Stabilisation of the Fatty Acid Decarboxylase from Chlorella variabilis by Caprylic Acid

Abstract The fatty acid photodecarboxylase from Chlorella variabilis NC64 A (CvFAP) catalyses the light‐dependent decarboxylation of fatty acids. Photoinactivation of CvFAP still represents one of the major limitations of this interesting enzyme en route to practical application. In this study we demonstrate that the photostability of CvFAP can easily be improved by the administration of medium‐chain length carboxylic acids such as caprylic acid indicating that the best way of maintaining CvFAP stability is ‘to keep the enzyme busy’.


Protein expression and purification
The E. coli BL21(DE3) cells containing the plasmid pET-28a(+) expressing CvFAP were cultivated in terrific broth (TB) medium containing 50 g mL -1 kanamycin at 37 °C and 180 rpm. When the optical density at 600 nm (OD600) reached 0.7-0.8, protein induction was initiated by adding 0.5 mM IPTG and cultivation temperature was decreased to 17 °C. After cultivation for about 20 h, cells were harvested by centrifugation (11000 × g at 4 °C for 10 min), washed twice with ice-cooled buffer A (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0). The cell pellet was resuspended in the same buffer containing 1 mM PMSF and 1 mM MgCl2 and then cells were lysed by passing them through a Multi Shot Cell Disruption System at 1.5 kbar. The lysates were centrifuged at 38000 × g at 4 °C for 1 h and then passed through a filter of 0.45 m to remove the particulate fraction. The purification was made on a His Trap Ni-NTA FF column (5 mL, GE Healthcare). After loading the lysate, the column was washed by 20 vol % buffer B (50 mM Tris-HCl, 300 mM NaCl, 200 mM imidazole, 10% glycerol, pH 8.0) and protein was then eluted by a step gradient using 40 vol% buffer B. The fractions were determined by SDS-PAGE and concentrated by ultrafiltration (50 kDa filters). The Ni-NTA column purification was performed by NGC system in the 10 °C fridge covered by aluminum foil. The concentrated purified protein was loaded on the desalting column (6 mL, PD10) to remove imidazole. Protein was eluted by buffer (100 mM Tris-HCl, pH 8.5). The yellow fractions were collected and protein concentration used for activity assay corresponded the protein containing FAD. FAD was quantified by measuring absorbance at 450 nm on the protein which was previously heated at 95 °C for 5 min in the addition of 1 w/w % SDS.

Activity assay
The activity of enzyme CvFAP was assayed at 37 °C by monitoring the increase of pentadecane in a 1-mL reaction by using the gas chromatography (Shimadzu GC-2014) equipped with the column CP Sil 5 CB (50 m × 0.53 mm × 1.0 µm), using flame ionization detection (FID), and N2 as the carrier gas. The standard assay mixture was composed of 13 mM palmitic acid as substrate, 30 vol % DMSO as cosolvent, buffer (100 mM Tris-HCl, pH 8.5), and CvFAP with an appropriate concentration and was under gentle magnetic stirring at 37 °C under the illumination of blue light (light intensity=14.5 E L -1 s -1 ) for 30 min. S3

Thermal stability assay
The residual activity of CFE CvFAP and purified CvFAP were determined by incubating the enzyme (18 M) in buffer (100 mM Tris-HCl, pH 8.5) at different temperature protected from light for a proper time and then performed the activity assay. The activity of CvFAP without any incubation before the activity assay was defined as 100% residual activity.

Photostability assay
The residual activity of CFE CvFAP and purified CvFAP were determined by incubating the enzyme CvFAP (18 M) in buffer (100 mM Tris-HCl, pH 8.5) at 30 °C under the illumination of LEDs for a proper time and then performed the activity assay. The activity of CvFAP without any incubation or pre-illumination before the activity assay was defined as 100% residual activity. The half-lives (t1/2) of enzyme under corresponding conditions were calculated according to the deactivation function: ln(residual activity)=-kD/t; t1/2=ln2/kD. kD here represents the deactivation rate constant.

Photoenzymatic decarboxylation of palmitic acid to pentadecane
The 1 mL reaction was composed of 13 mM palmitic acid as substrate, 30 vol % DMSO as cosolvent, 3 M purified CvFAP, and buffer (100 mM Tris-HCl, pH 8.5) and was under gentle magnetic stirring at 30 °C under the illumination of blue light (light intensity=14.5 E L -1 s -1 ). Samples were withdrawn and extracted with ethyl acetate (containing 5 mM 1-octanol) for gas chromatography analysis.

Light intensity measurement
The light intensity was determined by means of ferrioxalate actinometry. 1 mL ferrioxalate solution (37.5 mM in 50 mM H2SO4) in a 4-mL transparent glass vial was illuminated under LED light. At defined intervals, 25 µL samples of the illuminated solution were taken and mixed with 175 µL of another solution (7.5 mL 50 mM H2SO4, 2 mL 0.1% 1,10-phenantroline, 5 mL 1 M sodium acetate solution and 3 mL H2O). The absorbance of the mixture was measured under 510 nm at room temperature. FeSO4 was used as Fe(II) for the calibration curve. The light intensity was then calculated based on the Fe(II) generation rate in the irradiated ferrioxalate solution. S4

GC analyses
The enzyme activity and time course of decarboxylation reaction catalyzed by CvFAP were measured by monitoring the production of pentadecane by using the gas chromatography (Shimadzu GC-2014) with FID, equipped with column CP Sil 5 CB (50 m × 0.53 mm × 1.0 µm), 20 mL/min N2 as the carrier gas. The injection temperature was 340 °C.  3.2 Influence of temperature on purified CvFAP-catalysed decarboxylation reaction of palmitic acid to pentadecane  3.5 Photoreaction setup. Figure S5. The homemade photoreactor setup employed in this study.