Directed evolution of a β-N-acetylhexosaminidase from Haloferula sp. for lacto-N-triose II and lacto-N-neotetraose synthesis from chitin

Liu, Yihao; Yan, Qiaojuan; Ma, Jinlu; Wang, Yunlong; Jiang, Zhengqiang; Yang, Shaoqing

doi:10.1016/j.enzmictec.2022.110177

Cited by 5 publications

(24 citation statements)

References 39 publications

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“…SSM on residue F76 (amino acids in this study were all in this abbreviation) of TsGal48 was carried out using the pET28a-TsGal48 template and the primers designed by the degenerate mutation library (Table S1). All other 19 variants of this position was obtained according to the previous method …”

Section: Materials and Methodsmentioning

confidence: 99%

“…For the hydrolysis of chitin powder by PbChi70, the enzyme dosage (40–400 U/g) and reaction time (4–24 h) was optimized. For the synthesis of LNT2 in multienzyme system, the reaction conditions were optimized according to the previous method . The conditions for LNnT synthesis by mTsGal48 were optimized by the method of Section .…”

Section: Materials and Methodsmentioning

confidence: 99%

“…Then, penetrating fluid was followed by a nanofiltration membrane (500 Da) for removing monosaccharides. The active charcoal column was used to further remove the monosaccharides by the previous method . The fractions containing LNT2 were collected, concentrated, and freeze-dried.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…The preparation of (GlcNAc) 2 and LNT2 was performed by the previous method with minor modification. 16 In brief, 600.5 g of the pretreated chitin powder (3%, w/v) was mixed with 20 L of 5 mM citrate buffer pH 5.5 in a 30-L bioreactor, and then the PbChi70 (50 U/g) was added into the bioreactor to hydrolyze the chitin powder at 50 °C for 9 h. After centrifugation, the supernatant was concentrated about 10-fold to obtain suitable donor ((GlcNAc) 2 ) concentration. Subsequently, 0.8 M β-lactose and 4 U mL −1 mHaHex74 16 were poured into the concentrated solution for preparation of LNT2 at pH 7.0 and 40 °C for 8 h. The BHBgal2A could hydrolyze lactose but not LNT2 (data not shown).…”

Section: Isolation and Identification Of Lnntmentioning

confidence: 99%

“…Synthesis of LNnT from Chitin Powder. According to the previous method, 16 the multienzyme system was further studied to prepare LNnT from chitin powder in a 30-L bioreactor (Figure 4). The reaction route in multienzyme system is shown in Figure S6.…”

Section: Directed Evolution and Ssm Of β-Galactosidasementioning

confidence: 99%

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Protein Engineering of a Novel β-Galactosidase from Thermus scotoductus for Efficient Synthesis of Lacto-N-Neotetraose from Chitin Powder

Wang,

Xiang,

Liu

et al. 2024

J. Agric. Food Chem.

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A novel β-galactosidase (TsGal48) from Thermus scotoductus was cloned, and the enzyme was biochemically characterized. TsGal48 catalyzed the synthesis of lacto-N-neotetraose (LNnT) from lactose via the transglycosylation reaction with a maximal yield of 20%, which is the highest yield for the synthesis of LNnT so far. To further improve the yield of LNnT, TsGal48 was successfully engineered by directed evolution and site-saturation mutagenesis. A mutated β-galactosidase (mTsGal48) was selected and characterized. mTsGal48 produced LNnT with a yield of 27.7 g/L, which is 1.4-fold higher than that of TsGal48 (19.7 g/L). Then, a developed strategy for LNnT synthesis from chitin powder was provided in a 30 L bioreactor. The reaction process included chitin powder hydrolysis, lacto-N-triose II (LNT2) synthesis, and LNnT synthesis. The reaction time was reduced from 44 to 17 h in chitin powder hydrolysis and LNT2 synthesis. The content of LNnT was up to 25 g/L in the multienzyme system. The green and efficient route may be suitable for large-scale production of LNnT from chitin powder.

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Section: Materials and Methodsmentioning

confidence: 99%

Section: Materials and Methodsmentioning

confidence: 99%

Section: Materials and Methodsmentioning

confidence: 99%

Section: Isolation and Identification Of Lnntmentioning

confidence: 99%

Section: Directed Evolution and Ssm Of β-Galactosidasementioning

confidence: 99%

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