【作者】 刘娟；

【导师】 生举正；

【作者基本信息】 山东大学 ， 药学（专业学位）， 2023， 硕士

【摘要】 肝素(heparin,HP)是一种天然存在的糖胺聚糖,临床上用作抗凝和抗血栓类药物。目前,肝素类药物原料的来源依然依靠动物组织提取,来源受限而且质量不稳定。酶法合成反应条件温和,产物分子结构均一、确定,产量高,易于纯化。因此非动物源肝素的化学酶法合成为肝素的生产提供了一条安全的替代途径。肝素的化学酶法合成分为骨架构建和骨架修饰两个过程。骨架构建过程中,骨架合酶将核苷酸活化单糖供体中的单糖分子逐个连接到受体糖链的非还原端。合成寡糖骨架的步骤占合成全部步骤的一半以上,而且比例随着活性糖链分子的延长而增加。合成过程中所需的酶是影响酶法合成效率的关键因子,尽管酶具有优异的催化特性,但是游离酶在高温、强酸强碱等极端条件下稳定性差,易失活,无法回收再利用,产物的分离与纯化比较困难,导致生产成本提高,固定化酶与游离酶相比,酶性能更加稳定,可以均匀分布在载体上,减少酶的聚集和水解,实现昂贵酶的重复使用,且固定化酶便于与体系分离,反应体系中没有酶的残留,简化了后续目的产物的分离提纯工艺,为在工业上的广泛应用提供了可能性。因此,本课题希望利用固定化肝素骨架合酶建立一个高效连续合成肝素寡糖的体系,对肝素寡糖规模化生产与应用提供参考。目前肝素化学酶促合成常使用的肝素骨架合酶为来自Escherichia coli K5的N-乙酰氨基葡萄糖转移酶(α-1,4-N-acetylglucosaminytransferase,α-1,4-GlcNAc-T)KfiA和来自Pasteurella multocida的肝素骨架合酶 2(Pasteurella multocida heparosan synthases 2,PmHS2)。后者为具有N-乙酰氨基葡萄糖转移酶活性(α-1,4-N-acetylglucosaminytransferase,α-1,4-GlcNAc-T)和葡萄糖醛酸转移酶活性(β-1,4-Glucuronic acid transferase,β-1,4-GlcA-T)的双功能酶,本文简称为 PmHS2。本课题希望通过固定化酶建立一个高效连续合成肝素寡糖的体系。经过多个固定化酶层析柱的连续催化,骨架合酶将核苷酸活化的单糖供体中的单糖分子逐个连接到受体糖链的非还原末端,完成糖链的延伸。为了合成结构均一、确定的肝素寡糖,需要固定单一功能的肝素骨架合酶,交替反应,实现高效连续合成肝素寡糖。因此本课题以寻找微生物来源的单功能GlcA-T糖基转移酶为出发点,通过共表达合适的分子伴侣,实现大肠杆菌来源的EcKfiC在大肠杆菌中的可溶性表达,并对EcKfiC进行酶活测定和催化性质的研究。然后基于EcKfiC氨基酸序列进行同源序列比对检索,新发掘并表征了四种KfiC家族成员。随后,选择催化活性最高的酶作为单功能GlcA-T糖基转移酶,与课题组前期筛选获得的高活性GlcNAc-T糖基转移酶NaKfiA(C16L/S165K)配合组成完整的连续肝素寡糖合成固定化酶体系。最后,通过在固定化酶合成体系中引入固定化的肝素N位点硫酸基转移酶(N-sulfotransferase,NST),实现了N-硫酸化的肝素寡糖的连续固定化酶高效合成。研究内容概括如下:(1)大肠杆菌EcKfiC为典型的双功能肝素骨架合酶,且其与EcKfiA形成异源多聚体:①肝素骨架合酶EcKfiC的可溶重组表达:本实验以大肠杆菌触发因子(triggerfactor,TF)为分子伴侣,与EcKfiC形成融合蛋白,实现了 EcKfiC在大肠杆菌BL21(DE3)Codonplus RIL中的可溶性表达;并经Ni2+柱通过亲和层析纯化后得到目的蛋白 TF-EcKfiC。②TF-EcKfiC催化活性的研究:以自主酶法制备的结构均一、确定肝素骨架六糖(GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-pNP)和 五 糖(GlcA-GlcNAc-GlcA-GlcNAc-GlcA-pNP)为模式寡糖受体,进行活性测定。反应产物分子量和构型结构,表征数据明确证明EcKfiC同时具有β-1,4-GlcA-T和α-1,4-GlcNAc-T糖基转移酶活性。完成EcKfiC酶学性质研究,确定了其最优反应温度,分析了pH对酶分子催化活性的影响,测定了 PmHS2和EcKfiC的动力学参数Km/Vmax,相关数据表明EcKfiC催化效率(Kcat/Km=27.57)略高于PmHS2(Kcat/Km=8.43)。通过将EcKfiC与GT-A型糖基转移酶家族成员催化区域保守位点的信息比对,确定EcKfiC仅含有1个典型的GT-A催化结构区域,其位于第275-520氨基酸位点之间。随后,通过将该区域“DxD motif”中的2个关键天冬氨酸点突变为天冬酰胺,EcKfiC(D352N/D354N)同时失去β-1,4-GlcA-T和α-1,4-GlcNAc-T糖基转移酶活性,该结果表明EcKfiC依靠该催化结构域发挥两种肝素骨架合成催化活性。③基于EcKfiC氨基酸序列同源序列比对检索,分别完成五个不同物种来源的KfiC家族成员的重组表达与活性表征:除Hyphomicrobium methylovorum来源的HmKfiC外,EcKfiC、ApKfiC(来源于Avibacterium paragallinarum)、NaKfiC(来源于Neisseria animaloris)、MspKfiC(来源于Marinimicrobium sp.LS-A18)同时具有GlcNAc-T和GlcA-T活性。这些新来源KfiC家族成员活性数据再次证明了 KfiC是一个典型的双功能肝素骨架合酶,使KfiC成为开展GT-A型糖基转移酶催化机理的模型酶分子。④EcKfiC与EcKfiA在K5菌株中形成异源多聚体,协同发挥多糖合成活性:通过在K5菌种中协同超表达KfiC和KfiA,纯化得到了 KfiC和KfiA的复合体蛋白;分子排阻色谱分离复合体实验证明KfiC在体外主要与KfiA以1:1的复合体蛋白形式存在。在 K5 细胞内,借助双分子荧光互补(Bimolecular fluorescence complementation,BiFC)实验,证明了 KfiA和KfiC在K5细胞内同样形成异源多聚体。(2)基于固定化单功能肝素骨架合酶体系的寡糖的高效连续合成:①β-1,4-GlcA-T的单功能肝素骨架合酶的构建:完成了3个双功能肝素骨架合酶α-1,4-GlcNAc-T DxD motif结构域缺失的突变体残留GlcNAc-T活性的测定。其中,选择突变体中残留GlcNAc-T活性最低的PmHS2(D479N/D481N)为人工构建的β-1,4-GlcA-T的单功能肝素骨架合酶。