Polyphenol oxidase plays a critical role in melanin formation in the fruit skin of persimmon (Diospyros kaki cv. 'Heishi').

In this study, the melanin in persimmon and its formation were investigated. Melanin was found to be deposited on the cell walls of the upper epidermis and subepidermal cells in persimmon skin and the isolated pigment appears to have lamellar structures. Diagnostic analysis of the isolated pigment showed results that were similar to those of melanin from other sources. Ultraviolet-visible spectroscopy revealed that the extracted skin pigment displayed a broadband, structureless absorption profile that increased progressively towards shorter wavelengths. The Fourier transform infrared spectroscopy assay revealed that melanin in persimmon skin exhibits many characteristic absorption peaks. The phenolic profile analysis suggested that the precursors of this pigment may include gallic acid, procyanidin B1, procyanidin B2, ferulic acid and epigallocatechin gallate. The PPO activity and DkPPO expression significantly increased during melanin formation, and transient overexpression of DkPPO promoted melanin synthesis. These results indicate that the isolated pigment was a type of melanin and that PPO plays a critical role in its formation.

Overexpression of the persimmon abscisic acid ß-glucosidase gene (DkBG1) alters fruit ripening in transgenic tomato.

ß-Glucosidases (BG) are present in many plant tissues. Among these, abscisic acid (ABA) ß-glucosidases are thought to take part in the adjustment of cellular ABA levels, however the role of ABA-BG in fruits is still unclear. In this study, through RNA-seq analysis of persimmon fruit, 10 full-length DkBG genes were isolated and were all found to be expressed. In particular, DkBG1 was highly expressed in persimmon fruits with a maximum expression 95 days after full bloom (DAFD). We verified that, in vitro, DkBG1 protein can hydrolyze ABA-glucose ester (ABA-GE) to release free ABA. Compared with wild-type, tomato plants that overexpressed DkBG1 significantly upregulated the expression of ABA receptor PYL3/7 genes and showed typical symptoms of ABA hypersensitivity in fruits. DkBG1 overexpression (DkBG1-OE) accelerated fruit ripening onset by 3-4 days by increasing ABA levels at the pre-breaker stage and induced early ethylene release compared with wild-type fruits. DkBG1-OE altered the expression of ripening regulator NON-RIPENING (NOR) and its target genes; this in turn altered fruit quality traits such as coloration. Our results demonstrated that DkBG1 plays an important role in fruit ripening and quality by adjusting ABA levels via hydrolysis of ABA-GE.

DkWRKY interacts with pyruvate kinase gene DkPK1 and promotes natural deastringency in C-PCNA persimmon.

PAs, also known as condensed tannins, cause the astringency sensation in the persimmon fruit. The astringency of Chinese pollination-constant non-astringent (C-PCNA) persimmon (Diospyros kaki Thunb.) can be naturally removed on the tree, but the regulatory mechanisms of deastringency remain to be elucidated. In our previous research, DkPK1 was shown to be involved in the natural loss of astringency of C-PCNA persimmon fruit. In the present study, yeast one-hybrid (Y1H) library screening using the DkPK1 promoter as baits identified two DkWRKY transcription factor genes (DkWRKY3 and -15). The transcript levels of both DkWRKY3 and -15 exhibited a positive correlation with the decrease in soluble proanthocyanidin (PA) content during the last developmental stage in C-PCNA persimmon. Multiple sequence analysis and subcellular localization confirmed that DkWRKY3 and -15 belonging to the group II and I families, respectively, were both located in the nucleus. Dual-luciferase and Y1H assays demonstrated that DkWRKY3 and -15 can transactivate the DkPK1 promoters. The combination of DkWRKY3 and -15 most likely produced an additive activation effect compared to a single activator on DkPK1, although the two transcriptional activators were not capable of interacting. Notably, DkWRKY3 and -15 showed ubiquitous expression in various organs and abundant upregulation in seeds. Furthermore, transient overexpression of both DkWRKY3 and -15 in persimmon leaves led to a significant decrease in the content of soluble PAs but a significant increase in the expression levels of the acetaldehyde metabolism-related DkPK, DkPDC and DkADH genes. Thus, we suggest that DkWRKY3 and -15 are the upstream regulators of DkPK1 and positively regulate the natural deastringency in C-PCNA persimmon.

Functional characterization of persimmon ß-galactosidase gene DkGAL1 in tomato reveals cell wall modification related to fruit ripening and radicle elongation.

