Screening And Identification Of Novel Chondroitin Sulfate/Dermatan Sulfate Degrading Enzymes Derived From Intestinal Microbes

Chondroitin sulfate(CS)/Dermatan sulfate(DS)is a kind of polyanionic linear polysaccharides,which are repeatedly linked with disaccharides composed of glucuronic acid(GlcUA)/iduronic acid(IdoUA)and N-acetylgalactosamine(GalNAc)by β1-4 glycosidic bonds.Different sulfation pattern of disaccharide units complicates structures of polysaccharides,which seriously hinders the research on CS/DS.CS/DS-degrading enzymes are indispensable tools for the structural and functional studies of CS/DS.Currently,however,the available CS/DS-degrading enzymes are still very limited.Therefore,it is of great significance to find new CS/DS-degrading enzymes.There are a large number of polysaccharide-degrading bacteria in the mammalian intestine,which is a huge resource treasure for mining new polysaccharide-degrading enzymes.In our study,we acclimated the intestinal flora by long-term feeding of mouse with CS-A,combined with the unique carbon source culture in vitro to enrich the intestinal CS/DS-degrading bacteria,and used metagenomic sequencing to obtain a large number of suspected CS/DS degrading enzyme genes,and performed in-depth study on two novel CS/DS lyases(CSlyl and CSly2)and one novel CS/DS sulfatase(SF2)of them.The main results are as follows:CS/DS lyase CSlyl:The full length of cslyl gene is 3087 bp with a GC content of 54.10%,and it encodes a protein of 1068 amino acids with an isoelectric point of 6.99.Blast multiple sequence alignment showed that the primary sequence of CSlyl contains a lyase catalytic module.Although it belongs to the polysaccharide lyase superfamily 8(PL-8),it has very low sequence similarity with the identified CS/DS lyases(32%).The results of enzymatic properties and substrate degradation pattern analysis showed that CSlyl could use HA and CS/DS as substrates,and only produce unsaturated disaccharide products during the degradation process,which indicated that CSlyl was an exotype CSase ABC.The optimal reaction conditions of CSlyl were 40℃ and 50 mM NaAc-HAc(pH 6.0),and various metal ions K+,Ca2+,Mg2+and Ba2+had significant promoting effects on its enzyme activity.It was determined that CSlyl exhibited different specific enzyme activities for HA,CS-A,CS-C,CS-D,CS-E and DS substrates,which were 79 mU/mg,88 mU/mg,572 mU/mg,936 mU/mg,2574 mU/mg,2250 mU/mg,respectively.It is clear that this enzyme tends to degrade highly sulfated CS-D and CS-E as well as DS,very different from other CSase ABCs that have been identified,and is a great novelty.In addition,CSlyl can degrade CS-A octasaccharide fluorescently labeled by 2-AB at the reducing end,resulting in CS-A hexasaccharide and CS-A tetrasaccharide,but not CS-A tetrasaccharide and HA tetrasaccharide,indicating reducing end fluorescence The labeled pair inhibits the degradative activity of CSly1.In conclusion,as a novel exo-type CSase ABC,it has important application potential and research value in the study of the structure-activity relationship of CS/DS sugar chains and the enzymatic sequencing of oligosaccharides with special structures.CS/DS lyase CSly2:The csly2 gene sequence consists of 2250 bases with a GC content of 58.57%,encoding a protein with 2250 amino acids and an isoelectric point of 8.85.Blast analysis showed that CSly2 belongs to the polysaccharide lyase superfamily 8(PL-8).The optimum reaction conditions of CSly2 were 40℃ and 50 mM NaH2PO4-Na2HPO4(pH 8.0).Metal ions such as K+,Na+,Ca2+,Mg2+ could improve the rate of the enzymatic reaction.CSly2 can degrade HA and CS/DS,but it is different from typical CSase ABC with quite low activity to HA comparing with those to CS/DS substrates.The specific enzymatic activities of CSly2 against HA,CS-A,CS-C,CS-D and DS were 5.301 U/mg,1155 mU/mg,835 mU/mg,507 mU/mg,2028mU/mg,respectively.Different from CSly1,its products contain unsaturated disaccharides and larger oligosaccharides,which indicates that it is an endo-type CSase ABC,and it has important application value in the oligosaccharide preparation and structural study of CS/DS.Sulfatase SF2:The full-length sf2 gene sequence is 1467 bp,and its GC content is 47.03%.The isoelectric point of SF2 is 6.83.The homologous sequence alignment analysis showed that SF2 was clustered with CS/DS 2-O-sulfatase,but had very low homology with 4-O-sulfatase and 6-O sulfatase,and so it was speculated that SF2 was a 2-O-sulfatase.The optimal reaction conditions of SF2 were 40℃ and 50 mM Tris-HCl(pH 9.0).Metal ions such as Na+,K+,Ca2+,Ba2+,Mg2+ had a significant effect on the enzymatic activity of SF2,and the enzyme activity was is 139 mU/mg.Substrate degradation pattern analysis indicate that SF2 can only remove the 2-O-sulfate of the unsaturated disaccharide ΔHexUA(2S)β1-3GalNAc(6S),but not the sulfate at other positions,and thus belongs to CS/DS 2-O-sulfatase.SF2 has important application value in the sulfation pattern editing and functional study of CS/DS oligosaccharides.