Correlation between superior enzymatic properties of β-mannanase AuMan5A/Af and its residue Asp320

Abstract:

[Objective] AuMan5A is a glycoside hydrolase (GH) family 5 β-mannanase from Aspergillus usamii. To improve its enzymatic properties, we have previously constructed a mutant with loop substitution, AuMan5A/Af, by substituting a loop of seven residues (³¹⁶KSPDGGN³²²) in its substrate binding groove with the corresponding region (PSPNDHF) of A. fumigatus GH family 5 β-mannanase. To reveal the correlation between the superior enzymatic properties of AuMan5A/Af and its residue Asp³²⁰, site-directed mutagenesis was used to obtain a new mutant enzyme AuMan5A/Af D^320G. [Methods] Using megaprimer PCR method, we constructed a new mutant-encoding gene, Auman5A/Af ^D320G by mutating an Asp³²⁰-encoding codon GAC of Auman5A/Af into a Gly³²⁰-encoding GGT. Then, Auman5A/Af ^D320G was extracellularly expressed in Pichia pastoris GS115, and the enzymatic properties of the expressed product were analyzed. [Results] Analytical results indicated that the optimal and melting temperature of AuMan5A/Af D^320G was 70.0 ℃ and 71.5 ℃, repectively, higher than those of AuMan5A (T_opt=65.0 ℃, T_m=64.5 ℃) and lower than those of AuMan5A/Af (T_opt=75.0 ℃, T_m=76.6 ℃); its half-life at 70.0 ℃ was 40 min, 10 min longer than that of AuMan5A but greatly shorter than 480 min of AuMan5A/Af. Besides, its specific activity was 2.7 fold and 0.3 fold that of AuMan5A and AuMan5A/Af, respectively, and its catalytic efficiency (k_cat/K_m) was 3.9 fold and 0.3 fold that of AuMan5A and AuMan5A/Af. [Conclusion] The mutation of Asp³²⁰ into Gly³²⁰ greatly affected the temperature characteristics and catalytic activity of AuMan5A/Af, demonstrating that Asp³²⁰ plays an improtant role in temperature characteristics, specific activity and catalytic efficiency improving of AuMan5A after loop substitution.