纤维素前驱物6-~(13)C标记示踪研究纤维素与木质素连接方式
|
摘要
从细胞壁多糖的角度分析糖单元与木质素苯丙烷结构单元之间的共价键连接方式,合成了带6-13C标记的纤维素前驱物尿苷二磷酸葡萄糖,并将其与苯丙氨酸解氨酶(PAL)的抑制剂L-2-氨氧基-3-苯基丙酸(AOPP)及外源性木质素前驱物松柏醇-β-D-葡萄糖苷一起投入生长中的银杏植物体内,分析结果证明了(6-13C)尿苷二磷酸葡萄糖是对植物中纤维素进行选择性13C标记的合适的前驱物。从正常生长的银杏新生木质部组织提取木质素-碳水化合物复合体(LCC),并用纤维素酶和半纤维素酶酶解LCC得LCC酶解产物(EDLCC)。红外光谱及13C NMR检测LCC和EDLCC,分析证实了纤维素6位碳与木质素以苯甲醚键方式连接。
In this paper,carbohydrate part of the lignin-carbohydrate complexes( LCC) in cell wall was labelled with13C and traced with13C NMR. Cellulose precursor,i. e.,uridine diphoshphate glucose-[6-13C],was synthesized,and injected into a living ginkgo tree together with lignin inhibitor AOPP and exogenous lignin precursor. It is proved that uridine diphoshphate glucose-[6-13C] is a suitable precursor for selective13C-enrichment of cellulose in a plant. The specifically13C-enriched LCC was isolated from the newly formed xylem of ginkgo shoots. Then it was degraded by cellulase and hemicellulase. Thus the lignin-rich fractions,which were called enzyme-degraded LCC( EDLCC),were obtained. By determining their FT-IR and13C NMR spectra, information confirms that the bonds between C6position of glucose units in cellulose and α-carbons of lignin side chain are benzyl ether linkage.
In this paper,carbohydrate part of the lignin-carbohydrate complexes( LCC) in cell wall was labelled with13C and traced with13C NMR. Cellulose precursor,i. e.,uridine diphoshphate glucose-[6-13C],was synthesized,and injected into a living ginkgo tree together with lignin inhibitor AOPP and exogenous lignin precursor. It is proved that uridine diphoshphate glucose-[6-13C] is a suitable precursor for selective13C-enrichment of cellulose in a plant. The specifically13C-enriched LCC was isolated from the newly formed xylem of ginkgo shoots. Then it was degraded by cellulase and hemicellulase. Thus the lignin-rich fractions,which were called enzyme-degraded LCC( EDLCC),were obtained. By determining their FT-IR and13C NMR spectra, information confirms that the bonds between C6position of glucose units in cellulose and α-carbons of lignin side chain are benzyl ether linkage.
引文
[1]XIE Y M,YASUDA S,WU H,et al.Analysis of the structure of lignin-carbohydrate complexes by the specific13C tracer method[J].Journal of Wood Science,2000,46(2):130-136.
[2]MCCABE B J,PREVIS S F.Using isotope tracers to study metabolism:Application in mouse models[J].Metabolic Engineering,2004,6(1):25-35.
[3]ALLEN D K,YAIR SHACHAR-HILL Y,OHLROGGE J B.Compartment-specific labeling information in13C metabolic flux analysis of plants[J].Phytochemistry,2007,68(16/17/18):2197-2210.
[4]YANG H T,XIE Y M,YAO L,et al.On the Lignin-carbohydrate association in rice stalk by13C isotopic tracer technique.Cellulose chemistry and technology[J].Cellulose Chemistry and Technology 2007,41(7/8):363-369.
[5]顾瑞军,谢益民,曾绍琼,等.稻秆木素侧链13C同位素示踪及固体13C NMR分析[J].高等学校化学学报,2002,23(6):1073-1076
[6]XIE Y,TERASHIMA N.Selective carbon 13-enrichment of side chain carbons of ginkgo lignin traced by carbon 13 nuclear magnetic resonance[J].Mokuzai Gakkaishi,1991,37(10):935-941.
[7]XIE Y,GU R,WATANABE T.Studies on the linkages between dehydrogenation polymer and cell wall polysaccharide by carbon-13 isotopic tracer method[C]//Proceeding of 12th International Symposium on Wood and Pulping Chemistry,Madison:[s.n.],2003:207-210.
[8]王鹏,谢益民,范建云.果胶-木质素复合体化学结构13C同位素示踪法的研究[J].陕西科技大学学报,2006,24(5):17-24.
[9]XIE Y,DANIELLE R,TERASHIMA N.Selective carbon 13 enrichment of side chain carbons of ginkgo lignin traced by carbon 13 nuclear magnetic resonance[J].Plant Physiology and Biochemistry,1994,32(2):234-249.
[10]STEPHEN Y L,CARLTON W D(Eds.).Methods in Lignin Chemistry[M].Berlin Heidelberg:Springer-Verlag press,1992:68-74.
