Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69
Abstract
:1. Introduction
2. Results
2.1. Optimization of Extraction Conditions of CEP by RSM
2.1.1. Box–Behnken Design and Response Surface Method
2.1.2. The Influence of Time, Temperature and Buffer pH on the Response Value
2.1.3. Model Validation
2.2. Enzymatic Properties of CEP
2.2.1. Effects of pH and Temperature on Enzyme Activity
2.2.2. Effect of Metal Ions and Inhibitors on Enzyme Activity
2.2.3. Kinetic Studies of CEP
3. Discussion
4. Materials and Methods
4.1. Strains and Chemicals
4.2. Preparation of Cell-Free Extracts of L. Plantarum LP69
4.3. Determination of Enzyme Activity
4.4. Determination of Protein Concentration
4.5. Specific Activity Assay
4.6. Experimental Design
4.7. Enzymatic Properties of CEP from L. plantarum LP69
4.7.1. Effect of pH on the Enzyme Activity
4.7.2. Temperature Optimum
4.7.3. Influence of Metal Ions and Inhibitors on Enzyme Activity
4.7.4. Kinetic Studies of CEP
4.8. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Runs | Variables | Enzyme Activity (Y1, U/mL) | Specific Activity (Y2, U/mg) | ||
---|---|---|---|---|---|
Time (min, X1) | Temperature (°C, X2) | pH (X3) | |||
1 | 1 (90) | 0 (39) | 1 (7.0) | 17.6 ± 0.52 | 1.31 ± 0.02 |
2 | 0 (75) | 0 | 0 (6.5) | 20.64 ± 0.73 | 1.35 ± 0.04 |
3 | 1 | −1 (37) | 0 | 15.84 ± 0.59 | 1.22 ± 0.06 |
4 | −1 (60) | 0 | 1 | 11.75 ± 0.66 | 1.31 ± 0.02 |
5 | −1 | 0 | −1 (6.0) | 14.34 ± 0.71 | 1.27 ± 0.03 |
6 | 0 | 0 | 0 | 22.21 ± 0.67 | 1.39 ± 0.07 |
7 | 1 | 1 (41) | 0 | 21.42 ± 0.78 | 1.18 ± 0.06 |
8 | 0 | 1 | 1 | 13.43 ± 0.87 | 1.28 ± 0.03 |
9 | 1 | 0 | −1 | 22.16 ± 0.76 | 1.30 ± 0.10 |
10 | 0 | 1 | −1 | 16.29 ± 0.81 | 1.20 ± 0.03 |
11 | 0 | −1 | −1 | 13.72 ± 0.52 | 1.29 ± 0.07 |
12 | −1 | 1 | 0 | 11.21 ± 0.54 | 1.21 ± 0.11 |
13 | 0 | −1 | 1 | 16.43 ± 0.57 | 1.27 ± 0.08 |
14 | 0 | 0 | 0 | 20.77 ± 0.75 | 1.37 ± 0.04 |
15 | −1 | −1 | 0 | 15.94 ± 0.69 | 1.21 ± 0.05 |
Source | DF | Enzyme Activity (Y1, U/mL) | Specific Activity (Y2, U/mg) | ||||
---|---|---|---|---|---|---|---|
MS | F | Pr > F | MS | F | Pr > F | ||
Model | 9 | 20.73 | 10.99 | 0.0084 * | 0.0062 | 23.47 | 0.0014 * |
X1 | 1 | 70.69 | 37.47 | 0.0017 * | <0.0001 | 0.047 | 0.8366 |
X2 | 1 | 0.022 | 0.012 | 0.9181 | 0.0018 | 6.79 | 0.0479 * |
X3 | 1 | 6.66 | 3.53 | 0.1190 | 0.0015 | 5.71 | 0.0625 |
X1X2 | 1 | 26.57 | 14.09 | 0.0133 * | 0.0004 | 1.51 | 0.2739 |
X1X3 | 1 | 0.97 | 0.51 | 0.5054 | 0.0002 | 0.85 | 0.3991 |
X2X3 | 1 | 7.76 | 4.11 | 0.0984 | 0.0025 | 9.43 | 0.0277 * |
X12 | 1 | 12.02 | 6.37 | 0.0529 | 0.015 | 56.63 | 0.0007 * |
X22 | 1 | 40.20 | 21.31 | 0.0058 * | 0.038 | 142.84 | <0.0001 * |
X32 | 1 | 31.91 | 16.91 | 0.0092 * | 0.0002 | 1.07 | 0.3490 |
Residual | 5 | 1.89 | 0.0002 | ||||
Lack of fit | 3 | 2.64 | 3.47 | 0.2315 | 0.0001 | 0.44 | 0.7506 |
Pure error | 2 | 0.76 | 0.0004 | ||||
R2 | 0.9519 | 0.9769 | |||||
R2adj | 0.8652 | 0.9352 |
Inhibitors and Ions a | Relative Enzyme Activity (%) b | |
---|---|---|
Compound Concentration (mM) | ||
1.00 | 10.00 | |
None | 100.00 | 100.00 |
Ca2+ | 105.96 | 122.82 |
Na+ | 91.42 | 93.75 |
K+ | 94.23 | 106.01 |
Zn2+ | 53.59 | 42.34 |
PMSF | 40.94 | 25.73 |
EDTA | 45.20 | 34.93 |
Factors | Symbol | Coded Levels | ||
---|---|---|---|---|
−1 | 0 | 1 | ||
Time (min) | X1 | 60 | 75 | 90 |
Temperature (°C) | X2 | 37 | 39 | 41 |
Buffer pH | X3 | 6.0 | 6.5 | 7.0 |
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Chen, H.; Huang, J.; Cao, B.; Chen, L.; Song, N.; Lei, N. Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69. Catalysts 2018, 8, 325. https://doi.org/10.3390/catal8080325
Chen H, Huang J, Cao B, Chen L, Song N, Lei N. Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69. Catalysts. 2018; 8(8):325. https://doi.org/10.3390/catal8080325
Chicago/Turabian StyleChen, He, Jie Huang, Binyun Cao, Li Chen, Na Song, and Ni Lei. 2018. "Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69" Catalysts 8, no. 8: 325. https://doi.org/10.3390/catal8080325