HaHex74 Enzymatic Synthesis of Human Milk Oligosaccharides

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Solution Overview

Problem

Current methods for large-scale production of Lacto-N-triose II (LNT2) and Lacto-N-neotetraose (LNnT) are inefficient and costly due to low yields and the need for metabolic pathway modifications in whole-cell biotransformation, and chemical synthesis is not suitable due to low efficiency and safety issues with activated artificial donors.

Innovation Solution

Utilizing a β-N-acetylhexosaminidase from Haloferula sp. (HaHex74) to catalyze the synthesis of LNT2 from chitin hydrolysate and β-lactose, followed by β-galactosidase to produce LNnT, with optimized conditions for pH, temperature, and enzyme concentrations.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If whole-cell biotransformation is used for synthesis of LNT2 and LNnT, then production can be performed at large-scale, but production cost remains high due to low yield and need for metabolic pathway modification

Engineering Contradiction:

Improveproduction scaleVSAvoidproduction cost

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The invention extracts the key enzymatic function from the complex whole-cell system. By using purified β-N-acetylhexosaminidase and β-galactosidase enzymes instead of engineered whole cells, the process eliminates the need for metabolic pathway modification while maintaining large-scale production capability. The enzymes are produced in E. coli and then used in vitro for oligosaccharide synthesis.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention introduces purified enzymes as intermediaries between the starting materials (chitin, lactose) and the final products (LNT2, LNnT). This intermediary approach allows the synthesis to proceed without requiring the complex metabolic machinery of whole cells, thereby reducing production costs while maintaining scalability.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Ease of manufacture

If chemical synthesis with activated artificial donors is used, then synthesis can be performed, but efficiency is low and safety issues arise

Engineering Contradiction:

Improvesynthesis feasibilityVSAvoidsynthesis efficiency

Core Design Contradiction:

Ease of manufactureVSProductivity

Solution Approach 1:

The invention replaces chemical synthesis mechanisms with enzymatic catalysis. Instead of using activated artificial donors and chemical reagents, the process uses β-N-acetylhexosaminidase and β-galactosidase enzymes to catalyze the formation of LNT2 and LNnT from natural substrates (chitin and lactose), thereby improving both efficiency and safety.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The invention changes the reaction parameters from chemical to biological conditions. The enzymatic synthesis proceeds under mild physiological conditions (aqueous buffer, moderate temperature and pH) rather than requiring harsh chemical conditions, improving both safety and efficiency while using natural substrates instead of activated artificial donors.

Inventive Principle:

Principle #35Parameter changes

3Ease of manufacture

If activated artificial donors are used in enzymatic synthesis, then synthesis can proceed, but cost increases and food-grade quality is compromised

Engineering Contradiction:

Improvesynthesis capabilityVSAvoidfood-grade safety

Core Design Contradiction:

Ease of manufactureVSObject-affected harmful factors

Solution Approach 1:

The invention converts the limitation of using natural substrates (chitin and lactose) into an advantage. By demonstrating that enzymatic synthesis can efficiently produce LNT2 and LNnT from these natural, food-safe substrates without requiring activated artificial donors, the process simultaneously improves safety and maintains synthesis capability.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Achieves efficient and cost-effective synthesis of LNT2 and LNnT with high yields, addressing the inefficiencies of existing methods and ensuring food-grade quality.

Implementation Method 1

β-N-acetylhexosaminidases (EC 3.2.1.52) are a type of glycoside hydrolases (GHs) which catalyze the cleavage of β-linkages in N-acetylamino-β-D-hexosamines

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Implementation Method 2

Recently, GH family 20 β-N-acetylhexosaminidases have been used for the synthesis of functional oligosaccharides due to their high transglycosylation activity

Methodology Applied

Scientific EffectTransglycosylation: Catalysis

Data Source

PatentUS12577599B2Synthesis of human milk oligosaccharides by a beta-N-acetylhexosaminidase from <i>Haloferula </i>sp

Publication Date: 2026.03.17 CHINA AGRI UNIV

AI summary

The invention discloses the application of a β-N-acetylhexosaminidase (HaHex74) from Haloferula sp. in the synthesis of human milk oligosaccharides. The invention provides the use of HaHex74 protein or related biological materials thereof in any one of the following: synthesizing human milk oligosaccharides; synthesizing Lacto-N-triose II and/or Lacto-N-neotetraose; the HaHex74 protein having the amino acid sequence shown in SEQ ID No. 2 is derived from Haloferula sp. The β-N-acetylhexosaminidase HaHex74 disclosed by the invention possesses high-level expression, excellent hydrolysis properties and transglycosylation activity, which may make it potentially useful in the production of human milk oligosaccharides.