Journal
FEBS LETTERS
Volume 594, Issue 5, Pages 799-812Publisher
WILEY
DOI: 10.1002/1873-3468.13652
Keywords
activity; coevolving residues; directed evolution; evolutionary information; pullulanase; saturation mutagenesis
Funding
- National Natural Science Foundation of China (NSFC) [31872891, 21676120]
- 111 Project [111-2-06]
- High-end Foreign Experts Recruitment Program [G20190010083]
- Program for Advanced Talents within Six Industries of Jiangsu Province [2015-NY-007]
- National Program for Support of Top-notch Young Professionals
- Fundamental Research Funds for the Central Universities [JUSRP51504]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
- Jiangsu province 'Collaborative Innovation Center for Advanced Industrial Fermentation' industry development program
- National First-Class Discipline Program of Light Industry Technology and Engineering [LITE2018-09]
- NIH NIGMS [R01-GM120574]
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Pullulanases are well-known debranching enzymes hydrolyzing alpha-1,6-glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis-involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size. Using Bacillus naganoensis pullulanase as a target protein, here we introduce the 'evolutionary coupling saturation mutagenesis' (ECSM) approach: residue pair covariances are calculated to identify residues for saturation mutagenesis, focusing directed evolution on residue pairs playing important roles in natural evolution. Evolutionary coupling (EC) analysis identified seven residue pairs as evolutionary mutational hotspots. Subsequent saturation mutagenesis yielded variants with enhanced catalytic activity. The functional pairs apparently represent distant sites affecting enzyme activity.
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