Comparison of catalytic properties of multiple β-glucosidases of Trichoderma reesei
| Autor: | Nobuaki Sato, Kouichi Nozaki, Boyang Guo, Yoshihiko Amano, Peter Biely |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Cellobiose DNA Complementary Sophorose Aspergillus oryzae Molecular Sequence Data Disaccharides Applied Microbiology and Biotechnology Substrate Specificity Fungal Proteins 03 medical and health sciences chemistry.chemical_compound Glucosides Escherichia coli Cellulases Gentiobiose Glycosyl Amino Acid Sequence DNA Fungal Glucans Laminaribiose Trichoderma reesei Thermostability Trichoderma biology Temperature General Medicine Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Glucose 030104 developmental biology Biochemistry chemistry biology.protein Glucosidases Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology. 100:4959-4968 |
| ISSN: | 1432-0614 0175-7598 |
| DOI: | 10.1007/s00253-016-7342-x |
| Popis: | Ten putative Trichoderma reesei β-glucosidase (BGL) isozymes were heterologously expressed in Escherichia coli and Aspergillus oryzae and purified to homogeneity. Catalytic properties of nine enzymes which showed hydrolytic activity on cellobiose and p-nitrophenyl-β-D-glucopyranoside (pNPG) were investigated. Three BGLs, encoded by the genes cel3A, cel3B, and cel3E, contained a predicted signal peptide, showed higher hydrolytic activity on cello-oligosaccharides than on pNPG, and preferred longer oligosaccharides. Another three putative extracellular BGLs, Cel3B, Cel3F, and Cel3G, and two intracellular enzymes, Cel3C and Cel3D, exhibited preference for pNPG. Intracellular Cel1A showed the highest affinity for cellobiose as a typical cellobiase. Four BGLs, Cel3A, Cel3B, Cel3E, Cel1A, that showed high activity against cello-oligosaccharides were capable of catalyzing transglycosylation reactions from cellobiose, leading to formation of cellotriose and isomeric glucobioses. While Cel3A, Cel3B, and Cel3E synthesized mainly gentiobiose, glycosyl transfer reactions of Cel1A led mainly to sophorose and laminaribiose. Conversion of cellobiose to sophorose by Cel1A reached about 3.6 and 10 % at 1 and 10 % cellobiose concentration, respectively. The formation and persistence of individual cellobiose isomers in incubation mixtures of four BGLs (Cel3A, Cel3B, Cel3E, and Cel1A) with cellobiose correlated well with the k cat values for isomeric glucobioses. Cel1A also showed the lowest sensitivity to inhibition by glucose. Based on all studied catalytic properties, Cel1A appears to be unambiguously the best candidate for site-directed mutations or directed evolution toward improvement of activity, thermostability, and, eventually, efficiency of sophorose synthesis. |
| Databáze: | OpenAIRE |
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