Characterization of a novel sn1,3 lipase from Ricinus communis L. suitable for production of oleic acid-palmitic acid-glycerol oleate

The hydrolysis properties of lipase in castor was evaluated using two different substrate forms (tripalmitic glycerides and trioleic glycerides) to catalyze the reaction under different operational conditions. RcLipase was obtained from castor seeds and results show that RcLipase is a conservative serine lipase with a conserved catalytic center (SDH) and a conserved pentapeptide (GXSXG). This enzyme exhibited the greatest activity and tolerance to chloroform and toluene when it was expressed in Pichia pastoris GS115 at 40 ℃ and pH 8.0. Zn and Cu ions exerted obvious inhibitory effects on the enzyme, and displayed good hydrolytic activity for long-chain natural and synthetic lipids. HPLC analysis showed that this enzyme has 1,3 regioselectivity when glycerol tripalmitate and oleic acid are used as substrates. The fatty acid composition in the reaction product was 21.3% oleic acid and 79.1% sn-2 palmitic acid.

Expression of lipase gene in P. pastoris. The pMD19-T-RcLipase and pPICZ a-A vectors were digested by NcoI and XhoI, respectively, and recombined using T4 DNA ligase. The recombinant plasmid was linearized by SacI and transformed into P. pastoris X-33 by electroporation. The transformants were cultured on YPD-Zeocin agar plates for 2-3 days. Positive transformants were cultured in BMGY medium at 200 rpm at 30 ℃ until OD 600 reached 2-6. Cells were collected and suspended in BMMY medium for 5 days. During this period, induction was maintained at a final concentration of 0.5% (v/v) every 24 h. The supernatant was obtained from crude enzyme solution (supernatant) after centrifugation at 10,000 rpm for 10 min, thereafter, lipase activity was detected.
Fed-batch fermentation. In order to expand the production scale of lipase, a 3 L fermentation was adopted according to the method described 13 . Yeast seed liquid (100 ml) was added to BSM medium (900 ml). The fermentation process consisted of three phases, including the glycerol culture stage, glycerol feeding stage, and methanol induction stage. Glycerol culture was conducted at 30 ℃, with pH 6.0 maintained using NH 4 OH. The initial glycerol was depleted for about 20 h and entered the glycerol feeding stage. In the glycerol feeding stage, 50% glycerol (glycerol:water = 1:1, v/v) was added to the fermentation tank until OD 600 reached 110. After the exhaustion of glycerol in the medium, it entered the methanol induction phase, where the temperature was maintained at 28 ℃, and the concentration of methanol was maintained at 0.1 ± 0.02% (w/v). The fermented samples were centrifuged at 10,000 rpm for 10 min. The cell number, lipase activity, and protein concentration of the supernatant were measured. The concentration of protein was determined using the Bradford method, and bovine serum albumin (BSA) (Beijing Solarbio Science & Technology Co., Ltd.) was used as the standard.
Enzyme activity measurement. The activity of enzymes was determined using a spectrophotometer.
The mother liquor of pNP (p-nitrophenol) with 20 mM was diluted into different concentration gradients at 40 ℃ for 15 min, and the termination reaction of ethanol with 500 μl 95% was added. The absorbance value was determined at 410 nm, and the standard curve was drawn. 2 ml EP tube was mixed with 300 ml buffer solution (0.3% p-nitrophenol palmitate, pNPP) isopropanol, 20 mM Tris-HCl buffer solution (pH 8.0, 0.11% gum arabic), 25 ml crude enzyme solution, 20 mM Tris-HCl buffer solution (pH 8.0, 0.11% gum arabic), and 40 mM Tris-HCl buffer solution (25 ml, 0.11% gum arabic), respectively. The reaction was terminated by adding 500 μl 95% ethanol at 15 min. The absorbance value was determined at 410 nm, and the concentration of pNP was obtained according to the standard curve, followed by enzyme activity calculation. The amount of p-nitrophenol required to catalyze the production of p-nitrophenol by substrates within 1 min is defined as one unit of enzyme activity (U).

