【摘要】：木質(zhì)素是由三種單體通過C-C鍵與C-O鍵連接聚合而成,是一種含量豐富、結(jié)構(gòu)復(fù)雜的天然支鏈型芳香族聚合物,其被視為許多高價值的芳香族化合物的潛在替代品。由于木質(zhì)素中的p-醚鍵數(shù)量最為豐富,所以通過Sphingobium sp.strain SYK-6中以NAD+為輔酶的Ca-脫氫酶(LigD)、以谷胱甘肽為輔酶的β-醚酶(LigF)與谷胱甘肽裂合酶(LigG)斷裂p-0-4鍵成為木質(zhì)素降解中的研究熱點。本研究中同時利用還原酶AVR對LigDFG酶解體系中的較為昂貴的輔酶NAD+與GSH進行循環(huán)再利用。本研究中我們利用酶解體系LigDFG+AVR嘗試降解天然木質(zhì)素樣品,并且對木質(zhì)素降解酶LigD、LigF與木質(zhì)素之間的吸附機制進行研究。1、本研究中將基因ligD、ligF、ligG、AVR克隆至大腸桿菌并重組表達純化,通過優(yōu)化誘導(dǎo)條件(16℃, IPTG終濃度0.5 mmol·L-1,誘導(dǎo)12h)得到可溶性表達的LigD、LigF、LigG、AVR四種酶,純度都達到95%以上。利用模型化合物GGE通過高效液相色譜(HPLC)與薄層層析驗證了它們具有較高的體外活性。2、利用酶解體系嘗試降解利用膜分離、有氧氧化的手段得到重均分子量分別為24482 Da、1627 Da、859 Da、725 Da、679 Da等的木質(zhì)素磺酸鹽樣品及p-0-4鍵Cα位羥基被氧化的木質(zhì)素樣品,利用凝膠滲透色譜(GPC)、高效液相色譜(HPLC)及電噴霧質(zhì)譜(ESI-MS)等分析手段證明該酶解體系對天然木質(zhì)素有一定的降解趨勢,但是酶解效率很低。建立了針對LigF酶解活性的快速檢測方法——比色法與熒光測定法,并進行了綜合比對。熒光定量法精確度高出至少1000倍,而比色法操作簡便。3、通過表面等離子體共振(SPR)、等溫滴定量熱法(ITC)、高效液相色譜(HPLC)對木質(zhì)素降解酶LigD、LigF與木質(zhì)素磺酸鹽之間的吸附機制進行分析研究,發(fā)現(xiàn)木質(zhì)素能夠抑制LigF的活性(MPHPV轉(zhuǎn)化率由99.5%下降至32.6%),對LigD影響較小(GGE轉(zhuǎn)化率由41.7%變?yōu)?1%)；分子量較大的木質(zhì)素片段對酶的抑制作用明顯,分子量較小的木質(zhì)素片段對酶的抑制作用不明顯；疏水相互作用與靜電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，

本文編號：2200128