title: N-glycoform diversity of cellobiohydrolase I from Penicillium decumbens and synergism of nonhydrolytic glycoform in cellulose degradation.
abstract: Four cellobiohydrolase I (CBHI) glycoforms, namely, CBHI-A, CBHI-B, CBHI-C, and CBHI-D, were purified from the cultured broth of Penicillium decumbens JU-A10. All glycoforms had the same amino acid sequence but displayed different characteristics and biological functions. The effects of the N-glycans of the glycoforms on CBH activity were analyzed using mass spectrum data. Longer N-glycan chains at the Asn-137 of CBHI increased CBH activity. After the N-glycans were removed using site-directed mutagenesis and homologous expression in P. decumbens, the specific CBH activity of the recombinant CBHI without N-glycosylation increased by 65% compared with the wild-type CBHI with the highest specific activity. However, the activity was not stable. Only the N-glycosylation at Asn-137 can improve CBH activity by 40%. rCBHI with N-glycosylation only at Asn-470 exhibited no enzymatic activity. CBH activity was affected whether or not the protein was glycosylated, together with the N-glycosylation site and N-glycan structure. N-Glycosylation not only affects CBH activity but may also bring a new feature to a nonhydrolytic CBHI glycoform (CBHI-A). By supplementing CBHI-A to different commercial cellulase preparations, the glucose yield of lignocellulose hydrolysis increased by >20%. After treatment with a low dose (5 mg/g substrate) of CBHI-A at 50 °C for 7 days, the hydrogen-bond intensity and crystalline degree of cotton fibers decreased by 17 and 34%, respectively. These results may provide new guidelines for cellulase engineering.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China.
authors.forename: Le
authors.initials: L
authors.lastname: Gao
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Lushan
authors.initials: L
authors.lastname: Wang
authors.forename: Cunliang
authors.initials: C
authors.lastname: Geng
authors.forename: Lianli
authors.initials: L
authors.lastname: Chi
authors.forename: Jian
authors.initials: J
authors.lastname: Zhao
authors.forename: Yinbo
authors.initials: Y
authors.lastname: Qu
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D032561:Cotton Fiber; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014961:X-Ray Diffraction
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D011134:Polysaccharides; D001216:Asparagine; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase