Heterologous expression and characterization of class III chitinases from rice (Oryza sativa L.)

Park, Seung-Moon; Kim, Dae-Hyuk; Truong, Nam Hai; Itoh, Yoshifumi

doi:10.1016/s0141-0229(02)00042-x

Cited by 22 publications

(23 citation statements)

References 21 publications

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“…We incubated P. pastoris cells harboring a recombinant chitinase cDNA in BMMY medium (50 ml) at 309 C for 36 h to fully express the chitinase as described. 21) The chitinases were puriˆed by passage through a Ni 2＋ -a‹nity column (His-Trap column, Amersham Biotech; Buckinghamshire, UK) according to Park et al 21) Puriˆed enzymes were thoroughly dialyzed against 20 mM phosphate buŠer (pH 7.2) containing 1 mM EDTA. The induction and purity of the enzyme were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).…”

Section: Methodsmentioning

confidence: 99%

“…After the indicated incubation period, reaction products were resolved and detected on silica gel 60 plates (Merck; Darmstadt, Germany) as described. 21) Antifungal activity was assayed by disk-plate diŠusion 26) using Trichoderma reesei IFO31329 as the test fungus.…”

Section: )mentioning

confidence: 99%

“…25) Trichoderma reesei conidia (about 10 3 ) were inoculated onto paper disks (T) placed at the center of plates and incubated for 24 h at 259 C. Enzymes were applied in various amounts onto disks placed at edges of extending hyphae and plates were incubated for a further 24 h. Amounts of the enzymes applied: 1, 0 mg; 2, 1 mg; 3, 2 mg; 4, 5 mg; 5, 10 mg. N-Acetylchitooligosaccharides were incubated with OsChia1c or OsChia2b (1 mg each) at 379 C for 30 min and reaction products at indicated incubation times were analyzed by TLC. 21) The N-acetylchitooligosaccharides used as substrates are: 1, (GlcNAC) 4 ; 2, (GlcNAC) 5 ; 3, (GlcNAC) 6 . M, standard N-acetylchitooligosaccharides.…”

Section: Chitin-binding A‹nity Of Cbd and Its Increase Of Antifungal mentioning

confidence: 99%

“…After the indicated incubation periods, reaction products were analyzed by TLC. 21) M, Nacetylchitooligosaccharide standards.…”

Section: Catalytic Propertiesmentioning

confidence: 99%

“…To further understand the physiological roles of rice chitinases, a complete set of the relevant genes must be identiˆed and the properties of the corresponding products should be understood. We have identiˆed and characterized two family 18 chitinase (OsChib3a and OsChib3b) cDNAs 21) in the EST library (about 50,000 clones) of Nipponbare that had been constructed by the Rice Genome Project (http://rgp.dna.aŠrc.go.jp).…”

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confidence: 99%

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Structure, Heterologous Expression, and Properties of Rice (Oryza sativaL.) Family 19 Chitinases

Truong

Park

Nishizawa

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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Section: Methodsmentioning

confidence: 99%

Section: )mentioning

confidence: 99%

Section: Chitin-binding A‹nity Of Cbd and Its Increase Of Antifungal mentioning

confidence: 99%

“…After the indicated incubation periods, reaction products were analyzed by TLC. 21) M, Nacetylchitooligosaccharide standards.…”

Section: Catalytic Propertiesmentioning

confidence: 99%

mentioning

confidence: 99%

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Structure, Heterologous Expression, and Properties of Rice (Oryza sativaL.) Family 19 Chitinases

Truong

Park

Nishizawa

et al. 2003

Bioscience, Biotechnology, and Biochemistry

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A comparative proteomic approach to analyse structure, function and evolution of rice chitinases: a step towards increasing plant fungal resistance

et al. 2012

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Glycoside hydrolase family 19 chitinases (EC 3.2.1.14) widely distributed in plants, bacteria and viruses catalyse the hydrolysis of chitin and play a major role in plant defense mechanisms and development. Rice possesses several classes of chitinase, out of which a single structure of class I has been reported in PDB to date. In the present study an attempt was made to gain more insight into the structure, function and evolution of class I, II and IV chitinases of GH family 19 from rice. The three-dimensional structures of chitinases were modelled and validated based on available X-ray crystal structures. The structural study revealed that they are highly α-helical and bilobed in nature. These enzymes are single or multi domain and multi-functional in which chitin-binding domain (CBD) and catalytic domain (CatD) are present in class I and IV whereas class II lacks CBD. The CatD possesses a catalytic triad which is thought to be involved in catalytic process. Loop III, which is common in all three classes of chitinases, reflects that it may play a significant role in their function. Our study also confirms that the absence and presence of different loops in GH family 19 of rice may be responsible for various sized products. Molecular phylogeny revealed chitinases in monocotyledons and dicotyledons differed from each other forming two different clusters and may have evolved differentially. More structural study of this enzyme from different plants is required to enhance the knowledge of catalytic mechanism and substrate binding.

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Cloning and characterization of a chitinase gene Lbchi31 from Limonium bicolor and identification of its biological activity

Liu

Wang

et al. 2009

Mol Biol Rep

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Chitinases are digestive enzymes that break down glycosidic bonds in chitin. In the current study, an endochitinase gene Lbchi31 was cloned from Limonium bicolor. The cDNA sequence of Lbchi31 was 1,107 bp in length, encoding 322 amino acid residues with a calculated molecular mass of 31.7 kDa. Clustal analysis showed that there was a highly conserved chitin-binding domains in Lbchi31 protein, containing four sulfide bridges. The Lbchi31 gene was inserted into the pPIC9 vector and transferred into yeast Pichia pastoris GS115 and KM71 for heterologous expression. The transformant harboring the Lbchi31 gene showed a clearly visible protein band with a molecular mass of more than 31 kDa in the SDS-PAGE gel, indicating that it had been translated in P. pastoris. Enzyme characterization showed that the optimal reaction condition for chitinase LbCHI31 activity was: 40 degrees C, pH of 5.0 and 5 mmol l(-1) of Mn(2+). The maximum enzyme activity was 0.88 U ml(-1) following exposure to the cell wall chitin of Valsa sordida. The LbCHI31 enzyme can efficiently degrade cell wall chitin of the phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, V. sordida, Septoria tritici and Phytophthora sojae, suggesting that it has the biocontrol function to fungal phytopathogen.

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Heterologous expression and characterization of class III chitinases from rice (Oryza sativa L.)

Cited by 22 publications

References 21 publications

Structure, Heterologous Expression, and Properties of Rice (Oryza sativaL.) Family 19 Chitinases

Structure, Heterologous Expression, and Properties of Rice (Oryza sativaL.) Family 19 Chitinases

A comparative proteomic approach to analyse structure, function and evolution of rice chitinases: a step towards increasing plant fungal resistance

Cloning and characterization of a chitinase gene Lbchi31 from Limonium bicolor and identification of its biological activity

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