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

Jingyuan Yang,Chao Wang,Qunqun Guo,Wenjun Deng,Guicai Du, Ronggui Lia

Microbiology spectrum(2022)

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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 b- glucosidase to form genipin, which reacts with amino acids to produce GB. In this study, a bacterial strain which secreted thermostable beta - glucosidase (EC 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 degrees C for 45 h with additional sucrose. To obtain a large amount of beta- 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 beta- glucosidase (bgl) in B. altitudinis JYY-02 was screened from the genome and overexpressed in Escherichia coli BL21(DE3). The recombinant b- glucosidase was purified by affinity chromatography on a Ni Sepharose 6 fast flow (FF) column. The optimal temperature, pH, and Km values for the recombinant beta- glucosidase were 60 degrees C, pH 5.6, and 0.331 mM, respectively, when p-nitrophenyl- beta- D-glucopyranoside (pNPG) was used as the substrate. The recombinant beta- glucosidase catalyzed the deglycosylation reaction of geniposide, which was then used to produce GB. IMPORTANCE beta- Glucosidases are enzymes capable of hydrolyzing beta- 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 beta- glucosidases are still expensive, limiting their application in industry. In the present study, a thermostable beta- glucosidase-producing strain was obtained for GB production by fermentation, engineered bacteria were constructed for preparing recombinant b- glucosidase, and a one-step method to purify the recombinant enzyme was established. A large amount of purified beta- glucosidase was easily obtained from the engineered bacteria for industrial applications such as GB production.
Bacillus altitudinis JYY-02,beta-glucosidase,genome,gardenia blue pigment,fermentation,enzymatic characterization
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