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.

中文翻译：

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黑曲霉AS3.4523中高活性β-葡萄糖苷酶在大肠杆菌中的表达及其在ia子蓝制剂中的应用

ia子蓝是东亚多年使用的天然食品添加剂之一。它的生物合成依赖于关键的限速纤维素酶：β-葡萄糖苷酶（BGL），该酶主要存在于黑曲霉（A. niger）细胞中。本研究的目的是通过细胞工程方法获得活性的β-葡萄糖苷酶，并将其应用于garden子蓝的合成，这将有助于推广应用，降低β-葡萄糖苷酶和garden子蓝的成本。基于18S rRNA基因测序鉴定了黑曲霉。基于PCR和原核表达，克隆并表达了β-葡萄糖苷酶基因。根据对硝基苯基-β-D-吡喃葡萄糖苷法测定β-葡萄糖苷酶的酶活性。从土壤中鉴定出黑曲霉分离株（AS3.4523）。克隆并测序了AS3.4523的β-葡萄糖苷酶基因，编码了一种新型的β-葡萄糖苷酶突变体，该突变体包含两个特定的氨基酸取代（Asp154Gly和Ser163Pro）。野生型β-葡萄糖苷酶在大肠杆菌BL21中的原核表达表明其纤维素酶活性较低（0.29±0.13 U / mL）。然而，与野生型β-葡萄糖苷酶（12.59±1.07 U / mL）或其他A相比，黑曲霉AS3.4523的β-葡萄糖苷酶除去其信号肽后，表现出极高的活性（25.88±0.45 U / mL）。黑曲霉菌株M85（3.61±0.24 U / mL）和CICC2041（4.36±0.76 U / mL）。此外，将重组β-葡萄糖苷酶应用于子苷水解，通过with子苷和Lys的反应成功合成了garden子蓝色素。这项工作发现了一种新型的高活性β-葡萄糖苷酶，并为大规模生产β-葡萄糖苷酶提供了理论基础，