Immobilization Of Phospholipase Research Articles

Immobilization of Phospholipase D for Production of Phosphatidylserine via Enzyme-Inorganic Hybrid Nanoflower Strategy

Phosphatidylserine (PS) is a natural phospholipid with particular importance in the food, cosmetic, and pharmaceutical industries. Recently, the synthesis of PS mediated by phospholipase D (PLD) has drawn great attention. But the application of free PLD is limited by various drawbacks, including its instability under extreme conditions, difficulties in reuse and recovery, and high costs. In this work, saPLD-inorganic hybrid nanoflowers (saPLD@NFs) were synthesized with PLD from Streptomyces antibioticus (saPLD) as the organic component and Ca3(PO4)2 as the inorganic component. The saPLD@NFs demonstrated outstanding immobilization capability and achieved a 119% enzyme activity recovery rate. Furthermore, the saPLD@NFs exhibited better thermostability and pH stability in comparison to free saPLD. The PS yield of saPLD@NFs was about 57.4% in the first cycles and still reached 60.4% of its initial PS yield after four cycles. After 25 d storage at 4 °C, saPLD@NFs retained 66.5% of its original activity, but free saPLD only retained 38.3%, indicating that saPLD@NFs have excellent storage stability. Thus, this study established a new method of preparing PLD nanoflowers for effective PS synthesis, which might accelerate the practical utilization of this biocatalyst.

Immobilization of Phospholipase D for Production of Phosphatidylserine by a Pickering Emulsion Strategy

As a natural phospholipid, phosphatidylserine (PS) plays a key role in the food, cosmetic, and pharmaceutical industries. Recently, substantial attention has been focused on the phospholipase D (PLD)-mediated synthesis of PS. However, the application of free PLD is usually limited by high cost, poor reusability, and low stability. In this study, PLD from Streptomyces antibiotics (saPLD) was efficiently immobilized on SiO2 through physical adsorption to develop saPLD@SiO2. The stability of the saPLD@SiO2 was higher than that of the free saPLD over an extensive range of temperature and pH conditions. Furthermore, the PS yield of saPLD@SiO2 was approximately 41% in the first cycles, and still kept 60% of its initial PS yield after 14 cycles. After a 25-day storage period, the saPLD@SiO2 retained 62.5% of its initial activity, while the free saPLD retained only 34.3%, suggesting that saPLD@SiO2 has better stability than free saPLD. A Pickering emulsion was produced by dispersing saPLD@SiO2 in solutions (ethyl propanoate and acetate/acetic acid buffer) using ultrasound. The engineered Pickering emulsion demonstrated excellent catalytic activity, with a 62% PS yield after 6 h, while free saPLD had only 18%. The results indicated that a high-performance and sustainable biocatalysis method was established for the effective synthesis of PS.

Design of amino-functionalized hollow mesoporous silica cube for enzyme immobilization and its application in synthesis of phosphatidylserine

In this study, hollow mesoporous silica cube (HMSC) modified with amino (-NH2) were synthesized and applied in the immobilization of phospholipase D (PLD) via physical adsorption and chemical cross-linking strategy. The amino-functionalized nano carrier HMSC represented excellent immobilization ability and achieved 87.15 % immobilization rate. The immobilized PLD has wider pH application range and thermal stability, and maintained over 90% of the initial activity after incubation at 50 °C for 2 h. After 50 days of storage at 4 ℃, immobilized PLD retained 40.12 % of its initial activity while free PLD lost 88.28% of its initial activity. The modified HMSC with immobilized PLD (HMSC-NH2-PLD) retained 50.73% activities after 9 consecutive reuses. Using the HMSC-NH2-PLD, a high-efficient method for the conversion of phosphatidylserine (PS) from phosphatidylcholine (PC) and L-serine was proposed. The HMSC-NH2-PLD exhibited prominent enzymatic activity for PS synthesis, the maximal conversion of PS was 90.40% with a catalytic efficiency (CE) of 31.95 μmol / (g h under the optimal conditions. The research in this paper provides a sustainable and efficient biocatalysis application for PS synthesis.

