectin methylesterase

Pectin methylesterase (PME) from green bell peppers (Capsicum annuum) was extracted and purified by affinity chromatography on a CNBr-Sepharose-PMEI column. A single protein peak with pectin methylesterase activity was observed. For the pepper PME, a biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters for activity and thermostability was performed. The optimum pH for PME activity at 22 degrees C was 7.5, and its optimum temperature at neutral pH was between 52.5 and 55.0 degrees C. The purified pepper PME required the presence of 0.13 M NaCl for optimum activity. Isothermal inactivation of purified pepper PME in 20 mM Tris buffer (pH 7.5) could be described by a fractional conversion model for lower temperatures (55-57 degrees C) and a biphasic model for higher temperatures (58-70 degrees C). The enzyme showed a stable behavior toward high-pressure/temperature treatments.     

Functional expression in pichia pastoris of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

A cDNA fragment encoding an acidic pectin methylesterase (PME) of jelly fig achene was successfully expressed in Pichia pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase promoter. The recombinant PME was produced as a secretory protein by N-terminal fusion of a cleavable prepropeptide for signal trafficking, and thus easily harvested from the culture medium. Compared with native N-glycosylated PME (38 kDa) purified from jelly fig achenes, this recombinant PME (45 kDa) appeared to be hyperglycosylated. Activity staining indicated that the recombinant PME was functionally active. Yet the hyperglycosylated recombinant PME possessed thermostability and enzymatic capability over a broad pH range equivalent to those of the native PME. The success of functional production of this acidic jelly fig PME in P. pastoris has significantly broadened its applications in industry.     

Enzyme-induced gelation of extensively hydrolyzed whey proteins by alcalase: comparison with the plastein reaction and characterization of interactions

Extensive hydrolysis of whey protein isolate by Alcalase 2.4L produces a gel. The objectives of this study were to compare enzyme-induced gelation with the plastein reaction by determining the types of interactions involved in gelation. The average chain length of the peptides did not increase during hydrolysis and reached a plateau after 30 min to be approximately 4 residues, suggesting that the gel was formed by small molecular weight peptides held together by non-covalent interactions. The enzyme-induced gel network was stable over a wide range of pH and ionic strength and, therefore, showed some similarities with the plastein reaction. Disulfide bonds were not involved in the gel network. The gelation seems to be caused by physical aggregation, mainly via hydrophobic interactions with hydrogen bonding and electrostatic interactions playing a minor role.     

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.     

Formation of disulfide bonds in acid-induced gels of preheated whey protein isolate

Cold gelation of whey proteins is a two-step process. First, protein aggregates are prepared by a heat treatment of a solution of native proteins in the absence of salt. Second, after cooling of the solution, gelation is induced by lowering the pH at ambient temperature. To demonstrate the additional formation of disulfide bonds during this second step, gelation of whey protein aggregates with and without a thiol-blocking treatment was studied. Modification of reactive thiols on the surface of the aggregates was carried out after the heat-treatment step. To exclude specific effects of the agent itself, different thiol-blocking agents were used. Dynamic light scattering and SDS-agarose gel electrophoresis were used to show that the size of the aggregates was not changed by this modification. The kinetics of gelation as determined by the development of pH and turbidity within the first 8 h of acidification were not affected by blocking thiol groups. During gelation, formation of large, covalently linked, aggregates occurred only in the case of unblocked WPI aggregates, which demonstrates that additional disulfide bonds were formed. Results of permeability and confocal scanning laser microscope measurements did not reveal any differences in the microstructure of networks prepared from treated or untreated whey protein aggregates. However, gel hardness was decreased 10-fold in gels prepared from blocked aggregates. Mixing different amounts of blocked and unblocked aggregates allowed gel hardness to be controlled. It is proposed that the initial microstructure of the gels is primarily determined by the acid-induced noncovalent interactions. The additional covalent disulfide bonds formed during gelation are involved in stabilizing the network and increase gel strength.     

不同柑橘类果胶分子的理化特性、结构及凝胶性能

为了解不同果胶的理化性质、结构与其功能特性的区别,拓展不同性能果胶的针对化应用,提高柑橘加工副产物的附加值。该研究采用酸提法从上饶柚子皮、赣南脐橙皮、南丰蜜桔皮、日本柚子皮、小青柑皮、柠檬皮6种柑橘类水果果皮渣中分别提取果胶,依次为HMP-1、HMP-2、HMP-3、HMP-4、HMP-5和HMP-6,并对其理化特性、结构及凝胶性能进行比较分析。结果表明：不同来源的6种柑橘果胶酯化度在67.20%～75.45%,均为高酯果胶（high methoxyl pectin, HMP）,具备阴离子多糖特性,在提取过程中均出现了不同程度的降解,理化特性和分子结构上存在一定差别。果胶的分子量、黏度、分子链结构等因素共同作用于果胶的凝胶性能,6种HMP均能形成具有一定强度的酸/糖凝胶,将水分截留在三维凝胶网络中,经历冻融循环过程持水性仍在96%以上,具备较好的稳定性。其中分子量较低的HMP-1和HMP-6凝胶强度最高,胶凝度均在200°以上,这与HMP-1的高同型半乳糖醛酸聚糖（HG）含量、较完整的分子结构,以及HMP-6较高的鼠李糖半乳糖醛酸聚糖-I（RG-I）含量及分支度有关,而分子量及黏度较高的HMP-5的弱凝胶强度则与其较大的分子链降解程度及低RG-I含量有关,表明HG和RG-I结构域对凝胶的形成均有促进作用。原子力显微镜图像也显示,HMP-1和HMP-6中出现较为明显的分子链交联。对不同柑橘类果胶的理化性质、结构与凝胶特性进行比较分析,为确定凝胶性能更加优异的提取原料、果胶的扩大化生产及针对性应用提供了理论参考。     

Vacuum infusion of plant or fungal pectinmethylesterase and calcium affects the texture and structure of eggplant

The effect of vacuum infusion on eggplant quality of a commercial fungal (Aspergillus niger) and citrus pectinmethylesterase (PME) with calcium chloride (4000 ppm) was investigated after processing and during storage. Firmness of infused eggplants using fungal or citrus PME was significantly increased compared to controls (fresh noninfused and water-infused control) after processing and during storage for 7 days at 4 degrees C. Activity of fungal PME-infused eggplant increased almost 32 times, whereas activity of eggplant infused with Marsh grapefruit PME increased 2-fold. Degree of esterification of pectin of eggplants infused with fungal or citrus PME decreased slightly. Cryo-SEM showed that samples treated with fungal PME/ CaCl2 displayed more integrity among cells as compared with water-infused control. The change of pectin in the cell wall was visualized using monoclonal antibodies JIM5 (low-esterified pectin) and JIM7 (high-esterified pectin). JIM5 showed more binding than JIM7 with the cell walls of eggplant tissues from fungal PME/ CaCl2 treatment.