Glycosylation Of Cellobiohydrolase ? Induced By Different Carbon Sources In Trichoderma Reesei

Nowadays,the scarcity of non-renewable energy such as oil is severe,and environmental problems caused by the combustion of fossil fuel are becoming increasingly serious.So,it is urgent to develop clean renewable energy.Considering the huge storage and cheap price of lignocelluloses,the strategy of converting the lignocellulose into cellulosic ethanol will not only effectively alleviate the energy crisis,but also protect the ecological environment and make full use of potential resources.Cellulase plays a significant role in this biological refining process.Cellulase is highly glycosylated,and it will be a new breakthrough to improve the activity of cellulase by modifying the glycosylation of cellulase and then improve the hydrolysis efficiency of cellulose.In order to modify the glycosylation of cellulase,we need to understand the glycosylation site of cellulase and the structure of glycans systematically.In this study,the glycosylation modification of Trichoderma reesei cellobiose hydrolase I(TrCBH I)induced by lactose,cellobiose and rich medium was studied systematically using the cellulase high-yield strain Trichoderma reesei RutC-30 and cellulase low-yield strain Trichoderma reesei QM9414.Six target proteins were separated and purified,and then the target proteins were treated by mixed enzymatic hydrolysis(gluC+trypsin and chymotrypsin),and the obtained peptides were separated by ZIC-HILIC column.After the glycopeptides were collected,the glycopeptides were dissociated by collision-induced dissociation(CID)and electron transfer dissociation(ETD),respectively.Finally,the glycopeptides were analyzed by LC-MS/MS.Through the analysis of mass spectrometry data,the following experimental results were obtained:(1)There are high mannose type N-glycans modification on N64.Glycosylation on N64 was identified in both TrCBH I of T.reesei RutC-30 and T.reesei QM9414,and the glycans form are high mannose type.(2)In addition to the heterozygous glycans,N45 are also modified with high mannose type glycans.(3)O-glycosylation modifications are prevalent in the catalytic domain.In few previous studies on the O-glycosylation of the CD of TrCBH I,only one HexNAc modification on S20,S21 and T24 has been reported in a recent paper.In this study,11 new O-glycosylation sites,such as S8,T10,T232,S396,were identified.There are usually 1?4 Hex or 1?2 HexNAc modifications at these O-glycosylation sites.(5)There is a new O-glycosylation site T484 in the CBM of TrCBH I.Only one paper reported that there were 1?3 Man modifications on Thr462 and Ser464 of CBM.In this study,T.reesei RutC-30 was induced by lactose and a single Hex modification at position of 484 on the threonine in TrCBH I was found.(6)The glycosylation site of CD in TrCBH I tend to form glycosylation clusters:N270/T271/S272/S396,T232/T255,T383/N384/T389,S8/T10,and N45/S46.Among them,N45/S46 and S8/T10 are located near the tunnel entrance of the catalytic active site,while T383/N384/T389 is located on the peptide ring closest to the cellulose crystal.It is suggested that the three glycosylation clusters might be related to substrate recognition or the catalytic activity for substrate.Through the transverse analysis,the glycosylation modification of TrCBH I was different when the different strain induced by the same carbon source,not only that,the glycosylation modification of TrCBH I was also different when the same strain induced by the different carbon source.It was found that the glycosylation of T.reesei RutC-30 are more diverse and complicated compared with T.reesei QM9414,and the heterogeneity of the glycans at the same site is more obvious.In terms of induction methods,the glycosylation sites of TrCBH I induced by lactose are more abundant,and the forms of glycans are more diverse.The glycosylation of the cellulase high-yield strain T.reesei RutC-30 and the cellulase low-yield strain T.reesei QM9414 induced by different carbon sources was studied systematically,and more glycosylation sites and glycans were excavated.The understanding of glycosylation of TrCBH I are more comprehensive,which lays a solid theoretical foundation for the subsequent modification of glycosylation and then improve the hydrolysis activity of cellulase.