Cloning and characterization of a chitinase gene Lbchi31 from Limonium bicolor and identification of its biological activity.
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.
Liu
Zhi Hua
ZH
Key Laboratory of Forest Tree Genetic Improvement and Biotechnology, Ministry of Education, Northeast Forestry University, Harbin 150040, China.
Wang
Yu Cheng
YC
Qi
Xiao Tian
XT
Yang
Chuan Ping
CP
D000595:Amino Acid Sequence; D002688:Chitinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D017343:Genes, Plant / Q000235:genetics*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D010843:Pichia / Q000187:drug effects / Q000378:metabolism; D029606:Plumbaginaceae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D008670:Metals; D011994:Recombinant Proteins; D002688:Chitinases
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