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

BMC Biotechnology
Huang JiangNan Hu

Abstract

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. 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 ...Continue Reading

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Citations

Sep 27, 2016·Frontiers in Microbiology·Margarita SantiagoLoreto P Parra
Nov 5, 2016·International Journal of Biological Macromolecules·Yian HangZiduo Liu
May 26, 2017·Evolution; International Journal of Organic Evolution·Scott R Miller
Nov 4, 2017·Preparative Biochemistry & Biotechnology·Zhen RongXue-Wei Xu
Nov 28, 2018·Archives of Microbiology·Amit S DhaulaniyaDileep Kumar Singh
Oct 1, 2020·Journal of Food Biochemistry·Taiwo O AkanbiDominic Agyei

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Datasets Mentioned

BETA
AGQ31273.1
ATCC
27592
CP006252.1
KU362566

Methods Mentioned

BETA
circular dichroism
PCR

Software Mentioned

BLASTP
SYBYL
CDSSTR
SELCON3
CONTIN
MOE
GenScript
Jasco Standard Analysis
Graphpad Prism
LL

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