Modification of a Thermostable β-mannanase and Its High-Efficiency Expression

A β-mannanase from Aspergillus usamii (A. usamii) belonging to glycoside hydrolase (GH) family 5 (abbreviated as AuMan5A) was selected as the research subject to enhance the enzyme protein’s thermotolerance and catalytic efficiency by replacing a random coil (loop) structure on the enzyme molecule. Furthermore, the correlation between the structure and function of β-mannanase was clarified. Homology modeling was used to simulate the three-dimensional structure of AuMan5A, and mannopentaose was docked in the AuMan5A substrate binding groove. Based on the spatial characteristics and phylogenetic analyses of the complex structure, a non-conserved loop structure (Loop FG) was speculated to be involved in enzyme-substrate interactions. Therefore, Loop FG was selected as the modified region. Additionally, the corresponding fragments of Aspergillus nidulans, Aspergillus fumigatus, and Trichoderma harzianum β-mannanases were selected to replace the Loop FG sequence (316KSPDGGN322) of AuMan5A, respectively, based on the enzymological properties and structural characteristics of other fungal GH 5 family β-mannanases. Mutant enzyme bases were constructed by PCR, and using plasmid pPICZαA, the original and mutant enzymes were expressed in Pichia pastoris GS115 to analyze the enzymological properties of the expressed products. Recombinant enzymes re-AuMan5A, re-AuMan5A-Af, re-AuMan5A-An, and re-AuMan5A-Th had an optimum temperature of 65 °C, 75 °C, 65 °C, and 70 °C, respectively. Their activity half-lives at 70 °C were 10 min, 480 min, 5 min, and 25 min, respectively, and their melting temperatures were 64.5 °C, 76.6 °C, 63.2 °C, and 69.1 °C, respectively. Re-AuMan5A-Af, re-AuMan5A-An, and re-AuMan5A-Th had a kcat/km (catalytic efficiency) value that was 12.7, 6.0, and 11.0 times higher than re-AuMan5A, respectively, with re-AuMan5A-Af exhibiting the best temperature characteristics and catalytic efficiency. The G320-to-D320 mutation of AuMan5A during loop structure replacement significantly affected AuMan5A/Af’s enzymological properties, suggesting the vital role of G320 in improving AuMan5A/Af’s thermostability, specific activity, and catalytic efficiency.