Cell wall metabolism during fruit ripening is a highly organized process that involves complex interplay among various cell wall hydrolases. Among these cell wall hydrolases, ß-galactosidase has been identified to participate in cell wall metabolism via its ability to catalyze galactosyl metabolism from the large and complex side chains of cell walls. In this study, the galactose content in the pericarp increased during persimmon fruit ripening, but cell wall galactosyl residues decreased, indicating a relationship between galactose metabolism and persimmon fruit ripening. Expression of a previously isolated ß-galactosidase gene, DkGAL1, increased 25.01-fold during fruit ripening. Heterologous expression of DkGAL1 under the CaMV 35S promoter in tomato accelerated on-plant and postharvest fruits ripening. The fruit firmness of one of transgenic line, OE-18, was 23.83% lower than that of WT at the breaker stage. The transgenic fruits produced more ethylene by promoting the expression of ethylene synthesis-related genes and cell wall degradation-related genes. Overexpression of DkGAL1 in tomato also reduced cell-to-cell adhesion and promoted both wider intercellular spaces and less cell compaction in transgenic fruit structures. Moreover, DkGAL1 was involved in seed germination and radicle elongation in transgenic tomato seeds. These results confirm the role of DkGAL1 in fruit ripening and suggest that this gene alters galactose metabolism in the fruit, which can promote ripening and reduce cellular adhesion. In addition, the role of DkGAL1 is not limited to fruit softening; DkGAL1 was also involved in seed germination and radicle elongation in transgenic tomato seeds.

Hypoxia-responsive ERFs involved in postdeastringency softening of persimmon fruit.

Removal of astringency by endogenously formed acetaldehyde, achieved by postharvest anaerobic treatment, is of critical importance for many types of persimmon fruit. Although an anaerobic environment accelerates de-astringency, it also has the deleterious effect of promoting excessive softening, reducing shelf life and marketability. Some hypoxia-responsive ethylene response factors (ERFs) participate in anaerobic de-astringency, but their role in accelerated softening was unclear. Undesirable rapid softening induced by high CO2 (95%) was ameliorated by adding the ethylene inhibitor 1-MCP (1 µL/L), resulting in reduced astringency while maintaining firmness, suggesting that CO2 -induced softening involves ethylene signalling. Among the hypoxia-responsive genes, expression of eight involved in fruit cell wall metabolism (Dkß-gal1/4, DkEGase1, DkPE1/2, DkPG1, DkXTH9/10) and three ethylene response factor genes (DkERF8/16/19) showed significant correlations with postdeastringency fruit softening. Dual-luciferase assay indicated that DkERF8/16/19 could trans-activate the DkXTH9 promoter and this interaction was abolished by a mutation introduced into the C-repeat/dehydration-responsive element of the DkXTH9 promoter, supporting the conclusion that these DkERFs bind directly to the DkXTH9 promoter and regulate this gene, which encodes an important cell wall metabolism enzyme. Some hypoxia-responsive ERF genes are involved in deastringency and softening, and this linkage was uncoupled by 1-MCP. Fruit of the Japanese cultivar 'Tonewase' provide a model for altered anaerobic response, as they lost astringency yet maintained firmness after CO2 treatment without 1-MCP and changes in cell wall enzymes and ERFs did not occur.

DkXTH8, a novel xyloglucan endotransglucosylase/hydrolase in persimmon, alters cell wall structure and promotes leaf senescence and fruit postharvest softening.

Fruit softening is mainly associated with cell wall structural modifications, and members of the xyloglucan endotransglucosylase/hydrolase (XTH) family are key enzymes involved in cleaving and re-joining xyloglucan in the cell wall. In this work, we isolated a new XTH gene, DkXTH8, from persimmon fruit. Transcriptional profiling revealed that DkXTH8 peaked during dramatic fruit softening, and expression of DkXTH8 was stimulated by propylene and abscisic acid but suppressed by gibberellic acid and 1-MCP. Transient expression assays in onion epidermal cells indicated direct localization of DkXTH8 to the cell wall via its signal peptide. When expressed in vitro, the recombinant DkXTH8 protein exhibited strict xyloglucan endotransglycosylase activity, whereas no xyloglucan endohydrolase activity was observed. Furthermore, overexpression of DkXTH8 resulted in increased leaf senescence coupled with higher electrolyte leakage in Arabidopsis and faster fruit ripening and softening rates in tomato. Most importantly, transgenic plants overexpressing DkXTH8 displayed more irregular and twisted cells due to cell wall restructuring, resulting in wider interstitial spaces with less compact cells. We suggest that DkXTH8 expression causes cells to be easily destroyed, increases membrane permeability and cell peroxidation, and accelerates leaf senescence and fruit softening in transgenic plants.

