Improvement in salt-tolerance of Aspergillus oryzae γ-glutamyl transpeptidase via protein chimerization with Aspergillus sydowii homolog

Abstract

γ-Glutamyl transpeptidase is one of the key enzymes involved in glutamate production during high-salt fermentation of soy sauce and miso by koji mold, Aspergillus oryzae. However, the activity of γ-glutamyl transpeptidase from A. oryzae (AOggtA) is markedly reduced in the presence of NaCl, thus classifying it as a non-salt-tolerant enzyme. In contrast, the homologous protein from the xerophilic mold, A. sydowii (ASggtA) maintains its activity under high-salt conditions. Therefore, in this study, a chimeric enzyme, ASAOggtA, was designed and engineered to improve salt-tolerance in AOggtA by swapping the N-terminal region, based on sequence and structure comparisons between salt-tolerant ASggtA and non-salt-tolerant AOggtA. The parental AOggtA and ASggtA and their chimera, ASAOggtA, were heterologously expressed in A. oryzae and purified. The chimeric enzyme inherited the superior activity and stability from each of the two parent enzymes. ASAOggtA showed > 2-fold greater tolerance than AOggtA in the presence of 18% NaCl. In addition, the chimera showed a broader range of pH stability and greater thermostability than ASggtA. AOggtA and ASAOggtA were sy over the range pH 3.0 to pH 10.5. Thermal stability was found to be in the order AOggtA (57.5 °C, t1/2 = 32.5 min) > ASAOggtA (55 °C, t1/2 = 20.5 min) > ASggtA (50 °C, t1/2 = 12.5 min). The catalytic and structural characteristics indicated that non-salt-tolerant AOggtA would not undergo irreversible structural changes in the presence of NaCl, but rather a temporary conformational change, which might result in reducing the substrate binding and catalytic activity, on the basis of kinetic properties. In addition, the chimeric enzyme showed hydrolytic activity toward L-glutamine that was as high as that of AOggtA. The newly-designed chimeric ASAOggtA might have potential applications in high-salt fermentation, such as miso and shoyu, to increase the content of the umami-flavor amino acid, L-glutamate.