Study On The Catalytic Mechanism Of Chitin Deacetylase With Novel Deacetylation Mode

Chitin is a linear polysaccharide ofβ-1,4-linked N-acetylglucosamine（GlcNAc）units.It is the main component of the exoskeleton of crustaceans such as shrimp,crab and shellfish.Chitin,a renewable raw material whose annual production is about 10¹¹ tons,is the second most abundant natural biopolymer after cellulose.As a new functional material,chitin has attracted wide attention in various fields.However,it is insoluble in water and most organic solvents,and this property severely limits its development and application.Chitin can be converted to chitooligosaccharides（COS）by complete deacetylation or partial deacetylation and hydrolysis.COS is a family of functional biopolymers with diverse structures.The structure of COS can be described by three parameters:degree of polymerization（DP）,degree of acetylation（DA）,and pattern of acetylation（PA）.Although we have a basic understanding of the structural and functional relationships between DP and DA in COS molecules,little is known about the effects of PA.Due to the lack of COS with specific PA and appropriate analytical methods,the PA of COS is difficult to solve experimentally,so it is challenging to elucidate the relationship between the acetylation pattern of chitosan and the molecular structure and function.In addition the chemically produced COS always shows a random acetylation pattern.In contrast,COS produced by chitin deacetylase（CDA）can have a specific acetylation pattern,as shown by some bacterial CDAs.However,compared to our understanding of bacterial CDA,we know very little about the fungal CDA producing COS with a specific acetylation pattern.A chitin deacetylase from Saccharomyces cerevisiae,ScCDA₂,which can produce fully defined COS has been characterized which solves the randomization of acetylation patterns during chemical production.ScCDA₂ also exhibits about 8%and 20%deacetylation activity on crystalline chitin and colloid chitin,respectively.In addition,the enzyme productions were separated and detected by high performance liquid chromatography and electrospray ionization mass spectrometry（HPLC-ESI-MS）.Mass spectrometry sequencing revealed that ScCDA₂produced COS with specific acetylation patterns of DAAA,ADAA,AADA,DDAA,DADA,ADDA and DDDA,respectively.So ScCDA₂ does not deacetylate the GlcNAc unit that is closest to the reducing end of the oligomer.Therefore,ScCDA₂ has a multiple-attack catalytic mechanism on chitin oligosaccharides.This specific mode of action significantly enriches the existing limited library of chitin deacetylase deacetylation patterns.And these fully defined COS may be used in the study of COS structure and function.