A Novel Thermo-Alkaline Stable GDSL/SGNH Esterase with Broad Substrate Specificity from a Deep-Sea Pseudomonas sp

Abstract

Marine environments harbor a plethora of microorganisms that represent a valuable source of new biomolecules of biotechnological interest. In particular, enzymes from marine bacteria exhibit unique properties due to their high catalytic activity under various stressful and fluctuating conditions, such as temperature, pH, and salinity, fluctuations which are common during several industrial processes. In this study, we report a new esterase (EstGoM) from a marine Pseudomonas sp. isolated at a depth of 1000 m in the Gulf of Mexico. Bioinformatic analyses revealed that EstGoM is an autotransporter esterase (type Va) and belongs to the lipolytic family II, forming a new subgroup. The purified recombinant EstGoM, with a molecular mass of 67.4 kDa, showed the highest hydrolytic activity with p-nitrophenyl octanoate (p-NP C8), although it was also active against p-NP C4, C5, C10, and C12. The optimum pH and temperature for EstGoM were 9 and 60 °C, respectively, but it retained more than 50% of its activity over the pH range of 7-11 and temperature range of 10-75 °C. In addition, EstGoM was tolerant of up to 1 M NaCl and resistant to the presence of several metal ions, detergents, and chemical reagents, such as EDTA and β-mercaptoethanol. The enzymatic properties of EstGoM make it a potential candidate for several industrial applications.

Keywords: Pseudomonas; Autotransporter; GDSL/SGNH esterase; Marine bacteria.

Fig. 1

EstGoM is an autotransporter esterase belonging to family II of lipolytic enzymes. a Schematic representation of the type Va secretion system, which is composed of a signal peptide (SP), a passenger domain with specific activity, a linker, and a β-domain that forms a β-barrel that anchors the protein to the bacterial outer membrane. b Phylogenetic tree of EstGoM and homologs belonging to family II of lipolytic enzymes. The analysis was performed using the maximum likelihood tree test in MEGA X software and the amino acid sequences of EstGoM and its close homologs. The red circle shows the position in the phylogenetic tree and the secondary structure of EstGoM

Fig. 2

Modeling of the three-dimensional structure of EstGoM. a Predicted structure of the full-length EstGoM. The β-barrel (yellow) and passenger (blue and green) domains are connected by a linker (red) that resides in the lumen of the translocation domain. b Structure of the EstGoM esterase domain that presents a common α/β SGNH hydrolase fold, which consists of central β-sheets surrounded by α-helices. c The EstGoM linker is modeled as a long α-helix. d Structure of the β-barrel transporter of EstGoM that is composed of 12 antiparallel β-sheets. The EstGoM model was created from the EstA structure of P. aeruginosa (PDB: 3KVN) using SWISS-MODEL (https://swissmodel.expasy.org/)

Fig. 3

Biochemical characterization of purified EstGoM. a Determination of EstGoM substrate specificity. The relative activity was calculated assuming the highest activity observed with p-nitrophenyl caprylate (C8) as 100%. b Determination of the optimum pH for EstGoM activity with p-NP C8. c Optimum temperature for EstGoM activity with p-NP C8. d EstGoM activity in the presence of NaCl. In all panels, the data are the mean values of three independent experiments performed in triplicate. Error bars, standard deviation (SD)

Fig. 4

Effect of metal ions, detergents and chemical reagents on EstGoM activity. a Relative activity of EstGoM in the presence of several cationic metal ions (5 mM). b EstGoM activity in the presence of detergents (0.5%). c Activity of EstGoM in the presence of EDTA and β-mercaptoethanol at 1 and 5 mM. For all panels, the activity of EstGoM was determined with p-NP C8. The data are the mean values of three independent experiments performed in triplicate. Error bars (SD)

Fig. 5

Effects of pH, temperature, and detergents on EstGoM stability. a Thermal stability assays were performed in a range of 10–55 °C. b pH stability of EstGoM at pH values from 5 to 12. c EstGoM stability after incubation with different detergents (0.5%). For all panels, the activity of EstGoM was determined with p-NP C8. The data are the mean values of three independent experiments performed in triplicate. Error bars (SD)