Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223
Abstract
:1. Introduction
2. Results and Discussion
2.1. Expression and Purification of the Recombinant Enzyme
2.2. Biochemical Properties and Conformation Changes of the BkLAP under Different Conditions
2.2.1. Effect of pH on the Enzymatic Activity and Secondary Structure of BkLAP
2.2.2. Effect of Temperature on the Enzymatic Activity and the Secondary Structure of BkLAP
2.2.3. Effect of Various Divalent Cations on the Enzymatic Activity and Secondary Structure of BkLAP
2.2.4. The Substrate Selectivity of BkLAP
2.3. BkLAP Greatly Promoted the Hydrolysis of Anchovy Protein
3. Experimental Section
3.1. Total Gene Synthesis of Leucine Aminopeptidase and Construction of Expression Plasmid
3.2. Gene Expression and Enzyme Purification
3.3. Electrophoresis and Protein Assay
3.4. Enzyme Assay
3.5. Circular Dichroism Spectroscopy
3.6. Anchovy Hydrolytic Experiment
3.6.1. Materials
3.6.2. Production of Protein Hydrolysate with Commercial Enzymes Combined with BkLAP
3.6.3. Measurement of Degree of Hydrolysis (DH)
3.6.4. Free Amino Acid Analysis
3.6.5. Molecular Weight Distribution and Free Amino Acids Contents Anlaysis
3.7. Statistical Analysis
4. Conclusions
Acknowledgments
References
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pH | Citrate-phosphate buffer | McIlvanie buffer | HEPES buffer | Potassium-phosphate buffer | Tris-HCl buffer | Glycine-NaOH buffer |
---|---|---|---|---|---|---|
3.0 | 0.00 ± 0.00 | 0.06 ± 0.00 | — | — | — | — |
4.0 | 0.00 ± 0.00 | 0.10 ± 0.02 | — | — | — | — |
5.0 | 0.14 ± 0.07 | 0.49 ± 0.18 | — | — | — | — |
6.0 | 0.66 ± 0.08 | 0.75 ± 0.08 | — | 4.43 ± 0.56 | — | — |
7.0 | 0.31 ± 0.06 | 2.81 ± 2.79 | 44.70 ± 3.32 | 63.59 ± 3.05 | — | — |
7.5 | — | 12.38 ± 1.29 | 53.10 ± 2.15 | — | — | — |
8.0 | — | 56.26 ± 7.42 | 31.44 ± 6.67 | 100.00 ± 0.58 | 40.45 ± 0.94 | — |
9.0 | — | — | — | — | 39.32 ± 0.99 | 1.79 ± 1.63 |
10.0 | — | — | — | — | — | 1.86 ± 1.61 |
11.0 | — | — | — | — | — | 2.19 ± 1.65 |
12.0 | — | — | — | — | — | 0.00 ± 0.00 |
pH 4.0 | pH 6.0 | pH 8.0 | pH 10.0 | |
---|---|---|---|---|
Helix | 43.87% a | 32.40% b | 27.67% c | 25.57% c |
Sheet | 0.00% a | 37.27% b | 38.10% b | 40.27% b |
Turn | 32.83% a | 1.53% b | 5.50% b | 7.30% b |
Random | 23.27% a | 28.77% b | 28.67% b | 26.83% b |
Metal ions | Concentration (M) | Relative activity (%) |
---|---|---|
None | — | 100 |
Hg2+ | 1×10−4 | 6 |
Mg2+ | 1×10−4 | 39 |
Fe2+ | 1×10−4 | 99 |
Fe3+ | 1×10−4 | 48 |
Ca2+ | 1×10−4 | 32 |
Ba2+ | 1×10−4 | 101 |
Cu2+ | 1×10−4 | 48 |
Zn2+ | 1×10−4 | 22 |
Li2+ | 1×10−4 | 97 |
Co2+ | 1×10−5 | 141 |
5×10−5 | 175 | |
1×10−4 | 202 | |
5×10−4 | 243 | |
