(a) Biofuels contain alcohols that are produced by the fermentation of sugars derived from crop waste. This waste contains cellulose and other organic compounds in cell walls.
Scientists investigated the production of sugars from crop waste for biofuel production. The scientists discovered that a strain of the fungus Penicillium citrinum, isolated from soil, was a good source of three different extracellular enzymes, M, N and O. These enzymes break down polysaccharides in cell walls.
The scientists cultured P. citrinum in a liquid medium containing cell wall material. Samples of the liquid were taken, and the three enzymes were separated from the medium. Each enzyme was placed in a reaction mixture with an appropriate substrate. The activity of each enzyme was determined to give a measurement of the quantity of enzyme produced by P. citrinum.
The results are shown in Table 4.1.
| enzyme | maximum activity / arbitrary units |
|---|---|
| M | 292.83 |
| N | 111.72 |
| O | 6.54 |
Optimum conditions for each enzyme were used to obtain the results in Table 4.1. The conditions were different for each enzyme.
The scientists carried out further research so that a solution containing the three enzymes (enzyme mixture) could be used for the most efficient production of sugars from crop waste. Suggest what the scientists needed to find out in their research.
(b) Students investigated the composition of the cell wall of leaf cells of thale cress, Arabidopsis thaliana. The students began by isolating the cell wall components from the rest of the cell material.
The students used enzymes extracted from a fungal pathogen of A. thaliana to hydrolyse the cell wall components to smaller molecules. They prepared a reaction mixture containing the cell wall components and the enzymes. After 24 hours, they separated and identified the smaller molecules found in the reaction mixture.
Four types of molecule were identified:
- short chains of β-glucose
- β-glucose
- peptides
- amino acids
Explain the presence of these molecules in the reaction mixture after 24 hours of hydrolysis.
▶️ Answer/Explanation
(a)
The scientists needed to investigate several aspects to optimize the enzyme mixture for efficient sugar production:
- Optimal conditions for the enzyme mixture: They needed to determine the ideal pH and temperature that would allow all three enzymes (M, N, and O) to work together effectively, even though each has different individual optimal conditions.
- Substrate specificity: They should identify which crop wastes contain the highest concentration of substrates that enzyme M can act on, since M showed the highest activity (292.83 units).
- Enzyme ratios: They needed to find the most effective proportions of the three enzymes in the mixture to maximize sugar production, considering their different activity levels.
- Pre-treatment methods: Research was needed to determine if any physical or chemical pre-treatment of the crop waste would make the cellulose more accessible to the enzymes.
- Reaction time: They should investigate how long it takes for complete conversion of crop waste to sugars under optimal conditions.
(b)
The presence of these molecules after hydrolysis indicates:
- Cellulose breakdown: The short chains of β-glucose and individual β-glucose molecules show that cellulose (composed of β-1,4-linked glucose units) was present in the cell wall and was being broken down by cellulase enzymes. The short chains represent partial hydrolysis, while free glucose indicates complete breakdown of some cellulose chains.
- Protein components: The peptides and amino acids indicate that proteins were present in the cell wall (likely structural proteins like extensins) and were hydrolyzed by proteases. The peptides represent partially broken down proteins, while amino acids show complete hydrolysis of some peptide bonds.
- Enzyme activity: The mixture of breakdown products suggests that both cellulases (breaking glycosidic bonds in cellulose) and proteases (breaking peptide bonds in proteins) were active in the fungal enzyme extract.
- Time factor: After 24 hours, both complete and partial hydrolysis products were present, suggesting some cellulose and protein molecules were fully broken down while others were only partially digested.
This pattern of breakdown products demonstrates that the A. thaliana cell wall contains both cellulose (a polysaccharide) and structural proteins, and that the fungal enzymes were effective at hydrolyzing these components over the 24-hour period.