Abstract
Use of recombinant glycosidases is a promising approach for the production of minor ginsenosides, e.g., Compound K (CK) and F1, 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 F1. Using gram-scale ginseng extracts as substrates, the immobilized enzyme produced 7.59 g/L CK and 9.42 g/L F1 in 24 h. To the best of our knowledge, these are the highest reported product concentrations of CK and F1, 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.
Highlights
Ginseng has been widely used to treat various diseases in East Asia for more than a thousand years and is increasingly used in foods and dietary supplements worldwide (Chung et al 2011; Shi et al 2013)
We selected MT619, which exhibited the highest activity among all candidates, as a suitable enzyme for the production of Compound K (CK) and F1 from major ginsenosides
Recent studies have focused on the many pharmacological effects of ginsenoside CK and F1 owing to their potential food, cosmetics, and pharmaceutical applications
Summary
Ginseng has been widely used to treat various diseases in East Asia for more than a thousand years and is increasingly used in foods and dietary supplements worldwide (Chung et al 2011; Shi et al 2013). Most of the minor ginsenosides, which have fewer sugar moieties attached on aglycon, are absent or are present in smaller amounts than those of the major ginsenosides (Figure S1) (Shi et al 2013; Zhou et al 2014). Many minor ginsenosides have higher chemical reactivities than those of abundant major ginsenosides in raw materials (Smith et al 2014; Wong et al 2015). Among these minor ginsenosides, Compound K (CK)
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