Isolation of the Thermostable β-Glucosidase-Secreting Strain Bacillus altitudinis JYY-02 and Its Application in the Production of Gardenia Blue

Abstract

Gardenia blue (GB) is a natural blue pigment widely used in textiles and the pharmaceutical industry. The geniposide in gardenia fruits can be hydrolyzed by β-glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable β-glucosidase (EC 3.2.1.21) was isolated from soil and identified as Bacillus altitudinis JYY-02. This strain could potentially be used for GB production from geniposide by fermentation. Optimal fermentation results were achieved at pH 6.5 or 8.0 at 45°C for 45 h with additional sucrose. To obtain a large amount of β-glucosidase, the whole genome of B. altitudinis JYY-02 was sequenced and annotated; it is 3,727,518 bp long and contains 3,832 genes. The gene encoding β-glucosidase (bgl) in B. altitudinis JYY-02 was screened from the genome and overexpressed in Escherichia coli BL21(DE3). The recombinant β-glucosidase was purified by affinity chromatography on a Ni Sepharose 6 fast flow (FF) column. The optimal temperature, pH, and K_m values for the recombinant β-glucosidase were 60°C, pH 5.6, and 0.331 mM, respectively, when p-nitrophenyl-β-d-glucopyranoside (pNPG) was used as the substrate. The recombinant β-glucosidase catalyzed the deglycosylation reaction of geniposide, which was then used to produce GB. IMPORTANCE β-Glucosidases are enzymes capable of hydrolyzing β-glucosidic linkages present in saccharides and glycosides and have many agricultural and industrial applications. Although they are found in all domains of living organisms, commercial β-glucosidases are still expensive, limiting their application in industry. In the present study, a thermostable β-glucosidase-producing strain was obtained for GB production by fermentation, engineered bacteria were constructed for preparing recombinant β-glucosidase, and a one-step method to purify the recombinant enzyme was established. A large amount of purified β-glucosidase was easily obtained from the engineered bacteria for industrial applications such as GB production.

Keywords: Bacillus altitudinis JYY-02; enzymatic characterization; fermentation; gardenia blue pigment; genome; β-glucosidase.

FIG 1

Screening of bacterial strains for GB production. (A) Abilities of four different strains to produce gardenia blue by fermentation. (B) B. altitudinis JYY-02-produced GB on geniposide-glycine screening medium.

FIG 2

Phylogenetic tree constructed from the 16S rRNA gene sequences of B. altitudinis JYY-02 and other species.

FIG 3

Graphs showing the factors influencing GB production by fermentation of B. altitudinis JYY-02. Effect on production of time (A), pH (B), temperature (C), and the addition of an additional carbon source (D).

FIG 4

Genome circles representing (outer to inner) genome size; forward strand gene, colored according to the Cluster of Orthologous Groups (COG) classification; reverse strand gene, colored according to the COG classification; forward-strand ncRNA; reverse-strand ncRNA; repeat; GC; GC skew.

FIG 5

Prediction of the structure of β-glucosidase of B. altitudinis JYY-02. (A) Secondary structure prediction of β-glucosidase (blue, α-helix; green, β-turn; red, extension chain; purple, random curl). (B) Tertiary structure prediction of β-glucosidase (red, N-terminal; dark blue, C-terminal).

FIG 6

SDS-PAGE analysis of the expression and purification of β-glucosidase. (Lane M) Marker; (lane 1) cell lysate of E. coli BL21(DE3); (lane 2) cell lysate of E. coli BL21(DE3) harboring plasmid pET-15b-bgl; (lane 3) recombinant β-glucosidase purified by nickel ion affinity chromatography.

FIG 7

Graphs showing the factors influencing the activity of recombinant β-glucosidase. Effect on enzyme activity of pH (A), temperature (B), metal ions (C), and the thermal stability of β-glucosidase (D).

FIG 8

Graph showing a kinetic analysis of the recombinant β-glucosidase (pNPG as the substrate).

FIG 9

Effect of the content of the recombinant β-glucosidase on GB synthesis. (A) Different enzyme amounts influencing the production of GB. Aliquots 1 through 6 were the control, 25, 50, 100, 200, and 400 μg, respectively. (B) Graph showing the relationship between the different amounts of enzymes and the relative amounts of GB.