In this work, the lipase from
One of the main limitations for the use of enzymes in industrial processes is their low stability under operational conditions (e.g., high temperatures, organic solvents, and extremes of pH). Enzymes from thermophilic and hyperthermophilic microorganisms are inherently more stable towards a variety of enzyme denaturants and thus represent promising alternatives for the development of industrial biocatalytic processes [
Carboxylesterases (E.C. 3.1.1.1) and lipases (E.C. 3.1.1.3) are enzymes that catalyze the hydrolysis of ester bonds involving a carboxylic acid of variable chain length, that is, from C2 to C18 or more. In organic media, these two classes of enzymes also possess the ability to catalyze several other types of biotransformations such as esterification, transesterification, alcoholysis, aminolysis, and acidolysis [
A number of lipases and esterases from extremophilic organisms have been cloned in
In this work, we investigated the effects of the TRX tag on the lipase activity of the purified enzyme. To this end, the purification of the recombinant
Calf intestine enterokinase was purchased from Roche (Nutley, NJ, USA); Isopropyl-
The
A Hitrap Chelating resin (bed volume 1 mL) (Amersham Biosciences/GE Healthcare) was washed with 10 mL of sterile water and charged with Ni2+ using 5 mL of a 100 mM NiSO4 solution. After a second wash with 10 mL of sterile water, the charged resin was equilibrated with 10 mL of sodium phosphate buffer (50 mM, pH 7.0). Eight milliliters of the clarified supernatant of the cell lysate was applied directly to the column. A washing step with 100 mM imidazole in equilibration buffer was done in order to elute nonspecific ligands. Elution of the TRX-PF2001Δ60 was carried out with 150 mM imidazole in the equilibration buffer. Fractions of 1 mL were collected at a flow rate of 0.2 mL/min and monitored by their absorbance at 280 nm.
Twenty-five micrograms of purified enzyme were dialyzed against Tris-Cl buffer (50 mM, pH 7.5). The dialyzed enzyme was treated with 0.6
Nonreducing gel electrophoresis (SDS-PAGE) was carried out with a 10% separating gel on a vertical slab minigel apparatus (Bio-Rad) at 120 V for 1 h [
After the run, the gels were soaked for 30 min in 2.5% Triton X-100 at room temperature, briefly washed in 50 mM sodium phosphate buffer, pH 7.0, and covered by a solution of 100
Enzyme assays were carried out using 4-methylumbelliferone (MUF) derivatives as substrates (MUF-Ace, MUF-Hep and MUF-Pal) in a Varian Cary Eclipse spectrofluorimeter, as described elsewhere [
For optimum temperature determination, the reaction was assayed using MUF-Hep as substrate at 50, 60, 70, and 80°C with 50 mM phosphate buffer pH 7.0.
For optimum pH determination, the reactions were performed at 70°C with 100 mM BIS-TRIS-propane buffer at pH 6, 7, 8, and 9.
The enzymes TRX-PF2001Δ60 and PF2001Δ60, in sodium phosphate buffer (50 mM pH 7.0) containing 0.1% gum arabic and 0.4% Triton X-100, were preincubated for different times at 55, 75, and 95°C. Tubes were removed periodically and assayed for lipase activity using MUF-Hep at 70°C. The Triton X-100 influence on thermostability was determined for PF2001Δ60 at 70°C with and without 0.4% Triton X-100.
The substrate preferences of TRX-PF2001Δ60 and PF2001Δ60 were determined using 100
Lipase activity was analyzed using MINITAB v.14 package. The values expressed were based on the average of triplicate experiments. Experimental errors were between 5 and 10%, and averages were compared using
In a previous work, we reported the identification and cloning of a novel lipase from
The TRX-PF2001Δ60 purification results are summarized in Table
Summary of the purification of the recombinant TRX-PF2001Δ60 from
Total protein (mg) | Total activity (U) | Specific activity (U/mg) | Yield (%) | Purification fold | |
---|---|---|---|---|---|
Soluble Ext. | 16.9 | 22.5 | 1.34 | 100 | — |
Ni-NTA | 2.28 | 20.4 | 8.9 | 89.8 | 6.63 |
SDS-PAGE (a) and zymography (b), on a 15% polyacrylamide gel, of expressed and purified protein encoded by the PF2001Δ60 gene. (1) soluble extract; (2) flow-through; (3) recombinant lipase eluted with 150 mM imidazole; (4) lipase treated with enterokinase to remove the thioredoxin tag.
The purified enzyme was cleaved with enterokinase, and the hydrolyzed product showed two protein bands (about 48.8 and 26.5 kDa, representative of TRX-PF2001Δ60 and PF2001Δ60, resp.) in the SDS-PAGE analysis (line 4, Figure
The purified enzyme was stable at −20°C for several months (data not shown).
The activities of TRX-PF2001Δ60 and PF2001Δ60 increased linearly from 50 to 70°C. At 80°C, the measured activities were higher than at 70°C, but the slope of the curve over this range was less steep than between 50 and 70°C. Among the temperatures tested, the temperature at which both enzymes show maximum activity was 80°C. This temperature was considered as the optimal temperature for both enzymes (Figure
(a) Effects of temperature on lipase activity. The purified lipases, TRX-PF2001Δ60 and PF2001Δ60, were assayed at temperatures ranging from 50 to 80°C, in 50 mM phosphate buffer, pH 7.0. (b) Effects of pH on lipase activity. The purified lipases, TRX-PF2001Δ60 and PF2001Δ60, were assayed at pH ranging from 5.0 to 9.0, in 100 mM BIS-TRIS-propane buffer at 70°C. All assays were carried out without Triton X-100.
