Efficient sequential synthesis of lacto-N-triose II and lacto-N-neotetraose by a novel β-N-acetylhexosaminidase from Tyzzerella nexilis

Liu, Yihao; Wang, Ling; Huang, Ping; Jiang, Zhengqiang; Yan, Qiaojuan; Yang, Shaoqing

doi:10.1016/j.foodchem.2020.127438

Cited by 27 publications

(31 citation statements)

References 40 publications

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“…The optimal temperature was approximately 40°C at pH 5, and the activity was stable at temperatures of up to 30°C at pH 5 for 30 min. The optimal pH and temperature are similar to those of β‐ N ‐acetylhexosaminidase enzymes from many origins (Y. H. Liu et al., 2020; Rauvolfová et al., 2004; Weignerová et al., 2003; Zhang et al., 2020).…”

Section: Results and Disccusionsupporting

confidence: 56%

Characterization of a β‐N‐acetylhexosaminidase with transglycosylation activity from Metarhizium sp. A34

Kurakake

Amai

2022

Journal of Food Science

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In applications of chitin, one of the most abundant resources on earth, human milk oligosaccharides with many health functions were synthesized by transglycosylation of β‐N‐acetylhexosaminidase. Synthesis of new transfer products can be expected by other β‐N‐acetylhexosaminidases in nature. A total of 38 microorganisms that secrete β‐N‐acetylhexosaminidases with transglycosylation activity were isolated from a soil screen. Using N,N′‐diacetylchitobiose as the substrate, the transfer ratio increased with a decrease in substrate degradation when it was less than 60%. Metarhizium sp. A34 β‐N‐acetylhexosaminidase had high transglycosylation activity and showed a maximum production of the oligosaccharides against the substrate degradation where (GlcNAc)5 and (GlcNAc)4 were produced in addition to (GlcNAc)3. The maximum curve was attributed to a sequential reaction of transglycosylation followed by hydrolysis where oligosaccharides are an intermediate product and are hydrolyzed in a second step. The purified β‐N‐acetylhexosaminidase from Metarhizium sp. A34 had an optimal pH of 5 and was stable from pH 7 to 8. At pH 5, it had an optimal temperature of 40°C and was stable up to 30°C for 30 min. This enzyme had high thermostability up to 55°C when bound to the cell wall. The acceptor specificity for the transglycosylation reaction was enhanced for lower molecular weight sugar alcohols in the order of glycerin (C3), erythritol (C4), and xylitol (C5). The transfer product with glycerin was identified as 1‐O‐β‐d‐N‐acetylglucosaminyl glycerin, which may prove useful as a starting material for new glycolipids in food applications. Practical Application Metarhizium sp. A34 β‐N‐acetylhexosaminidase produced 1‐O‐β‐d‐N‐acetylglucosaminyl glycerin through the transglycosylation. Chitin oligosaccharides of the donor are obtained by hydrolysis of chitin. 1‐O‐β‐d‐N‐Acetylglucosaminyl glycerin may be useful to start material for the synthesis of new glycolipids. High thermostability of this enzyme is useful to prevention of contamination in the transglycosylation reaction.

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Section: Results and Disccusionsupporting

confidence: 56%

Characterization of a β‐N‐acetylhexosaminidase with transglycosylation activity from Metarhizium sp. A34

Kurakake

Amai

2022

Journal of Food Science

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“…Optimal pH (A), pH stability (B), optimal temperature (C), and thermostability (D) of HaHex74. The optimal pH and pH stability of purified HaHex74 were determined in 50 mM of different buffers at pH 4.0–10.5 based on the method described by Liu et al The optimal temperature and thermostability of purified HaHex74 were assessed in phosphate buffer (50 mM, pH 6.5) at different temperatures (20–70 °C) by the method described by Liu et al …”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…One unit of β-N-acetylhexosaminidase activity was defined as the enzyme amount requested to release 1 μmol pNP per min under the given conditions. Protein content was evaluated by the Lowry method according to the reference reported by Liu et al 21 Biochemical Characterization and Substrate Specificity of HaHex74. The optimal pH and pH stability of purified HaHex74 was determined in 50 mM different buffers at pH 4.0−10.5 based on the method of Liu et al 21 The optimal temperature and thermostability of purified HaHex74 was assessed in phosphate buffer (50 mM, pH 6.5) at different temperatures (20−70 °C) by the method described by Liu et al 22 The substrate specificity of HaHex74 was determined using different substrates.…”

Section: ■ Experimental Sectionmentioning

confidence: 99%

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Production of Lacto-N-triose II and Lacto-N-neotetraose from Chitin by a Novel β-N-Acetylhexosaminidase Expressed in Pichia pastoris

Liu

Yan

et al. 2020

ACS Sustainable Chem. Eng.

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An unstudied β-N-acetylhexosaminidase (HaHex74) from Haloferula sp. was expressed in Pichia pastoris. HaHex74 displayed the highest similarity (61%) with a β-N-acetylhexosaminidase from an uncultured Bacteroidetes bacterium. The recombinant P. pastoris secreted the highest enzyme activity of 3500 U mL–1 after incubation for 168 h in a 5-L fermenter. HaHex74 showed an optimal pH and temperature of 6.5 and 45 °C, respectively. HaHex74 displayed high transglycosylation activity, showing a maximal conversion ratio of 13.1% (7.1 g L–1), which is by far the highest conversion ratio for the synthesis of lacto-N-triose II (LNT2) from N-acetyl chitobiose. Thus, an efficient strategy for the production of high value-added LNT2 and lacto-N-neotetraose (LNnT) from chitin by enzyme cocktails was first developed in a 5-L reactor. As a result, the contents of LNT2 and LNnT reached 8.6 and 2.0 g L–1, respectively. The novel and environmentally friendly bioprocess may greatly promote the production of LNT2 and LNnT from chitin.

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“…Several studies have been carried out for the synthesis of LNT II in vivo and in vitro [ 13 ]. For example, 4.7 g L −1 of LNT II was obtained using purified β- N -acetylhexosaminidase from Tyzzerella nexilis towards p NP-NAG and β-lactose as substrates [ 14 ] (Table 1 ). However, the enzyme dosage and the expensive nucleotide donors limit their industrial applications.…”

Section: Introductionmentioning

confidence: 99%

Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin

Zhu

et al. 2021

Biotechnol Biofuels

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Background Lacto-N-triose II (LNT II), an important backbone for the synthesis of different human milk oligosaccharides, such as lacto-N-neotetraose and lacto-N-tetraose, has recently received significant attention. The production of LNT II from renewable carbon sources has attracted worldwide attention from the perspective of sustainable development and green environmental protection. Results In this study, we first constructed an engineered E. coli cell factory for producing LNT II from N-acetylglucosamine (GlcNAc) feedstock, a monomer of chitin, by introducing heterologous β-1,3-acetylglucosaminyltransferase, resulting in a LNT II titer of 0.12 g L−1. Then, lacZ (lactose hydrolysis) and nanE (GlcNAc-6-P epimerization to ManNAc-6-P) were inactivated to further strengthen the synthesis of LNT II, and the titer of LNT II was increased to 0.41 g L−1. To increase the supply of UDP-GlcNAc, a precursor of LNT II, related pathway enzymes including GlcNAc-6-P deacetylase, glucosamine synthase, and UDP-N-acetylglucosamine pyrophosphorylase, were overexpressed in combination, optimized, and modulated. Finally, a maximum titer of 15.8 g L−1 of LNT II was obtained in a 3-L bioreactor with optimal enzyme expression levels and β-lactose and GlcNAc feeding strategy. Conclusions Metabolic engineering of E. coli is an effective strategy for LNT II production from GlcNAc feedstock. The titer of LNT II could be significantly increased by modulating the gene expression strength and blocking the bypass pathway, providing a new utilization for GlcNAc to produce high value-added products.

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Efficient sequential synthesis of lacto-N-triose II and lacto-N-neotetraose by a novel β-N-acetylhexosaminidase from Tyzzerella nexilis

Cited by 27 publications

References 40 publications

Characterization of a β‐N‐acetylhexosaminidase with transglycosylation activity from Metarhizium sp. A34

Characterization of a β‐N‐acetylhexosaminidase with transglycosylation activity from Metarhizium sp. A34

Production of Lacto-N-triose II and Lacto-N-neotetraose from Chitin by a Novel β-N-Acetylhexosaminidase Expressed in Pichia pastoris

Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin

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