1,3-β-glucanase enzyme has been proved as antibiofilm by hydrolyzing the main component of extracellular matrix of C. albicans polymicrobial biofilm, to prevent resistancy during the use of antibiotics. The aim of this study is to construct a metagenomic expression library from Achatina fulica's digestive gland and to screen for a novel 1,3-β-glucanase genes by using its specific substrate of laminarin. A cDNA expression library was constructed using the λTriplEx2 vector in the E. coli strain XL1-Blue. Cre-recombinase circularization was used to convert λTriplEx2 to pTriplEx2 in the E. coli strain BM 25.8;then IPTG induction was used to express 1,3-β-glucanase. High-efficiency cDNA library of A. fulica's digestive gland was constructed, from where we obtained seventeen halo positive plaques, among them is a novel 1,3-β-glucanase gene designated MkafGlu1. Its nucleotide sequence has similarities to the endo-1,3-β-glucanase from Gossypium hirsutum, as well as the β-glucanases from Paenibacillus mucilaginosus, Verticillium alfalfa, and Cryptopygus antarcticus of 45%, 40%, 38% and 37%, respectively. An open reading frame of 717 bp encoded a protein of 239 amino acids. A novel 1,3-β-glucanase gene called MkafGlu1 was successfully expressed in E.coli BM 25.8 with activity of 1.07 U mL-1.
Keywords: 3-β-glucanase; Achatina fulica; Digestive gland; Expression library; Metagenomic; Novel 1.