Lactase Sequence Engineering for Low- and High-Temperature Hydrolysis
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Solution Overview
Problem
Existing lactases are not suitable for lactose hydrolysis at low or high temperatures, leading to microbial growth and enzyme inactivation during fermentation, and are not compatible with ultra-heat treated milk processes, requiring high doses and separate steps for lactose reduction.
Innovation Solution
Development of beta-galactosidases with improved stability and activity at a broad range of temperatures and pH values, allowing for efficient lactose reduction in dairy products using peptides and dimeric peptides with specific amino acid sequences, suitable for low-lactose or lactose-free product production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional lactases are used for lactose hydrolysis, then lactose can be broken down, but the enzyme activity drops significantly at low temperatures and high temperatures cause enzyme inactivation
Solution Approach 1:
The patent modifies the amino acid sequence of the lactase enzyme through site-directed mutagenesis, specifically changing residues in the calcium-binding loop region (positions 446-452). These parameter changes at the molecular level enable the enzyme to maintain structural stability and catalytic activity across a broader temperature range, from 4°C to 60°C, resolving the contradiction between productivity at low temperatures and reliability at high temperatures.
2Productivity
If high enzyme doses are used to achieve desired lactose reduction, then lactose hydrolysis efficiency improves, but production costs increase
Solution Approach 1:
By modifying the enzyme's amino acid sequence to enhance its catalytic efficiency and stability, the patent enables effective lactose hydrolysis at lower enzyme concentrations. The mutated enzyme maintains high activity over extended periods, reducing the total enzyme dosage required from conventional levels to optimized lower doses, thereby resolving the contradiction between productivity and quantity of substance.
3Productivity
If prolonged incubation is used to reduce lactose content, then lactose hydrolysis is more complete, but microbial growth increases and process time extends
Solution Approach 1:
The patent enhances the enzyme's temperature stability through amino acid mutations, enabling effective lactose hydrolysis at elevated temperatures (up to 60°C). By conducting the process at higher temperatures where microbial growth is suppressed, the patent achieves complete lactose hydrolysis in shorter incubation times (4-18 hours), resolving the contradiction between hydrolysis completeness and harmful microbial growth.
4Productivity
If separate enzyme treatment steps are used before fermentation, then lactose can be reduced effectively, but process complexity increases
Solution Approach 1:
The patent develops a lactase enzyme variant that functions effectively across diverse conditions including low temperature storage, pasteurization, and fermentation processes. This multi-functional enzyme can be added once to milk and remains active through subsequent processing steps, eliminating the need for separate pre-fermentation treatment steps and resolving the contradiction between effectiveness and process complexity.
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
Enables efficient lactose reduction in dairy products at various temperatures and pH levels, including high temperatures, reducing lactose concentration significantly with low enzyme doses, and maintaining enzyme activity through processes like pasteurization and UHT treatment.
Implementation Method 1
Lactase (beta-galactosidase; EC 3.2.1.23) is the enzyme that performs the hydrolysis step of the milk sugar lactose into monosaccharides
Implementation Method 2
the enzyme that performs the hydrolysis step of the milk sugar lactose into monosaccharides
Data Source
AI summary
The present invention relates to new improved peptides and dimeric peptides exhibiting beta-galactosidase enzyme activity as well as methods using them for reducing the lactose content of milk-based compositions.