Abstract
BackgroundIn 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.ResultsAn 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.ConclusionEstS 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.
Highlights
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
Our previous studies confirmed that a cold active esterase (Est11), produced from Psychrobacter pacificensis, was salt tolerant, highly active at low temperatures, and its biochemical characteristics made it important for commercial applications [17]
A multiple sequence alignment of EstS was performed with thermophilic carboxylesterase Est2 [PDB: 1EVQ_A] from Alicyclobacillus acidocaldarius, esterase PestE [PDB: 2YH2_A] from Pyrobaculum calidifontis (41 %), carboxylesterase Este1 [PDB: 2C7B_A] from a metagenomic library (40 %), esterase Lpest1 [PDB: 4C88_A] from Lactobacillus plantarum (29 %), and all of them were retrieved using BLASTP in the NCBI and PDB database (Fig. 1)
Summary
Cold-active esterases have received increased attention due to their attractive properties for some industrial applications such as high catalytic activity at low temperatures. Lipolytic enzymes produced from psychrophilic microorganisms in cold environment could be active and stable at low temperatures as compared to mesophilic enzymes. Esterases have gained immense importance in pharmaceutical, polymer, food, flavor, oleochemical, biofuel and detergent industries [2]. The high catalytic activity of these lipolytic enzymes at. Our previous studies confirmed that a cold active esterase (Est11), produced from Psychrobacter pacificensis, was salt tolerant, highly active at low temperatures, and its biochemical characteristics made it important for commercial applications [17]. Few reports of esterase from Serratia sp. Few reports of esterase from Serratia sp. are available in literature
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