Recombinant Expression And Molecular Engineering Of Lyases Of Glycosaminoglycan Chondroitin Sulfate And Heparin

Chondroitin sulfate and heparin are a large class of glycosaminoglycan that widely present on the surface of animal cells and surrounding matrices and have important biological functions.Especially,chondroitin sulfate combined with glucosamine and collagen can effectively relieve joint pain,and heparin is mainly used for the treatment of antithrombotic and postoperative anticoagulant drugs.In recent years,the studies found that low-molecular weight chondroitin sulfate and heparin are more susceptibly absorbed by the body and have higher biological activities.Currently,low-molecular weight chondroitin sulfate and heparin are mainly produced by chemical methods.Although the chemical process is mature,there are also problems such as desulfation,poor product composition,and so on.Moreover,these chemical processes are not environmentally-friendly as they require hazardous reagents.By contrast,the bio-enzymatic method has the advantages of mild reaction conditions,no pollution,simple process,easy control of the molecular weight of the product,and does not destroy the active groups of sulfuric acid.Thus,production of low-molecular weight chondroitin sulfate and heparin by bio-enzymatic method is a new mode of green production.This study aims to optimize the chondroitinase ABC I and heparinase ? from the perspective of enzyme expression and performance.The main findings are as follows:(1)Recombinant expression of chondroitinase ABC I and heparinase ? in Escherichia coli.The optimized P.vulgaris cs ABC I based on the prefered codons of E.coli was expressed in the form of inclusion bodies with high copy replicon RSF 1030 and T7 promoter.The soluble expression level of the protein was promoted by the fusion of maltose binding protein tag at the N-terminal.The catalytic activity was further improved by I309V mutation.Subsequently,the signal peptides were screened to achieve extracellular secretion of the recombinant protein.The secretion efficiency of omp A signal peptide was the highest,and the extracellular activity was 3.2×10² U·L^-1.The membrane lipoprotein gene lpp was further knocked out,and the extracellular activity was further increased to 5.0×10² U·L^-1.By optimizing inducer and inoculum,the extracellular activity reached to 1.4×10³ U·L^-1.Besides,the F.heparinum heparinase ?(Fhep ?)and B.thetaiotaomicron heparinase ?(Bhep ?)were all expressed well at the optimized culture conditions.Moreover,Bhep ? showed higher catalytic performance and thermostability compared with Fhep ?,and had great advantage for the enzymatic production of low molecular weight heparin in the future.(2)Engineering of chondroitinase ABC I with improved thermostability.By deleting the loop R166-L170,the truncated variant N?5 improved the thermostability with a half-time of18 min at 37?,which was 2.9-fold higher than that of control(4.6 min).Site-directed mutagenesis and site-saturation mutagenesis guided by Consensus mutation revealed that E694P mutation can significantly improve the thermostability of cs ABC I.The optimum temperature of variant N?5/E694P was 35?,which was 5?higher than that of variant N?5and control(30?).The half-time of variant N?5/E694P was 19 h,which was 62-fold higher than that of variant N?5 and 247-fold higher than that of control.This was much higher than that of the highest level reported by the literature.The specific activity of variant N?5/E694P is 57.6 U·mg^-1,which is 133%higher than that of control(24.7 U·mg^-1).The k_cat/K_m of variant N?5/E694P is 544.1 m L·mg^-1 s^-1,which is 346%higher than that of control(121.9m L·mg^-1 s^-1).Through batch fermentation in a 3-L fermenter,the chondroitinase activity reached to 1.0×10⁵ U·L^-1.(3)Engineering of the heparinase ? with improved performance.Asp321 and Ser264were identified as essential residues for catalytic efficiency and thermostability by semi-rational engineering of active pocket,respectively.The variant S264F increased the thermostability with a half-time of 3.8 h at 50?,which is 40%higher than that of WT(2.7 h).The k_cat/K_mof variant S264F is 124.7 m L·mg^-1 min^-1,which is 24%lower than that of WT(164.2 m L·mg^-1 min^-1).The half-time of variant D321Q is 2.2 h,which is 19%lower than that of WT.The k_cat/K_mis 276.6 m L·mg^-1 min^-1,which is 69%higher than that of WT.The k_cat/K_m of the combined variant S264F/D321Q is 566.8 m L·min^-1·mg^-1,which is 245%higher than that of WT.The half-time is 2.0 h,which is 26%lower than that of WT.E105R mutation that generated a 348%increase in k_cat/K_m(735.7 m L·mg^-1 min^-1)was further identified by electric potential engineering of the pocket tunnel.The half-time of variant E105R is 2.2 h,which is19%lower than that of WT.The k_cat/K_m of the combined variant E105R/S264F is 851.6m L·mg^-1 min^-1,which is 418%higher than that of WT.More importantly,the half-time is 2.7h,which is the same as WT.Through batch fermentation in a 3-L fermenter,the heparinase activity reached to 2.9×10⁴ U·L^-1.(4)High-level expression of chondroitinase ABC I in B.subtilis and its use in preparation of low-molecular weight chondroitin sulfate.The effect of free expression and integrant expression on N?5/E694P production were firstly compared,and the result revealed that free expression was preferable.By replacing the inducible promoter Pxyl A with constitutive promoter Pspov G,the intracellular enzyme activity was 9.8×10³ U·L^-1.Subsequently,an oligopeptide of gln A with 45 bp was attached to the N-terminal,and the the intracellular enzyme activity was 1.2×10⁴ U·L^-1 with a 22%increase.Then,5'UTR was further optimized,and the intracellular enzyme activity reached to 3.8×10⁴ U·L^-1 with a 217%increase.Through batch fermentation in a 3-L fermenter,the chondroitinase activity reached to 5.0×10⁴ U·L^-1,which was 32%higher than that of shake flask.By modulating enzyme concentrations and depolymerization time,low-molecular weight chondroitin sulfate with low polydispersity were produced.