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Identification of amino acid residues responsible for increased thermostability of feruloyl esterase A from Aspergillus niger using the PoPMuSiC algorithm

Shuai-Bing Zhang, Zhong-Liu Wu
Bioresource technology 2011 v.102 no.2 pp. 2093-2096
thermal stability, Aspergillus niger, energy, Komagataella pastoris, esterases, amino acid substitution, amino acids, hydrolysis, site-directed mutagenesis, algorithms, half life
Feruloyl esterases are key enzymes involved in the complete hydrolysis of hemicellulose. In order to improve the thermostability of feruloyl esterase A (FaeA) from Aspergillus niger CIB 423.1, the PoPMuSiC algorithm was applied to predict the folding free energy change (ΔΔG) of amino acid substitutions. Four amino acid substitutions (S92A, D93G, D174A and S187F) were introduced into the enzyme by site-directed mutagenesis and the enzymes were produced in Pichia pastoris KM71. No obvious changes in thermal stability resulted from substitutions S92A and D174A, but, compared to the wild-type enzyme which has a half-life of inactivation of 8min, the half-lives of enzymes with a D93G or S187F substitution increased to 9.4 and 60.5min, respectively. The double mutant D93G/S187F displayed a synergistic effect with a t₁/₂ value of 77.0min. It also displayed over 10-fold increase in catalytic turnover frequency. The result will benefit further investigation of the thermostability of feruloyl esterase A.