Immobilization of phospholipase D on macroporous SiO2/cationic polymer nano-composited support for the highly efficient synthesis of phosphatidylserine

Novel nano-composites were prepared by coating epoxy resin-based cationic polymer in nano-thickness via in-situ curing on the nano-wall of macroporous SiO2 with pore size of 0.5∼1 μm. By changing the thickness of polymer coating the specific surface area and porosity varied in range of 115∼74 m2/g and 90.4∼83.9 %, respectively. Through ion exchange phospholipase D (PLD, from Streptomyces sp) was efficiently immobilized on the nano-composites as support and the immobilized PLD was applied for the highly efficient synthesis of phosphatidylserine (PS). The loading amount of PLD on the nano-composited support reached to a maximum of 90.2 mg/gsupport, 4 times as high as that on the pure macroporous silica. The specific activity of the immobilized PLD reached as high as 16,230 U/gprotein, while that of free PLD was 18,780 U/gprotein. Under a wide range of temperature and pH the stability and activity of the immobilized PLD were greatly improved as compared with the free ones. Under optimized conditions at 45 °C and pH 7.0, the PS yield reached as high as 96.2 % within 40 min. After 28 days storage the immobilized PLD retained 82.2 % of original activity, and after 12 cycling reuses it retained 79.3 % of PS yield, which indicated that the immobilized PLD exhibited good stability.

Immobilization of Phospholipase D on Silica-Coated Magnetic Nanoparticles for the Synthesis of Functional Phosphatidylserine

In this study, silica-coated magnetic nanoparticles (Fe3O4/SiO2) were synthesized and applied in the immobilization of phospholipase D (PLDa2) via physical adsorption and covalent attachment. The immobilized PLDa2 was applied in the synthesis of functional phosphatidylserine (PS) through a transphophatidylation reaction. The synthesis process and characterizations of the carriers were examined by scanning electron microscope (SEM), transmission electron microscope (TEM), and Fourier-transform infrared spectroscopy (FT-IR). The optimum immobilization conditions were evaluated, and the thermal and pH stability of immobilized and free PLDa2 were measured and compared. The tolerance to high temperature of immobilized PLDa2 increased remarkably by 10°C. Furthermore, the catalytic activity of the immobilized PLDa2 remained at 40% after eight recycles, which revealed that silica-coated magnetic nanoparticles have potential application for immobilization and catalytic reactions in a biphasic system.

Immobilization of Phospholipase A1 and its Application in Soybean Oil Degumming

AbstractPhospholipase A1 (PLA1), or Lecitase® Ultra, was immobilized on three different supports, calcium alginate (CA), calcium alginate‐chitosan (CAC), and calcium alginate‐gelatin (CAG), and crosslinked with glutaraldehyde. The results indicated that PLA1–CA retained 56.2% of the enzyme's initial activity, whereas PLA1–CAC and PLA1–CAG retained 65.5 and 60.2%, respectively. Compared with free PLA1, the optimal pH of immobilized PLA1 shifted to the basic side by 0.5–1.0 pH units and the pH/activity profile range was considerably broadened. Similarly, the temperature‐optima of PLA1–CAC and PLA1–CAG increased from 50 to 60 °C, and their thermal stability increased with relative activities of more than 90% that covered a wider temperature range spanning 50–65 °C. In a batch oil degumming process, the final residual phosphorus content was reduced to less than 10 mg/kg with free PLA1, PLA1–CAC and PLA1–CA in less than 5, 6 and 8 h respectively while PLA1–CAG was only able to reduce it to 15 mg/kg in 10 h. When the PLA1–CAC was applied in a plant degumming trial, the final residual phosphorus content was reduced to 9.7 mg/kg with 99.1% recovery of soybean oil. The recoveries of immobilized PLA1–CAC and activity of PLA1 were 80.2 and 78.2% respectively. Therefore, it was concluded that PLA1–CAC was the best immobilized enzyme complex for the continuous hydrolysis of phospholipids in crude vegetable oils.

Immobilization of Streptomyces phospholipase D on a Dowex macroporous resin

The immobilization of phospholipase D produced by Streptomyces sp. YU100 was evaluated to see it would be practical for industrial applications. To accomplish this, the purified enzyme, which contained 53 unit/mg of protein, was subjected to immobilization on various matrices. When immobilization supports including calcium alginate gel, polyacrylamide gel, and macroporous resin were evaluated, the highest enzyme retention ratio (> 42%) was observed on a Dowex MSA-2 macro-porous resin. This may have occurred as a result of the ability of the hydrophobic domain of phospholipase D to interact with the polystyrene backbone of the resin, as well as the ability of the dimethylethanolamine group of the MSA-2 resin to retain the enzyme by forming hydrogen bonds with the acidic residues of the enzyme. Upon the operation of a reactor packed with enzyme that had been immobilized on a Dowex MSA-2 resin, greater than 80% of the initial enzyme activity was retained for 16 days. During the reaction, phosphatidylcholine became bound to the immobilized resin and interfered with the enzyme reaction, therefore, the resin was washed with ethyl ether every 2 h. A process for recovering excessive l-serine from phospholipids using the Dowex MR-3 resin was designed, and the separated l -serine was employed again after replacing the amount that was used.