Biochemical characterization of purified enzyme. Effect of pH on RcLipase activity and stability.
To determine the optimum lipase pH, pNPP activity was measured under the optimum reaction temperature and concentration of 20 mM buffer with various pH values (pH 4.0-9.0) for 15 min. All experiments were repeated three times. The effect of pH on lipase activity was expressed by relative enzyme activity. The maximum enzyme activity was determined to be 100%. www.nature.com/scientificreports/ For the lipase pH stability test, lipase protein samples were placed in 100 mM buffer with different pH and incubated at 40 ℃ for 12 h to determine the enzyme activity. The effect of pH on the stability of lipase was expressed by relative enzyme activity. The enzyme activity of untreated protein samples was determined to be 100%.
Effect of temperature on RcLipase activity and stability. Lipase activity was determined at 30, 35, 40, 45, 50, 55, 60, and 70 ℃ with pNPP as substrate in 20 mM Tris-HCl (pH 8.0) buffer solution for 15 min. All experiments were repeated three times. The effect of temperature on lipase activity was expressed by relative enzyme activity. The maximum enzyme activity was determined to be 100%.
For the determination of temperature stability of lipase, the lipase protein samples were incubated at 30, 40, 50, 60, and 70 ℃ for 12 h, and the enzyme activity was determined immediately after being cooled with ice water every 6 h. The effect of temperature on the stability of lipase was expressed by relative enzyme activity. The enzyme activity of untreated protein samples was determined to be 100%.

Effect of metal ions, surfactants, and organic solvents on RcLipase activity. Effect of metal ions
on RcLipase activity. NiCl 2 , ZnSO 4 , MnSO 4 , MgSO 4 , FeCl 2 , CaCl 2 , CoCl 2 , and CuCl 2 were added to the lipase protein samples, and the final concentration of metal ions in the system was 5 mM. The mixtures were incubated at 40 ℃ for 2 h and the activity of lipase was determined with pNPP as the substrate under the optimum reaction conditions. All experiments were repeated three times. The effect of metal ions on lipase activity was expressed by relative enzyme activity. The activity of lipase was determined to be 100% in the absence of metal ions.
Effect of surfactants on RcLipase activity. SDS, Tween 20, Tween 60, Tween 80, and Triton X-100 were added to the lipase protein samples, respectively, and the final concentration of surfactant in the system was 1% (m/v). The mixtures were incubated at 40 ℃ for 2 h and the activity of lipase was determined with pNPP as the substrate under the optimum reaction conditions. All experiments were repeated three times. The effect of surfactant on lipase activity was expressed by relative enzyme activity. The activity of lipase determined by protein samples without surfactant was 100%.
Effect of organic solvents on RcLipase activity. Methanol, ethanol, isopropyl alcohol, acetone, toluene, and chloroform were added to the lipase protein samples, respectively, and the final concentration of organic solvent was 30% (v/v). The mixtures were incubated at 40 ℃ for 2 h and the enzyme activity was determined with pNPP as the substrate under the optimum reaction conditions. All experiments were conducted three times. The effect of organic solvents on lipase activity was expressed by relative enzyme activity. The activity of lipase was determined to be 100% in the samples without organic solvents.
Regiospecific analysis. The determination of the regiospecificity of RcLipase was performed according to a previously described method 22 , In brief, triolein (500 mg) and 500 μl RcLipase (50 U) were dissolved in 20 ml of 20 mM Tris HCl (pH 8.0), the mixture was shaken at 180 rpm for 12 h at 40 ℃. Samples (0.05 ml) were withdrawn periodically and centrifuged at 10,000 rpm for 5 min, 0.02 ml upper layer was transferred into another centrifugation tube and was mixed with 0.5 g anhydrous sodium sulfate and 1 mL of n-hexane, 2-propanol and methanoic acid (20:1:0.003, by volume) were added and centrifuged at 10,000 rpm for 10 min. The supernatant was used for HPLC.
