Strains and chemicals
A. niger GZUF36 was isolated in our laboratory and deposited at the China Center for Type Culture Collection (CCTCC) under the CCTCC Preservation No. M2012538. Polyvinyl alcohol (PVA) and tert-butanol were purchased from Tianjin Kemiou Chemical Reagent Co. (Tianjin, China). GA (50%) and oleic acid were purchased from Chengdu Jinshan Chemical Reagent Co. (Chengdu, Sichuan, China). Anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Reagent Co. (Tianjin, China). Acetone was purchased from Chuandong Chemical Group Co. (Chongqing, China). TLC-grade porcine pancreatic lipase (PPL, 1000 U/g), triolein, 1,3-diglycedide, and 1,2-diglycedide were procured from Sigma-Aldrich Co. (USA). Olive oil was acquired from Sinopharm Chemical Reagent Co. (Shanghai, China). All other chemical reagents were of analytical grade and commercially available.
Production of EXANL1
Two-ring spores of A. niger GZUF36 were inoculated into the fermentation medium and incubated for 60 h at 30 °C at 180 rpm. Then the mycelium was filtered and the fermentation broth was centrifuged at 4000 g and 4 °C for 15 min to obtain the crude EXANL1 solution.
Purification of EXANL1
The crude EXANL1 was purified by reverse micelle extraction combined with acetone precipitation. 250 mL of acetone was added into 100 mL crude enzyme solution at –18 °C for 3 h. The mixture was then centrifuged at 6000 g and 4 °C for 15 min. The forward extraction and backward extraction were referred to our previous work [1].
Synthesis of CLEA-EXANL1
CLEA-EXANL1 was synthesized in accordance with the method described by Rehman et al. [38] with some modifications. CLEA-EXANL1 was prepared by adding some amounts of precipitants and cross-linker GA into 10 mL of purified enzyme solution. The mixture was agitated at 200 rpm and 25 °C for some time and then centrifuged at 4 °C for 15 min at 6000 g to collect the aggregates. And the effect of precipitants (acetone, saturated ammonium sulfate, tert-butanol, PVA, and a mixture of tert-butanol and acetone), volumes (one-, two-, three-, four-, five-, and six-folds) of precipitants, quantities of the cross-linker GA (10–60 mM) and cross-linking time (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4) on the activity of CLEA-EXANL1 were investigated.
The recovered CLEA-EXANL1 was thoroughly washed with the relevant solvents until enzyme activity in the supernatant was undetectable. CLEA-EXANL1 was then stored in 50 mM PBS buffer at 4 °C for further use.
Assay of EXANL1 activity
The activity of free EXANL1 and CLEAs was measured via alkali titration. The reaction mixtures consisted of 4 mL of olive oil emulsification, 5 mL of 25 mM sodium phosphate buffer (pH 6.4), and 1 mL of enzyme solution. The reaction mixtures were placed in 50 mL Erlenmeyer flasks with a glass stopper and incubated at 34 °C for 15 min with constant agitation. The reaction was terminated with 15 mL of 95% ethanol. One unit of lipase activity was defined as the amount of enzyme releasing 1 μmol of fatty acid per minute. Eq. (1) was used to calculate activity recovery:
Optimum temperature and pH of EXANL1 and CLEA-EXANL1
The effect of CLEAs strategy on optimum temperature of EXANL1 was studied by measuring the activity of free EXANL1 and CLEAs for 15 min at 25–55 °C. Enzyme activity was measured through the alkali titration method, as described in Section 2.5. The highest enzyme activity of free and CLEAs of EXANL1 within these temperature ranges was defined as 100%. The effect of CLEAs strategy on optimum pH of EXANL1 was determined by performing the activity assay under the pH range of 4.0–9.0. Furthermore, 0.1 M citrate buffer was used for pH 4.0–5.0, 0.2 M phosphate buffer was used for pH 5.0–7.0, and 0.2 M boric acid buffer was used for pH 8.0–9.0. The activities of free and CLEAs of EXANL1 were determined via the method described in section 2.5. Enzyme activity was defined as described above.
Thermal and pH stability of EXANL1 and CLEA-EXANL1
Free EXANL1 and CLEA-EXANL1 were incubated at 30 °C, 35 °C, 40 °C, 45 °C and 50 °C for 10 h to investigate the effect of CLEAs technique on the thermal stability of EXANL1. The relative activities of free EXANL1 and CLEA-EXANL1 without heat treatment were defined as 100%, and the relative activities of free lipase and CLEA-EXANL1 at different temperature values (30 °C–50 °C) were determined via the method described in Section 2.5. The effect of CLEAs technique on the pH stability of EXANL1 was estimated by incubating free lipase and CLEA-EXANL1 at different pH values (4.0–8.0). After vacuum freeze drying, free EXANL1 and CLEA-EXANL1 were added to buffer solutions with pH values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 for 2 h. The activities of untreated free EXANL1 and CLEA-EXANL1 were defined as 100%.