②完成 Zbasic-NaKfiA(C16L/S165K)和 Zbasic-PmHS2(D479N/D481N)固定参数测定:分别在两种单功能酶NaKfiA(C16L/S165K)和PmHS2(D479N/D481N)的N-端与阳离子结合模块Zbasic融合构建融合蛋白,采用阳离子交换树脂purolite MS/C作为载体,从细胞破碎上清中直接对酶进行固定化。测定了固定化酶的催化活性,稳定性,反应动力学,最佳负载量等,根据实验结果选择合适的参数进行固定化酶实验。以固定化酶Zbasic-PmHS2(D479N/D481N)为例,证明使用新的purolite MS/C树脂和再生处理的负载量和酶活基本无差别。③固定单功能肝素骨架合酶,通过交替反应,建立肝素骨架13糖连续高效合成体系:交替使用固定化单功能肝素骨架合酶NaKfiA(C16L/S165K)和PmHS2(D479N/D481N),成功在12h的反应时间里,以70%的转化率合成至13糖,酶及中间产物均不需要纯化。(3)N-硫酸化肝素寡糖的连续固定化酶合成:①完成四种α-1,4-GlcNTFA-T糖基转移酶催化活性的测定:结果表明EcKfiA催化活性最好,选择EcKfiA和PmHS2(D479N/D481N)组合,在固定化体系中交替使用完成糖链的延伸。②完成Zbasic-EcKfiA和Zbasic-NST固定参数测定:测定了固定化酶的催化活性,稳定性,反应动力学,最佳负载量等,根据实验结果选择合适的参数进行固定化酶实验。③多酶固定完成N-硫酸化肝素寡糖的连续合成:分别固定肝素骨架合酶EcKfiA、PmHS2(D479N/D481N)和硫酸基转移酶NST,交替使用,约14h,连续合成得到N-硫酸化的3糖(GlcA-GlcNS-GlcA-pNP,简称HP-3mer(NS)),产物生成率92%以上。该体系酶和中间产物不需要纯化,固定化酶实现连续合成,各固定化酶循环三次最终获得HP-3mer(NS)约1.1g。更多还原

【Abstract】 Heparin(HP),a naturally occurring glycosaminoglycan,is clinically used as an anti-coagulant and anti-thrombotic drug.At present,the production of heparin raw materials still relies on animal tissue extraction,which is limited in source and unstable in quality.Enzymatic synthesis of non-animal-derived heparin provides a safe alternative for the production of heparin due to its mild reaction conditions,uniform and definite molecular structure,high yield and easy purification.The chemoenzymatic synthesis of heparin consists of two processes:skeleton construction and skeleton modification.During skeleton construction,skeleton synthase connects monosaccharide molecules from nucleotide-activated monosaccharide donors to the non-reducing end of the acceptor sugar chain one by one.The steps of oligosaccharide synthesis accounted for more than one half of the all steps of synthesis,and the proportion increased with the extension of the active sugar chain molecules.The enzyme required in the synthesis process is the key factor affecting the synthesis efficiency of this method.Although the enzyme has excellent catalytic properties,the free enzyme has poor stability and easy inactivation under extreme conditions such as high temperature,strong acid and base,and cannot be recycled.The separation and purification of the product is difficult,leading to the increase of production cost.In order to make the industrial utilization of enzyme more widely,with the continuous development of immobilized enzyme technology,the stability of the enzyme is improved after immobilization,so that it can be evenly distributed on the carrier,reduce the aggregation and hydrolysis of enzymes,and realize the reuse of expensive enzymes.The immobilized enzyme is easy to separate and there is no enzyme residue in the reaction system,which simplifies the separation and purification process of the subsequent products and provides the possibility for wide application in industry.Therefore,this study hopes to use immobilized heparin skeleton synthase to establish an efficient and continuous synthesis system of heparin oligosaccharides,so as to provide a reference for the large-scale production and application of heparin oligosaccharides.At present,the heparin skeleton synthase commonly used in heparin chemical enzymatic synthesis is from E.coli K5 N-acetylglucosamine transferase(α-1,4-N-acetylglucosaminytransferase,α-1,4-GlcNAc-T)from KfiA and Pasteurella multocida heparin skeleton synthase 2(Pasteurella multocida heparosan synthases 2,PmHS2).The latter with N-acetylglucosamine transferase activity(α-1,4-N-acetylglucosaminytransferase,α-1,4-GlcNAc-T)and Glucuronic acid transferase activity(β-1,4-Glucuronic acid transferase,β-1,4-GlcA-T)was referred to as PmHS2 in this paper.The aim of this study is to establish an efficient and continuous system for the synthesis of heparin oligosaccharides by immobilized enzyme.Through the continuous catalysis of multiple immobilized enzyme chromatograph columns,skeleton synthase connects the monosaccharide molecules in nucleotide-activated monosaccharide donor to the extended ending of the acceptor sugar strands one by one to complete the extension of the sugar chain.In order to synthesize heparin oligosaccharides with uniform and definite structure,it is necessary to fix single function heparin skeleton synthase,and alternate reaction to achieve efficient and continuous synthesis of heparin oligosaccharides.Therefore,this topic is to find the single function GlcA-T glycosyltransferase from microbial source as a starting point,through the co-expression of appropriate molecular partner,to achieve the soluble expression of EcKfiC from Escherichia coli,and EcKfiC enzyme activity determination and catalytic properties of the study.Then homologous sequence matching was conducted based on EcKfiC amino acid sequence,and four KfiC family members were newly discovered and characterized.Subsequently,the enzyme with the highest catalytic activity was selected as the single function GlcA-T glycosyltransferase,which combined with the highly active GlcNAc-T glycosyltransferase NaKfiA(C16L/S165K)obtained by our research group in the previous stage to form a complete system of continuous heparin oligosaccharide synthesis immobilized enzyme.Finally,by introducing immobilized heparin N-sulfotransferase(NST)into the immobilized enzyme synthesis system,the continuous immobilized enzyme synthesis of N-sulfotransferase of heparin oligosaccharides was realized.The research content is summarized as follows:(1)Escherichia coli EcKfiC is a typical bifunctional heparin skeleton synthase,and it forms heteropolymers with EcKfiA:①Soluble recombinant expression of heparin skeleton synthase EcKfiC:In this study,Escherichia coli triggerfactor(TF)was used as a molecular partner to form a fusion protein with EcKfiC,and the soluble expression of EcKfiC in Escherichia coli BL21(DE3)Codonplus RIL was realized.The target protein TF-EcKfiC was purified by Ni2+affinity column.