Studies On Gene Cloning And Catalytic Characteristics Of A Novel Microbial Chondroitin Sulfate AC Lyase

Glycosaminoglycans (GAGs) are polysaccharides that play vital functional roles in numerous biological processes, and the compounds belonging to this class have been implicated in a wide variety of diseases. Chondroitin sulfate AC lyase (ChnAC) (EC 4.2.2.5) catalyzes the degradation of various GAGs, including chondroitin sulfate and hyaluronic acid, to give the corresponding disaccharides containing a 4-unsaturated uronic acid at their non-reducing terminus. ChnAC has been isolated from various bacteria and utilized as an enzymatic tool for studying and evaluating the sequencing of GAGs.Our group had screened an Arthrobacter sp. from the soil, named as SSAs-1, which could efficiently generate chondroitin sulfate lyase (ChSase). The main achievements of this thesis include:1. The coding gene (AsChnAC) of ChSase was cloned from SSAs-1 by means of molecular biology. Firstly we designed degenerate primers and amplified the middle part of AsChnAC using polymerase chain reaction(PCR); then the downstream and upstream sequences of AsChnAC were obtained by inverse PCR and genome walking technology separately; finally, the initiation codon (ATG), the termination codon (TAG) and the open reading frame of AsChnAC were determined by sequencing the PCR products.2. The recombinant ChSase was efficiently expressed in E. coli expression system.3. The ChSase cloned from SSAs-1 was identified as chondroitin sulfate AC lyase through the homology comparison analysis of amino acid sequence and the substrate specificity analysis of purified enzyme. So the purified enzyme was named AsChnAC.4. The enzymatic parameters of AsChnAC were analyzed systematically and the cleavage direction of it was determined using model substrates. Despite the substrate specificites and the crystal structures of ChnAC from microorganisms have been determined, the direction in which ChnAC catalyzes the cleavage of oligosaccharides remains unclear because of the lacking of model substrates. Herein, the structural cues of substrate depolymerization and the cleavage direction of ChnAC were determined using model substrates and the recombinant AsChnAC. Several structurally defined oligosaccharides were synthesized using a chemoenzymatic approach and subsequently cleaved using AsChnAC. The degradation products resulting from this process were determined by mass spectrometry. The results revealed that AsChnAC cleaved the β-1,4-glycosidic linkages between glucuronic acid and glucosamine units when these bonds were located on the reducing end of the oligosaccharide. In contrast, the presence of a-GlcNAc-α1,4-GlcA-unit at the reducing end of the oligosaccharide prevented AsChnAC from cleaving the -GalNAc-β1,4-GlcA-moiety located in the middle or at the non-reducing end of the chain. These interesting results therefore provide direct proof that ChnAC cleaves oligosaccharide substrates from their reducing end toward their non-reducing end. This conclusion will therefore enhance our collective understanding of the mode of action of ChnAC.