Directed evolution of a ß-N-acetylhexosaminidase from Haloferula sp. for lacto-N-triose II and lacto-N-neotetraose synthesis from chitin.
Enzyme Microb Technol
; 164: 110177, 2023 Mar.
Article
em En
| MEDLINE
| ID: mdl-36549095
ABSTRACT
In our previous study, a ß-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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
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Beta-N-Acetil-Hexosaminidases
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Quitina
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Evolução Molecular Direcionada
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Verrucomicrobia
Limite:
Humans
Idioma:
En
Revista:
Enzyme Microb Technol
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
China