Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution

Abstract

Background: In recent years, cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures.

Results: An esterase-encoding gene (estS, 909 bp) from Serratia sp. was identified, cloned and expressed in Escherichia coli DE3 (BL21). The estS encoded a protein (EstS) of 302 amino acids with a predicted molecular weight of 32.5 kDa. It showed the highest activity at 10 °C and pH 8.5. EstS was cold active and retained ~92 % of its original activity at 0 °C. Thermal inactivation analysis showed that the T1/2 value of EstS was 50 min at 50 °C (residual activity 41.23 %) after 1 h incubation. EstS is also quite stable in high salt conditions and displayed better catalytic activity in the presence of 4 M NaCl. To improve the thermo-stability of EstS, variants of estS gene were created by error-prone PCR. A mutant 1-D5 (A43V, R116W, D147N) that showed higher thermo-stability than its wild type predecessor was selected. 1-D5 showed enhanced T1/2 of 70 min at 50 °C and retained 63.29 % of activity after incubation at 50 °C for 60 min, which were about 22 % higher than the wild type (WT). CD spectrum showed that the secondary structure of WT and 1-D5 are more or less similar, but an increase in β-sheets was recorded, which enhanced the thermostability of mutant protein.

Conclusion: EstS was a novel cold-active and salt-tolerant esterase and half-life of mutant 1-D5 was enhanced by 1.4 times compared with WT. The features of EstS are interesting and can be exploited for commercial applications. The results have also provided useful information about the structure and function of Est protein.

Fig. 1

Multiple alignments of EstS and other four esterases. The four esterases are Est2 [PDB: 1EVQ_A] from Alicyclobacillus Acidocaldarius, PestE [PDB: 2YH2_A] from Pyrobaculum calidifontis, Este1 [PDB: 2C7B_A] from a metagenomic library and Lpest1 [PDB: 4C88_A] from Lactobacillus Plantarum. The identical and conserved residues are shaded. The conserved G–X–S–X–G motif and the catalytic triad (Ser, Asp, and His) were indicated by red box and black triangle, respectively

Fig. 2

SDS-PAGE analysis of purified EstS protein. M: Protein molecular weight marker; 1: Uninduced cell lysate of E. coli BL21 (DE3) harboring pGEX-6P-1; 2: IPTG-induced of cell lysate of E. coli BL21 (DE3) harboring pGEX-6P-1; 3: Uninduced cell lysate of E. coli BL21 (DE3) harboring pGEX-6P-estS; 4: IPTG-induced of cell lysate of E. coli BL21 (DE3) harboring pGEX-6P-estS; 5: Purified EstS. The protein GST-EstS is indicated by arrow

Fig. 3

Substrate specificity of the purified EstS. The esterase activity of EstS was tested with various chain lengths of p-NP esters (C2, C4, C6, C8, C12 and C16) in 50 mM Tris – HCl, pH 8.5, at 30 °C. The activity against p-NP acetate (C2) was taken as 100 %. All measurements were performed in triplicate

Fig. 4

Effect of temperature and pH on enzyme activity and stability of WT and mutant. a The effect of temperature on enzyme activity. The temperature-activity profile was measured at a temperature range of 0 to 80 °C in 50 mM Tris–HCl buffer (pH 8.5). Activity value obtained at 10 °C was defined as 100 %. b Temperature stability. The WT and mutant enzyme was incubated at 45 (○ WT; ● 1-D5), 50 (□ WT; ■ 1-D5) and 55 °C (▼ WT; ▲ 1-D5) for various time intervals and the residual activity was measured. The specific activity without incubation was taken as 100 %. c The effect of pH on enzyme activity. The pH-activity profile was determined in phosphate–citrate buffer (pH 5.0–7.0) and 50 mM Tris–HCl buffer (pH 7.0–10.0) at 10 °C. The activity at pH 8.5 was defined as 100 %. d pH stability. The activity was determined by pre-incubating enzyme solutions in different pHs buffers at 4 °C for 24 h and the residual activity was measured under standard condition. The residual activity after treatment with pH 7.0 buffer was shown as 100 %

Fig. 5

Effects of NaCl on activity and stability of EstS. The enzyme activity (●) was assayed in 50 mM Tris–HCl buffer (pH 8.5) containing 0–4 M NaCl and the residual activity (■) of EstS was measured after incubating with 0–4 M NaCl (pH 8.5) at 4 °C for 24 h

Fig. 6

Three-dimensional model of 1-D5. The catalytic sites and substitution sites were displayed with stick-ball model

Fig. 7

The surface electrostatic potential of EstS. The most negative and most positive electrostatic potentials are indicated by purple and red, respectively. The right image is the 180° rotated view of the left one

Fig. 8

CD spectra of the EstS and 1-D5 in the far-UV spectral region (195–250 nm). CD spectra of Est WT and 1-D5 was more or less similar and an increase in the percentage of β-sheets was reported by CD analysis