Screening, whole genome analysis, and fermentation condition optimization of a β-glucosidase-producing endophyte from Glycyrrhiza uralensis Fisch

Abstract:

[Background] Bacillus rugosus A78.1 isolated from the fibrous roots of healthy Glycyrrhiza uralensis Fisch. has high β-glucosidase activity. [Objective] To investigate the genetic information of the isolate for enzyme production and provide data support for the further research and industrial application of the strain. [Methods] A β-glucosidase-producing Bacillus strain was screened by the medium supplemented with esculin. Nanopore PromethION and Illumina NovaSeq were used for genome sequencing and assembly, and the potential β-glucosidase genes of the strain were predicted by gene prediction and functional annotation. In addition, the effects of carbon source, nitrogen source, inoculum amount, temperature, and initial pH on the enzyme-producing activity of the strain were investigated with β-glucosidase activity as an indicator. [Results] A strain capable of producing β-glucosidase was isolated from the fibrous roots of G. uralensis. It was identified as a strain of Bacillus by morphological observation, physiological and biochemical tests and named B. rugosus A78.1. The genome of this strain was 4 146 938 bp in length, with the G+C content of 43.86%, encoding 4 255 genes. In the genome, 192 carbohydrate-active enzyme genes were annotated, including 10 β-glucosidase genes belonging to the GH1 and GH3 families. A total of 2 896, 4 019, and 3 657 genes were annotated in GO (gene ontology), KEGG (Kyoto encyclopedia of genes and genomes), and COG (clusters of orthologous groups of proteins), respectively. The genome sequencing data were submitted to NCBI and obtained the GenBank accession No. CP096590. For the production of β-glucosidase, the strain should be fermented with 0.5% glucose as the carbon source, 1.0% yeast extract as the nitrogen source at 37℃ and pH 6.0 with the inoculum amount of 3%. Under these conditions, the activity of β-glucosidase produced by this strain reached (5.640±0.085) U/mL. [Conclusion] We confirmed the excellent β-glucosidase-producing ability of B. rugosus A78.1 and the potential of this strain in carbohydrate metabolism by whole genome sequencing and fermentation condition optimization for enzyme production. The findings provide a basis for the research and application of this strain in biochemical and food fields such as cellulose degradation and glycoside hydrolysis.