②Study on catalytic activity of TF-EcKfiC:The homogeneous structure of heparin skeleton hexaosaccharides(GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-pNP)and pentasaccharides(GlcA-GlcNAc-GlcA-GlcNAc-GlcA-pNP)were prepared by enzymic method as model oligosaccharide receptors,and activity determination.Molecular weight and configurational structure of the reaction products,the characterization data clearly demonstrated that EcKfiC had both β-1,4-GlcA-T and α-1,4-GlcNAc-T glycosyltransferase activities.The enzymatic properties of EcKfiC were studied,the optimal reaction temperature was determined,and the influence of pH on the catalytic activity of enzyme molecules was analyzed.The kinetic parameters Km/Vmax of PmHS2 and EcKfiC were determined.The relevant data show that the catalytic efficiency of EcKfiC(Kcat/Km=27.57)is slightly higher than that of PmHS2(Kcar/Km=8.43).By comparing the information of EcKfiC with that of GT-A glycosyltransferase family members,it was determined that EcKfiC contains only one typical GT-A catalytic structure region,which is located between the 270-520 amino acid sites.Subsequently,EcKfiC(D352N/D354N)lost β-1,4-GlcA-T and α-1,4-GlcNAc-T glycosyltransferase activities simultaneously by mutating two key aspartic acid sites in DxD motif to asparagine in this region.The results indicate that EcKfiC can exert the catalytic activity of two heparin frameworks by this catalytic domain.③Recombinant expression and activity characterization of KfiC family members from five different species were completed based on homologous sequence comparison retrieval of EcKfiC amino acid sequences:With the exception of Hyphomicrobium methylovorum source HmKfiC,EcKfiC,ApKfiC(from Avibacterium paragallinarum),NaKfiC(from Neisseria animaloris),MspKfiC(from Marinimicrobium sp.LS-A18)exhibited both GlcNAc-T and GlcA-T activity.The activity data of KfiC family members from these new sources once again prove that KfiC is a typical bifunctional heparin skeleton synthase,making KfiC a model enzyme for the catalytic mechanism of GT-A glycosyl transferase.④EcKfiC and EcKfiA formed heteropolymers in K5 strains,which showed synergistic polysaccharide synthesis activity:KfiC and KfiA complex proteins were purified by synergistic overexpression in K5 strains.The results of molecular exclusion chromatography showed that KfiC and KfiA were mainly 1:1 complex proteins in vitro.Within K5 cells,it was demonstrated that KfiA and KfiC also formed heteropolymers through Bimolecular fluorescence complementation(BiFC)experiment.