Enhanced thermostability and catalytic efficiency of glucose oxidase in Pichia Pastoris

Abstract

Glucose oxidase (GOD) has many practical applications, but its poor thermostability limits its broader use. In this research, three primary mutants of wild-type GOD were designed using rational mutagenesis, and the GODm mutant was constructed by combinatorial design. The expression, purification, and enzymatic properties of the mutants were studied. The specific enzyme activity of GODm was 2.10-fold higher than that of wild type, and the (k cat /K m ) value was increased by 1.45-fold. After treatment at 55 ℃ for 3 h, GODm retained 37.5% of its enzymatic activity, and the half-life (t 1/2 ) of GODm at 55 ℃ and 65 ℃ was 2.28-fold and 3.36-fold higher than that of wild type, respectively. By analyzing the three-dimensional structure of wild type and the GODm mutant, it was found that T30V formed a new hydrogen bond with FAD and strengthened the hydrophobic interaction, D315K optimized the surface electrostatic interaction, and A162T improved the efficiency of the electron pathway. Thus, a novel mutant with improved thermostability and catalytic efficiency was obtained in this research.

Purification and characterization of aspartic protease from Aspergillus niger and its efficient hydrolysis applications in soy protein degradation

“…The correctly expression plasmids were linearized with Sal I endonuclease, and the DNA was purified using a PCR product recovery kit and transformed into P. pastoris X33 competent cells [30,31]. After quickly adding 1.0 mL of pre-chilled 1.0 M sorbitol, the cells were incubated at 30 °C, 220 r/min for 2 h before being inoculated in yeast extract peptone dextrose (YPD) plates containing 0.1% zeocin antibiotic, and randomly selecting single colonies for shake flask fermentation re-screening.…”