title: N-linked glycosylation of recombinant cellobiohydrolase I (Cel7A) from Penicillium verruculosum and its effect on the enzyme activity.
abstract: Cellobiohydrolase I from Penicillium verruculosum (PvCel7A) has four potential N-glycosylation sites at its catalytic module: Asn45, Asn194, Asn388, and Asn430. In order to investigate how the N-glycosylation influences the activity and other properties of the enzyme, the wild type (wt) PvCel7A and its mutant forms, carrying Asn to Ala substitutions, were cloned into Penicillium canescens PCA10 (niaD-) strain, a fungal host for production of heterologous proteins. The rPvCel7A-wt and N45A, N194A, N388A mutants were successfully expressed and purified for characterization, whereas the expression of N430A mutant was not achieved. The MALDI-TOF mass spectrometry fingerprinting of peptides, obtained as a result of digestion of rPvCel7A forms with specific proteases, showed that the N-linked glycans represent variable high-mannose oligosaccharides and the products of their sequential enzymatic trimming, according to the formula (Man)0-13 (GlcNAc)2 , or a single GlcNAc residue. Mutations had no notable effect on pH-optimum of PvCel7A activity and enzyme thermostability. However, the mutations influenced both the enzyme adsorption ability on Avicel and its activity against natural and synthetic substrates. In particular, the N45A mutation led to a significant increase in the rate of Avicel and milled aspen wood hydrolysis, while the substrate digestion rates in the case of N194A and N388A mutants were notably lower relative to rPvCel7A-wt. These data, together with data of 3D structural modeling of the PvCel7A catalytic module, indicate that the N-linked glycans are an important part of the processive catalytic machinery of PvCel7A.
authors: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow, 119899, Russia.
authors: Anna S
authors: AS
authors: Dotsenko
authors: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow, 119899, Russia. avgusakov@enzyme.chem.msu.ru.|A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Pr. 33, Moscow, 119991, Russia. avgusakov@enzyme.chem.msu.ru.
authors: Alexander V
authors: AV
authors: Gusakov
authors: A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Pr. 33, Moscow, 119991, Russia.
authors: Pavel V
authors: PV
authors: Volkov
authors: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow, 119899, Russia.|A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Pr. 33, Moscow, 119991, Russia.
authors: Aleksandra M
authors: AM
authors: Rozhkova
authors: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow, 119899, Russia.|A. N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky Pr. 33, Moscow, 119991, Russia.
authors: Arkady P
authors: AP
authors: Sinitsyn
mesh_terms: D019943:Amino Acid Substitution; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010407:Penicillium / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000032:analysis*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: N-glycosylation; Penicillium verruculosum; cellobiohydrolase (Cel7A); cloning; enzyme activity; site-directed mutagenesis