[11]SENIOR D J,HAMILTON J.Biobleaching with xylanases brings biotechnology to reality[J].Pulp&Paper,1992,66(9):111-114.
[12]DE J E,WONG K Y,SADDLER J N.The mechanism of xylanase prebleaching of kraft pulp:An examination using model pulps prepared by depositing lignin and xylan on cellulose fibers[J].Holzforschung,1997,51(1):19-26.
[13]DURANOVA M,HIRSCH J,KOLENOVA K,et al.Fungal glucuronoyl esterases and substrate uronic acid recognition[J].Bioscience,Biotechnology,and Biochemistry,2009,73(11):2483-2487.
[14]DINEV Z,AHMAD Z W,ROBERT T C,et al.A convenient gram-scale synthesis of uridine diphospho(13C6)glucose[J].Carbohydrate Research,2006,341(10):1743-1747.
[15]IMAI T,TERASHIMA N.Determination of the distribution and reaction of polysaccharide in wood cell wall by the isotopic tracer technique I.Selective radio-labeling of cell wall polysaccharide in Magnolia kobus[J].Mokuzai Gakkaishi,1990,36(11):917-922.
[16]BJRKMAN A.Lignin and lignin-carbohydrate complexes:Extraction from wood meal with neutral solvents[J].Industrial&Engineering Chemistry,1957,49(9):1395-1398.
[17]AZUMA J,KOSHIJIMA T.13C NMR spectroscopy of cellulose and related carbohydrates[J].Wood Research,1981,16:63-69.
[2]MCCABE B J,PREVIS S F.Using isotope tracers to study metabolism:Application in mouse models[J].Metabolic Engineering,2004,6(1):25-35.
[3]ALLEN D K,YAIR SHACHAR-HILL Y,OHLROGGE J B.Compartment-specific labeling information in13C metabolic flux analysis of plants[J].Phytochemistry,2007,68(16/17/18):2197-2210.
[4]YANG H T,XIE Y M,YAO L,et al.On the Lignin-carbohydrate association in rice stalk by13C isotopic tracer technique.Cellulose chemistry and technology[J].Cellulose Chemistry and Technology 2007,41(7/8):363-369.
[5]顾瑞军,谢益民,曾绍琼,等.稻秆木素侧链13C同位素示踪及固体13C NMR分析[J].高等学校化学学报,2002,23(6):1073-1076
[6]XIE Y,TERASHIMA N.Selective carbon 13-enrichment of side chain carbons of ginkgo lignin traced by carbon 13 nuclear magnetic resonance[J].Mokuzai Gakkaishi,1991,37(10):935-941.
[7]XIE Y,GU R,WATANABE T.Studies on the linkages between dehydrogenation polymer and cell wall polysaccharide by carbon-13 isotopic tracer method[C]//Proceeding of 12th International Symposium on Wood and Pulping Chemistry,Madison:[s.n.],2003:207-210.
[8]王鹏,谢益民,范建云.果胶-木质素复合体化学结构13C同位素示踪法的研究[J].陕西科技大学学报,2006,24(5):17-24.
[9]XIE Y,DANIELLE R,TERASHIMA N.Selective carbon 13 enrichment of side chain carbons of ginkgo lignin traced by carbon 13 nuclear magnetic resonance[J].Plant Physiology and Biochemistry,1994,32(2):234-249.
[10]STEPHEN Y L,CARLTON W D(Eds.).Methods in Lignin Chemistry[M].Berlin Heidelberg:Springer-Verlag press,1992:68-74.
[11]SENIOR D J,HAMILTON J.Biobleaching with xylanases brings biotechnology to reality[J].Pulp&Paper,1992,66(9):111-114.
[12]DE J E,WONG K Y,SADDLER J N.The mechanism of xylanase prebleaching of kraft pulp:An examination using model pulps prepared by depositing lignin and xylan on cellulose fibers[J].Holzforschung,1997,51(1):19-26.
[13]DURANOVA M,HIRSCH J,KOLENOVA K,et al.Fungal glucuronoyl esterases and substrate uronic acid recognition[J].Bioscience,Biotechnology,and Biochemistry,2009,73(11):2483-2487.
[14]DINEV Z,AHMAD Z W,ROBERT T C,et al.A convenient gram-scale synthesis of uridine diphospho(13C6)glucose[J].Carbohydrate Research,2006,341(10):1743-1747.
[15]IMAI T,TERASHIMA N.Determination of the distribution and reaction of polysaccharide in wood cell wall by the isotopic tracer technique I.Selective radio-labeling of cell wall polysaccharide in Magnolia kobus[J].Mokuzai Gakkaishi,1990,36(11):917-922.
[16]BJRKMAN A.Lignin and lignin-carbohydrate complexes:Extraction from wood meal with neutral solvents[J].Industrial&Engineering Chemistry,1957,49(9):1395-1398.
[17]AZUMA J,KOSHIJIMA T.13C NMR spectroscopy of cellulose and related carbohydrates[J].Wood Research,1981,16:63-69.