Cloning, heterologous expression, and functional characterization of a chitinase gene, Lbchi32, from Limonium bicolor.
In the present study, an endochitinase gene, Lbchi32, was cloned from Limonium bicolor. The cDNA sequence of Lbchi32 was 1,443 bp in length and encoded 319 amino acid residues. The DNA sequence of Lbchi32 was 2,512 bp in length and contained three exons and two introns. The Lbchi32 gene was inserted into a pPIC9 vector and transferred into Pichia pastoris strains GS115 and KM71 for heterologous expression. SDS-PAGE analyses indicated that LbCHI32 was expressed in both GS115 and KM71 and that it was secreted extracellularly. The optimal reaction conditions for LbCHI32 activity are 45 degrees C, pH 5.0, and 5 mM Ba(2+). The LbCHI32 enzyme can efficiently degrade chitin, chitin derivatives, and the cell walls of different pathogenic fungi, including phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Valsa sordida, Septoria tritici, and Phytophthora sojae. These findings suggest that Lbchi32 has potential use in the degradation of chitin and chitin derivatives.
Liu
Zhi Hua
ZH
Northeast Forestry University, Ministry of Education, Harbin, China.
Yang
Chuan Ping
CP
Qi
Xiao Tian
XT
Xiu
Li Li
LL
Wang
Yu Cheng
YC
D000595:Amino Acid Sequence; D055162:Biocatalysis / Q000187:drug effects; 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 / 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
D008670:Metals; D011994:Recombinant Proteins; D002688:Chitinases