Expression and Characterization of a Novel Thermo-Alkalistable Lipase from Hyperthermophilic Bacterium Thermotoga maritima

Abstract

A gene coding for lipase (Tm1350) from the hyperthermophilic bacterium Thermotoga maritima MSB8 was cloned and overexpressed by Escherichia coli. The enzyme can degrade substrates with both short and long acyl chain lengths. The apparent Km and Vmax values for p-nitrophenyl butyrate were 8 mM and 333 U/mg, respectively. The enzyme displayed optimal activity at pH 7.5 and 70 °C, maintained 66 % of the original activity after 8 h of incubation, and its half-lives at pHs 9 and 10 were 8 and 1 h. The activity of Tm1350 was stimulated up to 131 or 151 % of the original activity by incubating with 4 M urea or 20 % (v/v) methanol, and 90.1 or 70.2 % of the activity was maintained after 8 h incubation of the enzyme in 20 or 75 % (v/v) of the methanol, showing potential for biodiesel production. The activity of the enzyme without cysteine residue was stimulated up to 618 and 550 % of the original activity by incubating with dithiothreitol (DTT) and reduced glutathione (GSH) at a concentration of 1 mM. However, the circular dichroism spectra of the enzyme have no obvious change after DTT treatment. It is speculated that DTT interacts with potential residues in some key active sites without influence of structure.

Identification and characterization of a novel alkalistable and salt‐tolerant esterase from the deep‐sea hydrothermal vent of the East Pacific Rise

“…According to previous reports, an alkalistable enzyme may possess more positively charged amino acids, which are acquired to form ion pairs to maintain the stability of the conformation under high‐alkaline conditions (Shirai et al., ). Similar to other alkalistable esterases such as Tm1350 from Thermotoga maritima (13.8%) (Tian et al., ) and EST‐SL3 from Alkalibacterium sp. SL3 (13.7%) (Wang et al., ), est‐OKK has a high proportion (13.8%) of positively charged amino acids.…”

Characterization of a Hyperthermostable Alkaline Lipase fromBacillus sonorensis4R

“…Bacillus sonorensis produced lipase was more active in temperature range of 80–120°C with more than 50% of its original activity remaining above 90°C up to 120°C after 30 min exposure ( Figure 5(b) ). The optimum temperature recorded for the lipase activity of TM12350, a recombinant lipase from a hyperthermophilic bacterium Thermotoga maritima , was 70°C [ 22 ] with maximum activity retained for 60 min at 70°C while maintaining more than its 50% activity within 8 h. At higher temperature the confirmation of enzyme is disrupted which results in reduced affinity sites for substrate [ 23 ]. Hence, in the present study when the temperature was increased from 80 to 120°C, a gradual decrease in catalytic activity of BSL was observed.…”

Characterization of Thermotoga maritima Esterase Capable of Hydrolyzing Bis(2-hydroxyethyl) Terephthalate

“…28 Esterase activity was monitored by the release of p-nitrophenol (pNP) with p-nitrophenyl esters as the substrate, as described previously. 29 The reaction volume was 200 μL, initiated by adding appropriate diluted recombinant TM1022 into 50 mM Tris-HCl buffer (pH 8.0), including 20 μL of 10 mM p-nitrophenyl butyrate (pN−C4) at 70 °C. After cultivation at 70 °C for 1 min, 200 μL of 0.5 M trichloroacetic acid (TCA) was added to the solution to end the reaction, and 200 μL of 0.5 M sodium carbonate was added for color development.…”

“…Esterase activity was monitored by the release of p -nitrophenol ( p NP) with p -nitrophenyl esters as the substrate, as described previously . The reaction volume was 200 μL, initiated by adding appropriate diluted recombinant TM1022 into 50 mM Tris-HCl buffer (pH 8.0), including 20 μL of 10 mM p -nitrophenyl butyrate ( p N–C4) at 70 °C.…”

“…As shown in Figure 4e,f, by analyzing the threedimensional interactions, it was found that BHET formed strong hydrogen-bonding interactions with protein amino acids Ser, 231 His, 30 Tyr, 175 Leu, and 130 Ile, while the hydrophobic functional group formed hydrophobic interactions with protein amino acids 125 Val and 203 Met. MHET exhibited strong hydrogen-bonding interactions with protein amino acids 98 Ser, 30 Tyr, 235 Asn, 164 Trp, and 31 Thr, and the hydrophobic functional group formed hydrophobic interactions with protein 232 Val and 29 Gly. These interactions promoted the binding of the small molecule ligand BHET/MHET to the active pocket of the protein, yielding a complex.…”