Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction†

doi:10.7503/cjcu20150358

Abstract

Abstract:

Acylaminoacyl peptidase has been named acyl-peptide releasing enzyme(AARE), catalyses the removal of an N-acylated amino acid from N-α-acylpeptides. In this research, a novel acyl-peptide releasing enzyme gene(BSU32230) from Bacillus subtilis 168 was cloned and expressed in Escherichia coli BLP DE3 codon plus to produce acyl-peptide releasing enzyme. The recombinant enzyme was purified in two steps: ammonium sulfate precipitation and Ni²⁺-column affinity chromatography. The optimum temperature and pH of enzyme were 50 ℃ and 8.0, respectively. The half-life of recombinant enzyme at 40 ℃ was 29 h, the enzyme was stable at pH range from 4 to 10. This report demonstrated the acylaminoacyl peptidase from Bacillus subtilis peptidase can catalyze aldol reaction and showed high enantioselectivity. The reaction provided optically active secondary alcohol with satisfying enantioselectivity(84.6% e.e.).

Key words: Acylaminoacyl peptidase, Bacillus subtili, Aldol reaction

CLC Number:

O629.8

TrendMD:

YU Xiaoxiao, WANG Qi, ZHOU Ye, GAO Renjun, WANG Yingwu. Cloning and Application of a New Acylaminoacyl Peptidase from Bacillus subtilis 168 for Aldol Reaction^†[J]. Chem. J. Chinese Universities, 2015, 36(12): 2454.

Figures/Tables 8

Scheme 1 Aldol reaction catalyzed by BSU32230

Fig.1 Sequcence aligment of prolyl oligopeptidases

Fig.2 Substrate specificity of purified BSU32230 against different p-nitrophenyl esters(A) and various short peptides(B) (A) a. pNPC2; b. pNPC3; c. pNPC4; d. pNPC8; e. pNPC10; f. pNPC12; g. pNPC16.(B) a. Ala2; b. (D-Ala)2; c. Ala-phe; d. Asp-Ala; e. Ala3; f. Ac-Ala3.

Table 1 Purification of BSU32230

Sample	Total protein/mg	Total activity/U	Specific activity/(U·mg^-1)	Purification fold	Yield(%)
Crude extract	198.00	84.26	0.43	1	100
Ammonium sulfate	78.00	52.61	0.67	1.59	62.44
Ni²⁺ affinity column	4.48	30.73	6.86	16.14	36.47

Fig.3 Effects of temperature(A) and pH(B) on BSU32230 activity

Fig.4 Thermostability(A) and pH stability(B) of BSU32230 pH=4.0—8.0, 50 mmol/L Na2HPO4-NaH2PO4, ■ 1 h, ◆ 24 h; pH=7.0~10.0, 50 mmol/L Tris-HCl, ● 1 h, ▲ 24 h.

Fig.5 Effect of temperature on the asymmetric aldol addition of Acetone and 4-nitrobenzaldehyde catalyzed by BSU32230

Table 2 Comparison of Acyl-peptide releasing enzyme(BSU32230) and Porcine Pancreatic Lipase(PPL) catalyzed aldol additions between 4-nitrobenzaldehyde and Acetone

Enzyme	Temperature/℃	Time/h	Yield(%)	e.e.(%)
BSU32230	50	24	30.9	84.6
PPL	37	24	24.6	82.1
Denatured BSU32230	50	24	<0.5	—
Denatured PPL	37	24	<0.5	—
Control/No enzyme	50	24	<0.5	—

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