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

Yihao Liu; Qiaojuan Yan; Junwen Ma; Jianyu Wang; Zhengqiang Jiang; Shaoqing Yang

doi:10.1016/j.enzmictec.2022.110177

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

Enzyme Microb Technol. 2023 Mar:164:110177. doi: 10.1016/j.enzmictec.2022.110177. Epub 2022 Dec 15.

Authors

Yihao Liu¹, Qiaojuan Yan¹, Junwen Ma¹, Jianyu Wang¹, Zhengqiang Jiang², Shaoqing Yang³

Affiliations

¹ Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, No.17 Qinghua East Road, Haidian, Beijing 100083, China.
² 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.
³ 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.

PMID: 36549095
DOI: 10.1016/j.enzmictec.2022.110177

Abstract

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 (GlcNAc₂), 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 L¹ and 4.9 g L¹, respectively. Therefore, mHaHex74 maybe a good candidate for enzymatic synthesis of HMOs.

Keywords: Lacto-N-neotetraose; Lacto-N-triose II; Protein engineering; Transglycosylation; β-N-acetylhexosaminidase.

MeSH terms

Bacterial Proteins* / chemistry
Bacterial Proteins* / genetics
Chitin*
Directed Molecular Evolution*
Humans
Milk, Human
Oligosaccharides / chemistry
Verrucomicrobia* / enzymology
beta-N-Acetylhexosaminidases* / chemistry
beta-N-Acetylhexosaminidases* / genetics

Substances

beta-N-Acetylhexosaminidases
Chitin
lacto-N-neotetraose
lacto-N-triose II
Oligosaccharides
Bacterial Proteins