Abstract: Glutaminase is an important food enzyme. In order to improve the thermal stability of glutaminase, Discovery studio 2017 was used to virtually mutate forty-nine amino acids in Bacillus subtilis glutaminase (YbgJ) that have adverse interactions. Key amino acid loci E3, E55, and D213 that affect the thermal stability of YbgJ were identified and saturated mutated. Compared with wild type enzyme, half-lives (t1/2, 55 ℃) of the mutants E3C, E55F, and D213T increased by 58%, 69%, and 41%, respectively. The t1/2 of the compounded mutants E3C/E55F/D213T, E3C/E55F, E3C/D213T, and E55F/D213 were 1.73, 1.44, 1.22, and 0.97 times higher than that of the wild type enzyme, respectively. Among them, the specific activity of E3C/E55F raised by 23%, reaching 693 U/mg. Structural analysis indicated that E3C/E55F/D213T, E3C/E55F, E3C/D213T, and E55F/D213T had thirty, twenty-three, eleven, and eight more hydrogen bonds, respectively, than that in wild type enzyme. Meanwhile, their adverse interactions decreased by 5, 4, 4, and 3, respectively. The results revealed that substituting key amino acids in the enzyme molecule had great influences on the thermal stability of YbgJ. Decreases in intramolecular adverse interactions and increases in hydrogen bonds may account for enhanced thermal stability of YbgJ.
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