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

Abstract

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. 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.