Biotechnology Bulletin ›› 2022, Vol. 38 ›› Issue (6): 245-251.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0927
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FU Qiao1(), LIN Qi-lan1, XUE Qiang1, XIONG Hai-rong1, WANG Ya-wei1,2()
Received:
2021-07-17
Online:
2022-06-26
Published:
2022-07-11
Contact:
WANG Ya-wei
E-mail:1196840811@qq.com;253182556@qq.com
FU Qiao, LIN Qi-lan, XUE Qiang, XIONG Hai-rong, WANG Ya-wei. Effects of CBM41 N-terminal Truncation on the Enzymological Properties of the Pullulanase from Bacillus subtilis 168[J]. Biotechnology Bulletin, 2022, 38(6): 245-251.
Primer name | Nucleotide sequence(5'-3') | Gene |
---|---|---|
WT-F(Nco I) | CATGCCATGGTCAGCATCCGCCGCAG- CTTC | PulB |
M1-F(Nco I) | CATGCCATGGATAGCATCCGCCGCAG- CTTCGAAG | PulBΔN2 |
M2-F(Nco I) | CATGCCATGGATCGCCGCAGCTTCGA- AGCGTATG | PulBΔN4 |
M3-F(Nco I) | CATGCCATGGATAGCTTCGAAGC- GTATGTCGATG | PulBΔN6 |
WT-R(Xho I) | CCGCTCGAGAGCAAAACTCTTAAGAT- CTGATGC |
Table 1 Primers for amplification of wild-type and trun-cated mutants
Primer name | Nucleotide sequence(5'-3') | Gene |
---|---|---|
WT-F(Nco I) | CATGCCATGGTCAGCATCCGCCGCAG- CTTC | PulB |
M1-F(Nco I) | CATGCCATGGATAGCATCCGCCGCAG- CTTCGAAG | PulBΔN2 |
M2-F(Nco I) | CATGCCATGGATCGCCGCAGCTTCGA- AGCGTATG | PulBΔN4 |
M3-F(Nco I) | CATGCCATGGATAGCTTCGAAGC- GTATGTCGATG | PulBΔN6 |
WT-R(Xho I) | CCGCTCGAGAGCAAAACTCTTAAGAT- CTGATGC |
Fig.4 Optimal pH and pH stability of wild-type pullulan-ase and the truncated mutants A:Optimal pH. The reaction temperature and time:40℃ and 30 min. B:pH stability. pH:4.0-9.0;treatment time:1 h;reaction temperature:40℃. WT:Wild-type pullulanase;M1,M2 and M3:different mutants of pullulanase. The same below
Fig.5 Optimal temperature and thermostability of wild-type pullulanase and the truncated mutants A:Optimal temperature. The reaction pH and time:6.0 and 30 min. B:Thermostability. Temperature:30-60℃;treatment time:1 h;reaction temperature:40℃
比酶活Specific enzyme activity/(U·mg-1) | Km/(mg·mL-1) | Vmax/(U·mg-1) | Tm/℃ | |
---|---|---|---|---|
WT | 2.30±0.09 | 23.89±0.66 | 4.06±0.09 | 48.57±0.42 |
M1 | 2.72±0.08 | 29.01±1.21 | 2.35±0.12 | 50.03±0.25 |
M2 | 3.69±0.07 | 17.29±0.73 | 3.92±0.06 | 48.43±0.22 |
M3 | 5.62±0.06 | 19.08±0.41 | 7.24±0.11 | 49.50±0.32 |
Table 2 Comparison of specific activity,kinetic parameters and Tm of pullulanase and the truncated mutants
比酶活Specific enzyme activity/(U·mg-1) | Km/(mg·mL-1) | Vmax/(U·mg-1) | Tm/℃ | |
---|---|---|---|---|
WT | 2.30±0.09 | 23.89±0.66 | 4.06±0.09 | 48.57±0.42 |
M1 | 2.72±0.08 | 29.01±1.21 | 2.35±0.12 | 50.03±0.25 |
M2 | 3.69±0.07 | 17.29±0.73 | 3.92±0.06 | 48.43±0.22 |
M3 | 5.62±0.06 | 19.08±0.41 | 7.24±0.11 | 49.50±0.32 |
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