Evaluation of synthetic gene encoding α-galactosidase through metagenomic sequencing of paddy soil

Many genes of industrial relevance can be found in soil. In this study, metagenome sequencing of paddy soil was performed with 55.68 Gb sequences and 1,787,113 putative open reading frames (ORFs). The functional profiles and metabolic pathway of soil metagenomes were examined using Gene Ontology, Metagenomics RAST, and Kyoto Encyclopedia of Genes and Genomes. To verify the protein function and assembly of ORFs, a putative gene encoding α-galactosidase, namely GalR, which shares 65% identity with an unpublished glycoside hydrolase (GH) 27 family protein, was synthesized using its optimal codon for overexpression in Escherichia coli. GalR was successfully obtained and characterized. The optimal temperature and pH for GalR activity were 30°C and pH 9, respectively. Enzymatic activity indicated that GalR was alkaliphilic and different from acidophilic α-galactosidase in the GH 27 family. Furthermore, 50% of the relative activity of GalR can be attained for 1.7 and 0.7 h preincubation at 40°C and 50°C, respectively. Significant inhibition of GalR was observed in the presence of ethylenediaminetetraacetic acid (EDTA), MgCl₂, sodium dodecyl sulfate (SDS), and H₂O₂; however, it was resistant to 0.1% methanol and ethanol and was slightly activated with NaCl and KCl. The specific activity of GalR was achieved at 11.6 and 0.59 μmol/min/mg of protein using p-nitrophenyl-α-d-galactopyranoside and raffinose as substrates, respectively. Consequently, the metagenomic sequencing-based strategy can provide information for mining novel genes.