Determination of kinetic parameters. The kinetic parameters of lipase were determined using glycerol ester substrates (triolein, tripalmitate, and castor oil). The substrate concentration range was controlled from 0 to 1 mM, and the reaction conditions were 40 ℃, 20 mM Tris-HCl buffer with pH 8.0. The kinetic parameters V max , K m , and K cat of the lipase enzymatic reaction were calculated using the non-linear regression and Michaelis-Menten equation.

Results
Cloning and sequence analysis of a novel lipase gene from Ricinus communis L.. The ORF of the RcLipase gene was 987 bp, without introns. It encodes 329 amino acids and does not contain signal peptides (Fig. S1). The molecular weight of the predicted mature protein was 38.24 KDa and the inferred pI was 8.47. The multiple comparison between RcLipase and other lipases showed that the highly conserved catalytic trimer consisted of Ser (146 site), Asp (211 site), and His (276 site). RcLipase had a conservative ester box GxSxG, which conformed to the conservative domain structure of lipase in class III (Fig. 1). There were Gly-X1-Ser-X2 oxygen ion holes, X1 was threonine (63 site), X2 was leucine (147 site).
Expression and purification of RcLipase. RcLipase was expressed in Pichia pastoris. After 7 days of fermentation in a 3 l fermentor, the extracellular lipase activity reached 1492 U/ml (for pNPP) and the protein concentration was 7.65 mg/ml ( Fig. 2A). After purification using affinity chromatography, the recovery of crude enzyme was 80.2%, and the specific activity was 147.2 U/mg. The purified RcLipase was detected using SDS-PAGE with a band at 38 KDa ( Fig. 2B-D).
To determine the optimal temperature of RcLipase, the enzyme activity was assayed at temperatures ranging from 30 to 70 ℃ in 20 mM Tris-HCl buffer (pH 8.0) for 15 min. The relative activity of RcLipase was the strongest at 40 ℃ (Fig. 3C). To determine the thermostability of RcLipase, the enzyme activity was assayed at temperatures ranging from 30 to 70 ℃ in 20 mM Tris-HCl buffer (pH 8.0) for 12 h. After incubation at 40-45 ℃ for 12 h, it retains more than 80% of its relative activity; after incubation at 50 ℃ for 12 h, the relative activity drops to 13.57%; after incubation at 60-70 ℃ for 12 h, the activity is completely lost. Therefore, the enzyme is stable below 40-45 ℃ (Fig. 3D).
Formaldehyde and ethanol exerted significant inhibitory effects on RcLipase. The activity of RcLipase decreased by 88.2% and 88.4% in 2 h, followed by isopropanol and acetone. The activity of RcLipase decreased by 77.2% and 78.7% in 2 h, respectively. Chloroform and toluene exerted the lowest effect (10% and 15%, respectively) (Fig. 3E).
The inhibitory effect of Zn 2+ and Cu 2+ ions on RcLipase was obvious. After 2 h, the activity of RcLipase decreased by 53% and 40%, respectively. However, the inhibitory effect of other metal ions on RcLipase was not obvious (Fig. 3G).  Table 1, the enzyme displays hydrolytic activity to both pNPC and natural lipids, but both display hydrolytic activity to pNPC and natural lipids. The hydrolysis activity of long-chain substrates (C14/C16/C18) was stronger than that of short-chain substrates. The hydrolysis activity of two basic lipids, tripalmitic glycerides, and trioleic glycerides, was also strong for artificial breast milk (88.35% and 90.1%). This indicated that the enzyme had the potential to synthesize artificial breast milk lipids. In order to study the location specificity, the hydrolysate was analyzed using high performance liquid chromatography with RcLipase as a catalyst ( Table 1). The results showed that it could hydrolyze triglycerides to produce 1, 2(2, 3)-DAG and 1,3-DAG. The ratio of 1, 2(2, 3)-DAG to 1,3-DAG in the hydrolysate was 24.52, 16.54 and 8.52 in 10 min, 1 h, and 4 h, respectively, for sn-1,3 regiospecific lipases, the reported ratios of 1, 2(2, 3)-DAG to 1,3-DAG hydrolysis generally range from 7 to 26.71 22 . The agreement with previous studies indicates that that RcLipase has sn1,3-specific lipase.
