?-O-4 Aryl Ether Of Lignin Enzymolysis Via The Ligninolytic Enzyme System LigDFG And The Mechanism Of Absorption Between The Ligninolytic Enzymes LigD, LigF And Sulfonated Lignin

Lignin is a branched aromatic polymer of high complexity that consists of three phenolic monomers called monolignols joined by carbon-carbon and ether linkages and is regarded as a potential substitute for phenolic and other aromatic, oil-based chemicals in the upcoming post oil age. The ?-O-4 aryl ether linkages are the most abundant among various intermolecular linkages between phenylpropane units, so cleavage of the ?-aryl ether linkage by NAD+-dependent Ca-dehydrogenase (LigD), glutathione-dependent (3-etherases (LigF) and glutathione-dependent glutathione lyase (LigG) from Sphingobium sp. strain SYK-6 is the most important process in lignin degradation. Allochromatium vinosum reductase (AVR) is also used to recycle and supply this enzyme system with the expensive co-substrates NAD+and glutathione in this research. We tried to break down the real lignin sample via the enzyme system LigDFG+AVR and investigated the mechanism of absorption between the ligninolytic enzymes LigD, LigF and sulfonated lignin.1. We cloned and recombinantly expressed genes ligD, ligF, ligG and AVR in E. coli and determined their activity on the lignin model substratel-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propane diol (GGE) by HPLC and silica gel thin-layer chromatography. The concentration of purified LigD, LigF, LigG and AVR were 0.12 mmol·L-1, 0.05 mmol·L-1,0.08 mmol·L-1, and 0.08 mmol·L-1, respectively. The purity of the purified enzymes were all 95% or more.2. The enzyme system LigDFG+AVR were used to try to break down the different real lignin samples obtained by ultrafiltration process and aerobic oxidation. The weight-average molecular weight of the lignin samples obtained by ultrafiltration process were 24482 Da,1627 Da,859 Da,725 Da and 679 Da, respectively. The Ca-hydroxyls of ?-O-4 aryl ether linkages in the lignin samples obtained by aerobic oxidation were oxidized to ketone. It found that the catalytic efficiency of the two enzymes is low when they are used to break down the (3-aryl ether linkage in natural lignin analyzed by gel permeation chromatography (GPC), high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). Two rapid detection methods for LigF activity, the colorimetric method and the fluorometric method were established and made a comprehensive comparison between them. The fluorometric method is 1000 times more precise than the colorimetric micro-method, but the colorimetric method is more convenient than the fluorometric method.3. The mechanism of absorption between the ligninolytic enzymes LigD, LigF and sulfonated lignin was investigated by surface plasma resonance (SPR), isothermal titration calorimetry (ITC) and HPLC. When sulfonated lignin was added to LigF hydrolysis reactions, the conversion rate of MPHPV decreased significantly from 99.5% to 32.6%. On the contrary, sulfonated lignin has little affection on LigD, which the conversion rate of GGE only decreased from 41.7% to 41%. Sulfonated lignin could be inhibitory for the catalytic efficiency of ?-etherase LigF, not for Ca-dehydrogenase LigD. Hydrophobic interactions and ionic bond interactions were responsible for the increased adsorption of the enzymes to sulfonated lignin.