Lactase Expression in Altered Regulation Strains for Low-Lactose Dairy
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Solution Overview
Problem
Existing lactases are not suitable for low-lactose dairy product production due to pH and temperature limitations, and they are not effective in ultra-heat treated (UHT) milk or pasteurization processes, with commercial lactases requiring high dosages and separate enzyme treatments.
Innovation Solution
Altered regulation strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus with mutations in the glucokinase and glucose transporter genes, allowing for high lactase production and activity in a wide pH and temperature range, suitable for dairy product applications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If commercial neutral lactases (from K. lactis, B. coagulans, etc.) are used for lactose hydrolysis in dairy products, then lactose can be broken down into monosaccharides, but the enzyme activity drops significantly as pH decreases during fermentation and they are not suitable for high temperature processing
Solution Approach 1:
The patent applies parameter changes by modifying the enzyme's structural and functional parameters through directed evolution and rational design. Specifically, the lactase enzyme was engineered to change its pH optimum from neutral (pH 6-8) to acidic conditions and to gain thermostability, allowing it to function reliably across the pH range of dairy fermentation (pH 3-6) and withstand high temperature processing (up to 70°C).
Solution Approach 2:
The patent uses copying by expressing the engineered lactase gene in heterologous host organisms (such as E. coli, B. subtilis, or other bacteria) to produce large quantities of the enzyme. The gene is cloned into expression vectors and transferred to host cells that can be easily cultivated industrially, enabling scalable production of the engineered enzyme without requiring the original organism (K. lactis) to be cultivated under complex conditions.
2Reliability
If high enzyme dosages are used to compensate for reduced activity at low pH, then lactose hydrolysis can be maintained, but the cost and process complexity increase due to separate enzyme treatment steps
Solution Approach 1:
The patent merges the lactose hydrolysis function with the existing dairy fermentation process by using an acid-stable lactase that remains active during fermentation. Instead of adding enzyme separately before or after fermentation, the engineered lactase can be introduced during the fermentation process itself, combining two functions (fermentation and lactose hydrolysis) into a single integrated process step, thereby reducing process complexity.
Solution Approach 2:
The patent applies preliminary action by pre-engineering the lactase enzyme to have broad pH and temperature stability before the actual dairy production process. The enzyme is designed in advance to withstand the specific conditions of dairy fermentation and high-temperature processing, so that when added to the production line, it immediately functions effectively without requiring subsequent adjustments or separate treatment steps.
3Productivity
If wild type lactic acid bacteria are used for lactose hydrolysis, then they can grow on lactose and excrete glucose, but they produce only low titers of lactase enzyme
Solution Approach 1:
The patent applies parameter changes by modifying the regulatory parameters of the lactase gene (lacZ) in the bacterial host. Through mutations in promoter regions, operator sequences, or regulatory genes (such as lac repressor), the expression level of the lactase enzyme is dramatically increased. This allows the bacteria to produce high titers of lactase while maintaining their ability to utilize lactose as a carbon source.
Solution Approach 2:
The patent uses an intermediary approach by employing plasmid vectors or chromosomal integration systems as mediators to overexpress the lactase gene in the host bacteria. These intermediaries (expression vectors, promoters, ribosome binding sites) are designed to maximize transcription and translation of the lactase gene, thereby increasing enzyme production efficiency while maintaining bacterial viability and lactose utilization.
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
The altered regulation strains produce 3-10 folds more lactase than wild type strains, enabling efficient lactose hydrolysis in dairy products, including low-lactose and UHT milk, without the need for additional enzyme treatments.
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
lactose hydrolysis is a good way for lactic acid bacteria to obtain glucose and galactose as carbon source
Data Source
AI summary
The present invention relates to new improved methods for expressing native lactases in their native hosts. Methods for homologous as well as heterologous expression of lactase in lactic acid bacteria with altered expression dynamics are comprised by present invention.