Preparation And Structure Analysis Of Pectin Domain From 7 Species Of Plants

Pectin substances are a class of complex polysaccharides present in plant cell walls.It has many biological activities,such as anti-tumor,anti-fatigue,immune regulation.To study its structure-activity relationship,it is important to prepare pectin domain from plant.In this study,biotransformation and enzymolysis were chosed and successfully prepared pectin domain.The strains,B4,B14,F2 and F5,which could degrade ginseng pectin were screened by ginseng pectin as the sole carbon source,and they were used to transform ginseng pectin.The products were purified by ion exchange chromatography and gel filtration chromatography and 10 homogeneous fractions were obtained.The bacteria transformation products WGPAY-2B,WGPAY-3A and WGPAZ-2A were mainly composed of GalA with the molecular weight of 3.3,3.5 and 5 KDa.Structural analyses by enzymolysis and NMR found that they were HG domain covalently cross-linked to RG-II domain.The bacteria transformation products WGPAY-2A and WGPAZ-1A were mainly composed of GalA,Rha,Gal,Ara and Glc,among which the ratio of Rha/GalA were 1 and 0.9 suggesting they were pure RG-I domain with the molecular weight of 4 and 22.9 KDa,respectively.Structure analysis by partial hydrolysis,enzymolysis and NMR found that they were used Rha and GalA as backbone with AG-II domain as side chain.The fungi transformation products were all proved as RG-II domains with the molecular weight ranged from 4 KDa to 10 KDa.Structural analyses by partial hydrolysis,ion-exchange chromatography and ESI-MS found that the side chain A of RG-II domains from ginseng pectin was composed of eight sugar residues with 0 to 1 methyl group and Gal substitution.The side chain B contained 9 residues with 0 to 2 acetyl groups.In this part,pectins were transformed by Penicillium oxalicum from Panax ginseng,Viscum coloratum,Veratrum nigrum,Apocynum venetum,Helianthus annuus,Codonopsis pilosula and Scrophularia ningpoensis.The products were purified by ion exchange chromatography and gel filtration chromatography and 16 homogeneous fractions were obtained from the above-mentioned plant.The fractions were proved as RG-II domain by TBA test.Structural analyses by partial hydrolysis,ion-exchangechromatography and ESI-MS found that the side chain A of RG-II domains from different plants were composed of eight sugar residues with Gal substitution.They were composed of 0 to 2 methyl ether group in Apocynum venetum,Codonopsis pilosula and Scrophularia ningpoensis,0 to 1 methyl ether group in Panax ginseng,Veratrum nigrum and Helianthus annuus,and non methyl ether group in Viscum coloratum.The side chain B contained 8 residues with 0 to one acetyl group in Helianthus annuus,10 residues with 0 to 2 acetyl groups in Apocynum venetum and 9residues with 0 to 2 acetyl groups in other plant.Pectins were treated with NaOH and pectinase from Panax ginseng,Viscum coloratum,Veratrum nigrum,Apocynum venetum,Helianthus annuus,Codonopsis pilosula and Scrophularia ningpoensis.The homogeneous fractions were obtained after purification by ion exchange chromatography and gel filtration chromatography and proved as RG-I domain and RG-II domain.The yield of RG-II domain in these plants were ranged from 4.0% to 9.9%,with 4.0% in Codonopsis pilosula,9.9% and8.4% in Apocynum venetum and Panax ginseng,and about 6% in other plants.The yield of RG-I domain in these plants were ranged from 2.5% to 12.2%,with 2.5% in Apocynum venetum and Scrophularia ningpoensis,12.2% and 9.1% in Viscum coloratum and Veratrum nigrum,and about 5% in other plants.Structural analysis by partial hydrolysis,enzymolysis and NMR found that the molecular weight of RG-I domain ranged from 17.6 KDa to 96.6 KDa.They were mainly composed of GalA,Rha,Gal and Ara,and the content of Rha and Gal A were ranged from 10% to 30%respectively and the ratio of Rha/GalA was ranged from 0.7 to 1,suggesting they were pure RG-I domain.The content of Gal was ranged from 20% to 50% and the content of Ara was ranged from 5% to 20%.Structure analysis also showed that they were used Rha and GalA as backbone with AG-II domain as side chain,which may also contained AG-I and Arabinan as side chain in individual plants.In this research work,the methods for preparing pectin domain were established.Further more,their structure features were studied.Our results have significant impact in studying pectin structure-activity relationships.