(2)Efficient continuous synthesis of oligosaccharides based on immobilized mono-functional heparin skeleton synthase system:①Construction of mono-functional heparin skeleton synthase of β-1,4-GlcA-T:Residual GlcNAc-T activity was determined in three bifunctional heparin skeleton synthase α-1,4-GlcNAc-T DxD motif domain deletion mutants.In the mutant,PmHS2(D479N/D481N)with the lowest GlcNAc-T catalytic activity was selected as β-1,4-GlcA-T mono-functional heparin skeleton synthase.②Determination of fixed parameters of Zbasic-NaKfiA(C16L/S165K)and Zbasic-PmHS2(D479N/D481N):Two enzymes NaKfiA(C16L/S165K)and PmHS2(D479N/D481N)were fused with the cation-binding module Zbasic to construct the N-terminal fusion protein.The cation exchange resin purolite MS/C was used as the carrier to immobilize the enzyme directly from the cell crushing supernatant.The catalytic activity,stability,reaction kinetics,optimum loading capacity,resin recovery and utilization of immobilized enzyme activity determination.According to the experimental results,appropriate parameters were selected for immobilized enzyme experiment.The immobilized enzyme Zbasic-PmHS2(D479N/D481N)was used as an example to show that there was little difference in load and enzyme activity between the use of a new purolite MS/C resin and two repeated uses.③The continuous and alternating reaction of single functional heparin skeleton synthase was fixed to establish the continuous and efficient synthesis system of heparin skeleton 13 sugar:The immobilized mono-functional heparin skeleton synthase NaKfiA(C16L/S165K)and PmHS2(D479N/D481N)were used to synthesize 13 sugars with a 70%conversion rate.The enzyme and intermediate products did not require purification,and the total reaction time was about 12h.(3)Continuous immobilized enzyme synthesis of N-sulfated heparin oligosaccharides:①The catalytic activity of four α-1,4-GlcNTFA-T glycosyltransferases was determined.The results showed that EcKfiA had the best catalytic activity.The combination of EcKfiA and PmHS2(D479N/D481N)was used alternately in the immobilized system to complete the extension of the sugar chain.②Determination of fixed parameters of Zbasic-EcKfiA and Zbasic-NST:the catalytic activity,stability,reaction kinetics and optimal loading capacity of the immobilized enzyme were determined,and the appropriate parameters were selected for the immobilized enzyme experiment according to the experimental results.③Continuous synthesis of N-sulfated heparin oligosaccharides by multi-enzyme fixation:The heparin skeleton synthase EcKfiA,PmHS2(D479N/D481N)and sulfatyltransferase NST were fixed respectively,and were used interchangingly for about 14h.N-sulfurated 3-sugar(GlcA-GlcNS-GlcA-pNP,HP-3mer(NS))was synthesized continuously.The product formation rate was more than 92%.In this system,the enzymes and intermediates do not need purification,and the immobilized enzymes achieve continuous synthesis,and each immobilized enzyme cycle three times to finally obtain about 1.1 g HP-3mer(NS).更多还原