Study On The Connection Between Cellulose And Lignin In Gymnosperm By ²H/¹³C Dual Isotope Tracer

In the pulping process,due to the connection between cellulose and lignin,lignin cannot be completely removed.Therefore,investigation on the assocition between cellulose and lignin is an important work.The position of connection is on which carbon atom of lignin and cellulose is of great significance for finding an efficient enzyme or biomimetic system for biological pulping.In this paper,anhydrous D-glucopyranose-6-D₂ was used as a raw material to synthesize the cellulose precursor with a ²H label at the C₆ position,i.e.UDP-glucose-6-D₂.Technical route was six-step reaction as follows:glucose acetylation,deacetylation at position 1,phosphorylation of hydroxyl groups,hydrogenation cleavage of bisphenyl ring,coupling condensation with UMP-morpholidate reagent and deacetylation at positions 2,3,4,and 6.Melting point analyzer,Fourier transform infrared spectrometer and nuclear magnetic resonance spectrometer were used to characterize and analyze the synthesis products in each step.Ginkgo biloba L.is a typical gymnosperm tree.This work focus on the analysis of the structure of cellulose-lignin complexes in ginkgo tree.Deuterium-labeled cellulose precursor—UDP-glucose-6-D₂,¹³C-labeled lignin precursor—coniferin-¹³Cαand 4CL inhibitor—3,4-methylenedioxycinnamic acid were injected into the five-year-old ginkgo plant.Microscopic observation of the ginkgo cambium tissue at the end of the culture revealed that the cambium structure was relatively wide,indicating that ginkgo tree grows well.The element analyzer combined with stable isotope ratio mass spectrometer was used to detect the ¹³C and ²H abundances in the ginkgo newly-formed xylem.It was found that the ¹³C and ²H abundances were significantly increased in the ginkgo newly-formed xylem.It was showed that the6-²H₂ labeled cellulose precursors and theα-¹³C labeled lignin precursors were efficiently metabolized and deposited in the newly-formed xylem.Meanwhile,the deposition routes of lignin and cellulose on the cell wall were also understood.The deposition of lignin and cellulose start from primary wall and middle lamella,followed by the secondary wall.CP/MAS ¹³C-NMR was used to determine the ginkgo newly-formed xylem tissue and to analyze the differential spectrum.The results showed that the signals related with Cαin lignin of the ginkgo newly-formed xylem labeled with ¹³C were significantly enhanced.This indicates that the lignin precursor was transformed into a lignin macromolecule.The lignin structural units were mainly connected byβ-5,β-O-4 andβ-1 linkages.Moreover,the lignin and carbohydrates were mainly connected byα-ether bond andα-acetal bonds.The method of Bj（?）rkman was used to isolate 6-²H/α-¹³C dual isotope-labeled lignin-carbohydrate complex（LCC）and milled wood lignin（MWL）from ginkgo tree.The 6-²H/α-¹³C dual isotope-labeled LCCs were enzymatic hydrolyzed with cellulase and hemicellulase to obtain enzymatic degraded LCC（EDLCC）.The FT-IR was used to detect and analyze the Ginkgo LCC,MWL and EDLCC respectively,and found the absorption peaks of typical Ginkgo LCC structure,MWL structure and EDLCC structure.Through ¹³C-NMR of MWL,it was found that lignin structural units are mainly connected byβ-O-4,β-5,β-1,andβ-βlinkages.The analysis of ¹H-NMR differential spectrum of 6-²H/α-¹³C-labeled ginkgo LCC showed that the connection between cellulose and lignin was mainly benzyl ether bond and ester bond.The¹H-NMR difference spectrum of the ginkgo EDLCC showed signals from glucose,which further proved the connection between cellulose and lignin.The carbon atom at the 6th position of cellulose forms a benzene methyl ether bond with Cαon the side chain of lignin.In addition,between the carbon atoms at the 6-position of cellulose and the Cγof ferulic acid and p-coumaric acid in lignin,ester bond was also formed.During the suspension culture of ginkgo cambium tissue,the coniferin-¹³Cαwas added.Through CP/MAS ¹³C-NMR analysis,As compared with the cambium tissue adminstered with unlabeled lignin precursor,it was found that several signals related to Cαin the cambium tissue administered with ¹³C-labeled lignin precursor were enhanced significantly.Differential spectrum analysis confirmed that the connection modes between lignin structural units were mainlyβ-5,β-β,β-O-4 andβ-1 linkage.The association between lignin and carbohydrates was mainly Cαacetal linkage,benzyl ehter linkage and benzyl ester linkage.