Synthesis of human milk oligosaccharides by a beta-N-acetylhexosaminidase from Haloferula sp

A preparation method of HaHex74 protein, comprising the following steps:

• introducing a nucleic acid molecule encoding the HaHex74 protein into a yeast receptor to obtain recombinant yeast;
• performing fermentation culture on the recombinant yeast according to the following steps and Pichia Fermentation Process Guidelines to obtain the HaHex74 protein from a fermentation product;
• (d1) a basic culture stage:
• inoculating the recombinant yeast into BSM medium supplemented with 50 g/L glycerol for culture, adjusting the temperature to 30° C. and pH to 4.0, and starting the glycerol fed-batch culture stage when the glycerol concentration is below 10 g/L,
• (d2) a glycerol fed-batch culture stage:
• feeding 500 g/L glycerol, maintaining the glycerol concentration at 10-25 g/L, adjusting a temperature to 28° C., pH to 5.0, and maintaining the dissolved oxygen content to 10-20% until the end of fermentation;
• the HaHex74 protein is any one of the following;
• (A1) a protein consisting of an amino acid sequence at positions 94-745 of SEQ ID NO. 2 or shown in SEQ ID NO. 2;
• (A2) a protein having 99% or more identity with the amino acid sequence defined in (A1) and having the same function and is derived from Haloferul a sp.;
• and
• (A3) a fusion protein obtained by attaching a tag to the N-terminus and/or the C-terminus of the protein defined in any one of (A1) and (A2).