Stability of EXANL1 andCLEA-EXANL1 in organic solvents
To study the effect of CLEAs strategy on the organic solvent stability of EXANL1, free EXANL1 and CLEA-EXANL1 in selected organic solvents were treated as follows. Free EXANL1 and CLEA-EXANL1 were weighed after vacuum freeze-drying in a test tube containing 10 mL of organic solvent, such as methanol, acetone, ethanol, dichloromethane, tetrahydrofuran, toluene, n-hexane, and acetic acid, at room temperature for 20 h. The above mixtures were centrifuged at 4000 g and 4 °C for 15 min. Then, free EXANL1 and CLEA-EXANL1 treated with organic solvents were suspended in a sodium phosphate buffer solution (pH 7.0). The activities of free EXANL1 and CLEA-EXANL1 were determined in accordance with the method presented in Section 2.5, and the definition of enzyme activity was the same as that mentioned in Section 2.6.2.
Operational stability
A certain amount of vacuum freeze-dried CLEA-EXANL1 was reacted with an olive oil emulsion in a constant-temperature water bath oscillator at 34 °C and 180 rpm for 15 min. After a cycle of the hydrolytic activity assay, insoluble CLEA-EXANL1 was separated from the reaction medium by centrifugation at 6000 g for 15 min. Then, CLEA-EXANL1 was removed and placed in the next batch of fresh substrates to continue the reaction. The relative activity of CLEA-EXANL1 in the first cycle was defined as 100% and measured in accordance with the method described in Section 2.5.
Storage stability of EXANL1 and CLEA-EXANL1
Free EXANL1 and CLEA-EXANL1 were stored at 4 °C for one month to investigate the effect of CLEAs strategy on storage stability of EXANL1, and relative activities of free EXANL1 and CLEA-EXANL1 was measured once every 3 days in accordance with the method described in Section 2.5. The activities of free EXANL1 and CLEA-EXANL1 before storage were defined as 100%. The stability of relative activity at different storage times was studied to ensure permanent dissolution and the retention of preorganized superstructure to maintain catalytic activity.
Kinetic parameters of EXANL1 and CLEA-EXANL1
Olive oil was emulsified with 4% (w/v) polyvinyl alcohol solution at a ratio of 1:3 (v/v) under a high stirring rate. Approximately 4 mL of olive oil emulsion (30–120 g/L), 5 mL of 25 mM PBS buffer (pH 6.4), and 1 mL of EXANL1 or CLEA-EXANL1 were allowed to react at 34 °C for 15 min with constant agitation to determine the effect of CLEAs strategy on the kinetic parameters of EXANL1. The apparent kinetic parameters, namely, Michaelis–Menten constant (Km) and maximum reaction rate (Vmax), were calculated using the Lineweaver–Burk method and Michaelis–Menten model, as shown in Eq. (2):
where v is the reaction rate, [S] (g/L) is the olive oil concentration of the surface of drops, Vmax (mmol/L/min) is the maximum reaction rate at saturating substrate concentration and Km (g/L) is the Michaelis–Menten constant.
Surface morphology analysis
Free EXANL1 and CLEA-EXANL1 were ground into powder after vacuum freeze-drying and evenly sprinkled on the SEM sample table. Then, the surface of the sample was sprayed with gold under vacuum. Subsequently, the surface morphology and particle size of the free EXANL1 and CLEA-EXANL1 were characterized via SEM (S-3400N, Hitachi, Japan).
FTIR analysis
Changes in the secondary structure of EXANL1 through CLEAs immobilization were analyzed via FTIR. Vacuum freeze-dried free EXANL1 and CLEA-EXANL1 were mixed with KBr at room temperature, ground and pressed into transparent sheets. A Nicolet is5 infrared spectrometer (Nicolet is5, Thermo fisher, America) was used to scan free EXANL1 and CLEA-EXANL1 within the wave number range of 400–4000 cm-1. Absorbance data were converted, and spectra were processed by Peakfit 4.12. The amide I spectrum with a wavelength range of 1600–1700 cm-1 was selected for baseline correction, and the Peakfit 4.12 software was used for peak segmentation fitting to obtain six subpeaks.
Positional selectivity analysis
Hydrolysis was conducted as described by Yamamoto et al. [39] with some modifications. 0.1 mL of triolein and 1 mL of enzyme (free EXANL1, CLEA-EXANL1 and porcine pancreas lipase [PPL]) were placed in 50 mL Erlenmeyer flasks with a glass-stopper and incubated at 30 °C for 15 min at 180 rpm for hydrolysate extraction. Then, 20 mL of n-hexane was added to the extracted hydrolysate for 30 min. The upper phase solution was used for TLC analysis.
Statistical analysis
All analytical experiments were performed in triplicate, and the results were reported as the mean values of replicates along with standard deviation. Collected data were subjected to analysis of variance by using the Statistical Analysis System software, where P<0.05 was considered statistically significant.