Thermal Inactivation of a Cold-Active Esterase PMGL3 Isolated from the Permafrost Metagenomic Library

Abstract

PMGL3 is a cold-adapted esterase which was recently isolated from the permafrost metagenomic library. It exhibits maximum activity at 30 °C and low stability at elevated temperatures (40 °C and higher). Sequence alignment has revealed that PMGL3 is a member of the hormone-sensitive lipase (HSL) family. In this work, we demonstrated that incubation at 40 °C led to the inactivation of the enzyme (t1/2 = 36 min), which was accompanied by the formation of tetramers and higher molecular weight aggregates. In order to increase the thermal stability of PMGL3, its two cysteines Cys49 and Cys207 were substituted by the hydrophobic residues, which are found at the corresponding positions of thermostable esterases from the HSL family. One of the obtained mutants, C207F, possessed improved stability at 40 °C (t1/2 = 169 min) and increased surface hydrophobicity, whereas C49V was less stable in comparison with the wild type PMGL3. Both mutants exhibited reduced values of Vmax and kcat, while C207F demonstrated increased affinity to the substrate, and improved catalytic efficiency.

A Broad Temperature Active Lipase Purified From a Psychrotrophic Bacterium of Sikkim Himalaya With Potential Application in Detergent Formulation

“…This property can be valuable in many industrial processes which require low to high temperature. Most of the cold-active lipases isolated and purified till now doesn't show activity at a higher temperature (Joseph et al, 2012 ; Kryukova et al, 2019 ; Li et al, 2019 ; Salwoom et al, 2019a ). The lipase isolated in the current study is remarkably active in a broad temperature range of 5–65°C and could be a valuable asset in many industrial processes like in detergent formulations.…”

Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure

“…DNA manipulations were performed by standard techniques using enzymes from Thermo Fisher Scientific. Expression and purification of the wild type PMGL3 was described previously [ 16 , 17 ]. The mutant genes were constructed by two-step Splicing by Overlap Extension (SOE)-PCR using Pfu DNA polymerase and primers listed in Table S1 .…”

“…The resulting products were cloned into the pET32a vector and mutations were confirmed by DNA sequencing (Evrogen). The mutant proteins were obtained as described for the wild type PMGL3 [ 17 ].…”

“…Esterase activity assay was conducted at 30 °C as described previously [ 17 ] using 0.25 mM p -nitrophenyl butyrate ( p -NPB) as a substrate in 50 mM Tris-HCl pH 8.0, 100 mM NaCl. Kinetic parameters of the reaction were determined from Michaelis-Menten equation using p -NPB substrate concentrations ranging from 0.2 to 2 mM.…”

“…The PMGL3 esterase demonstrated low thermal stability with rapid inactivation upon incubation at 40 °C, which is typical for the cold-active enzymes. In particular, the enzyme was shown to possess a dimeric state in solution, with a tendency to oligomerize upon heating [ 17 ].…”