Purification and some properties of polyphenol oxidase from the yam tubers, Dioscorea bulbifera

In a comparison of the polyphenol oxidase activity of various species of yam tubers the greatest enzyme activity was found in D. bulbifera. The enzyme was purified from acetone powder extracts of this plant. Ammonium sulphate fractionation, followed by ion exchange chromatography and gel filtration gave 22-fold purification. The final product gave a single band on polyacrylamide disc gel electrophoresis. The purified enzyme showed activity towards catechol, pyrogallol and dl-β-3,4-dihydroxyphenylalanine (dl-DOPA) and had a MW 115000 ± 2000. It was characterized by response to various inhibitors. β-Mercaptoethanol, dithioerythritol, l-cysteine, sodium metabisulphite and KCN inhibited strongly.     

Spirostanic diosgenin precursors from Dioscorea composita tubers

The main saponin from the fresh tuber of Dioscorea composita was dioscin and from the fermented material 3-O-[α-l-rhamnopyranosyl(1→4)-β-d-glucopyranosyl]diosgenin. The ¹³C NMR chemical shifts of saponins were used in the determination of their structure. No free sapogenin was isolated from the fresh tuber.     

Norclerodane diterpenoids from rhizomes of Dioscorea bulbifera

Three norclerodane diterpenoids, diosbulbins K-M, and one analogous enolglycoside, diosbulbinoside G, together with four norclerodane diterpenoids, diosbulbins B, E, F and G, were isolated from rhizomes of Dioscorea bulbifera. Their structures were established by spectroscopic and chemical methods, including ¹H and ¹³C NMR, NOESY, HSQC, HMBC, and HRMS analyses. The relative configurations of diosbulbins K and L, and diosbulbin F were confirmed by X-ray crystallographic diffraction analysis, and the absolute stereochemistry of diosbulbin K was determined by a modified Mosher’s method. The ¹³C NMR spectroscopic data for diosbulbins E, F and G were also measured for the first time. The compounds did not show significant cytotoxic and anti-bacterial activities.     

Antifungal phenanthrenes in yam tubers

An extract from the peel of yams (Dioscorea rotundata) showed anti-fungal activity towards both Cladosporium cladoporioides and a variety of ya     

Structure and synthesis of the growth inhibitor batatasin I from Dioscorea batatas

The synthesis of 6-hydroxy-2,4,7-trimethoxyphenanthrene is described and its identity with the growth inhibitor, batatasin I, is confirmed.     

Transformation associated with catecholase in Dioscorea alata during storage

The Michaelis constant of catecholase in 3 cultivars of Dioscorea alata was determined. Oxidative browning was related to the activity of the enzyme in the yams. Inhibition by cysteine was competitive, while 2-mercaptoethanol caused an uncompetitive type of inhibition. On DEAE column chromatography a minor peak preceded the major peak in the fresh yam while a minor peak was eluted after the major peak in the stored yam.     

Effect of storage under nitrogen on ethanol,lactate, malate and their dehydrogenases in yam tubers

Under anaerobiosis in white yam tubers, lactate and malate increase and decrease in opposing directions for the first 4 days. Ethanol appears in yams after 7 days under nitrogen, coinciding with the simultaneous rise of both lactate and malate. However ethanol does not occur in white yam tubers on storage in air.     

Diosbulbin d and 8-epidiosbulbin e acetate,norclerodane diterpenoids from dioscorea bulbifera tubers

From tubers of Dioscorea buibifera L. var sativa a new 18-norclerodane diterpenoid, 8-epidiosbulbin E acetate, has been isolated, besides the previously known norditerpenoid, diosbulbin D, and its structure established by spectroscopic means.     

The phenolics and a hydrolysable tannin polyphenol oxidase of Medinilla magnifica

