Study On The Recombinant Expression And Enzymatic Properties Of Coprinus Spp. Glucosidase BGL2

Fungal cell wall is a rigid structure and it is associated with various physiological activities of the fungus.The cell wall is mainly composed of polysaccharides filled with proteins.Various polysaccharides form a skeletal structure of the fungal cell wall.The ?-(1,6)-branched-?-(1,3)-glucan is the main chain,and other polysaccharides such as chitin and galactomannan are covalently attached.Coprinopsis cinerea is an ideal object for studies of the developmental processes in basidiomycetes,mainly because its short life cycles.The growth of fruiting body begins with the elongation of the stipe to the end of the autolysis of the pilei releasing of the spores.During the above process,glucans in the cell wall undergone a series of dynamic changes,including extension,branching,hydrolysis and cross-linking,and a variety of glucanases involved in the above remodeling process.In order to investigate which glucanases were involved in cell wall remodeling,some proteins with glucan hydrolase activity were extracted from the stipe and pilei of the Coprinopsis cinerea.The molecuLar masses of the protein from the stipe is 302.2kDa,which is likely a trimer consisting of three polypeptide subunits of 165.2,87.9 and 56.8 kDa.The molecuLar masses of the protein from the pilei is 109kDa,which is likely a dimer consisting of two polypeptide subunits of 60kDa and 53kDa.Identified by mass spectrometry,the two proteins both are extracelluLar beta-glycosidase(BGL2).In order to further study the physiological function of BGL2,we expressed different variants of BGL2 in vitro.We successfuLly expressed the fuLl length of ?-glucosidase(BGL2)with a eukaryotic expression system and used Pichia pastoris as the host cell.The recombinant protein was purified by Ni-column and the pNpG acticity of the purified BGL2 was 4.18U/mg and the protein yield was 21.83%.SDS-PAGE showed that the molecuLar size of BGL2 was 152.3 kDa,which was much higher than the theoretical molecuLar size of 96.6 kDa,indicating that the protein was glycosylated.The BGL2 couLd degrade oligosaccharides,and add a glucose residue to the non-reducing end of the glucan chains with 1,6 glycosidic bond,forming a branched glucan chains.The transglycoside activity of BGL2 is higher,when using the longer oligosaccharide as substrate.The low concentration of enzyme(0.63X10-4U,0.13x10-3U)had only hydrolytic activity on the oligosaccharides,the products of transglycosidation appers with a higher concentration(0.63X10-3U).With the increasing of the concentration(1.26x10-3U?2.52x10-3U),the hydrolyzate and transglycoside products will continue to increase until they are finally hydrolyzed to glucose.Glucose has some influence on the transglycoside activity of BGL2.After adding glucose,the glucose can inhibit the hydrolysis of the original hydrolysis products,and also can further inhibit the hydrolysis of the transglycoside products.We expressed different variants of BGL2 using Pichia pastoris and E.coli as host cells.However,we do not acquire the active variants.Therefore,we used protease to hydrolyze the recombinantly expressed BGL2 and found that the hydrolysis activity of BGL2 after treated with trypsin increased.SDS-PAGE showed that BGL2 was hydrolyzed by trypsin into two small proteins with a molecuLar size of 84.9 kDa and 58.1 kDa,respectively,which are similar to the size of two small subunits obtained from the stipe.N-terminal sequencing of 58.1 kDa small band was Thr-Gln-Thr-Leu-Val-Ala-Lys-Glu-Ala,which may be a cut site of BGL2.We found that BGL2 in the Coprinopsis cinerea had a branching enzyme activity and could be involved in the synthesis of a branched ?-1,3 glucan.After treated with trypsin,BGL2 was hydrolyzed to two small subunits and the hydrolytic activity increased.Therefore,we propose that the activity of BGL2 was regulated by posttranslational cleavage.