Enzymatic Yeast Lysis for Solvent-Free Bioproduct Extraction
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Solution Overview
Problem
Current methods for extracting bioproducts from industrious yeasts, particularly those with recalcitrant cell walls like Rhodotorula and Rhodosporidium, are costly, time-consuming, and often result in product perturbation or solvent contamination, lacking efficiency and environmental friendliness.
Innovation Solution
Employing low doses of β-1,3-glucomannanase and live cellulolytic fungi, or genetically modified organisms expressing cellulolytic enzymes, to enzymatically lyse yeast cells, followed by solvent-free or non-solvent extraction methods to isolate bioproducts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If conventional acid treatment and solvent recovery methods are used for lysis, then cell wall breakdown is achieved, but product quality is perturbed and solvent contamination occurs
Solution Approach 1:
The patent replaces mechanical/chemical lysis methods (acid treatment, solvent recovery) with enzymatic lysis using beta-glucanase and other cell wall degrading enzymes. This substitution eliminates the need for harsh chemicals and organic solvents that cause product degradation and contamination, while achieving effective cell wall breakdown through specific enzymatic action on beta-1,3-glucan and other cell wall components.
Solution Approach 2:
The patent changes the chemical parameters of the lysis process by using enzymatic reactions at controlled temperatures (37-50°C) and pH levels (6.5-7.5), replacing the extreme conditions of acid treatment. This parameter change maintains lysis effectiveness while preventing product oxidation and degradation that occur with conventional methods.
2Ease of manufacture
If high concentrations of enzyme are used for lysis, then complete cell wall breakdown is achieved, but extraction cost increases significantly
Solution Approach 1:
The patent applies partial action by using sub-optimal but sufficient enzyme concentrations (0.01-0.1 mg/mL beta-glucanase) combined with extended incubation times (12-24 hours) and mild physical pretreatments (autoclaving, bead beating). This approach achieves adequate lysis for effective extraction without the excessive enzyme costs that would result from using high concentrations for short durations.
Solution Approach 2:
The patent employs preliminary physical pretreatments (autoclaving at 121°C for 20-30 minutes, bead beating, or sonication) before enzymatic lysis. These preliminary actions partially disrupt the recalcitrant cell walls of oleaginous yeasts, making them more accessible to enzymatic degradation and reducing the overall enzyme quantity needed for complete lysis.
3Ease of manufacture
If recalcitrant cell walls with beta-1,3-glucomannose composition are targeted, then complete lysis of oleaginous yeast is achieved, but conventional enzymatic reagents have little effect
Solution Approach 1:
The patent uses a composite enzymatic system comprising multiple enzymes with complementary specificities: beta-glucanase (for beta-1,3-glucan), mannanase (for mannoprotein), and protease (for protein crosslinks). This composite approach addresses the complex cell wall composition of oleaginous yeasts containing beta-1,3-glucan, mannoprotein, and other polymers, achieving complete lysis where single enzymes fail.
Solution Approach 2:
The patent introduces autolytic enzymes produced by the yeast itself (such as alkaline phosphatase and other endogenous hydrolytic enzymes) as intermediaries that work synergistically with externally added beta-glucanase. The yeast's own enzymatic systems are activated or enhanced to facilitate cell wall breakdown, bridging the gap between external enzyme treatment and internal cell wall degradation pathways.
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, cost-effective extraction of bioproducts with minimal solvent contamination, preserving product quality and reducing environmental impact.
Implementation Method 1
treating yeast cells with a β-1,3-glucomannanase, wherein the β-1,3-glucomannanase is in an amount of less than 1.0e-4 g enzyme protein/g dry cell weight, thereby producing an enzymatically lysed sample
Implementation Method 2
inoculating the inactive biomass with live cellulolytic fungi and/or an organism engineered to express at least one cellulolytic enzyme, and incubating the live cellulolytic fungi and/or an organism engineered to express at least one cellulolytic enzyme for at least 5 hours to generate a lysed biomass
Implementation Method 3
separating the lipid phase of the enzymatically lysed sample via solvent or non-solvent extraction, thereby producing a separated sample
Data Source
AI summary
The disclosure relates to novel methods, compositions, and genetically modified microorganisms for extracting and/or isolating bioproducts from microorganisms having recalcitrant cell walls. In some aspects, the disclosure relates to solvent-free methods of extracting and/or isolating bioproducts. The disclosure further relates to bioproducts having less than 10 ppm of a solvent.