Wet Lab
The Wet-Lab module serves as an extension of the main project, including both the Project and Software modules (particularly the AI model content within the Software module). Its primary objective is to validate the accuracy of the HEATMAP model's predictions for the optimal temperature of enzymes by exploring their catalytic efficiency at different temperatures. In this module, we selected the widely used tool enzyme, Proteinase K, and measured its catalytic efficiency in cleaving α-casein from bovine milk to release tyrosine at varying temperatures. This preliminary validation of the HEATMAP model's accuracy also lays a foundation for the subsequent modification of key enzymes involved in the production of spinosad, aligning their optimal working temperatures with fermentation conditions.
Proteinase K is derived from Tritirachium album Limber and is named for its ability to degrade keratin. It is a serine protease with broad substrate specificity, cleaving peptide bonds at the carboxyl ends of aliphatic and aromatic amino acids to yield free amino acids. The typical serine active site for Proteinase K is Asp39-His69-Ser224. In this experiment, we used the sequence information of Proteinase K as input for the HEATMAP model, predicting an optimal temperature of 43.71°C. Following the National Standard of the People's Republic of China GB/T 33410-2016 for determining the activity of Proteinase K in biochemical reagents, we used α-casein as the substrate. The enzyme catalyzed the hydrolysis to L-tyrosine, and after terminating the reaction and centrifuging to separate proteins and other impurities, we measured the absorbance at 275 nm as a representation of the enzyme's catalytic efficiency. Based on the HEATMAP-predicted optimal temperature, we designed three experimental groups at 33.5°C, 43.5°C, and 53.5°C. By comparing the optimal working temperature of the enzyme determined in wet experiments with the predicted optimal temperature from dry experiments, we aimed to validate the accuracy of the HEATMAP model regarding the enzyme's optimal temperature.
20 mg/ml Proteinase K Stock Solution (provided by Professor Feng Yan, with a fasta file containing sequence information)
α-Casein from Bovine Milk
Tris Base (Tris Hydroxymethyl Aminomethane Hydrochloride)
6M HCl (diluted from concentrated hydrochloric acid)
Methanol
Double-distilled Water (ddH₂O)
Thermo Scientific NanoDrop Spectrophotometer
Finnpipette™ F3 Variable-Volume Single-Channel Pipette (1-10 µl, 20-200 µl, 100-1000 µl)
GeneSpeed GS-X1-A2 Microcentrifuge
Electronic Balance
pH Meter
Water Bath
250 ml Graduated Erlenmeyer Flask
200 ml 0.01 mol/L Tris·HCl Buffer (pH 8.0): Dissolve 2.4228 g of Tris base in an appropriate amount of deionized water and bring to a final volume of 200 ml. Adjust pH to 8.0 using concentrated HCl. Then, take 20 ml of the 0.1 mol/L Tris·HCl buffer and dilute it with deionized water to 200 ml to obtain 0.01 mol/L Tris·HCl buffer.
1% α-Casein from Bovine Milk: Dissolve 0.5 g of bovine milk casein in 30 ml of 0.01 mol/L Tris·HCl buffer (pH 8.0), incubate at 55°C for 15 minutes with stirring, cool to room temperature, adjust pH to 8.0, and bring to a final volume of 50 ml.
60 µg/mL Proteinase K Working Solution: Mix 20 mg/ml Proteinase K with 10 ml of 0.01 mol/L Tris·HCl buffer (pH 8.0).
Transfer 3 groups of 1 ml of 1% α-casein from bovine milk into three 2 ml microcentrifuge tubes and place them in water baths at 33.5°C, 43.5°C, and 53.5°C for 10 minutes of preheating.
Transfer 3 groups of 1 ml of 60 µg/mL Proteinase K into three 15 ml microcentrifuge tubes and place them in the same water baths for 10 minutes of preheating.
In a new microcentrifuge tube, mix 750 µl of pre-heated 1% α-casein with 250 µl of Proteinase K solution, gently mix, and incubate in the corresponding temperature water bath for 10 minutes (33.5°C, 43.5°C, 53.5°C).
Add 1 ml of methanol to the reaction mixture, mix well, let it sit at room temperature for 5 minutes, and then centrifuge at 4000 rpm for 5 minutes.
Use the Thermo Scientific NanoDrop spectrophotometer to perform full-wavelength scans from 190.0 nm to 850.0 nm, using 0.01 mol/L Tris·HCl buffer (pH 8.0) as a blank. Measure the absorbance of the supernatant from the reaction systems at 33.5°C, 43.5°C, and 53.5°C, particularly noting the absorbance values at 270 nm, 275 nm, and 280 nm (the characteristic peaks for L-tyrosine).
HEATMAP Prediction of Optimal Temperature for Proteinase K:
Using the sequence of Proteinase K as input for the HEATMAP model, we predicted an optimal temperature of 43.71°C, which served as the basis for the temperature gradient design in the wet experiments.
