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Sequence Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity

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Abstract

Tannin acyl hydrolases or tannases (E.C.3.1.1.20) are enzymes that hydrolyze the ester bond of tannins to produce gallic acid and glucose. We engineered the Aspergillus niger GH1 tannase sequence and Pichia pastoris strains to produce and secrete the enzyme as a single-chain protein. The recombinant tannase was N-glycosylated, had a molecular mass after N-deglycosylation of 65.4 kDa, and showed activity over broad pH and temperature ranges, with optimum pH and temperature of 5.0 and 20 °C. Furthermore, the single-chain tannase had an 11-fold increased specific activity in comparison to the double-chain A. niger GH1 tannase, which was also produced in P. pastoris. Structural analysis suggested that the high specific activity may be due to the presence of a flexible loop in the lid domain, which can control and drive the substrate to the active site. In contrast, the low specific activity of the double-chain tannase may be due to the presence of a disordered and flexible loop that could hinder the substrate’s access to the binding site. Based on its biochemical properties, high specific activity, and the possibility of its production in P. pastoris, the tannase described could be used in food and beverage processing at low and medium temperatures.

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Acknowledgements

We are thankful for Grant CN1207-20 (PAICYT) from the Universidad Autónoma de Nuevo León. We thank Glen D. Wheeler and Samira Hosseini for their stylistic suggestions in the preparation of the manuscript. D. O.-P. thanks CONACYT for her fellowship.

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  1. Instituto de Biotecnología, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, UANL, Av. Universidad S/N, Col. Ciudad Universitaria, 66455, San Nicolás de los Garza, Nuevo León, Mexico

    Daniela Ordaz-Pérez, José Antonio Fuentes-Garibay, Martha Guerrero-Olazarán & José María Viader-Salvadó

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  1. Daniela Ordaz-Pérez
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  2. José Antonio Fuentes-Garibay
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  3. Martha Guerrero-Olazarán
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  4. José María Viader-Salvadó
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DO-P participated in the methodology, formal analysis, investigation, data curation, preparation of the original draft, reviewing and editing of the manuscript, and visualization. JAF-G participated in the investigation, data curation, and reviewing and editing of the manuscript. MG-O participated in the conceptualization, methodology, resources, reviewing and editing of the manuscript, supervision, and project administration. JMV-S participated in the conceptualization, methodology, formal analysis, data curation, preparation of the original draft, reviewing and editing of the manuscript, supervision, project administration, and funding acquisition.

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Correspondence to José María Viader-Salvadó.

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Ordaz-Pérez, D., Fuentes-Garibay, J.A., Guerrero-Olazarán, M. et al. Sequence Engineering of an Aspergillus niger Tannase to Produce in Pichia pastoris a Single-Chain Enzyme with High Specific Activity. Mol Biotechnol 64, 388–400 (2022). https://doi.org/10.1007/s12033-021-00416-6

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