Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.

Abstract

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.

Identification and Characterization of a Novel Thermostable GDSL-Type Lipase from Geobacillus thermocatenulatus

“…While the SGNH hydrolases are well known in eukaryotic organisms, the isolation and characterization of SGNH hydrolases from bacteria remain to be limited [7]. In bacteria, GDSL motif enzymes are generally known as esterase type which has preference to short chain fatty acids [8][9][10][11]. To date, there have been few reports of GDSL family lipases in bacteria [12,13].…”

Biodiesel and flavor compound production using a novel promiscuous cold-adapted SGNH-type lipase (HaSGNH1) from the psychrophilic bacterium Halocynthiibacter arcticus

“…As shown in Fig. 5a, the optimal temperature of HaS-GNH1 was about 20 °C, which is comparable to estSL3 [11], slightly lower than estS9 [29] and EstA [30], and higher than a GDSL family esterase from Photobacterium sp. J15 [13].…”

“…In addition, HaSGNH1 exhibited high relative activities at low temperatures, retaining ~ 70% of its maximum activity even at 0 °C. The value is higher than other cold-adapted SGNH-type lipases like estSL3 [11], estS9 [29], or EstA [30]. HaSGNH1 thermostability was investigated over a temperature range from 15 to 100 °C ( Fig.…”

Peer Review #2 of "Biochemical characterization and inhibition of thermolabile hemolysin from Vibrio parahaemolyticus by phenolic compounds (v0.1)"

“…Meanwhile, Wicka et al 2016, reported similar substrate affinity and k cat for cold-adapted GDSL-lipase from Pseudomonas sp. S9 using p-nitrophenyl butyrate which has short carbon chain in fatty acids substituent than PNPL (Wicka et al 2016).…”