The production of viable meristem cultures of Medinilla magnifica has proved to be very difficult. This may be due, in part, to a pronounced ‘browning’ response of the tissues on cutting. For this reason the phenolic compounds and the hydrolysable-tannin polyphenol oxidase from Medinilla were studied. The distribution of the compounds was: simple phenols 19% , flavonoids 5% , hydrolysable tannins 69% , condensed tannins 7%. Amongst the simple phenols and phenolic acids, the following were identified: phloroglucinol, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, gallic acid (both in free and bound form the most abundant simple phenol), syringic acid, trans-p-coumaric acid, trans-ferulic acid and trans-caffeic acid. No kaempferol or quercetin or their derivatives were detected but condensed tannins are present. Methods for the extraction, fractionation and quantitative determination of phloroglucinol and the phenolic acids, as well as correction factors for losses during the extraction, alkali treatment and derivatization, are presented in a supplementary publication. With regard to the hydrolysable tannin polyphenol oxidase activity of Medinilla stems, the enzyme(s) is rather specific since at neither of its two pH optima (6 and 7) could a classical polyphenol oxidase activity be detected. The enzyme was strongly inhibited by 2-mercaptoethanol. Preliminary experiments have further shown that in addition to the hydrolysable tannins of the tissue, the ferrous ions of the medium, and oxygen together with the hydrolysable tannin polyphenol oxidase could play a role in the browning response. Ways to overcome this difficulty have been suggested.     

Effects of dithiothreitol on dopa oxidase activity from potato tubers

A filtrate, prepared from potato tuber by grinding in an isotonic medium, has been separated into a particulate and a ‘soluble’ fraction by ultracentrifugation. Following dialysis and lyophilization, both fractions catalysed the oxidation of l-DOPA, with approximately 30% of the l-DOPA: oxygen-oxidoreductase (EC 1.14.18.1; DOPA oxidase) activity being associated with the particulate fraction. When dithiothreitol (DTT, 10^?2M was included in the grinding medium, much lower yields of DOPA oxidase were obtained and 80% appeared to be associated with the particulate fraction. DTT proved to be a powerful inhibitor of DOPA oxidase. With concentrations of DTT causing only partial inhibition, the kinetics of the inhibited rate of dopachrome formation from l-DOPA were complex. When oxygen consumption was measured inhibition was not transient. The degree of inhibition was inversely related to the DOPA oxidase activity, indicating interaction of a product of this activity with DTT. Direct determination of -SH groups in DTT using 5,5′-dithiobis(2-nitrobenzoic acid (DTNB) showed that they were all oxidised during the initial phase of inhibition of dopachrome formation. It is concluded that the first phase of inhibition involves oxidation of DTT by an intermediate between l-DOPA and dopachrome. The second phase of inhibition also appeared to require -SH groups initially, since trans-4,5-dihydroxy-1,2-dithiane (oxidized DTT) caused very little inhibition at all.     

Purification of a polyphenol oxidase isoform from potato (Solanum tuberosum) tubers

A different expression pattern of polyphenol oxidases has been observed during storage in cultivars of potato (Solanum tuberosum L.) featuring different length of dormancy: a short-dormant cultivar showed, at the end of the dormancy, both the highest polyphenol oxidase activity and the largest number of enzyme isoforms. An isoform of polyphenol oxidase isolated at the end of the physiological dormancy from a short-dormant cultivar has been purified to homogeneity by means of column chromatography on phenyl Sepharose and on Superdex 200. The purification factor has been determined equal to 88, and the molecular mass of the purified isoform has been estimated to be 69 and 340 kDa by SDS polyacrylamide gel electrophoresis and gel filtration on Superdex 200, respectively, indicating this PPO isoform as a multimer. The corresponding zymogram features a diffused single band at the cathodic region of the gel and the pI of this polyphenol oxidase has been calculated equal to 6.5.     

Diosgenin saponins from Dioscorea floribunda

Five spirostanol glycosides and two furostanol glycosides were isolated from Dioscorea floribunda. In addition to the IR spectra of the free glycosides and the MS of the peracetates and permethyl ethers, the most effective method for structural determination proved to be the NMR spectra of the free saponins in pyridine-d₅.     

苹果多酚氧化酶的纯化与表征

运用丙酮浸漬干燥、磷酸盐缓冲液提取、低温离心、硫酸铵沉淀、DEAE-Sephadex(A-50)、Sephadex(G-75) 和DEAE-celluse(DE-52)层析等方法从苹果中分离获得一种新的含铜酶蛋白,该酶被命名为多酚氧化酶Ⅱ(polyphenol oxidase Ⅱ, PPOⅡ),纯化倍数是215,纯化收率是23%.PAGE、SDS-PAGE和MALDI-TOF 等技术用于测定所获的酶的纯度和分子量.在PAGE和SDS-PAGE 均显示一条带,表明PPOⅡ只由一个亚基组成,且已达到单一组分(MALDI-TOF的结果更证实了这一点).SDS-PAGE 和 MALDI-TOF 的结果都表明PPO的分子量为 38204 Da.pH值对酶活性和稳定性研究的结果显示,从pH值4.0～7.0随着pH值的增加,酶活性也不断增加;从pH值 7.0～11.0, 酶活性不断降低.PPOⅡ的最适pH值为6.6最适温度为30℃.     

