High-density immobilization of a ginsenoside-transforming β-glucosidase for enhanced food-grade production of minor ginsenosides

Abstract

Use of recombinant glycosidases is a promising approach for the production of minor ginsenosides, e.g., Compound K (CK) and F 1 , which have potential applications in the food industry. However, application of these recombinant enzymes for food-grade preparation of minor ginsenosides are limited by the lack of suitable expression hosts and low productivity. In this study, Corynebacterium glutamicum ATCC13032 , a GRAS strain that has been used extensively for the industrial-grade production of additives for foodstuffs, was employed to express a novel β-glucosidase (MT619) from Microbacterium testaceum ATCC 15829 with high ginsenoside-transforming activity. A cellulose-binding module was additionally fused to the N-terminus of MT619 for immobilization on cellulose, which is an abundant and safe material. Via one-step immobilization, the fusion protein in cell lysates was efficiently immobilized on regenerated amorphous cellulose at a high density (maximum 984 mg/g cellulose), increasing the enzyme concentration by 286-fold. The concentrated and immobilized enzyme showed strong conversion activities against protopanaxadiol- and protopanaxatriol-type ginsenosides for the production of CK and F 1 . Using gram-scale ginseng extracts as substrates, the immobilized enzyme produced 7.59 g/L CK and 9.42 g/L F 1 in 24 h. To the best of our knowledge, these are the highest reported product concentrations of CK and F 1 , and this is the first time that a recombinant enzyme has been immobilized on cellulose for the preparation of minor ginsenosides. This safe, convenient, and efficient production method could also be effectively exploited in the preparation of food-processing recombinant enzymes in the pharmaceutical, functional food, and cosmetics industries. Electronic supplementary material The online version of this article (10.1007/s00253-019-09951-4) contains supplementary material, which is available to authorized users.

SGL 121 Attenuates Nonalcoholic Fatty Liver Disease through Adjusting Lipid Metabolism Through AMPK Signaling Pathway

“…Minor ginsenoside, in low-molecular-weight form (622.9–785 g/mol), is known for its higher absorption rate and high physiological activity compared to major ginsenoside (801–1109.3 g/mol) [ 29 ]; however, it is either absent or found in trace amounts in ginseng; therefore, producing minor ginsenoside has become a popular topic of research [ 30 , 31 ]. Minor ginsenoside F2 is known to have anti-cancer effects and anti-obesity effects [ 32 , 33 ].…”

Characterization of a Novel Ginsenoside MT1 Produced by an Enzymatic Transrhamnosylation of Protopanaxatriol-Type Ginsenosides Re

“…Recombinant MT619 was expressed in Escherichia coli BL21 as described previously [15]. Briefly, the MT619-harboring pEX vector was inserted into the E. coli BL21 strain using heat-shock transformation.…”

“…MT619 was purified by Ni column and DEAE column chromatography as described in a previous study [15]. To analyze the transformation pathways, ginsenosides Re and Rg 2 were reacted as substrates with purified MT619 ginsenoside and F 1 and the conversion products were subjected to TLC analysis.…”

“…Many enzymes have been explored to efficiently convert major ginsenosides into pharmacologically active, rare minor ginsenosides [5,13,14]. Prior to this study, our research group cloned, characterized and applied many efficient glycoside hydrolases for the gram-scale production and purification of minor ginsenosides [15][16][17][18][19][20][21].…”

“…In our investigation into the enzymatic conversion of major ginsenosides, a new transformation product of ginsenoside Re that is different from the other hydrolyzing products (ginsenoside PPT, F 1 , Rg 1 and Rg 2 ) was accidentally found using a novel recombinant glucosidase (MT619) [15]. This compound was prepared at the gram scale using recombinant MT619 and isolated from the product mixture using chromatographic methods.…”