生物技术通报 ›› 2022, Vol. 38 ›› Issue (6): 245-251.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0927
付巧1(), 林啟兰1, 薛强1, 熊海容1, 王亚伟1,2()
收稿日期:
2021-07-17
出版日期:
2022-06-26
发布日期:
2022-07-11
作者简介:
付巧,女,硕士研究生,研究方向:微生物酶工程;E-mail: 基金资助:
FU Qiao1(), LIN Qi-lan1, XUE Qiang1, XIONG Hai-rong1, WANG Ya-wei1,2()
Received:
2021-07-17
Published:
2022-06-26
Online:
2022-07-11
摘要:
采用N端截短方式对Bacillus subtilis 168来源普鲁兰酶进行蛋白结构改造,构建不同形式的截短突变体,考察N端结构对酶学特性的影响。利用基因工程的手段分别删去CBM41结构域N端前2、4和6个氨基酸,获得突变体M1(ΔN2)、M2(ΔN4)和M3(ΔN6)。3种突变体最适反应温度(40-45℃)和最适pH(6.0)均与WT一致,WT、M1、M2和M3的Tm值分别为48.57℃、50.03℃、48.43℃和49.50℃,M1、M2和M3的比活力均高于野生型,是WT的1.18、1.60和2.44倍,WT、M1、M2和M3的Km值分别为23.89、29.01、17.29和19.08 mg/mL。上述结果表明,通过截短普鲁兰酶N端氨基酸可获得性质得到改良的突变体,为提高该酶的热稳定性、比活力和底物结合能力提供新的方法和思路。
付巧, 林啟兰, 薛强, 熊海容, 王亚伟. N端截短CBM41对枯草芽孢杆菌来源普鲁兰酶酶学性质的影响[J]. 生物技术通报, 2022, 38(6): 245-251.
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 |
表1 野生型及其截短突变体扩增用引物
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 |
图4 野生型及其截短突变体的最适pH及pH稳定性 A:最适pH,反应温度40℃,反应时间30 min;B:pH稳定性,pH 4.0-9.0处理时间1 h,反应温度40℃。WT为野生型普鲁兰酶;M1,M2和M3为该普鲁兰酶的不同突变体。下同
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
图5 野生型及其截短突变体的最适温度及热稳定性 A:最适温度,反应pH 6.0,反应时间30 min;B:热稳定性,30-60℃处理时间1 h,反应温度40℃
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 |
表2 普鲁兰酶及其截短突变体比酶活、动力学参数及Tm值比较
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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