Enzymatic Synthesis Of Chondroitin Sulfate And Heparin

Glycosaminoglycan,a linear polysaccharide,is widely distributed on the cell surface or in the extracellular matrix.Glycosaminoglycans consist of a disaccharide repeating unit?GlcUA-GalNAc,or GlcUA-GluNAc,GlcUA=glucuronic acid,GalNAc=N-acetylgalactosamine,GluNAc=N-acetylglucosamine?with N-sulfate,O-sulfate modification,or GlcUA epimerized modification.Their diversity structure gives rise to a wide range of biological functions,such as regulation of cellular growth,proliferation,and differentiation.To data,glycosaminoglycans have been applicated in cosmetics,cosmetic medicine,medicine,and so on.For example,chondroitin sulfate?CS?was used as drug of osteoarthritis in clinical trials.Heparin?HP?was used as anticoagulant and antithrombus drug.Currently,the availability of CS and HP are still dependent on their extraction from animal tissues.During this process,NaOH,and HCl were required to hydrolyze tissues and trichloroacetic acid for deproteinization.Moreover,the presence of other glycosaminoglycans also restricts their application and even causes deaths of patients.Therefore,a cost-effective method for producing safety,efficiency and homogeneous CS and HP were desirable.In this study,we constructed 3?-phosphoadenosine-5?-phosphosulfate?PAPS?regeneration system and sulfation systems to achieve enzymatic production of CS and HP.The rat aryl sulfotransferase IV?ASSTIV?was expressed in Escherichia coli,which is responsible for the regeneration of PAPS from 3?-phosphoadenosine-5?-phosphate?PAP?.The sulfotransferases and epimerase were comparatively expressed with the P.pastoris expression system and E.coli expression system and used for modification of heparoson and chondroitin to yield HP and CS.Main results were shown below.?1?Construction of PAPS regeneration system by optimizing the expression of rat ASSTIV.The rat ASSTIV encoding gene was optimized and expressed in E.coli BL21?DE3?with the pColdIII expression system.To facilitate the high-throughput screening of ASSTIV mutants and lateral protein purification,ASSTIV was firstly engineered to be secreted via fusion with signal peptides.Among the tested ten signal peptides,Cex,YebF and PelB allow secretory expression of ASSTIV to0.07?0.01 U/mL,0.14?0.01 U/mL and 0.36±0.01 U/mL,respectively.Further modification of PelB at the signal peptidase cleavage site yielded positive transformant?AGA?with higher expression?1.49?0.02 U/mL?.To increase the sulfotransferases activity,we performed random mutation to the PAP-binding pocket gate loop of ASSTIV and identified a positive mutant L89S/E90L.Subsequent site-saturation mutation to the residues Leu⁸⁹ and Glu⁹⁰ found that the mutant L89M/E90Q has higher specific activity?5.98?0.15 U/mg?,and catalytic efficiency(k_cat/K_m=1816.32?12.72 s^-1·M^-1),which was 1.76 times and 12.5 times,of that of the wild type ASSTIV,respectively.?2?Construction of specific sulfate modification system by expressing chondroitin sulfotransferase,heparin sulfotransferases and epimerases.First,6 sulfotransferases,including N-deacetylase-N-sulfotransferase?NDST?,heparan sulfate 2-O-sulfotransferase?HS2ST?,heparan sulfate 6-O-sulfotransferase?HS6ST?,heparan sulfate 3-O-sulfotransferase?HS3ST?,chondroitin 4-sulfotransferase?C4ST?,chondroitin 6-sulfotransferase?C6ST?and epimerase were comparatively expressed in recombinant E.coli and Pichia pastoris strains.HS3ST,C4ST,and C6ST were successfully expressed after induced by methanol with P.pastoris system while no enzymes were activately expressed with E.coli system.After fusion with the maltose-binding protein?MBP?,especially fusion with both MBP and TrxA,all the enzymes were activately expressed with the P.pastoris system while only HS2ST,HS6ST,and HS3ST were expressed with E.coli system.?3?Enzymatic synthesis of CS by sulfating chondroitin.After establishing an efficient PAPS regeneration system and specific sulfation transformation systems,enzymatic modification of animal-source CS by C4ST,or C6ST could increase the rate of GlcUA-GalNAc?4S?,or GlcUA-GalNAc?6S?,respectively and increased the rate of sulfate group of CS.What's more,chondroitin was efficiently transformed into CSA or CSC which was confirmed by LC-IT-TOF-MS,FTIR,and NMR.Chondroitin sulfate A?CSA?and chondroitin sulfate C?CSC?were produced from chondroitin by modification with the sulfotransferases C4ST and C6ST in the presence of PAPS.?4?Enzymatic synthesis of HP by sulfating and epimerizating heparosan.The biosynthesis of HP with anticoagulant activity from heparosan involves numerous enzymes,including NDST?containing both N-deacetylase and N-sulfotransferases activities?,C5epi,HS2ST,HS6ST,and HS3ST.Those enzymes have expressed successfully in P.pastoris.Besides,modification with the recombinant HS2ST,HS6ST and HS3ST,increased the anticoagulant activity of animal-derived heparin from?76?2?IU/mg to?189?17?IU/mg.What's more,all the enzymes,including NDST,C5epi,HS2ST,HS6ST,and HS3ST were used for the HP production with PAPS as the sulfo-group donor,resulted in trisaccharide residues GlcNS?6S?-IdoA?2S?-GlcNS?6S,3S?formation,with the anticoagulant activity of 23?4 IU/mg.