Determination of kinetic parameters. Due to the need to synthesize oleic acid-palmitic acid-oleic acid, the enzymatic kinetic parameters of trioleic acid glyceride and tripalmitic acid glyceride were analyzed by RcLipase. The lower the km, the stronger the affinity between the enzyme and the substrate. The higher the catalytic activity, the stronger the catalytic activity of K cat /K m . As shown in Table 2, RcLipase exhibited K m and K cat values towards tripalmitin of 0.17 ± 0.01 mM and 36.81 ± 5.94 s −1 , respectively; RcLipase exhibited K m and K cat values towards triolein of 0.21 ± 0.01 mM and 36.46 ± 4.89 s −1 , respectively. The two parameters in the substrate of castor oil displayed no significant difference between tripalmitin and triolein, although they displayed a 1.34-and 1.68-fold decrease in the K cat /K m values compared to the castor oil substrates. The non-linear regression curve analysis of RcLipase with different substrates were shown in Fig. S2. Results showed that RcLipase displayed high www.nature.com/scientificreports/ affinity and catalytic activity to triolein and tripalmitate, which further indicates that RcLipase can be used as an enzyme to produce artificial breast milk lipids.
Synthesis of "oleic-palmitic-oleic". Effect of substrate ratio on incorporation of oleic acid and stability of 2-palmitic acid. We attempted synthesis of oleic acid-palmitic acid-oleic acid and compared the conditions of the reaction process. It was found that the highest incorporation of oleic acid was 19.5% at the substrate concentration ratio (oleic acid: triglyceride) of 1:8 for 6 h, and the lowest migration of palmitic acid at sn-2 position was 77.6% at the concentration of 1:8 for 6 h (Fig. 4A,B).

Effect of reaction time on incorporation of oleic acid and stability of 2-palmitic acid.
Based on our results, we measured the stability of sn-2 palmitic acid at various time periods. As shown in Fig. 5, when the substrate ratio is 1:8, the stability of sn-2 palmitic acid was 78.1% in 6 h. The result showed that oleic acid was the most stable at 6 h.
Effect of enzyme levels on incorporation of oleic acid and stability of 2-palmitic acid. As shown in Fig. 6, when the enzyme concentration was 10%, the incorporation rate of oleic acid was the highest (21.3%) and the palmitic acid was the most stable (79.1%). Therefore, the optimal enzyme concentration was 10%.

Discussion
It is reported that many germinating seeds contain large amounts of lipase, which is mainly considered to hydrolyze triglycerides (TAG) and release carbon skeletons for seed germination and growth 23 . A new lipase gene (RcLipase) was cloned from Castor seeds and expressed as an extracellular protein in Pichia pastoris. After the homology comparison, it was found that there were Gly-X1-Ser-X2 oxygen ion holes and a conservative ester box GxSxG, which agreed with the conservative region structure of lipases in class III, which are widely distributed in plants, animals, and prokaryotes. They can hydrolyze long chain acyl triglycerides to binary glycerides and free fatty acids at the water/lipid interface 24 . RcLipase is a lipase in the class III lipase superfamily identified from castor. www.nature.com/scientificreports/ RcLipase in Ricinus communis was cloned from seeds, which were germinated for 2 d. The optimum pH value was 8.0, which was lower than that of Thermomyces lanuginosus TlLipase (pH 9.0) 25 and higher than that of Malbranchea cinnamea McLipase (pH 7.5) 26 and Neosartorya fischeri NfLipase (pH 5.0) 27 . Moreover, the optimum temperature of RcLipase was 40 ℃, which was lower than that of McLipase (45 ℃) 26 and TtLipase (60 ℃) from Talaromyces thermophilus 28 .
