Study On The Heterologous Recombinant Expression, Characteristics And Functions Of Chitin Deacetylase In Coprinus Chinensis

The enzyme chitin deacetylase belongs to carbohydrate esterase family 4(CE-4s),it can convert the cell wall chitin into the chitosan with differing degree of acetylation.According to the different pathway,chitin deacetylases were divided into two types,the one is called exo-type chitin deacetylases that have the multiple attack mechanism,another is called endo-type chitin deacetylase which have the multiple chain mechanism.During some certain fungus infect host cells,the organism will insect chitin deacetylase which can make the cell wall chitin into chitosan in order to assist in self defense from host chitinase and protect integrity of the cell wallIn order to explore the role of chitin deacetylation in the process of cell wall maturation,we attempted to clone,express and characterize the putative chitin deacetylases in C.cinerea genome.Four chitin deacetylases,Cda2 and 429 that have a high level in the apical and have the deacetylation activity,0.06× 10-6 U/mg and 0.03 × 10-6 U/mg,respectively,Cda2 and 884 that have a high level in the basal and have the deacetylation activity,1.08 × 10-6 U/mg and 0.35 × 10-6 U/mg,respectively,are expressed successfully in yeast cells.We studied Cdal that have higher enezyme activity than 884 and Cda2 that have higher enezyme activity than 429.Using the high performance anion exchange chromatography(HAPEC-PAD)and Q-TOF-MSMS,we study the effect of Cdal and Cda2 on chitin oligosaccharides(dp 1-6)and find that two enzymes have no effect on GlcNAc,but have part influence on(GlcNAc)2.Cdal preferably deacetylates the non-reducing end residue of(G1cNAc)2,the internal or non-reducing end residue of(GlcNAc);,and the non-reducing residue of(GlcNAc)4-6 after deacetylating the internal residues.In contrast,Cda2 preferably deacetylates the reducing end residue of(GlcNAc)2,the internal or reducing end residue of(GlcNAc)3,and the reducing residue of(GlcNAc)4-6 after deacetylating the internal residues.Furthermore,Cdal prefers chitohexaose with higher degrees of acetylation for deacetylation,while Cda2 shows a weaker preference for chitohexaose with varying degrees of acetylation.The predicted Cdal structure shows more hydrophobic aromatic amino acids on the surface near subsite+ 1 in the active site than on the surface near subsite-1,whereas the predicted Cda2 structure has more hydrophobic aromatic amino acids on the surface near subsite-1 than on the surface near subsite +1,which may be the molecular basis of the distinctive catalytic features between Cdal and Cda2.In order to study further the physiological function of these two enzymes in the growth of the stipe,we analyzed the expression levels of these two enzymes.By the research,Cdal has a high transcription level in the non-elongating basal stipe region,whereas Cda2 has a high transcription level in the elongating apical stipe region,and the transcription level of the former is approximately five times that of the latter.Furthermore,using the 3-methyl-2-benzothiazone hydrochloride to determine the content of GlcNAc and GlcN in the cell wall of different regions.We find that the molar ratio of GlcN/GlcNAc increased from 0.15 in the cell wall of the apical stipe region to 0.22 in the cell wall of the basal stipe region,it shows the cell wall of stipe contains chitosan with differing degree of acetylation,furthermore,the chitosan in the cell wall increased along the length of the stipe from the rapidly elongation apical region to the nonelongating basal region of the stipe,implying that deacetylation may play a role in the maturation of the basal stipe call wall.The study finds that two chitin deacetylases Cdal and Cda2 in the action in deacetylation and the relative expression in different stipe regions have some differences,we propose that the differences may be related to the function in the different regions of stipe during the development of Coprinopsis cinerea.Cda2,a high expression in the apical,is favour to deacetylase the reducing end residue of free chitin in the growing stipe region.While,in the mature stipe regions,Cdal preferentially deacetylases non-reducing end residues of chitin chains which be linked to(3-1,3-glucan;deacetylation of Cdal may break out the balance between plasticity and rigidity of cell wall,resulting in the cell wall maturation and cessation of stipe elongation.