Identification and Modification of Enzymatic Substrate Specificity through Residue Alteration in the Cap Domain: A Thermostable Zearalenone Lactonase.

Abstract

Zearalenone (ZEN) and its derivatives are prevalent contaminants in cereal crops. This study investigated a novel thermostable ZEN lactonase (ZENM) from Monosporascus sp. GIB2. ZENM demonstrated its highest activity at 60 °C, maintaining over 90% relative activity from 50 to 60 °C. Notably, efficient hydrolysis of ZEN and its two derivatives was achieved using ZENM, with specific activities of 333 U/mg for ZEN, 316 U/mg for α-zearalenol (α-ZOL), and 300 U/mg for α-zearalanol (α-ZAL). The activity of ZENM toward α-ZOL is noteworthy as most ZEN lactonases rarely achieve such a high degradation rate of α-ZOL. Based on the sequence-structure analysis, five residues (L123, G163, E171, S199, and S202) conserved in other ZEN lactonases were substituted in ZENM. Of interest was the G163S mutant in the cap domain that displayed enhanced activity toward α-ZOL compared to the wild-type enzyme. Notably, the mutant G163S exhibited higher catalytic activity toward α-ZOL (kcat/Km 0.223 min-1 μM-1) than ZEN (kcat/Km 0.191 min-1 μM-1), preferring α-ZOL as its optimum substrate. In conclusion, a thermostable ZEN lactonase has been reported, and the alteration of residue G163 in the cap domain has been shown to modify the substrate specificity of ZEN lactonase.