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Takehiko Shimada - One of the best experts on this subject based on the ideXlab platform.
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Characterization of γ‐terpinene synthase from Citrus Unshiu (Satsuma mandarin)
Biofactors, 2020Co-Authors: Yukiko Suzuki, Takehiko Shimada, Mitsuo Omura, Hiroshi Sakai, Shigenori Kumazawa, Tsutomu NakayamaAbstract:γ-Terpinene is a monoterpene and a major component of essential oils made from Citrus fruits and shows strong antioxidant activity in various assay systems. Plant γ-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of γ-terpinene synthase from Citrus Unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be γ-terpinene by GC/MS.
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parental diagnosis of satsuma mandarin Citrus Unshiu marc revealed by nuclear and cytoplasmic markers
Breeding Science, 2016Co-Authors: Hiroshi Fujii, Mitsuo Omura, Tomoko Endo, Satoshi Ohta, Keisuke Nonaka, Yuichi Katayose, Toshimi Matsumoto, Terutaka Yoshioka, Takehiko ShimadaAbstract:Satsuma mandarins (Citrus Unshiu Marc.) are the predominant cultivated Citrus variety in Japan. Clarification of its origin would prove valuable for Citrus taxonomy and mandarin breeding programs; however, current information is limited. We applied genome-wide genotyping using a 384 Citrus single nucleotide polymorphism (SNP) array and MARCO computer software to investigate the satsuma mandarin parentage. Genotyping data from 206 validated SNPs were obtained to evaluate 67 Citrus varieties and lines. A total of five parent–offspring relationships were newly found by MARCO based on the 206 SNP genotypes, indicating that ‘Kishuu mikan’ type mandarins (Citrus kinokuni hort. ex Tanaka accession ‘Kishuu mikan’ and ‘Nanfengmiju’) and ‘Kunenbo’ type mandarins (Citrus nobilis Lour. var. kunip Tanaka accession ‘Kunenbo’ and ‘Bendiguangju’) are possible parents of the satsuma mandarin. Moreover, cleaved amplified polymorphic sequences analysis showed that the genotypes of four regions in chloroplast DNA of ‘Kishuu mikan’ type mandarins were identical to that of the satsuma mandarin. Considering the historical background, satsuma mandarins may therefore derive from an occasional cross between a ‘Kishuu mikan’ type mandarin seed parent (derivative or synonym of ‘Nanfengmiju’) and a ‘Kunenbo’ type mandarin pollen parent (derivative or synonym of ‘Bendiguangju’).
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isolation and characterization of germacrene a synthases gene in Citrus Unshiu marc
Scientia Horticulturae, 2012Co-Authors: Takehiko Shimada, Ana M Rodriguez, Michiharu Nakano, Aiko Sugiyama, Tokuro Shimizu, Leandro Pena, Tomoko Endo, Hiroshi Fujii, Mitsuo OmuraAbstract:Abstract CuSTS1 cDNA clone was isolated from Satsuma mandarin ( Citrus Unshiu Marc.) and functionally characterized. Genomic clone corresponding to CuSTS1 consists of 7 exons and 6 introns which is typical structure of angiosperm sesquiterpene synthase genes. Their predicted proteins possess a general feature of plant sesquiterpene synthases and RR motif and DDXXD motifs were conserved. Phylogenetic tree analysis showed that the molecular evolutions of Citrus germacrene synthase might occur according to enzymatic function before the divergence of Citrus species, in contrast to Citrus monoterpene synthase genes. Functionally analysis indicated that CuSTS1 protein produces mainly germacrene A from farnesyl diphosphate (FPP) in accompany with side product. In Satsuma mandarin, transcription of CuSTS1 were comparatively abundant in flowers and young fruit at 60 days after flowering (DAF), and peel at 120 DAF. The transcription was decreasing toward fruit maturing. In young fruit at 120 DAF, CuSTS1 is induced by methyl jasmonic acid, salicylic acid and ethylene treatments, suggesting it would be involved in plant defense response.
