木质素β-O-4键酶解及酶与木质素吸附作用的研究
发布时间：2018-08-24 07:51
【摘要】：木质素是由三种单体通过C-C键与C-O键连接聚合而成,是一种含量丰富、结构复杂的天然支链型芳香族聚合物,其被视为许多高价值的芳香族化合物的潜在替代品。由于木质素中的p-醚键数量最为丰富,所以通过Sphingobium sp.strain SYK-6中以NAD+为辅酶的Ca-脱氢酶(LigD)、以谷胱甘肽为辅酶的β-醚酶(LigF)与谷胱甘肽裂合酶(LigG)断裂p-0-4键成为木质素降解中的研究热点。本研究中同时利用还原酶AVR对LigDFG酶解体系中的较为昂贵的辅酶NAD+与GSH进行循环再利用。本研究中我们利用酶解体系LigDFG+AVR尝试降解天然木质素样品,并且对木质素降解酶LigD、LigF与木质素之间的吸附机制进行研究。1、本研究中将基因ligD、ligF、ligG、AVR克隆至大肠杆菌并重组表达纯化,通过优化诱导条件(16℃, IPTG终浓度0.5 mmol·L-1,诱导12h)得到可溶性表达的LigD、LigF、LigG、AVR四种酶,纯度都达到95%以上。利用模型化合物GGE通过高效液相色谱(HPLC)与薄层层析验证了它们具有较高的体外活性。2、利用酶解体系尝试降解利用膜分离、有氧氧化的手段得到重均分子量分别为24482 Da、1627 Da、859 Da、725 Da、679 Da等的木质素磺酸盐样品及p-0-4键Cα位羟基被氧化的木质素样品,利用凝胶渗透色谱(GPC)、高效液相色谱(HPLC)及电喷雾质谱(ESI-MS)等分析手段证明该酶解体系对天然木质素有一定的降解趋势,但是酶解效率很低。建立了针对LigF酶解活性的快速检测方法——比色法与荧光测定法,并进行了综合比对。荧光定量法精确度高出至少1000倍,而比色法操作简便。3、通过表面等离子体共振(SPR)、等温滴定量热法(ITC)、高效液相色谱(HPLC)对木质素降解酶LigD、LigF与木质素磺酸盐之间的吸附机制进行分析研究,发现木质素能够抑制LigF的活性(MPHPV转化率由99.5%下降至32.6%),对LigD影响较小(GGE转化率由41.7%变为41%)；分子量较大的木质素片段对酶的抑制作用明显,分子量较小的木质素片段对酶的抑制作用不明显；疏水相互作用与静电相互作用在吸附中起到重要作用。
[Abstract]:Lignin is a kind of natural branched aromatic polymer with rich content and complex structure. Lignin is a kind of natural branched aromatic polymer which is composed of three kinds of monomers, which are linked with C-C bond and C-O bond. Lignin is regarded as a potential substitute for many high value aromatic compounds. Since the number of p-ether bonds in lignin is the most abundant, p-0-4 bond breaks between 尾 -etherase (LigF) and glutathione co-enzyme (LigG) through Ca- dehydrogenase (LigD), with NAD as coenzyme in Sphingobium sp.strain SYK-6 has become a hot topic in lignin degradation. In this study, reductase AVR was used to recycle the more expensive coenzymes NAD and GSH in LigDFG hydrolysis system. In this study, we try to degrade natural lignin samples by enzymatic hydrolysis system LigDFG AVR, and study the adsorption mechanism between lignin degrading enzyme LigD,LigF and lignin. In this study, we cloned the gene ligD,ligF,ligG,AVR into Escherichia coli and expressed and purified it. Four kinds of soluble LigD,LigF,LigG,AVR were obtained by optimizing the induction conditions (16 鈩，
本文编号：2200128
[Abstract]:Lignin is a kind of natural branched aromatic polymer with rich content and complex structure. Lignin is a kind of natural branched aromatic polymer which is composed of three kinds of monomers, which are linked with C-C bond and C-O bond. Lignin is regarded as a potential substitute for many high value aromatic compounds. Since the number of p-ether bonds in lignin is the most abundant, p-0-4 bond breaks between 尾 -etherase (LigF) and glutathione co-enzyme (LigG) through Ca- dehydrogenase (LigD), with NAD as coenzyme in Sphingobium sp.strain SYK-6 has become a hot topic in lignin degradation. In this study, reductase AVR was used to recycle the more expensive coenzymes NAD and GSH in LigDFG hydrolysis system. In this study, we try to degrade natural lignin samples by enzymatic hydrolysis system LigDFG AVR, and study the adsorption mechanism between lignin degrading enzyme LigD,LigF and lignin. In this study, we cloned the gene ligD,ligF,ligG,AVR into Escherichia coli and expressed and purified it. Four kinds of soluble LigD,LigF,LigG,AVR were obtained by optimizing the induction conditions (16 鈩，
本文编号：2200128
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