Steroidal saponins from Dioscorea floribunda: Structures of floribundasaponins A and B

Two steroidal saponins, floribundasaponins A and B isolated from the yams of Dioscorea floribunda, have been characterized as pennogenin-3-O-β-d-glucopyranoside and pennogenin-3-O-α-l-rhamnopyranosyl(1→4)-β-d-glucopyranoside.     

烟草中多酚氧化酶(PPO)的特征

烟草中的多酚氧化酶介导的褐变会影响烟叶和烟丝的色泽和内在质量,因此对其特性的研究,以及活性的控制成为多年来的研究热点。本文从其生物发生模型、分子结构、生物化学和光谱学特征与植物抗病和机械损伤的关系,多酚氧化酶的抑制、多酚氧化酶的应用等方面着手,对近几年来烟草中PPO研究的最新成果进行总结和回顾,对一些有争议的问题进行了探讨,并对未来PPO研究的方向和领域进行了展望。     

Alterations in Solanum tuberosum polyphenol oxidase activity induced by gamma irradiation

On gamma irradiation of potato tubers at sprout-inhibiting dose (10 krad) the cresolase activity showed a 45% increase while catecholase was reduced by 25%. This reduced the ratio of catecholase to cresolase from 11–12 in unirradiated to 5–6 in irradiated potatoes. Chlorogenic acid oxidation was enhanced by about 25% on irradiation. The increase in the oxidation of p-cresol corresponded with the production of diphenolic compounds. The process of activation of cresolase was slow, reversible and oxygen dependent. A comparative study of the isoenzyme pattern suggested that this activation was due to conformational change, rather than synthesis of new protein.     

槐尺蠖多酚氧化酶的纯化及酶学特征

经40%饱和度硫酸铵分级沉淀,Sephadex G-100凝胶过滤等步骤,将槐尺蠖Semiothisa cinerearia Bremer et Grey 多酚氧化酶纯化,纯化倍数为6.96倍。该酶对焦性没食子酸,邻苯二酚和L多巴的Km值分别为0.23 mmol/L, 0.48 mmol/L和0.49 mmol/L。多酚氧化酶在pH 7.0,37℃时活性最高,并在40℃以上条件下,随着保温时间的延长酶活力下降。用槲皮苷和硫脲作抑制剂对该酶活性的抑制结果表明,这两种抑制剂分别属于竞争性和非竞争性抑制剂。     

Molecular cloning and characterization of the polyphenol oxidase gene from sweetpotato

Polyphenol oxidase is the enzyme responsible for enzymatic browning in sweetpotato that decreases the commercial value of sweetpotato products. Here we reported the cloning and characterization of a new cDNA encoding PPO from sweetpotato, designated as IbPPO (GeneBank accession number: AY822711). The full-length cDNA of IbPPO is 1984 bp with a 1767 bp open reading frame (ORF) encoding a 588 amino acid polypeptide with a calculated molecular weight of 65.7 kDa and theoretical pI of 6.28. The coding sequence of IbPPO was also directly amplified from the genomic DNA of sweetpotato that demonstrated that IbPPO was an intron-free gene. The computational comparative analysis revealed that IbPPO showed homology to other PPOs of plant origin and contained a 50 amino acid plastidial transit peptide at its N-terminal and the two conserved CuA and CuB copper-binding motifs in the catalytic region of IbPPO. A highly conserved serine-rich motif was firstly found in the transit peptides of plant PPO enzymes. Then the homology based structural modeling of IbPPO showed that IbPPO had the typical structure of PPO: the catalytic copper center was accommodated in a central four-helix bundle located in a hydrophobic pocket close to the surface. Finally, the results of the semiquantitative RT-PCR analysis of IbPPO in different tissues demonstrated that IbPPO could express in all the organs of sweetpotato including mature leaves, young leaves, the stems of mature leaves (petioles), the storage roots, and the veins but at different levels. The highest-level expression of IbPPO was found in the veins, followed by storage roots, young leaves and mature leaves; and the lowest-level expression of IbPPO was found in petioles. The present researches will facilitate the development of antibrown sweetpotato by genetic engineering. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 6, pp. 1006–1012. The article was submitted by the authors in English.