Study On Continuous Degumming Process Of Crude Oil By Restrictive Enzymolysis

Soybean crude oil contains about 3% of phospholipids, it absorbs water easily and causes rancidity of oil, otherwise, phospholipids are not able to bear high temperature, and it is need to remove phospholipids from oil. Phospholipids in soybean crude oil are divided into hydratable phospholipids and non-hydratable phosphatides(NHPs), hydratable phospholipids have hydrophilic properties and can be removed by hydration degumming process, non-hydrophilic substance of NHPs can be enzymolyzed by phospholipase in order to increase the hydrophilic and remove it from crude oil. With the rapid development of processing technology using enzyme and the emergence of immobilized enzyme technology, the application scope of enzyme is extended, and the number of repeated use of biological enzyme is improved, however, there are some problems should be solved in the enzymatic degumming, such as low efficiency of enzymolysis, difficulty for continuous production, increase of free fatty acids(FFA) in refined oil and others. In this study the NHPs is the research subject, through immobilizing free enzyme on magnetic carrier, to prepare magnetic immobilized enzyme and apply in magnetic fluidized bed in order to solve the difficult problem of continuous production of immobilized enzyme, by analyzing the changes of product composition in the enzymatic hydrolysis process, to reduce the free fatty acid in the oil from enzymolysis, to practice crude oil degumming process with continuous restriction enzymatic hydrolysis using magnetic immobilized phospholipase.Free phospholipase A1(PLA1) and A2(PLA2) were used for enzymolysis of phosphatidic acid in simulation of crude oil, the catalytic mechanism of "Ser-His-Asp" triplet and mechanism of acyl transfer of Sn-2-lysophosphatidic acid(Sn-2-HPA) product were researched using PLA1. It was found that empty track on inhibitor boric acid(H3BO3) and hydroxyl oxygen lone pair electrons on Sn-2-HPA formed coordination bonds; and five-membered ring intermediate was blocked; the stability of Sn-2-HPA was ensured by inhibiting acyl transfer. Restrictive enzymolysis of phosphatidic acid was effective. It was shown that suitable p H, temperature and relative enzyme activity of PLA1 was better than PLA2 by researching enzymatic properties on PLA1 and PLA2. Processing conditions of restrictive enzymolysis of phospholipid acid by PLA1 were: 2%(w/w) of H3BO3, p H 5.0, reaction temperature(50 ℃), reaction time(2 h). At similar phosphorus content in the oil after enzymolysis, the hydrolysis time was shortened by 0.80 h compared with the non-restrictive enzymolysis process, where the enzymolysis efficiency was increased by 28.60±0.07%.Super-paramagnetic Fe3O4 nanoparticles were used as magnetic core. It was prepared as a magnetic carrier by combining four kinds of organics polymers, respectively. Free PLA1 was immobilized on magnetic carrier by crosslinking method, adsorption, covalent coupling method, and enzymatic properties. In addition, the kinetics and thermodynamics of the four kinds of magnetic enzyme particles were researched. Nanoparticles of Fe3O4/Si Ox-g-P(GMA) magnetic immobilized PLA1 were screened out as optimum in comprehensive performance by extensive comparison. Compared with the free PLA1, Ea increased by 4.26±0.02 KJ/mol, Vmax only reduced to(2.90±0.02)×10-3 mol/min·mg, the results indicated a better enzymatic activity(at 2070±23 U/g), enzyme loading(at 123.30±1.10 mg/g), thermal stability increased(by 10 ℃), and suitable p H range became wider. The nanomagnetic enzymatic particles had an average particle size of 100.50±1.30 nm and the saturation magnetization strength was 15.80±0.30 emu/g.At intermittent conditions and without magnetic field, when phosphorus contents in the oil were similar after enzymolysis, technological effect of restrictive enzymolysis process was better than effects of non-restrictive enzymolysis. The selected conditional process and optimal reaction parameters were: H3BO3 of 2.20%(w/w), p H 6.30, temperature(57 ℃), time(3.70 h), and FFA(1.12±0.01 g/100 g) after the reaction. FFA content decreased by 0.14±0.01 g/100 g compared with non-restrictive enzymolysis process. The hydrolysis time was shortened by 1.30 h, enzymolysis efficiency was increased by 26±1.20%. The enzyme activity of magnetic enzyme nanoparticles was lower than 80% and were regenerated by ultrasonic assisted with tertbutyl alcohol. The use of magnetic enzyme was prolonged and the magnetic enzyme nanoparticles could be recycled 9 times after 3 h of regeneration. It was found that the enzyme protein α-helical content were increased, and surface hydrophobic residues were exposed after tertbutyl alcohol treatment by circular dichroism spectroscopy analysis. The results showed that the regeneration treatment was useful for recovery of phospholipase activity.Motion state distribution of magnetic enzyme nanoparticles, flow liquid phase, and gas phase were simulated based on Euler-Lagrange continuous-discrete model. Trajectory of magnetic enzyme nanoparticles in two-phases magnetic fluidized bed were simulated by a computer aided simulation technique besides FISH language program. When dispersion type and distribution of stable motion state of magnetic enzyme nanoparticles were reached in two-phases fluidized bed by adjusting external magnetic field strength and different velocity of liquid phase and gas phase. The optimal operation parameters of two-phases fluidized bed were determined: the external magnetic field strength of fluid-solid magnetic fluidized bed was 0.022 T, and liquid flow rate was 0.0025 m/s. The external magnetic field strength of gas-solid magnetic fluidized bed was of 0.022 T, and gas flow rate was 0.020 m/s.The experimental operation parameters of two-phases magnetic fluidized bed applied to the three-phases magnetic fluidized bed were practiced. A ten-stage and 3-phases magnetic fluidized bed were designed and manufactured. The optimum operational parameters of three-phases magnetic fluidized bed at stable distribution state of magnetic enzyme nanoparticles were obtained by researching the change of pressure drop and flow state of fluid in three-phases magnetic fluidized bed. By applying the process parameters of batch restrictive enzymolysis of NHPs to the dynamic three-phases magnetic fluidized bed, the optimized process time of continuous restrictive enzymolysis of magnetic enzyme nanoparticles in 3-phases magnetic fluidized bed was shortened by 0.30 h than that of traditional batch enzymolysis time. FFA content in oil was reduced to 0.12±0.01 g/100 g. Overall, the crude oil continuous degumming process was practiced.