Screening,Identification And Application Of Novel Glycosaminoglycan-degrading Enzymes From Marine Bacteria

Glycosaminoglycans(GAGs)also known as mucopolysaccharides,are a family of liner heteropolysaccharides that composed of repeating disaccharide units.Based on the chemical structure,GAGs are classified into four basic families:hyaluronic acid(HA),heparin/heparan sulfate(Hep/HS),chondroitin sulfate/determatan sulfate(CS/DS)and keratan sulfate(KS).Glycosaminoglycan chains are usually complicated due to the modification of sugar chains by various enzymes,which mainly includes epimerization of D-glucuronic acid(GlcUA)residues to L-iduronic acid(IdoUA)by the action of glucuronyl C-5 epimerase,sulfation of different-OH or/and-NH2 groups by specific sulfotransferases,acetylation of-NH2 on the C-2 of glycoaylamine,and so on.The GAG chains have the potential to display an enormous microheterogeneity,which is the structure basis for various biological functions.GAGs are ubiquitously distributed on cell surfaces and in extracellular matrices of animals,and are involved in a wide array of biological processes such as cell proliferation and differentiation,cell recognition,cell adhesion,tissues morphogenesis,oncogenesis and other physiological and pathological processes.Glycosaminoglycans,have a series of important biological functions,and are widely used in medicine and functional food.GAGs,especially sulfated GAGs,have prominent structural heterogeneity,which leads same GAG from different sources or batches to be different in their activity.A large number of studies have shown that GAG polysaccharides exert their biological function by interaction with specific proteins through special structure of functional domains.Different functional domains with specific structure in same GAG polysaccharide chains interact with different proteins.Therefore,specific functional domains with uniform or relative homogenous structure can be prepared by digestion using substrate specific GAG-degrading enzymes,modification using glycosaminoglycans sulfatase,and sequencing using GAG exo-lyases.These GAG-degrading enzymes with specific activity are not only useful tools in GAGs structure-activity relationship study and bioactive oligosaccharide preparation of GAG,but also potential therapeutic agents for treatment of injuries to thr central nervious system.However,there are only few GAG enzymes commercially available,and thus the screening and indentification of novel GAG enzymes are important for GAG structure-activity relationship research.The ocean is regarded as the origin of the life on the earth and hosts most life forms.There are several types of GAGs were found from marine animals,and the abundand presence of GAGs indicates the existence of GAG-degrading microbes.However,no GAG-degrading enzyme from marine bacteria has yet been characterized in detail.In the present study,a marine Vibrio strain with high GAG-degrading capacity was isolated using CS-C from shark cartilage as the sole carbon source.The bacterial genome was sequenced to identify the genes of GAG-degrading enzymes.The main results are summarized as follows:(a)Screening of GAG-degarding bacteria:Chondroitin sulfate from shark cartilage as sole carbon source was used to isolate CS-degrading bacteria from the marine sediments and the resulted 15 isolates could degrade more than three type of polysaccharides.The Virbrio sp.FC509 strain,one of the 15 polysaccharide-degarding marine bacteria,could degrade and utilize alginate?CS?DS?HA and Hep for its growth.Thus,FC509 strain was chosen for the following in-depth study.The genome of Vribio sp.509 was sequenced and several putative GAG-degrading enzyme genes were found and indentified in detail.(b)Study of a novel GAG endo-lyase HCLase:A novel GAG-degrading enzyme gene from Virbrio sp.FC509 was cloned,heterologously expressed,purified and characterized in detail.It showed strong lyase activity toward HA and CS,and was designated as HA and CS lyase(HCLase).As with most other marine enzymes,HCLase is a halopilic enzyme.In the degradation experiments of GAGs,HCLase shows that its minimum substrate is tetrasaccharide and minimum product is disaccharide.A HCLase-resistant tetrasaccharide ?4,5HexUAal-3GalNAc(6S)?1-4GlcUA(2S)?1-3 GalNAc(6S)was isolated from CS-D,the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated GlcUA in CS chains.HCLase,the first GAG lyase from marine bacterium which has super high enzyme activities,is a useful tool for GAG-ralated research and application.(c)Study of a novel GAG exo-lyase HCDLase:The gene of HCDLase was cloned and heterologous expressed in E.coli.Native enzyme was loaded onto nikel-Sepharose column for farther purification,and then analyzed its biological activities.Since it showed exolytic lyases activity towards towards HA,CS and DS,it was designated as HCDLase.HCDLase can degrade all types of natural CS,DS and HA oligosaccharides.It should be noted,however,that HCDLase cannot digest 2-AB-labeled DS/HA oligosaccharides,suggesting that disaccharide structures and bond types can affect the activity of HCDLase when oligosaccharides are labeled at the reducing ends.Time course experiments of 2-AB-labeled CS oligosaccharides digested by HCDLase showed that HCDLase is an exo-lyase that digests GAG from their reducing ends.Most notably,exosequencing of CS hexasaccharides and an octasaccharide was accomplished using HCDLase and CSase ABC.The identification of HCDLase provides a useful tool for CS-related research and applications.(d)Study of a CS/DS 4-O-Endosulfatase:The gene of 4-O-Endosulfatase was cloned and heterologous expressed in E.coli.Native enzyme was also loaded onto nikel-Sepharose column for farther purification,and then analyzed its biological activities.The recombinant protein showed a specific N-acetylgalactosamine-4-O-sulfatase activity that removes 4-O-sulfate from both disaccharides and polysaccharides of CS/DS.The novel endosulfatase exhibited maximal reaction rate in a phosphate buffer(pH 8.0)at 30? and effectively removed 65-17%of 4-O-sulfates from various CS and DS.As the first CS endosulfatase whose enzyme activities were analyzed in detail,this 4-O-Endosulfatase will play an important role in investigating the structure-activity relationship study and regulating the sulfation pattern of CS polysaccharides.