Studies On Efficient Preparation Of Several Key Enzymes And Its Muti-enzymatic Reaction Engineering For Heparin Biosynthesis

Heparin, a sulfated polysaccharide composed of uronic acid and D-glucosamine, is a widely used clinical anticoagulant. The pollution of heparin raw materials in 2008 has prompted the research of the production of non-animal origin heparin to ensure the safety of product.There are two strategies to prepare non-animal origin heparin. One strategy is started with heparosan prepared by E.coli K5 strain fermentation, which is converted to anticoagulant heparin by chemoenzymatic modification. The other is started with disaccharide, which is elongated to heptasaccharide using UDP-glucuronic acid, UDP-N-acetylglucosamine and UDP-N-trifluoacetylglucosamine as monosaccharide donors. Then heptasaccharide is converted to anticoagulant ultra-low molecular weight heparin (ULWMH). However, only milligram heparin can be prepared, which is far away industrial production. There are many limitations:1)3’-phosphoadenosine-5’-phosphosulfate (PAPS) is the "active" sulfo group donor in heparin modification, which is expensive and easily degraded.2) It is difficult to obtain large mounts of heparin modification enzymes because of their low expression levels.3) The preparation of heparin is using purified free enzymes, which is expensive and can not be reused.4) Monosaccharide donor UDP-GlcNAc and UDP-GlcNTFA in ULMWH preparation are expensive and their yields are low. In order to solve the problems mentioned above, the following research work has been carried out in this paper:In this study, adenosine triphosphate (ATP) sulfurylase and adenosine 5’-phosphosulfate (APS) kinase were cloned and expressed, and an enzymatic synthesis pathway of PAPS has been constructed. Under the optimal reaction conditionsop, the reaction was carried out with fed batch of ATP, PAPS reaches 10.32 g·L-1 and the conversion percentage is 81.4%.Through codon optimization, fusion expression with maltose-binding protein and the optimization of expression conditions, five key heparin modification enzymes C5 epimerase, heparan sulfate 2-O-sulforanferase (2OST), heparan sulfate 6-O-sulforanferase-1(6OST1), heparan sulfate 6-O-sulforanferase-3 (6OST3) and heparan sulfate 3-O-sulforanferase-1 (3OST1) were efficiently expressed in E.coli BL21(DE3). The expression levels were 305.325, 215,225 and 300 mg/L, respectively.Immobilization of heparin modification enzymes using aldehyde tag (LCTPSR) was further carried out. Through protein rational design, LCTPSR was introduced to fusion partner mbp at four different sites (E38H39, K170Y171, D209Y210 and S352G353). By comparing the expression level of Ald6-mbp and the binding ability with amino resin, the best fusion partner (pMAl-mbp7-FGE) was obtained. The results showed that the coupled yield for GFP and arginase was 90% and 71%, respectively using green fluorescent protein and arginase as reporter proteins. The immobilization greatly improved the operational stability of arginase,53% of activity remained after 14 cycles. This method was applied for immobilization of heparin modification enzymes.The process for chemoenzymatic synthesis of heparin was established using immobilized heparin enzymes. Heparosan was chemically deacetylated/N-sulfated to get N-sulfo heparosan. Using unpurified PAPS as sulfo group donor, N-sulfo heparosan was modified by immobilized C5 epimerase,2OST,6OST1 and 6OST3 in one pot reaction system. Then the reaction product was further modified by immobilized 3OST1. The heparin obtained by 3OST1 modification had anticoagulant activity and the titer was 1.4 IU.The heparin structure was analyzed by strong anion exchange high performance liquid chromatography (SAX-HPLC). Heparin was enzymatic lysis to disaccharides by Hep Ⅰ, Ⅱ and Ⅲ. Then heparin disaccharides were analyzed using SAX-HPLC, and the mass percentage of different disaccharide in heparin was calculated. Based on quantitative analysis, Ⅳ-A is 100% in heparosan. Ⅳ-A is 8.26% and Ⅳ-S is 91.74% in N-sulfo heparosan. In porcine heparin, he mass percentage of Ⅳ-A, Ⅳ-S, Ⅱ-A, Ⅲ-A, Ⅱ-S, Ⅲ-S, Ⅰ-A and Ⅰ-S is 1.98%,2.14%,3.65%, 1.10%,10.44%,5.64%,0.76% and 74.29%, respectively. This work provides a basis for further characterization of the bioengineering heparin structure.At last N-acetylhexosamine 1-kinase, N-acetylglucosamine-1-phosphate uridyltransferase and inorganic pyrophosphatase were cloned and expressed, and an enzymatic synthesis pathway of UDP-GlcNAc and UDP-GlcNTFA has been constructed. Under the optimized conditions, the reaction was carried out with fed batch of substrate, high production of UDP-GlcNAc (59.51 g/L) and UDP-GlcNTFA (46.54 g/L) were achieved in this three-enzyme one-pot system. This work provides an efficient technique for the production of ULMWH key precursors.