Analysis of xyloglucan endotransglycosylase/hydrolase (XTH) genes and diverse roles of isoenzymes during persimmon fruit development and postharvest softening.

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes have played a role in the remodeling of cell wall hemicelluloses. To investigate the function of XTHs in persimmon (Diospyros kaki L.) fruit development and postharvest softening, five cDNAs (DkXTH1 to DkXTH5), whose putative proteins contained the conserved DEIDFEFLG motif of XTH, were cloned. Real time quantitative PCR analysis revealed that DkXTH1, DkXTH4, and DkXTH5 peaked in immature expanding fruit, and their higher expression was observed along with higher fruit firmness in cold-treated fruit or firmer cultivar fruit during storage. The opposite gene expression patterns were observed in DkXTH2 and DkXTH3, which reached maxima concomitance with pronounced fruit softening. Meanwhile, the xyloglucan endotransglycosylase (XET) enzymes play important roles in both the rapid growth and ripening of persimmon fruit. Furthermore, the recombined DkXTH1 and DkXTH2 proteins showed significant XET activity without any detected XEH activity. However, the XET activity of recombined DkXTH2 protein had a higher affinity for small acceptor molecules than that of recombined DkXTH1 protein. The former might prefer to participate in cell wall restructuring, and the latter is more inclined to participate in cell wall assembly. Besides, DKXTH proteins could function by targeting to the cell wall under regulation of a signal peptide. The data suggested that individual DKXTHs could exhibit different patterns of expression, and the encoded products possessed specific enzymatic properties conferring on their respective functions in growth and postharvest softening of persimmon fruit.

Purification and characterisation of a novel chitinase from persimmon (Diospyros kaki) with antifungal activity.

A novel chitinase from the persimmon fruit was isolated, purified and characterised in this report. The Diospyros kaki chitinase (DKC) was found to be a monomer with a molecular mass of 29 kDa. It exhibited optimal activity at pH 4.5 with broad pH stability from pH 4.0-9.0. It has an optimal temperature of 60°C and thermostable up to 60°C when incubated for 30 min. The internal peptide sequences of DKC showed similarity with other reported plant chitinases. It has the ability to hydrolyse colloidal chitin into chito-oligomers such as chitotriose, chitobiose and into its monomer N-acetylglucosamine. It can be used to degrade chitin waste into useful products such as chito-oligosacchaarides. DKC exhibited antifungal activity towards pathogenic fungus Trichoderma viride. Chitinases with antifungal property can be used as biocontrol agents replacing chemical fungicides.

Identification of xyloglucan endotransglucosylase/hydrolase genes (XTHs) and their expression in persimmon fruit as influenced by 1-methylcyclopropene and gibberellic acid during storage at ambient temperature.

Xyloglucan endotransglucosylase/hydrolase (XTH) is thought to contribute to fruit softening by degrading xyloglucan that is a predominant hemicellulose in the cell wall. In this study, two full-length XTH genes (DKXTH1 and DKXTH2) were identified from 'Fupingjianshi' persimmon fruit, and the expression level of both XTH genes was investigated during softening for 18-24 d using RT-qPCR. Sequence analysis showed that DKXTH1 and DKXTH2 contained a putative open reading frame of 861 and 876 bp encoding polypeptides of 287 and 292 amino acid residues, respectively, which contained the conserved DEIDFEFLG motif of XTH, a potential N-linked glycosylation signal site. RT-qPCR analysis showed that DKXTH1 and DKXTH2 in untreated fruit had different expression patterns during fruit softening, in which maximum expression occurred on days 3 and 12 of ripening, respectively. 1-Methylcyclopropene (1-MCP) and gibberellic acid (GA(3)) treatments delayed the softening and ethylene peak of persimmon fruit, as well as suppressed the expression of both XTH genes, especially DKXTH1. These results indicated that the expression of both XTH genes might be ethylene dependent action, and closely related to softening of persimmon in the early (DKXTH1) and later (DKXTH2) ripening stages.

Isolation and characterization of a laccase gene potentially involved in proanthocyanidin polymerization in Oriental persimmon (Diospyros kaki Thunb.) fruit.