Most lipases showed high catalytic activity towards medium and long chain substrates, such as McLipase (pNPM, C14) 26 and N. fischeri lipase (pNPL, C12) 27 . RcLipase showed higher hydrolytic activity to natural fatty acids, such as castor oil (C9), palmitic acid (C16), and oleic acid (C18). It also showed strong hydrolytic activity for pNPC8, pNPL12, and pNPM14, indicating that the enzyme was a medium long chain neutral lipase. K m is an important kinetic parameter of the enzyme, which indicates the ratio of the disappearance rate constant to the formation rate constant of the ES complex. The reciprocal of K m (1/K m ) refers to the affinity of substrate to enzyme. RcLipase is 5.88 mm/min for 1/K m of palmitate, which is higher than Pseudomonas lipase (0.7 mM). The overall efficiency of the K cat /K m catalyst was 275.36 (s −1 mM −1 ) for the reaction of palmitate, which is higher than Staphylococcus xylosus SxLipase (164.4 s -1 mM −1 ) 29 . This shows that the enzyme exerts good hydrolytic activity for palmitate and oleate, which are important substrates for the synthesis of artificial human milk fat.
The main type of triglyceride, "oleic-palmitic-oleic", is formed by the fatty components of human milk, In recent years, it has become a research hotspot to select sn1,3-specific lipase for transesterification of vegetable oil and recombination of intramolecular fatty acid position to synthesize breast milk fat substitutes and formulas similar to breast milk fat 19 . At present, many lipases with sn1,3 site specificity have been cloned from microorganisms, such as Rhizomucor miehei RmLipase 30 and Malbranchea cinnamea McLipA, which are sn1,3-specific lipases 26 . Most lipases from G. candidum are sn1,3-specific 31 . In general, the ratio of 1, 2(2, 3)-DAG to 1,3-DAG  www.nature.com/scientificreports/ in the hydrolysate of triglyceride is used to evaluate the specificity of lipase location. For the lipase with sn1,3 site specificity, the ratio is usually 3.28-31.83 13,28 . The ratio of 1,2-DAG to 1,3-DAG is 10.52-26.81, which indicates that RcLipase displays sn1,3 regiospecificity, with glycerides and oleic acid as substrates, and 10% of the substrate concentration of RcLipase, the molar ratio of the substrate is 1:8 and the reaction time is 6 h. The composition of fatty acids in the reaction product is 19.5% oleic acid and 79.1% palmitic acid at sn-2. Therefore, RcLipase can potentially be used to synthesize human milk fat (oleic acid palmitate oleic acid). These results provide a reference for enriching sources of sn1,3 lipase and improving the application value of castor. Regional specificity of lipase depends on many factors, such as conserved pentapeptide residues and catalytic site distance between serine, residues of active sites 13 , size and/or ratio of affinity, and sparsity of binding sites 32 . Detailed mechanisms regarding RcLipase region specificity requires further study. In addition, both hydrolyzate products alcohols and carboxylic acids can act as enzyme inhibitors, and the in-depth study of this part will have a strong significance for the subsequent industrial production of the enzyme. Furthermore, our future research goal is to enhance the regional specificity of RcLipase to prepare products with high infant nutritional value. Therefore, in the future, it is necessary to study the reverse esterification reaction in order to have irreversible proof of this mechanism.

Conclusions
This study provides the catalytic properties of lipase from castor in hydrolysis reactions for the production of oleic acid-palmitic acid-glycerol oleate. A neutral lipase was successfully expressed in Pichia pastoris X-33. The lipase belongs to the lipase III superfamily and displays sn1,3 regiospecificity. Furthermore, RcLipase exhibited high affinity and hydrolytic activity to the substrates of tripalmitin and triolein. Overall, RcLipase has the potential to be used to synthesize human milk fat (oleic palmitic acid oleic acid).