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isolation and characterization of a new d limonene synthase gene with a different expression pattern in Citrus Unshiu marc
Scientia Horticulturae, 2005Co-Authors: Takehiko Shimada, Tomoko Endo, Hiroshi Fujii, Mitsuo OmuraAbstract:Abstract We isolated a new d-limonene synthase gene (CitMTSE2) from Satsuma mandarin (Citrus Unshiu Marc.) and compared its mRNA expression pattern with that of the previously isolated CitMTSE1. The predicted protein of CitMTSE2 showed 84.2% identity to that of CitMTSE1 at the amino acid level and possessed the typical structure and chemical features of general monoterpene synthases, such as transit peptides in the N-terminus, sequence motifs of RR and DDXXD, and cation-dependent reactions. A functional test demonstrated that CitMTSE2 is a d-limonene synthase gene. Under different expression regulations in response to the fruit developmental stage, the mRNA expression of CitMTSE2 indicated a specificity to fruit tissue and was different from that of CitMTSE1. Occurring mainly in peel at an early stage of fruit development, the mRNA expression profile of CitMTSE2 was similar to that of d-limonene biosynthesis in C. Unshiu.
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isolation and characterization of e beta ocimene and 1 8 cineole synthases in Citrus Unshiu marc
Plant Science, 2005Co-Authors: Takehiko Shimada, Tomoko Endo, Hiroshi Fujii, Masakazu Hara, Mitsuo OmuraAbstract:Abstract We isolated and functionally characterized CitMTSL1 and CitMTSL4 cDNA clones from Satsuma mandarin (Citrus Unshiu Marc.), which were identified as the 1,8 cineole and (E)-beta-ocimene synthase genes in Citrus species. Their predicted proteins possessed the general characteristics of plant monoterpene synthases, such as transit peptides in the N-terminus, RR sequences, and DDxxD motifs. Acyclic monoterpene synthase of CitMTSL4 had a unique structural feature that differentiated it from Citrus cyclic monoterpene synthase genes. Phylogenetic tree analysis showed that the molecular evolutions of Citrus monoterpene synthases might occur according to product specificity and enzymatic function before the divergence of Citrus species. In Satsuma mandarin, 1,8 cineole and (E)-beta-ocimene were comparatively abundant in flowers and then decreased toward fruit development. The transcript accumulation patterns of CitMTSL1 and CitMTSL4 were in good agreement with the biosynthesis of 1,8 cineole and (E)-beta-ocimene. We considered that the regulation of these gene expressions might be controlled according to some functional roles in plant development and that the different volatile profiles in tissues might depend on the gene expression.
Mitsuo Omura - One of the best experts on this subject based on the ideXlab platform.
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Characterization of γ‐terpinene synthase from Citrus Unshiu (Satsuma mandarin)
Biofactors, 2020Co-Authors: Yukiko Suzuki, Takehiko Shimada, Mitsuo Omura, Hiroshi Sakai, Shigenori Kumazawa, Tsutomu NakayamaAbstract:γ-Terpinene is a monoterpene and a major component of essential oils made from Citrus fruits and shows strong antioxidant activity in various assay systems. Plant γ-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized. It is necessary to prepare large amounts of γ-terpinene synthase from Citrus Unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E. coli) cells. As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E. coli cells harboring extra copies of the argU gene. The purified enzyme was incubated with GPP and the main product was confirmed to be γ-terpinene by GC/MS.
