Effects of CBM41 N-terminal Truncation on the Enzymological Properties of the Pullulanase from Bacillus subtilis 168

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

Abstract

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

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.

Figures/Tables 8

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.1 Structure of pullulanase from B. subtilis 168

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

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

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℃

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

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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