Insight into the glycosylation and hydrolysis kinetics of alpha-glucosidase in the synthesis of glycosides

Chen, Hanchi; Yang, Shanshan; Xu, Anjie; Jiang, Ruini; Tang, Zhuance; Wu, Jiamin; Zhu, Linjiang; Liu, Shijie; Chen, Xiaolong; Lu, Yuele

doi:10.1007/s00253-019-10205-6

Cited by 18 publications

(14 citation statements)

References 39 publications

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“…In addition to glycosidic bond cleavage, glycoside hydrolases (GHs) can be used for the synthesis of glycoside bonds in vitro via reverse hydrolysis reaction without the need for cofactors, such as uridine diphosphate (Lu et al 2015;Perugino et al 2004;Rosengren et al 2019;Xu et al 2016). Recently, several glycosidases, such as β-glucosidase, α-glucosidase, α-L -rhamnosidase, β-mannosidase and β-galactosidase, have been used to synthesize glycosides via the reverse hydrolysis reaction (Lu et al 2015;Arthornthurasuk et al 2018;Chen et al 2019;Ajisaka et al 2012;Tan et al 2016). Gentiooligosaccharides, octyl glucoside, laminaribiose, sophorose, cellobiose, and gentiobiose have been synthesized by β-glucosidase via reverse hydrolysis reaction (Semenova et al 2015;Arthornthurasuk et al 2018;Wang et al 2018;Ravet et al 1993).…”

Section: Introductionmentioning

confidence: 99%

A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

Niu

Liu

Feng

et al. 2020

Bioresour. Bioprocess.

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Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1b I177S , TrCel1b I177S/I174S , and TrCel1b I177S/I174S/W173H , and one negative variant TrCel1b N240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1b I177S/I174S/W173H was accelerated and then the maximum total production (195.8 mg/mL/ mg enzyme) of the synthesized disaccharides was increased by 3.5-fold compared to that of wild type. On the contrary, TrCel1b N240I lost reverse hydrolysis activity. The results demonstrate that the average hydropathy index of the key amino acid residues in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.

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Section: Introductionmentioning

confidence: 99%

A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

Niu

Liu

Feng

et al. 2020

Bioresour. Bioprocess.

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“…Among the results of reactions, a typical ‘n’‐shaped kinetic profile of the AA‐2G formation was observed. By understanding the glucosidase‐catalyzed glycosylation process in water‐rich environment, [ 50 ] it can be concluded that glycosylation is dominant at the beginning of reaction, which leads to rapid synthesis of AA‐2G. At the same time, the synthesized AA‐2G started to degrade with time.…”

Section: Discussionmentioning

confidence: 99%

Biocatalytic synthesis of 2‐O‐α‐D‐glucopyranosyl‐L‐ascorbic acid using an extracellular expressed α‐glucosidase from Oryza sativa

Shao

Cheng

et al. 2021

Biotechnology Journal

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Background 2‐O‐α‐D‐Glucopyranosyl‐L‐ascorbic acid (AA‐2G) is an important derivative of L‐ascorbic acid (L‐AA), which has the distinct advantages of non‐reducibility, antioxidation, and reproducible decomposition into L‐AA and glucose. Enzymatic synthesis is a preferred method for AA‐2G production over alternative chemical synthesis owing to the regioselective glycosylation reaction. α‐Glucosidase, an enzyme classed into O‐glycoside hydrolases, might be used in glycosylation reactions to synthesize AA‐2G. Main Methods and Major Results Here, an α‐glucosidase from Oryza sativa was heterologously produced in Pichia pastoris GS115 and used for biosynthesis of AA‐2G with few intermediates and byproducts. The extracellular recombinant α‐glucosidase (rAGL) reached 9.11 U mL–1 after fed‐batch cultivation for 102 h in a 5 L fermenter. The specific activity of purified rAGL is 49.83 U mg–1 at 37°C and pH 4.0. The optimal temperature of rAGL was 65°C, and it was stable below 55°C. rAGL was active over the range of pH 3.0–7.0, with the maximal activity at pH 4.0. Under the condition of 37°C, pH 4.0, equimolar maltose and ascorbic acid sodium salt, 8.7 ± 0.4 g L–1 of AA‐2G was synthesized by rAGL. Conclusions and Implications The production of rAGL in P. pastoris was proved to be beneficial in providing enough enzyme and promoting biocatalytic synthesis of AA‐2G. These studies lay the basis for the industrial application of α‐glucosidase.

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“…Among the results of reactions, a typical 'n'-shaped kinetic profile of the AA-2G formation was observed. By understanding the glucosidase-catalyzed glycosylation process in waterrich environment [59] , it can be concluded that glycosylation is dominant at the beginning of reaction, which leads to rapid synthesis of AA-2G. At the same time, the synthesized AA-2G started to degrade with time.…”

Section: Figure 6 4 Discussionmentioning

confidence: 99%

Extracellular expression and characterization of an α-glucosidase from Oryza sativa and its transglycosylation for synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid

Shao

Cheng

et al. 2021

Preprint

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2-O-α-D-Glucopyranosyl-L-ascorbic acid (AA-2G) is an important industrial derivative of L-ascorbic acid (AA), which has the distinct advantages of non-reducibility, antioxidation, and reproducible decomposition into L-ascorbic acid and glucose. Enzymatic synthesis is a preferred method for AA-2G production over alternative chemical synthesis owing to the regioselective glycosylation reaction. α-Glucosidase, an enzyme classed into O- glycoside hydrolases, may be used in glycosylation reactions to synthesize AA-2G. Here, one α-glucosidase from Oryza sativa (rAGL) was recombinantly produced in Pichia pastoris GS115 and used for biosynthesis of AA-2G with few intermediates and byproducts. The extracellular rAGL reached 9.11 U/mL after fed-batch cultivation for 102 h in a 5-L fermenter. The specific activity of purified rAGL is 49.83 U/mg at 37 °C and pH 4.0. The optimal temperature of rAGL was 65 °C, and it was stable below 55 °C. rAGL was active over the range of pH 3.0–7.0, with the maximal activity at pH 4.0. Under the condition of 37 °C , pH 4.0, equimolar maltose and AA·Na, 8.7±0.4 g/L of AA-2G was synthesized by rAGL. These studies lay the basis for the industrial application of recombinant α-glucosidase. Keywords: α-Glucosidase; Oryza sativa; 2-O-α-D-glucopyranosyl-L-ascorbic acid; Transglycosylation; Pichia pastoris

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Insight into the glycosylation and hydrolysis kinetics of alpha-glucosidase in the synthesis of glycosides

Cited by 18 publications

References 39 publications

A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

A novel strategy for efficient disaccharides synthesis from glucose by β-glucosidase

Biocatalytic synthesis of 2‐O‐α‐D‐glucopyranosyl‐L‐ascorbic acid using an extracellular expressed α‐glucosidase from Oryza sativa

Extracellular expression and characterization of an α-glucosidase from Oryza sativa and its transglycosylation for synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid

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