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parental diagnosis of satsuma mandarin Citrus Unshiu marc revealed by nuclear and cytoplasmic markers
Breeding Science, 2016Co-Authors: Hiroshi Fujii, Mitsuo Omura, Tomoko Endo, Satoshi Ohta, Keisuke Nonaka, Yuichi Katayose, Toshimi Matsumoto, Terutaka Yoshioka, Takehiko ShimadaAbstract:Satsuma mandarins (Citrus Unshiu Marc.) are the predominant cultivated Citrus variety in Japan. Clarification of its origin would prove valuable for Citrus taxonomy and mandarin breeding programs; however, current information is limited. We applied genome-wide genotyping using a 384 Citrus single nucleotide polymorphism (SNP) array and MARCO computer software to investigate the satsuma mandarin parentage. Genotyping data from 206 validated SNPs were obtained to evaluate 67 Citrus varieties and lines. A total of five parent–offspring relationships were newly found by MARCO based on the 206 SNP genotypes, indicating that ‘Kishuu mikan’ type mandarins (Citrus kinokuni hort. ex Tanaka accession ‘Kishuu mikan’ and ‘Nanfengmiju’) and ‘Kunenbo’ type mandarins (Citrus nobilis Lour. var. kunip Tanaka accession ‘Kunenbo’ and ‘Bendiguangju’) are possible parents of the satsuma mandarin. Moreover, cleaved amplified polymorphic sequences analysis showed that the genotypes of four regions in chloroplast DNA of ‘Kishuu mikan’ type mandarins were identical to that of the satsuma mandarin. Considering the historical background, satsuma mandarins may therefore derive from an occasional cross between a ‘Kishuu mikan’ type mandarin seed parent (derivative or synonym of ‘Nanfengmiju’) and a ‘Kunenbo’ type mandarin pollen parent (derivative or synonym of ‘Bendiguangju’).
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isolation and characterization of germacrene a synthases gene in Citrus Unshiu marc
Scientia Horticulturae, 2012Co-Authors: Takehiko Shimada, Ana M Rodriguez, Michiharu Nakano, Aiko Sugiyama, Tokuro Shimizu, Leandro Pena, Tomoko Endo, Hiroshi Fujii, Mitsuo OmuraAbstract:Abstract CuSTS1 cDNA clone was isolated from Satsuma mandarin ( Citrus Unshiu Marc.) and functionally characterized. Genomic clone corresponding to CuSTS1 consists of 7 exons and 6 introns which is typical structure of angiosperm sesquiterpene synthase genes. Their predicted proteins possess a general feature of plant sesquiterpene synthases and RR motif and DDXXD motifs were conserved. Phylogenetic tree analysis showed that the molecular evolutions of Citrus germacrene synthase might occur according to enzymatic function before the divergence of Citrus species, in contrast to Citrus monoterpene synthase genes. Functionally analysis indicated that CuSTS1 protein produces mainly germacrene A from farnesyl diphosphate (FPP) in accompany with side product. In Satsuma mandarin, transcription of CuSTS1 were comparatively abundant in flowers and young fruit at 60 days after flowering (DAF), and peel at 120 DAF. The transcription was decreasing toward fruit maturing. In young fruit at 120 DAF, CuSTS1 is induced by methyl jasmonic acid, salicylic acid and ethylene treatments, suggesting it would be involved in plant defense response.
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isolation and characterization of a new d limonene synthase gene with a different expression pattern in Citrus Unshiu marc
Scientia Horticulturae, 2005Co-Authors: Takehiko Shimada, Tomoko Endo, Hiroshi Fujii, Mitsuo OmuraAbstract:Abstract We isolated a new d-limonene synthase gene (CitMTSE2) from Satsuma mandarin (Citrus Unshiu Marc.) and compared its mRNA expression pattern with that of the previously isolated CitMTSE1. The predicted protein of CitMTSE2 showed 84.2% identity to that of CitMTSE1 at the amino acid level and possessed the typical structure and chemical features of general monoterpene synthases, such as transit peptides in the N-terminus, sequence motifs of RR and DDXXD, and cation-dependent reactions. A functional test demonstrated that CitMTSE2 is a d-limonene synthase gene. Under different expression regulations in response to the fruit developmental stage, the mRNA expression of CitMTSE2 indicated a specificity to fruit tissue and was different from that of CitMTSE1. Occurring mainly in peel at an early stage of fruit development, the mRNA expression profile of CitMTSE2 was similar to that of d-limonene biosynthesis in C. Unshiu.
