Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

Abstract

The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5U/mL was observed at 30°C and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150rpm. The optimized conditions from the shake flask experiments were validated in a 3L lab scale bioreactor, and the lipase production increased to 48U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50°C and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98U/mg, 0.51mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Keywords: Acinetobacter sp.; MALDI-TOF; Organic solvent tolerant lipase; Response surface methodology; Thermostable lipase.

Supplemental Fig. S1

Effect of different oils on the Acinetobacter sp. AU07 lipase activity. The enzyme production was carried out with different oils as inducers, viz. castor oil, palm oil, coconut oil, sesame oil, and olive oil, at (1%, v/v) with a 1% inoculum size at 30 °C for 20 h in a rotary shaker (150 rpm). The media containing oils were emulsified with 0.25% gum acacia. The initial pH of the media was adjusted to 7.0. To the individual effects of pH, temperature and inducers were monitored and optimized. The cell-free supernatant was recovered by centrifugation at 12,500 × g for 10 min at 4 °C and used to determine the extracellular lipase activity.

Supplemental Fig. S2

2D contour plots of the optimization of lipase production by Acinetobacter sp. AU07. Each figure depicts the impact of specific factors on lipase production. The variables investigated were temperature, pH, agitation, inducer concentration, and inoculum volume. (A) Temperature and pH, (B) temperature and agitation, (C) temperature and inducer, (D) temperature and inoculum volume, (E) pH and agitation, (F) pH and inducer, (G) pH and inoculum volume, (H) agitation and inducer, (I) agitation and inoculum volume, and (J) inducer and inoculum volume.

Supplemental Fig. S3

RP-HPLC analysis of the purified Acinetobacter sp. AU07 lipase. The column was eluted with the following buffers: 0.1% (v/v) trifluroacetic acid (TFA) in water and 0.1% (v/v) TFA in acetonitrile. The bound proteins were eluted with an increasing gradient of 2–100% acetonitrile for 40 min at a flow rate of 1 mL/min.

Supplemental Fig. S4

MALDI-TOF analysis of observed mass, experimentally determined mass, calculated mass and corresponding peptide sequence.

Supplemental Fig. S5

CLUSTAL 2.1 multiple sequence alignment. Ser 175, Asp 317, His 348 → catalytic triad; the lipases are from A. calcoaceticus PHEA-2, A. baumanii, A. oleivorans DR1, A. radioresistens WC-A-157, A. lwoffii SH145, Rhodococcus sp. EsD8, and the active site consensus sequence, ‘GXSXG’, is conserved in all the sequences.

Supplemental Fig. S6

Enzyme kinetics analysis – determination of K_m and V_max values of enzyme by Lineweaver–Burk and Woolf–Hanes plots for different substrates.

Fig. 1

Response surface plots of the impact of various factors on optimal production of the AU07 lipase. Each figure depicts the impact of specific factors on lipase production. The variables investigated were temperature, pH, agitation, inducer concentration, and inoculum volume. (A) Temperature and pH, (B) temperature and agitation, (C) temperature and inducer, (D) temperature and inoculum volume, (E) pH and agitation, (F) pH and inducer, (G) pH and inoculum volume, (H) agitation and inducer, (I) agitation and inoculum volume, and (J) inducer and inoculum volume.

Fig. 2

Growth curve and lipase production from Acinetobacter sp. AU07 in shake flask and bioreactor. (A) Growth curve and lipase production from Acinetobacter sp. AU07 in a shake flask (30 °C, pH 7.0, 0.5%, v/v inoculum size, 2%, v/v inducer concentration, 150 rpm). The lipase activity was measured using 1 mM 4-NP as the substrate. The reaction was incubated at 30 °C for 10 min, and the absorbance was measured at 410 nm. The OD was converted to lipase activity (U/mL) and the graph was plotted. Each data point is the mean of three replicates. (B) Growth curve and production of lipase from Acinetobacter sp. AU07 in a bioreactor (3 L) (30 °C, pH 7.0, 0.5%, v/v inoculum size, 2%, v/v inducer concentration, 150 rpm, 1.5 vvm aeration). The lipase activity was measured using 4-NP (1 mM) as the substrate. The reaction was incubated at 30 °C for 10 min and OD was measured at 410 nm. The OD was converted to lipase activity (U/mL) and the graph was plotted. Each data point is the mean of three replicates.

Fig. 3

SDS-PAGE (10%) of the purified lipase from Acinetobacter sp. AU07. Lane A: Molecular mass marker proteins: phosphorylase β (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and α-lactalbumin (14 kDa). Lane B: Purified lipase (10 μg) after ion-exchange chromatography showing single band.

Fig. 4

Partial amino acid sequence of the isolated lipase showing homology with Acinetobacter calcoaceticus (55% identity).

Fig. 5

(A) Effect of temperature on the activity of Acinetobacter sp. AU07. The reaction mixture was incubated at different temperatures (30–80 °C), and the enzyme activity was assayed under standard assay conditions. (B) Effect of temperature on the stability of the Acinetobacter sp. AU07 lipase. To determine the thermal stability, the enzyme was pre-incubated at various temperatures (40–70 °C) for 5 h, and the enzyme activity was assayed under standard assay conditions. (C) Effect of pH on the activity and stability of the Acinetobacter sp. AU07 lipase. For the enzyme activity assays, the reaction was assayed at various pH levels under standard assay conditions. For the enzyme stability assays, the enzyme was pre-incubated with various pH buffers (50 mM) of pH (5–11) at 30 °C for 1 h, and then the residual activity was determined by assaying under standard assay conditions.

Fig. 6

Effect of inhibitors on Acinetobacter sp. AU07 lipase activity. The enzyme was incubated with different inhibitors at 30 °C for 1 h, and the enzyme activity was assayed under standard assay procedure conditions. The enzyme incubated without inhibitors was used as a reference to calculate the relative activity. Each value presented here is an average of three replicates.