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

Abstract

Purpose: 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. Methods: 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. Results: 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. Conclusions: 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.