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isolation and characterization of e beta ocimene and 1 8 cineole synthases in Citrus Unshiu marc
Plant Science, 2005Co-Authors: Takehiko Shimada, Tomoko Endo, Hiroshi Fujii, Masakazu Hara, Mitsuo OmuraAbstract:Abstract We isolated and functionally characterized CitMTSL1 and CitMTSL4 cDNA clones from Satsuma mandarin (Citrus Unshiu Marc.), which were identified as the 1,8 cineole and (E)-beta-ocimene synthase genes in Citrus species. Their predicted proteins possessed the general characteristics of plant monoterpene synthases, such as transit peptides in the N-terminus, RR sequences, and DDxxD motifs. Acyclic monoterpene synthase of CitMTSL4 had a unique structural feature that differentiated it from Citrus cyclic monoterpene synthase genes. Phylogenetic tree analysis showed that the molecular evolutions of Citrus monoterpene synthases might occur according to product specificity and enzymatic function before the divergence of Citrus species. In Satsuma mandarin, 1,8 cineole and (E)-beta-ocimene were comparatively abundant in flowers and then decreased toward fruit development. The transcript accumulation patterns of CitMTSL1 and CitMTSL4 were in good agreement with the biosynthesis of 1,8 cineole and (E)-beta-ocimene. We considered that the regulation of these gene expressions might be controlled according to some functional roles in plant development and that the different volatile profiles in tissues might depend on the gene expression.
Keishi Shimokawa - One of the best experts on this subject based on the ideXlab platform.
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A Water Soluble Ethylene-Induced Chlorophyllase in Peel of Citrus Unshiu Marc
Journal of The Japanese Society for Horticultural Science, 1999Co-Authors: Rie Azuma, Masaru Adachi, Keishi ShimokawaAbstract:To elucidate the mechanism of ethylene-enhanced degreening in satsuma mandarin (Citrus Unshiu Marc. c.v. Nichinan No. 1) fruits, a water-soluble chlorophyllase (chlorophyll-chlorophyllide-hydrolase, E.C. 3.1.1.14) was prepared from the peel of ethylene-treated fruits. The maximum catabolic rate of chlorophyll a (Chl a) to chlorophyllide a occurred in the presence of 30a40% concentration of acetone at 30°C. Optimum concentrations of substrate and protein for the reaction were 12.4 μM and 2.6 mg/4.5ml, respectively. The optimum pH value was 7.6 with a phosphate buffer, 7.86 with a borate buffer, and 7.5 with an acetate buffer. At pH 7.6 (phosphate buffer) and 25°C in the presence of 33% acetone, the tentative Km value for Chl a hydrolysis activity was approximately 6.5 μM. Moreover, when the reaction mechanism of Chl a degradation was investigated using the 3-dimensional fluorescence spectral measurements, fluorescent Chl catabolites (FCC, Ex/Em : 440/670 nm) were detected but the fluorescent Chl-ring cleavage catabolites (Ex/Em : around 320a360/440a460 nm) were not. The FCC formation was inhibited by p-chloromercuribenzoate (PCMB), an inhibitor of chlorophyllase, but the inhibition was reversible by the addition of reduced glutathione. These results suggest that Chl a degradation occurred through chlorophyllide a formation catalyzed by a water-soluble chlorophyllase from ethylene-treated Citrus Unshiu fruits. A possible participation of the enzyme in ethylene-enhanced degreening of the fruit peel is briefly discussed.
