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

Autor: Liu Y; Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China., Yan Q; Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China., Ma J; Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China., Wang J; Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China., Jiang Z; Department of Nutrition and Health, College of Food Science and Nutritional Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China. Electronic address: zhqjiang@cau.edu.cn., Yang S; Department of Nutrition and Health, College of Food Science and Nutritional Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China. Electronic address: ysq@cau.edu.cn.
Jazyk: angličtina
Zdroj: Enzyme and microbial technology [Enzyme Microb Technol] 2023 Mar; Vol. 164, pp. 110177. Date of Electronic Publication: 2022 Dec 15.
DOI: 10.1016/j.enzmictec.2022.110177
Abstrakt: In our previous study, a β-N-acetylhexosaminidase (HaHex74) from Haloferula sp. showing high human milk oligosaccharides (HMOs) synthesis ability was identified and characterized. In this study, HaHex74 was further engineered by directed evolution and site-saturation mutagenesis to improve its transglycosylation activity for HMOs synthesis. A mutant (mHaHex74) with improved transglycosylation activity (HaHex74-Asn401Ile/His394Leu) was obtained and characterized. mHaHex74 exhibited maximal activity at pH 5.5 and 35 °C, respectively, which were distinct from that of HaHex74 (pH 6.5 and 45 °C). Moreover, mHaHex74 showed the highest LNT2 conversion ratio of 28.2% from N,N'-diacetyl chitobiose (GlcNAc 2 ), which is 2.2 folds higher than that of HaHex74. A three-enzyme cascade reaction for the synthesis of LNT2 and LNnT from chitin was performed in a 5-L reactor, and the contents of LNT2 and LNnT reached up to 15.0 g L 1 and 4.9 g L 1 , respectively. Therefore, mHaHex74 maybe a good candidate for enzymatic synthesis of HMOs.
Competing Interests: Declaration of Competing Interest The authors declare that there are no conflict of interest in the manuscript.
(Copyright © 2022 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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