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

doi:10.13560/j.cnki.biotech.bull.1985.2021-0927

摘要/Abstract

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T_m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K_m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。

关键词: N端截短, 普鲁兰酶, Bacillus subtilis 168, 酶学特性

Abstract:

Different N-terminal truncated variants were constructed using N-terminal truncation to modify the protein structure of Bacillus subtilis 168 pullulanase,the effects of truncated mutation on enzymatic properties were studied. Three variants,M1（ΔN2）,M2（ΔN4）and M3（ΔN6）,by deleting the first 2,4 and 6 residues from the N-terminus respectively,were cloned and expressed in Escherichia coli BL21 cells by genetic engineering method. The optimal temperature of three variants was 40-45℃,and the optimal pH was 6.0,which was consistent with the wild-type pullulanase（WT）. T_m values of WT,M1,M2 and M3 were 48.57,50.03,48.43 and 49.50℃,respectively. The specific activity of M1,M2 and M3 were all higher than those of wild-type,increasing by 1.18,1.60 and 2.44 times,respectively. K_mvalues of WT,M1,M2,and M3 were 23.89,29.01,17.29 and 19.08 mg/mL,respectively. These results indicate that variants with the improved properties can be obtained by N-terminal truncation of pullulanase,which provides new methods and ideas for improving the thermal stability,specific activity and substrate binding capacity of this enzyme.

Key words: N-terminal truncation, pullulanase, Bacillus subtilis 168, enzymatic property

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.

图/表 8

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1 枯草芽孢杆菌168来源普鲁兰酶结构示意图

Fig.1 Structure of pullulanase from B. subtilis 168

图2 截短突变体菌落PCR鉴定图

Fig.2 PCR identification of truncated mutants colonies M：DL5000 DNA marker;1：M1;2：M2;3：M3

图3 野生型普鲁兰酶及其截短突变体纯化后SDS-PAGE

Fig.3 SDS-PAGE analysis of the purified wild-type pull-ulanase and the truncated mutants M：Marker;1：M1;2：M2;3：M3;4：WT

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℃

图6 野生型及其截短突变体Tm值测定

Fig.6 Tm determination of wild-type pullulanase and the truncated mutants

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）	K_m/（mg·mL^-1）	V_max/（U·mg^-1）	T_m/℃
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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