At 50°C, TRX-PF2001Δ60 and PF2001Δ60 exhibited the same level of activity (3 U/mg), but the difference between them increases with temperature. At 80°C, the enzyme without the thioredoxin tag is twice as active as the enzyme with the thioredoxin tag.
Thioredoxin is a peptide of 11.7 kDa, responsible for 24% of the total molecular weight of the recombinant fusion protein. Although this tag is considered a thermostable protein [
The enzymes TRX-PF2001Δ60 and PF2001Δ60 showed optimum activity in pH 7.0. The enzyme with and without the thioredoxin tag demonstrated 73 and 90% of its maximum activity, respectively, at pH 8.0 (Figure
The resistance to heating in the presence of Triton X-100 was investigated at the temperatures of 55, 75, and 95°C and showed that the enzyme is endowed with high thermostability. Around 100% activity was recovered after 6 h pre-incubation at 55 and 75°C. However, less than 50% activity was retained after 10 min at 95°C. No differences were observed between PF2001Δ60 and TRX-PF2001Δ60 (Figure
Thermal stability of the purified enzyme, with (a) and without (b) the thioredoxin tag. The enzyme samples in phosphate buffer (50 mM pH 7.0) containing 0.1% gum arabic and 0.4% Triton X-100 were incubated at the indicated temperatures for 5 min, 30 min, 2, and 6 h. The residual enzyme activity was assayed at 70°C, pH 7.0, using the substrate MUF-Hep.
Among the different substrates tested, the enzyme was more active towards MUF-Hep. The activities towards MUF-Ace and MUF-Pal were, respectively, equivalent to 20.8 and 6.4% of the activity towards MUF-Hep.
In order to define the ion and inhibitor effects on the PF2001Δ60, enzyme the activity assays were performed in the presence of the above-mentioned substrates.
The influence of mono- and divalent metal ions was studied at a concentration of 10 mM. K+ did not affect the activity of the enzyme significantly, but significant activity loss was observed with Mg2+ and Ca2+ (20 and 45%, resp.). This suggests that PF2001Δ60 enzyme is not a metalloenzyme. This was reinforced by a significantly higher activity of the enzyme in the presence of 10 mM of metal chelators: EDTA (activity increased by 96%) and EGTA (69%) (Table
Effects of metal ions, chemicals, and detergents on PF2001Δ60 lipase activity. The enzyme (0.5
Chemical | Concentration (mM) | Relative activity (%) |
---|---|---|
KCl | 10.0 | 107.5 |
MgCl2 | 10.0 | 77.9 |
CaCl2 | 10.0 | 54.8 |
SDS | 1.0 | 37.0 |
DTT | 1.0 | 94.6 |
Triton X-100 | 6.4 | 47.0 |
EDTA | 1.0 | 97.8 |
10.0 | 195.7 | |
EGTA | 1.0 | 103.4 |
10.0 | 169.0 | |
PMSF | 1.0 | 0.0 |
PF2001Δ60 retained full activity in the presence of the disulfide bond reducing agent DTT. The enzymatic stability against DTT suggests that the Cys166 and Cys167 are not disulfide bonded, or that this covalent bond does not affect the protein structure.
In the presence of the detergents: SDS (1mM) and Triton X-100 (0.4%–0.6 mM), the activity decreased by 63% and 53%, respectively. Similar inhibitory effects on microbial lipases and esterases activity have been observed by other groups [
Because Triton X-100 has traditionally been used in a concentration of 0.4% as an emulsifier to measure lipase and esterase activities, its effects were further investigated.
Figure
(a) Effect of Triton X-100 on optimal temperature. The lipase PF2001Δ60 was assayed at temperatures ranging from 50 to 80°C, in 50 mM phosphate buffer, pH 7.0, in the presence and absence of Triton X-100. (b) Effects of Triton X-100 on thermal stability of the PF2001Δ60. The enzyme samples in phosphate buffer (50 mM pH 7.0) containing 0.1% gum arabic were incubated at 70°C for 85 min and in the same conditions with 0.4% Triton X-100. The residual enzyme activity was assayed at 70°C, pH 7.0, using the substrate MUF-Hep.
The Triton X-100 cloud point temperature is probably one of the main reasons for shift in optimal temperature of the enzyme. At 60°C, most non-ionic surfactants in aqueous solutions form micelles and become turbid when heated. Above this temperature, the micellar solution separates into a surfactant-rich phase, in which the surfactant concentration is close to the critical micellar concentration.
Although the presence of Triton X-100 affects the activity of the enzyme at higher temperatures, the thermostability was higher when the enzyme was incubated with the detergent. At 70°C, 89% of enzyme activity was recovered after 85 min of incubation in the presence of Triton, but only 40% was recovered when Triton X-100 was not used in the incubation mixture (Figure
In spite of its high optimal temperature and thermostability, it is curious to observe the rapid denaturation of PF2001Δ60 at 95°C with or without Triton X-100, a temperature close to the optimal growth temperature of
The purified recombinant lipase exhibits its highest activity at 80°C—one of the highest temperatures described for a lipase with medium chain length substrate preference. Furthermore, the TRX tag did not influence the optimal pH and optimal temperature of the enzyme; however, the temperature profile was influenced by the TRX tag, reducing its specific activity at 80°C by 50%.
In addition, we show here that Triton X-100, a commonly used detergent in lipase essays, influenced the enzymatic performance; it shifted the optimal temperature to 60°C, diminished the enzyme activity by 50, and stabilized the enzyme towards the temperature.
CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior), FAPERJ (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro), and PETROBRAS supported this work. The authors thank Dr. Welington Almeida and Dr. Martha Sorenson for discussion and comments on the paper.