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

• Published in: Annals of microbiology Vol. 70; no. 1
• Main Authors: Hao, Shuai, Liu, Yuanpu, Qin, Yu, Zhao, Lei, Zhang, Jiawen, Wu, Tingting, Sun, Baoguo, Wang, Chengtao
• Format: Journal Article
• Language: English
• Published: Springer 19.05.2020
• Subjects: Amino acids; Enzymes; Escherichia coli; Food additives; Hydrolysis; Peptides; RNA; Niger; China
• ISSN: 1590-4261; 1869-2044
• DOI: 10.1186/s13213-020-01576-7

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: [beta]-glucosidase (BGL), which mainly exists in Aspergillus niger (A. niger) cells. The purpose of this study was to obtain active [beta]-glucosidase by cell engineering method and applied to gardenia blue synthesis, which would help to promote the application and reduce the cost of [beta]-glucosidase and gardenia blue. A. niger was identified based on 18S rRNA gene sequencing. [beta]-Glucosidase gene was cloned and expressed based on PCR and prokaryotic expression. The enzyme activity of [beta]-glucosidase was measured based on p-nitrophenyl-[beta]-D-glucopyranoside method. An A. niger isolate (AS3.4523) was identified from soil. The [beta]-glucosidase gene of AS3.4523 was cloned and sequenced, which encoded a new type of [beta]-glucosidase mutant containing two specific amino acid substitutions (Asp154Gly and Ser163Pro). Prokaryotic expression of wild-type [beta]-glucosidase in Escherichia coli BL21 showed low cellulase activity (0.29 [+ or -] 0.13 U/mL). However, after removing its signal peptide, the [beta]-glucosidase of A. niger AS3.4523 exhibited extremely higher activity (25.88 [+ or -] 0.45 U/mL) compared with wild type [beta]-glucosidase (12.59 [+ or -] 1.07 U/mL) or other A. niger strains M85 (3.61 [+ or -] 0.24 U/mL) and CICC2041 (4.36 [+ or -] 0.76 U/mL). Furthermore, recombinant [beta]-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 [beta]-glucosidase and provided a theoretical basis for large-scale producing [beta]-glucosidase, which lays a brand-new foundation for gardenia blue preparation with high efficiency and low cost.