Cloning, Expression And Enzymatic Properties And Cell Wall Expansion Activity Of The Endo-?-1,6-glucanase GH30A From C

The cell wall of the model fungus Coprinopsis cinerea is mainly composed of?-glucan,chitin and protein,among which?-glucan is the main component.In this thesis,by studying the enzymatic properties of a novel Coprinopsis cinerea?-1,6glucanase,we further understand the extensibility activity of?-1,6-glucanase in the cell wall of Coprinopsis cinerea.We identified a hypothetical protein(XP?001831978.2),named as the?-1,6-glucanase GH30A hereafter,from the Coprinopsis cinerea okayama7#130genome,which has 58.32%identity to the amino acid sequence of the endo-?-1,6-glucanase Le Pus30A(BAK52530)from L.edodes Af293 in a protein BLAST search at NCBI.The conserved domains analysis at NCBI showed that the protein belongs to a member of the glycoside hydrolase(GH)family 30 in the Carbohydrate-Active en Zymes(CAZy)database.In order to study the?-1,6-glucanase GH30A of Coprinopsis cinerea,the sequence of N-terminal amino acids 1-21 was characterized as the signal peptide using the Signal P 5.0 server(http://www.cbs.dtu.dk/services/Signal P).Thus,c DNA for mature GH30A without the signal peptide was cloned.The specific primers were designed to amplify the GH30A gene,and it was ligated to the recombinant expression vector p PICZ?A using a one-step cloning method to obtain a positive transformant.After sequencing,compared to the sequence of the GH30A c DNA announced in the NCBI database,the cloned GH30A c DNA lost 12 bp(bp 211-223)and had 15 altered bp.Since we obtained the same results in the four independent cloning and sequencing experiments for GH30A c DNA,the cloned sequence was confirmed as a revised sequence of GH30A c DNA.Thus,GH30A from Coprinopsis cinerea ATCC 56838 consists of 496 amino acids,which four fewer amino acids and two different amino acids(A²⁰¹and S²⁶⁰are changed to T²⁰¹and N²⁶⁰,respectively)compared with the previously described protein at NCBI.We used the Pichia pastoris eukaryotic expression system to heterologously recombinantly express GH30A.Due to the use of the histidine tag of the recombinantly expressed protein,nickel column affinity chromatography was not used for purification.Therefore,the recombinant GH30A protein was precipitated from the culture medium with ammonium sulfate to 85%saturation.The precipitate was dissolved in 50 m M Tris-HCl buffer(p H 7.5)and 50 m M Tris-HCl buffer(p H7.5)dialyze overnight and load on a DEAE-Sepharose open column.The DEAE-Sepharose column adsorbs and removes foreign proteins,but the flow-through fraction of the DEAE-Sepharose open column still exhibited?-1,6-glucanase activity,with a yield of 47.03%,showed only one protein band at about 53 k Da via SDS-PAGE.Subsequently,an amino acid sequence analysis of trypsinized protein fragments assayed by MALDI TOF/TOF MS showed that this protein band represented GH30A from Coprinopsis cinerea.Research on the enzymatic properties of GH30A,we found that GH30A has an enzymatic activity up to 776.50 U mg^-1on pustulan,and is thermophilic.The enzyme activity reaches the highest at 60°C,at 60°C,Tris-HCl buffer at p H 7.5 retained88.85%of activity after 1 h incubation.Substrate specificity analysis showed that the optimal substrate of GH30A was pustulan.The reaction kinetic parameters of pustulan were Km=0.89 g L^-1and Vmax=1236.66 U mg^-1.By measuring whether different metal ions and metal chelator EDTA have an effect on the enzyme activity of GH30A,the hydrolytic activity of GH30A was enhanced(11-29%)toward pustulan by 1 m M of Mn²⁺,Co²⁺,Ni²⁺,Ca²⁺,Mg²⁺,Al³⁺,Ba²⁺,Mo O₄^2-,Cr₂O₇^-or EDTA,whereas it was completely inhibited by 1 m M Cu²⁺or Fe²⁺or 2 m M EDTA.GH30A did not hydrolyze laminarin from Laminaria digitata,which consists of a?-1,3-glucan backbone with branches of only one?-1,6-linked glycoside,whereas it hydrolyzed laminarin from Eisenia bicyclis,in which the?-1,3-glucan backbone contains 1-3 of?-1,6-linked glycosides in its branched chains,which is shown in studying fungal cell walls in structure,?-1,6-glucanase GH30A has great potential.The enzymatic digestion products of pustulan reacted with GH30A from Coprinopsis cinerea were analyzed by HPAEC-PAD.When reacted for 5 min,GH30A hydrolyzed pustulan to yield glucose,gentiobiose,and a series of other1,6-?-linked oligoglucosides,exhibiting an endo-action mode.After allowing the reaction to proceed for 20 min,glucose,gentiobiose,and gentiooligosaccharides with dp of 3-10 further accumulated,whereas the levels of gentiooligosaccharides with dp of>10 were reduced or disappeared.These results indicated that the enzyme preferably hydrolyzed the larger gentiooligosaccharides to produce the smaller gentiooligosaccharides.Furthermore,GH30A did not hydrolyze gentiobiose,while the amount of glucose was much higher than that gentiobiose in the GH30A digested products of pustulan.These results indicated that the glucose and gentiobiose in the reaction mixture were derived from the cleavage of both gentiotriose and gentiooligosaccharides with dp of>3 rather than only gentiotriose by the Coprinopsis cinerea GH30A.GH30A was used to study whether it can induce the extensibility activity of the heat-inactivated stipe cell wall to elucidate the chemical composition and structure of polysaccharides in the stipe cell wall.Studies have shown that GH30A can reconstruct the extensibility of the heat-inactivated stipe cell wall,showing the initial rapid extensibility,and then the extensibility rate decreases rapidly,similar to the extensibility profile of?-1,3-glucanase ENG.The extensibility of the heat-inactivated stipe cell wall reconstructed by GH30A and ENG has a similar extensibility curve when the two are acting separately,which is different from the continuous and stable extension curve of the stipe cell wall reconstructed by chitinase,indicating that the stipe cell wall chitin is cross-linked with?-1,6-glucan.In addition,chitin is crosslinked with?-1,3-glucan and?-1,6-glucan,as well as other polysaccharide components.In order to understand the expression of GH30A in the stipes and pileis of Coprinopsis cinerea,we studied it using RT-PCR technology.The analysis of m RNA expression levels showed that GH30A was almost not expressed in the rapidly growing apical stipe region and the slowly growing median stipe region,whereas it expressed in the non-growing basal and the basal swollen stipe regions.GH30A was also almost not expressed in the pilei with 0°opening angles,whereas the expression levels of GH30A were reached to 20.63-fold and 16.29-fold m RNA abundance,respectively,in the pilei with 90°opening angles and the pilei with 180°full opening angles.The differential expression of GH30A in various parts of the stipe and its pilei at different periods indicates that it is consistent with the requirement to stop the growth of the basal region of the stipe to reshape the cell wall,and also participates in the mature and cell wall degradation during autolysis.