Expression of a highly active β-glucosidase from Aspergillus niger AS3.4523 in Escherichia coli and its application in gardenia blue preparation

Gardenia blue is one of the natural food additives used in East Asia for many years. Its biosynthesis relies on a key rate-limiting cellulase: β-glucosidase (BGL), which mainly exists in Aspergillus niger (A. niger) cells. The purpose of this study was to obtain active β-glucosidase by cell engineering method and applied to gardenia blue synthesis, which would help to promote the application and reduce the cost of β-glucosidase and gardenia blue. A. niger was identified based on 18S rRNA gene sequencing. β-Glucosidase gene was cloned and expressed based on PCR and prokaryotic expression. The enzyme activity of β-glucosidase was measured based on p-nitrophenyl-β-D-glucopyranoside method. An A. niger isolate (AS3.4523) was identified from soil. The β-glucosidase gene of AS3.4523 was cloned and sequenced, which encoded a new type of β-glucosidase mutant containing two specific amino acid substitutions (Asp154Gly and Ser163Pro). Prokaryotic expression of wild-type β-glucosidase in Escherichia coli BL21 showed low cellulase activity (0.29 ± 0.13 U/mL). However, after removing its signal peptide, the β-glucosidase of A. niger AS3.4523 exhibited extremely higher activity (25.88 ± 0.45 U/mL) compared with wild type β-glucosidase (12.59 ± 1.07 U/mL) or other A. niger strains M85 (3.61 ± 0.24 U/mL) and CICC2041 (4.36 ± 0.76 U/mL). Furthermore, recombinant β-glucosidase was applied to geniposide hydrolysis, and gardenia blue pigment was successfully synthesized with the reaction of genipin and Lys. This work has discovered a new type of highly active β-glucosidase and provided a theoretical basis for large-scale producing β-glucosidase, which lays a brand-new foundation for gardenia blue preparation with high efficiency and low cost.