I need to develop an enzyme assay to evaluate the degradation of gluten using proteases. Previously, I conducted tests with finished products, which was straightforward. However, I now have to work with actual enzymes, which are in limited quantity. Consequently, I'm facing challenges in devising an assay method. The total enzyme quantity available is 100ml, and I need to conduct multiple tests to assess the enzyme's activity in degrading gluten. Could you please provide assistance in formulating an appropriate assay method for this task?
What methods and equipment do you have available to you to detect formation of the products of gluten hydrolysis by the enzyme?
Previously, I conducted tests using finished products by combining 25g of gluten with 25ml of water and 10g of enzyme product. The mixture was left at room temperature for observation, with visual readings of gluten degradation and periodic measurements of gluten weight. However, this method proved inefficient due to the lengthy degradation process and associated costs. To address this, we opted to use actual enzymes, specifically proteases.
The challenge now lies in adapting the testing procedure for actual enzymes, given the limited quantity available (100ml). Scaling down the amount of gluten for testing poses difficulties in accurately measuring degradation and determining efficiency. Additionally, some enzymes are in powder form, further complicating the process.
Questions arise regarding whether these enzymes can be diluted and how to assess their efficacy in degrading gluten. While it's known that many enzymes exhibit peak activity at specific temperatures, determining the optimal conditions for each enzyme remains essential. Fortunately, we have an incubator to control temperature.
To tackle this situation effectively, we need a revised methodology that addresses the constraints of enzyme quantity, facilitates accurate measurements, and ensures reliable assessment of enzyme efficiency in degrading gluten. Could you provide guidance on devising such a methodology? Adam B Shapiro
If there are alternative methods available, please inform me, even if they necessitate additional equipment. I believe I can arrange for any necessary resources. Our objective is to assess the efficacy of these enzymes in degrading gluten, so it's imperative to accurately measure their capabilities.
You have been working on a scale that I consider to be enormous. I'm used to micro-scale assays of enzyme activity, conducted in volumes on the order of 50 microliters. Also, the gluten and enzyme concentrations you use seem enormous to me at 1 g/mL and 0.4 g/mL, respectively.
You haven't described the method of measuring gluten hydrolysis. The products will be smaller peptides. A straightforward way to follow the reaction would be to run the sample through an HPLC column that separates peptides on the bases of either size or hydrophobicity (size exclusion or reverse phase, respectively). The samples would probably have to be diluted and centrifuged prior to analysis, and you would need a blank of enzyme without gluten to identify which peaks were from the enzyme. Detection would be by UV absorbance at 214 nm.
I found a method in this paper
Article Performance of enzymatic wheat gluten hydrolysis in batch an...
in which the hydrolysis of gluten was followed by the creation of new N-termini using the o-phthaldialdehyde (OPA) reaction, with detection at 340 nm with a spectrophotometer or absorbance plate reader. No chromatography would be needed, which would speed up the experimental testing of different conditions.
Both of these ideas could be performed in very small volumes, such as in 1.5-ml polypropylene microcentrifuge tubes containing no more than a few hundred microliters of solution. This would also simplify the centrifugation step, since the samples would already be in centrifuge tubes. The tubes could be incubated at various temperatures in temp-blocks designed to hold that kind of tube. The supernatants would be transferred to separate containers and either mixed with OPA for detection, or analyzed by chromatography.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA