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.

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.