Figure 1: HEATMAP Prediction of the Optimal Working Temperature for Proteinase K. Absorbance Results of Enzyme Reaction Systems at Different Temperature Gradients： We conducted full-wavelength absorbance scans on the supernatants from the reaction systems at 33.5°C, 43.5°C, and 53.5°C. Notably, all three groups exhibited a significant absorbance peak between 250 nm and 290 nm, with a peak position around 275 nm. This corresponds to the characteristic absorption peak of L-tyrosine, indicating that Proteinase K successfully cleaved α-casein in all three experimental groups.
Figure 2: Full Wavelength Absorbance Scan of the Supernatant from the Enzyme Reaction System at 33.5°C, covering the range from 190.0 nm to 850.0 nm.
Figure 3: Full Wavelength Absorbance Scan of the Supernatant from the Enzyme Reaction System at 43.5°C, covering the range from 190.0 nm to 850.0 nm.
Figure 4: Full Wavelength Absorbance Scan of the Supernatant from the Enzyme Reaction System at 53.5°C, covering the range from 190.0 nm to 850.0 nm.
Absorbance Values at 270 nm, 275 nm, and 280 nm under Different Temperature Gradients:
The enzyme reaction at 43.5°C showed absorbance values of 0.243, 0.285, and 0.283 at 270 nm, 275 nm, and 280 nm, respectively. These values were higher than those observed at 33.5°C (0.221, 0.265, 0.270) and 53.5°C (0.236, 0.277, 0.280). This strongly suggests that the 43.5°C reaction group produced more L-tyrosine, indicating that Proteinase K likely exhibits its highest catalytic efficiency at this temperature, which may represent its optimal temperature. This finding aligns with the HEATMAP model's predicted optimal temperature, thereby validating the model's accuracy based on the wet experimental data.temperature and confirming the model's accuracy through wet experiments.
Figure 5: Comparison of Absorbance at 270 nm, 275 nm, and 280 nm in the Supernatants of Enzyme Reaction Systems at 33.5°C, 43.5°C, and 53.5°C.
By measuring the efficiency of Proteinase K in cleaving casein at different temperature gradients using the NanoDrop, we determined a possible \( T_{opt} \) of 43.5°C, which closely matches the HEATMAP model's predicted optimal working temperature of 43.71°C. This preliminary validation confirms the accuracy of the HEATMAP model's predictions. The measured optimal temperature data will be used alongside the amino acid sequence to further optimize the HEATMAP model.
In the expansion of the iGEM project, we will continue to address the practical issues of fermentation production of spinosad in conjunction with the etcGEM of Saccharopolyspora spinosa. We aim to modify the sequences of key enzymes whose optimal temperatures do not match the organism's growth temperature, thereby improving production yields. This will involve enzyme purification, assessment of protein thermal stability and efficiency, and ultimately the transfer of the modified key enzymes into Saccharopolyspora spinosa to enhance spinosad production, contributing to the goal of using synthetic biology for a better world.
We would like to thank Professor Bai Linquan and Professor Feng Yan from the School of Life Sciences and Biotechnology, State Key Laboratory of Microbial Metabolism at Shanghai Jiao Tong University, for their guidance in planning the overall experimental approach and for providing the necessary reagents, enzymes, consumables, and other materials for the experiments. We would also like to thank Researcher Zhang Xia and Technician Zhang Ping from the National Experimental Teaching Center for Life Sciences and Biotechnology at Shanghai Jiao Tong University for their valuable suggestions regarding experimental details, as well as for providing the wet lab experimental platform and equipments.
[1] Bertelsen AB, Hackney CM, Bayer CN, Kjelgaard LD, Rennig M, Christensen B, Sørensen ES, Safavi-Hemami H, Wulff T, Ellgaard L, Nørholm MHH. DisCoTune: versatile auxiliary plasmids for the production of disulphide-containing proteins and peptides in the E. coli T7 system. Microb Biotechnol. 2021 Nov;14(6):2566-2580. doi: 10.1111/1751-7915.13895. Epub 2021 Aug 18. PMID: 34405535; PMCID: PMC8601162.
[2] Pähler A, Banerjee A, Dattagupta JK, Fujiwara T, Lindner K, Pal GP, Suck D, Weber G, Saenger W. Three-dimensional structure of fungal proteinase K reveals similarity to bacterial subtilisin. EMBO J. 1984 Jun;3(6):1311-4. doi: 10.1002/j.1460-2075.1984.tb01968.x. PMID: 6378621; PMCID: PMC557514.
[3] Kraus E, Femfert U. Proteinase K from the mold Tritirachium album Limber. Specificity and mode of action. Hoppe Seylers Z Physiol Chem. 1976 Jul;357(7):937-47. doi: 10.1515/bchm2.1976.357.2.937. PMID: 992572.
[4] Masuda T, Suzuki M, Inoue S, Song C, Nakane T, Nango E, Tanaka R, Tono K, Joti Y, Kameshima T, Hatsui T, Yabashi M, Mikami B, Nureki O, Numata K, Iwata S, Sugahara M. Atomic resolution structure of serine protease proteinase K at ambient temperature. Sci Rep. 2017 Mar 31;7:45604. doi: 10.1038/srep45604. PMID: 28361898; PMCID: PMC5374539.
[5] National Standardization Administration. GB/T 33410-2016. Method for Detecting the Activity of Protease K in Biochemical Reagents. Beijing: Standards Press of China, 2016.