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A Pyropheophorbide a Degrading Enzyme of Citrus Unshiu Fruit Peel
Journal of The Japanese Society for Horticultural Science, 1999Co-Authors: Yoshiyuki Maeda, Masaru Adachi, Keishi ShimokawaAbstract:Acetone powder extracts (APE), prepared from Citrus Unshiu fruit, contained a pyropheophorbide a degrading enzyme. The enzyme catalyses the degradation of pyropheophorbide a in the presence of H2O2 and 2, 4-dichlorophenol (DCP). The optimum pH values were 6.2 with potassium phosphate buffer and 4.2 with acetate buffer. Linearity between the rate of the degradation and protein concentration existed over the range 0.0a250μg protein per 3.0ml of the reaction mixture. The Km for the pyropheophorbide a, DCP and H2O2 was ca. 140μM, ca. 1.3mM, and ca. 45μM, respectively. The enzymatic degradation of pyropheophorbide a was inhibited by ascorbate, hydroquinone, n-propyl gallate. We confirmed that tiron and potassium cyanide (KCN) are involved with free radicals. Pyropheophorbide a degradation by APE resulted in the opening of the chlorophyll-ring and a decrease in the red and Soret bands of the UV/VIS differential spectrum of the reaction mixture. Moreover, pyropheophorbide a and pheophorbide a degradation by horseradish peroxidase (HRP) followed a similar reaction pattern. The velocity of pheophorbide a degradation by APE was slower than that of pyropheophorbide a under the same reacting condition. These results suggest that pyropheophorbide a-degrading enzyme from Citrus Unshiu fruit is a peroxidase isozyme.
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Degreening of Citrus Unshiu Fruits Via Ethylene-induced Soluble Chlorophyllase
Journal of The Japanese Society for Horticultural Science, 1999Co-Authors: Rie Azuma, Hirofumi Kurata, Masaru Adachi, Keishi ShimokawaAbstract:The mechanism of ethylene action on degreening in satsuma mandarin (Citrus Unshiu Marc. cv. Nichinan No.1) fruits was investigated using inhibitors of protein synthesis and soluble chlorophyllase (chlorophyll-chlorophyllide hydrolase, Chlase ; E.C. 3.1.1.14). This enzyme which catalyzes the dephytylation of chlorophyll (Chl) to chlorophyllide (Chlide), was partially purified. Chlase catalyzes the first step in the Chl breakdown process. Ethylene treatments promoted the degreening and increased soluble Chlase activity of C. Unshiu fruit peels held in the dark. Cycloheximide (CH), an inhibitor of cytoplasmic protein synthesis, inhibited the effects of ethylene, whereas chloramphenicol (CP), an inhibitor of plastid protein synthesis, also weakly inhibited the Chlase activity. These results suggest that ethylene appears to enhance the degreening of the peels through de novo synthesis of Chlase. Moreover, de novo cytoplasmic protein synthesis and chloroplast-dependent enzyme synthesis may regulate Chlase activity in the peel of ethylene-treated C. Unshiu fruits. Soluble Chlase was precipitated with acetone from C. Unshiu fruit peels and purified by the following method : after ammonium sulfate fractionation, gel-filtration through Sephadex G-25 and Phenyl Sepharose CL-4B (hydrophobic chromatography) were used. The soluble Chlase was purified ca. 203.5-fold with a yield of 45.5%.
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A Possible Participation of Pyropheophorbide a in Chlorophyll Catabolism during Ripening of Citrus Unshiu Fruits
Journal of The Japanese Society for Horticultural Science, 1998Co-Authors: Hirofumi Kurata, Yoshiyuki Maeda, Masaru Adachi, Keishi ShimokawaAbstract:Chlorophyll (chl) derivatives in the peel of green satsuma mandarin (Citrus Unshiu Marc. cv Nichinan No.1) fruits were studied to clarify the mechanism of chl degradation. High performance liquid chromatography (HPLC) and high performance thin-layer chromatography (HPTLC) were used to separate chl derivatives in acetone extracts from green satsuma mandarin fruits. Pyropheophorbide (pyrophed) a was present in the green skin but it gradually disappeared with ripening ; it was undetectable in the last harvest. A possible participation of pyrophed a in chl catabolism is discussed.