Proanthocyanidins (PAs, condensed tannins) are important health-promoting phytochemicals that are abundant in many plants. Oriental persimmon (Diospyros kaki Thunb.) is an excellent source of PAs because of its unique ability to accumulate large quantities of these compounds in its young fruit. There are two different spontaneous mutant phenotypes of oriental persimmons which lose their astringent taste naturally on the tree; while plants without these mutations remain rich in soluble PAs until the fruit fully ripened. The mutations are referred to as pollination-constant non-astringent genotypes named J-PCNA and C-PCNA, and are from Japan and China respectively. In this work we speculated that the loss of astringency in C-PCNA fruit is due to the soluble PAs transferred into insoluble upon polymerization, which was quite different from that of the J-PCNA. A DkLAC1 gene was isolated by the homology-based clone method. The predicted protein product of this gene showed that the DkLAC1 is a plant laccase which is phylogenetically related to the known enzyme AtLAC15 involved in the polymerization of PAs. Expression patterns of PAs biosynthetic genes associated with soluble PAs contents in three types of Oriental persimmons. Expression levels of DkLAC1 in C-PCNA type plants were linked with the reduction of soluble PAs in the flesh of the fruit. In addition the cis-elements in the DkLAC1 promoter regions indicated that the gene might also be regulated by the DkMYB4 as is seen with other well-known structural genes in Oriental persimmon. We conclude that DkLAC1 is potentially involved in PA polymerization in C-PCNA during normal ripening in C-PCNA persimmon.

Purification and chemical characterisation of a cell wall-associated ß-galactosidase from mature sweet cherry (Prunus avium L.) fruit.

Using four different chromatographic steps, ß-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium ß-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and ß-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing ß-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry ß-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple ß-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry ß-galactosidase exhibited a strict specificity towards p-nitrophenyl ß-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening.

Molecular cloning and expression of squalene synthase and 2,3-oxidosqualene cyclase genes in persimmon (Diospyros kaki L.) fruits.

Oleanolic acid (OA) and ursolic acid (UA) are the main triterpene acids in persimmon fruit, and squalene synthase and 2,3-oxidosqualene cyclases are important enzymes in pentacyclic triterpene biosynthesis. In order to study their relationship, DkSQS and DkOSC were cloned from persimmon fruits in the present study. The full-length cDNA of DkSQS was 1647 bp, containing an open reading frame (ORF) of 1245 bp that encoded a peptide of 415 amino acids (AA). The 3'-end of DkOSC cDNA fragment contained 522 bp, including a partial ORF of 298 bp, a full poly A tail that encoded 98 AA. Two cultivars of persimmon, i.e. cv. Nishimurawase and cv. Niuxinshi, were used to study the content of OA and UA and the related gene expression. Results showed that OA and UA contents changed in both cultivars during fruit development, the difference in cv. Nishimurawase was greater than that in cv. Niuxinshi. The expression of DkSQS and DkOSC had no obvious correlation with the biosynthesis of OA and UA in the flesh. There may be two main reasons. Firstly, different enzymes involved in the biosynthesis of triterpenes and mutual adjustment were existed in different gene expressions. Secondly, it was not clear that the DkOSC cloned in this research belonged to which subfamily. Therefore, the real relationship between triterpenes and DkSQS and DkOSC in persimmon fruits is still to be revealed.

Cloning of phytoene desaturase and expression analysis of carotenogenic genes in persimmon (Diospyros kaki L.) fruits.

Persimmon is a commercially important fruit crop, and the fruit is rich in different kinds of bioactive compounds, among which carotenoids contribute significantly to its color and nutritional value. In this study, the cDNA of phytoene desaturase gene (PDS) was isolated by rapid amplification of cDNA ends (RACE) technique. Sequence analysis indicated that the full-length cDNA of PDS was 2064 bp, encoding 586 amino acids and containing one open reading frame (ORF) of 1761 bp. Homology analysis showed that DkPDS, which had been submitted in GenBank with accession number GU112527, shared high similarities of 80-86% with PDS cloned from other plants. Prediction of deduced proteins showed that there was no signal peptide and transmembrane topological structure in DkPDS. It was a hydrophilic and stable protein, and located in chloroplast. To examine the specific expression patterns of carotenogenic genes we had cloned from persimmon, including phytoene synthase (DkPSY), DkPDS, ζ-carotene desaturase (DkZDS), lycopene ß-cyclase (DkLCYB) and ß-carotene hydroxylase (DkBCH), real-time quantitative PCR (Q-PCR) was performed in flesh at five different developmental stages. The results revealed that the expression levels of DkPSY, DkPDS and DkZDS gradually increased. Nevertheless, the expression level of DkLCYB was very low and maintained relatively stable. The expression level of DkBCH was also at a low level from stage 1 to 4, and then reached the maximum at stage 5. In addition, the expression level of DkZDS was higher than that of other genes. Carotenoid detection demonstrated that both ß-cryptoxanthin and total carotenoids increased with fruit development, and zeaxanthin had little change, but with a sudden increase in final stage.

Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (Diospyros kaki Thunb.) fruit.

Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3'5' hydroxylase (F3'5'H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3' hydroxylation (F3'H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3'5'H to F3'H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F3'5'H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.

Molecular identification of 1-Cys peroxiredoxin and anthocyanidin/flavonol 3-O-galactosyltransferase from proanthocyanidin-rich young fruits of persimmon (Diospyros kaki Thunb.).

Fruits of persimmon (Diospyros kaki Thunb.) accumulate large amounts of proanthocyanidins (PAs) in the early stages of development. Astringent (A)-type fruits remain rich in soluble PAs even after they reach full-mature stage, whereas non-astringent (NA)-type fruits lose these compounds before full maturation. As a first step to elucidate the mechanism of PA accumulation in this non-model species, we used suppression subtractive hybridization to identify transcripts accumulating differently in young fruits of A- and NA-type. Interestingly, only a few clones involved in PA biosynthesis were identified in A-NA libraries. Represented by multiple clones were those encoding a novel 1-Cys peroxiredoxin and a new member of family 1 glycosyltransferases. Quantitative RT-PCR analyses confirmed correlation of the amount of PAs and accumulation of transcripts encoding these proteins in young persimmon fruits. Furthermore, the new family 1 glycosyltransferase was produced in Escherichia coli and shown to efficiently catalyze galactosylation at 3-hydroxyl groups of several anthocyanidins and flavonols. These findings suggest a complex mechanism of PA accumulation in persimmon fruits.

Involvement of negative feedback regulation in wound-induced ethylene synthesis in 'Saijo' persimmon.

Wounding is one of the most effective stress signals to induce ethylene synthesis in persimmon (Diospyros kaki Thunb.). We found that wound-induced ethylene biosynthesis is subjected to negative feedback regulation in mature 'Saijo' persimmon fruit since ethylene production was enhanced by 1-methylcyclopropene (1-MCP) (an inhibitor of ethylene perception) pretreatment, which was approximately 1.8 fold of that in control tissues (without 1-MCP pretreatment). Wound-induced 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and DK-ACS2 gene expression were substantially increased by 1-MCP pretreatment after 12 h, which resulted in much higher ACC content in 1-MCP pretreated tissues than that in a control after 24 h. These results indicated that wound-induced DK-ACS2 gene expression was negatively regulated by ethylene in mature persimmon fruit. However, 1-MCP pretreatment had no effect on DK-ACO1 gene expression, suggesting the independence of wound-induced DK-ACO1 on ethylene. Out of accord with DK-ACO1 gene expression, ACC oxidase activity was enhanced 48 h after wounding in 1-MCP pretreated tissues, reaching a peak 1.5-fold higher than that in control tissues at 60 h.

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.

beta-Galactosidase and its significance in ripening of "Saijyo" Japanese Persimmon fruit.

The fruit extracts of ripening cv. Japanese Persimmon, "Saijyo", contained a number of glycosidases and glycanases. Among them, beta-galactosidase appeared to be the most significant, and the activity increased in parallel with tissue ripening. Persimmon beta-galactosidase was presented in at least three isoforms, beta-galactosidase-I (pI = 4.88), beta-galactosidase-II (pI = 6.76), and beta-galactosidase-III (pI = 7.05). beta-Galactosidase-III had exo-type galactanase activity, while the others did not. The activity of endo-type glycanases was a maximum in immature green or yellow fruits. The firmness of the pulp tissue decreased dramatically, and the amount of water-soluble polysaccharide (WSS) increased. The enzyme activities of exo-type glycosidases, especially beta-galactosidase, appeared maximal in mature red fruits. The amount of extractable pectin remained unchanged, although the galactose content of the high-molecular-weight fraction in WSS decreased dramatically. These results suggest that the ripening of persimmon was caused by the solubilization of pectic polysaccharide by endo-type glycanases and digestion by exo-type glycosidases. beta-Galactosidase, in particular, seemed to play a major role in ripening the fruit.

Partial purification of latent persimmon fruit polyphenol oxidase.

Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).