High-level stable expression of gene for preparation of chlorothalonil hydrolytic dehalogenase and its application in elimination of chlorothalonil inhibition on bioconversion of lignocellulosic biomass

Abstract

To achieve the high-level stable expression of chlorothalonil hydrolytic dehalogenase (Chd), the gene chd was first integrated into the chromosome of Bacillus subtilis WB800. High generation stability was achieved by almost no gene lost after six generations but Chd activity decreased. aprE promoter alteration, translation initiation region modification and multi-copy chromosome integration were studied and these modifications could increase Chd activity by 270%, 2304% and 25%. Chlorothalonil residual exhibited inhibition on bioconversion of lignocellulosic biomass. The addition of Chd crude enzyme (60 μL per g wheat straw) could increase glucose production by 36.10% and 39.65% in synergistic hydrolysis and separate hydrolysis by laccase and cellulase with 120 mg/L residual chlorothalonil. Filter paper activity and carboxymethyl cellulase activity were enhanced by 12.84% and 23.95%, and biomass of Trichoderma reesei was increased by 76.67% under 50 μg chlorothalonil/g dry straw in solid-state fermentation. Thus, the high-level stable expressed Chd effectively eliminated chlorothalonil inhibition on enzymatic hydrolysis and solid-state fermentation. It showed promising potential for bioremediation of chlorothalonil pollution and improving conversion efficiency of lignocellulose.

Keywords: Chlorothalonil hydrolytic dehalogenase; High-level stable expression; Inhibition; Lignocellulose bioconversion; Solid-state fermentation.