Enzymatic Preparation of Gentiooligosaccharides by a Thermophilic and Thermostable β-Glucosidase at a High Substrate Concentration

Abstract

Gentiooligosaccharides (GnOS) are a kind of oligosaccharide formed by glucose with β-1-6 glycosidic bonds, which has become a new type of functional oligosaccharide for its unique refreshing bitter taste and valuable probiotic effects. However, the research on the enzymatic preparation of GnOS is not thorough enough. In this study, a GH1 thermophilic β-glucosidase from Thermotoga sp. KOL6 was used as a biocatalyst for the synthesis of GnOS. TsBgl1 exhibited excellent thermophilic and thermostable properties by possessing a melting temperature of 101.5 °C and reacting at 80–90 °C efficiently. Its half-life at 90 °C was approximately 5 h, suggesting its high heat resistance as well. TsBgl1 also showed excellent glucose tolerance with an inhibition constant (Ki) of 1720 mM and was stimulated in the presence of 0–900 mM glucose. TsBgl1 showed the highest hydrolytic activity on laminaribiose (Glc-β-1,3-Glc), but mainly synthetized gentiobiose (Glc-β-1,6-Glc) during transglycosylation. By optimizing the reaction conditions and substrate concentration, the highest yield of GnOS synthesized by TsBgl1 reached 144.3 g·L−1 when 1000 g·L−1 glucose was used as a substrate, which was higher than the highest yield ever reported. The thermophilic and thermostable properties of TsBgl1 were considered to be significant advantages in the industrial production of GnOS, where long periods of high-temperature reactions are required. This study was expected to provide an excellent candidate enzyme for industrial production of GnOS and also provide a reference for studying the transglycosylation of GH1 β-glucosidases.

Effect of deep eutectic solvents on the biocatalytic properties of β-glucosidase@ZnOFe nano-biocatalyst

“…More specifically, by using hydrolytic enzymes as βglycosidases, food industries can improve the organoleptic properties of their products (juice, wine) or enrich them with nutritional aglycones (o-diphenols, coumarins, flavonoids, isoflavones), which mostly demonstrate enhanced bioactivities and aroma (Fernández-Pacheco et al, 2021;Monteiro et al, 2019); Several aglycone derivatives are of high pharmaceutical interest too (Cheng and Zhang, 2017;Ma et al, 2020). BGLs can also catalyze transglycosylation reactions (Ahmed et al, 2017;Xia et al, 2022); Especially immobilized β-glucosidases are ideal candidates for targeted enzymatic glycosylation of natural compounds due to the decreased enzyme inhibition by the substrates (Geronimo et al, 2019). To date, diverse BGLs have been explored to produce valuable compounds benefiting from the inclusion of DESs as co-solvents (Cheng and Zhang, 2017;Uhoraningoga et al, 2021).…”

Conversion of β‐1,6‐Glucans to Gentiobiose using an endo‐β‐1,6‐Glucanase PsGly30A from Paenibacillus sp. GKG

“…The main disadvantages of chemical synthesis [8] are the multiple reactions required: protecting, activating and deprotecting, and low yields. Trans-glycosylation reactions produce non-specific disaccharides, [4,9,10] oligosaccharides with higher DPs [6,11] or mixed type glycosidic linkage oligosaccharides [12] in addition to the target disaccharide gentiobiose. In contrast, treatment of β-1,6-glucans with endoβ-1,6-glucanases [13,14] allows the production of glucose and gentiobiose as the main reaction products, with glucose being easily removed using microbiological techniques.…”

“…In addition, gentiobiose promotes the growth of Lactobacillus acidophilus and Bifidobacterium animalis [2] and has a prebiotic index (PI) equal to 3.70 [3] . Secondly, gentiobiose has a refreshing bitter taste similar to chocolate, cocoa, coffee and beer and can reduce the astringency of fruits and vegetables, which is why gentiobiose and gentiooligosaccharides (GnOS) are used as flavour enhancers in a variety of foods, such as chocolate, ice cream, jam, coffee [4] . In some cases, gentiobiose is used as a blend with sweet tasting sugars (e. g. glucose, isomaltose) to improve the taste of foods such as soft drinks, dairy drinks, chocolate paste, jam, confectionery, spices and sauces [5] .…”

“…In some cases, gentiobiose is used as a blend with sweet tasting sugars (e. g. glucose, isomaltose) to improve the taste of foods such as soft drinks, dairy drinks, chocolate paste, jam, confectionery, spices and sauces [5] . Gentiobiose is responsible for the bitter taste of GnOS products as gentiobiose is five times more bitter than gentiotriose [6] and gentiobiose is the main ingredient in commercial GnOS products Gentose#45 and Gentose#80 [4] . In general, gentiobiose and GnOS are not freely available in nature, e. g., only a small amount of free gentiobiose (≈2 mg/kg) has been detected in fresh ripe tomato locule [7] …”

“…[3] Secondly, gentiobiose has a refreshing bitter taste similar to chocolate, cocoa, coffee and beer and can reduce the astringency of fruits and vegetables, which is why gentiobiose and gentiooligosaccharides (GnOS) are used as flavour enhancers in a variety of foods, such as chocolate, ice cream, jam, coffee. [4] In some cases, gentiobiose is used as a blend with sweet tasting sugars (e. g. glucose, isomaltose) to improve the taste of foods such as soft drinks, dairy drinks, chocolate paste, jam, confectionery, spices and sauces. [5] Gentiobiose is responsible for the bitter taste of GnOS products as gentiobiose is five times more bitter than gentiotriose [6] and gentiobiose is the main ingredient in commercial GnOS products Gentose#45 and Gentose#80.…”