Mn2+ | 1×10−5 | 100 |
5×10−5 | 420 | |
1×10−4 | 491 | |
5×10−4 | 755 | |
Ni2+ | 1×10−6 | 380 |
1×10−5 | 880 | |
5×10−5 | 1,007 | |
1×10−4 | 1,722 | |
5×10−4 | 3,500 |
None | Co2+ | Ni2+ | Zn2+ | Cu2+ | Mn2+ | Hg2+ | Fe3+ | Ca2+ | |
---|---|---|---|---|---|---|---|---|---|
Helix | 27.67% a | 29.40% a | 30.73% a | 28.93% a | 30.33% a | 29.87% a | 37.07% b | 31.97% a | 28.23% a |
Sheet | 38.10% a | 35.83% a | 36.47% a | 36.10% a | 35.23% a | 36.83% a | 0.00% b | 32.50% a | 37.77% a |
Turn | 5.50% a | 6.20% a | 3.77% a | 5.87% a | 6.10% a | 4.43% a | 28.77% b | 6.67% a | 6.47% a |
Random | 28.67% a | 28.60% a | 29.07% a | 29.07% a | 28.37% a | 28.83% a | 34.17% b | 28.87% a | 27.53% a |
Groups | Enzyme | Temperature (°C) | pH |
---|---|---|---|
1 | Endogenous enzyme | 55.0 | 7.0 |
2 | Flavourzyme + Endogenous enzyme | 50.0 | 6.5 |
3 | Endogenous enzyme + BkLAP | 55.0 °C for 6 h and then 70.0 °C for 12 h | 7.0 for 6 h and then 8.0 |
4 | Flavourzyme + Endogenous enzyme + BkLAP | 50.0 °C for 6 h and then 70.0 °C for 12 h | 6.5 for 6 h and then 8.0 |
Group 1 | Group 2 | Group 3 | Group 4 | |
---|---|---|---|---|
Asp | 139.03 | 179.62 | 249.74 | 305.93 |
Glu | 181.22 | 203.05 | 387.26 | 461.61 |
Ser | 135.09 | 225.65 | 159.20 | 294.60 |
Gly | 60.06 | 90.83 | 120.17 | 203.97 |
His | 115.55 | 235.00 | 149.35 | 253.23 |
Arg | 347.31 | 398.17 | 434.78 | 541.84 |
Thr | 95.54 | 182.94 | 168.62 | 241.27 |
Ala | 173.87 | 230.44 | 283.05 | 379.32 |
Pro | 27.48 | 45.99 | 50.40 | 75.08 |
Tyr | 85.78 | 104.84 | 782.47 | 887.89 |
Val | 138.53 | 211.30 | 221.81 | 324.37 |
Met | 95.27 | 129.20 | 125.29 | 169.00 |
Cys | 5.88 | 15.40 | 39.56 | 58.87 |
Ile | 131.57 | 197.37 | 198.73 | 285.70 |
Leu | 268.26 | 372.34 | 388.64 | 530.67 |
Phe | 137.05 | 174.54 | 187.47 | 268.58 |
Lys | 300.50 | 362.33 | 447.30 | 541.61 |
Total | 2,437.99 | 3,359.03 | 4,393.82 | 5,823.54 |
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Shen, Y.; Wang, F.; Lan, D.; Liu, Y.; Yang, B.; Wang, Y. Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223. Int. J. Mol. Sci. 2011, 12, 7609-7625. https://doi.org/10.3390/ijms12117609
Shen Y, Wang F, Lan D, Liu Y, Yang B, Wang Y. Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223. International Journal of Molecular Sciences. 2011; 12(11):7609-7625. https://doi.org/10.3390/ijms12117609
Chicago/Turabian StyleShen, Yanfei, Fanghua Wang, Dongming Lan, Yuanyuan Liu, Bo Yang, and Yonghua Wang. 2011. "Biochemical Properties and Potential Applications of Recombinant Leucine Aminopeptidase from Bacillus kaustophilus CCRC 11223" International Journal of Molecular Sciences 12, no. 11: 7609-7625. https://doi.org/10.3390/ijms12117609