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Comparison of catalytic properties of multiple β-glucosidases of Trichoderma reesei

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Abstract

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.

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Authors and Affiliations

  1. Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 4-17-1 Wakasato, Nagano, 380-8553, Japan

    Boyang Guo, Nobuaki Sato, Yoshihiko Amano & Kouichi Nozaki

  2. Institute of Chemistry, Slovak Academy of Sciences, 845 38, Bratislava, Slovakia

    Peter Biely

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  1. Boyang Guo
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  2. Nobuaki Sato
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  3. Peter Biely
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  4. Yoshihiko Amano
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  5. Kouichi Nozaki
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Correspondence to Kouichi Nozaki.

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This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

This study was funded by Grant-in-Aid for the Global COE Program by the Ministry of Education, Culture, Sports, Science and Technology of Japan and was also sponsored by New Energy and Industrial Technology Development Organization (NEDO).

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The authors declare that there are no conflicts of interest.

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Guo, B., Sato, N., Biely, P. et al. Comparison of catalytic properties of multiple β-glucosidases of Trichoderma reesei . Appl Microbiol Biotechnol 100, 4959–4968 (2016). https://doi.org/10.1007/s00253-016-7342-x

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  • DOI: https://doi.org/10.1007/s00253-016-7342-x

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