Immobilization of Phospholipase C for the Production of Ceramide from Sphingomyelin Hydrolysis

AbstractThe immobilization of Clostridium perfringens phospholipase C was studied for the first time and the catalytic properties of the immobilized enzyme were investigated for the hydrolysis of sphingomyelin to produce ceramide. Ceramide is of great commercial value in the cosmetic and pharmaceutical industries for use in, for example, hair and skin care products, owing to its major role in maintaining the water‐retaining properties of the epidermis. The feasibility of enzymatic production of ceramide through hydrolysis of sphingomyelin has previously been proven. In order to improve the reusability of the enzyme, the present study focused on the immobilization of phospholipase C in the production of ceramide from sphingomyelin. By screening nine different carriers, we found that the enzyme immobilized on Lewatit had the highest catalytic activity towards sphingomyelin hydrolysis. Prewetting Lewatit with ethanol led to higher enzyme fixation on the carrier, but the activity of the enzyme was decreased. Increasing the initial enzyme concentration resulted in more enzyme adsorption on the carrier, where the specific activity was increased. Through optimization of the reaction using the immobilized enzyme, the optimal temperature was around 46 °C and the optimal water volume was 3.5%. The reaction had little dependence on pH. After seven recycles, immobilized enzyme retained around 70% of the initial activity. Immobilized enzyme was deactivated irregularly when stored at room temperature, but followed first‐order deactivation when stored at 40 °C.

Immobilization of phospholipase A2 on porous glass and its application for lowering serum cholesterol concentration.

Phospholipase A2 (PLA2; EC 3.1.1.4) is a lipolytic enzyme that hydrolysis the ester bond in sn-2 position of phospholipids. In this work, the PLA2 from hog pancreas was covalently coupled to porous glass. The properties of free and immobilized enzyme were also investigated and compared. The optimum pH and temperature were found as 8.5 and 50 degrees C, respectively for both free and immobilized enzyme. The immobilized enzyme had good properties that potential for medical application is considerable. Its use in lowering plasma cholesterol concentrations in blood samples was also demonstrated.

Inmovilización de enzima fosfolipasa A<sub>2</sub>

A series of several supports were tested for phospholipase A 2 enzyme (Lecitase 100 S- NOVO) immobilization. This system would find a number of industrial applications such as the selective hydrolysis of soybean lecithin to obtain lysophospholipids (modified lecithins) of great importance in pharmaceuticals and bread making. Successful results have been obtained from the immobilization of Phospholipase A 2 on Alumina, Silica gel, Amberlite IRA-400S, Dowex and Sephadex A-25 via adsorption (the results were in the range 15-30 % of enzyme adsorption). The immobilization reaction was carried out at ambient temperature, with slow magnetic agitation such that the enzyme and 50 mg support were present in the total reaction volume of 5 ml in Tris-ClH buffer (pH 8.65). Several enzyme/supports ratio and the adsorption percentage with respect to pH and temperature and time were investigated. We have compared the behaviour of the phospholipase A 2 in soluble form and immobilized form toward pure powder soybean lecithin as substrate.

The adsorptive immobilization of phospholipids D mediated by calcium ions

Immobilization of phospholipase D from cabbage was studied with the aim of stabilizing the enzyme for its use in synthesis of phospholipids. It was shown that phospholipase D can be immobilized by adsorption to polymeric carriers containing long chain anchor groups such as octadecyl, octyl, or other alkyl residues. Starting from the crude enzyme, phospholipase D activity is preferentially bound (up to 100%) in competition with contaminating proteins. A prerequisite of high binding rates is the presence of calcium ions, which play a mediating role in the adsorption process. The maximum activity of the carrier-enzyme complexes depends upon the calcium concentration in the immobilization process and the carrier material (> or = 10 mM CaCl(2) with octadecyl-Si40, > or = 40 mM CaCl(2) with octyl-sepharose and butyl-fractogel). Immobilization of phospholipase D to octyl-sepharose was shown to result in a distinctly increased storage stability and an enlarged pH-optimum range for the catalytic activity. Operational stability of different phospholipase D-carrier complexes was compared.