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Chlorophyll catabolism in ethylene-treated Citrus Unshiu fruits
Journal of The Japanese Society for Horticultural Science, 1998Co-Authors: Yoshiyuki Maeda, Hirofumi Kurata, Masaru Adachi, Keishi ShimokawaAbstract:Changes in the levels of chlorophylls (Chls) and their derivatives were determined to clarify the Chl metabolic pathway in ethylene-treated satsuma mandarin (Citrus Unshiu Marc. cv. Nichinan No.1) fruits held in the dark. The fruits were treated with or without 200 ppm ethylene for 16 hr in a 20-1 desiccator at 25°C in darkness. After the treatment, the fruits were further stored in fresh air at 25°C in the dark for 72 hr. Chl pigments in the peel monitored with HPLC revealed that they remained unchanged during ethylene treatment. After 40 hours of treatment, a decrease occurred in the levels of Chl a, Chl b, and pheophytin a. With or without ethylene, low levels of chlorophyllide a (Chlide a), 132-Hydroxy (HO) Chl a, pheophorbide a (Phed a), and pyropheophorbide a (Pyrophed a) were eluted as Chl a derivatives. Chlide b and its derivatives could not be detected. The ratio of Chl a to b in the ethylene-treated fruits increased significantly with the advance of degreening. The ethylene-induced Chl metabolic process in Citrus Unshiu fruits is discussed.
Shou Hiasa - One of the best experts on this subject based on the ideXlab platform.
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isolation of cellulose nanofibrils from mandarin Citrus Unshiu peel waste
Industrial Crops and Products, 2014Co-Authors: Shou Hiasa, Shinichiro Iwamoto, Takashi Endo, Yusuke EdashigeAbstract:Abstract Cellulose obtained from mandarin (Citrus Unshiu) peel waste was purified by the removal of oil, coloring substances, and pectin. Two pectin-removing methods, namely multistep and hydrothermal treatments, were investigated to compare their purification efficiencies. The multistep treatment consisted of three steps: removal of metal in pectin, depolymerization, and dissolution of pectin. In contrast, the hydrothermal treatment comprises a single step process involving a solution of 0.18 wt% hydrochloric acid. Fourier-transform infrared spectroscopy and neutral sugar content analysis of the purified cellulose from the mandarin peel waste indicated that the hydrothermal treatment was more effective for the purification of cellulose, as compared to the multistep treatment. The crystal width of the purified cellulose was determined from X-ray diffraction analysis, and it revealed a smaller crystal width (2.5 nm) than that of wood cellulose (3.9 nm). After the pectin removal, the purified cellulose from the mandarin peel waste was fibrillated by sonication to obtain cellulose nanofibrils, yielding cellulose fibers with widths of 2–3 nm, as observed by atomic force microscopy. The observed fiber width corresponded to the crystal width, indicating that the cellulose nanofibrils were completely individualized by sonication.
Nagaraj Basavegowda - One of the best experts on this subject based on the ideXlab platform.
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synthesis of silver nanoparticles using satsuma mandarin Citrus Unshiu peel extract a novel approach towards waste utilization
Materials Letters, 2013Co-Authors: Nagaraj BasavegowdaAbstract:Abstract Silver nanoparticles (AgNPs) were synthesized using Satsuma mandarin (Citrus Unshiu) peel extract without adding external surfactant and capping agent. Aqueous silver (Ag+) ions when exposed to Satsuma mandarin peel extract were reduced and formed silver nanoparticles in the size range 5–20 nm. The AgNPs produced were characterized by UV-visible, X-ray diffraction (XRD), transmission electron microscopy (TEM), and by energy dispersive X-ray analysis (EDX). This study demonstrates the feasibility of using fruit wastes for the synthesis of AgNPs.