Immobilization Of Phospholipase By Magnetic Nanoparticles And Its Application In The Rapeseed Oil Degumming

Enzymatic degumming is an emerging degumming process with the advantages of mild reaction conditions,complete degumming,high oil yield,low energy consumption and low pollution.A large number of scholars at home and abroad have applied the phospholipase to vegetable oil degumming and have achieved good degumming effect.However,the free phospholipase is inactivated easily and difficult to be recycled,which increases the costs and limits the industrial applications.The phospholipase immobilization can improve these deficiencies.Choosing a suitable material of enzyme immobilization is the key to achieving high enzyme activity and high stability.Magnetic nanoparticles?MNPs?have become a research hotspot for enzyme immobilization materials due to their superparamagnetism,high specific surface area and low toxicity.In this paper,epoxy-modified MNPs were synthesized and phospholipase A₁?PLA₁?and phospholipase A₂?PLA₂?were immobilized using the prepared particles,respectively.The effects of immobilization conditions on relative enzyme activity and immobilization rate were investigated.The enzymatic properties of immobilized PLA₁and PLA₂ were explored.The immobilized PLA₁ and PLA₂ were applied to the degumming experiment of rapeseed oil.And the degumming conditions were optimized by single factor and response surface test to provide support for industrialized application of immobilized enzyme degumming.The specific research results were as follows:?1?MNPs were prepared by conventional coprecipitation method.MNPs were modified by 3-?2,3-epoxypropoxy?propyltrimethoxysilane?GPTMS?to prepare epoxy-modified magnetic nanoparticles?MNPs-GPTMS?.The surface epoxy group content of MNPs-GPTMS was 0.012 5 mmol/g by titration.X-ray diffraction?XRD?and scanning electron microscopy?SEM?showed that epoxy groups were modified on the surface of MNPs.Therefore,MNPs-GPTMS has the potential for enzyme immobilization.?2?By measuring the enzyme activity curves of three free phospholipases?PLA₁,PLA₂ and phospholipase B?PLB??under different pH and temperature conditions,the effects of temperature and pH on the activity of the free phospholipase were obtained.PLA₁,PLA₂ with higher enzyme activity,better pH and temperature tolerance were selected as the subsequent research objects.?3?The preferred process conditions for PLA₁ immobilization were obtained by single factor test:buffer pH 4.0,reaction time 2.0 h,reaction temperature 20°C,enzyme solution addition 3.0 mL/g.Under this condition,the enzyme activity was 3600 U/g and the immobilization rate was 61.1%.Compared with the free PLA₁,immobilized PLA₁had better thermal stability and storage stability.At the same time,the immobilized PLA₁ was used in the degumming of rapeseed oil.The optimum degumming reaction conditions were obtained according to single factor and response surface test:immobilized PLA₁ addition amount 90.00 mg/kg,reaction pH 6.0,reaction temperature54°C,degumming time 4.5 h.Under this condition,the obtained phosphorus content was reduced to 1.450 mg/kg.The immobilized PLA₁ had good reusability.The immobilized PLA₁ retained 54.1%of enzyme activity after repeated use of 10 batches in the degumming under the optimal degumming condition.?4?The preferred process conditions for PLA₂ immobilization were obtained by single factor test:buffer pH 2.5,reaction time 2.0 h,reaction temperature 20°C,enzyme solution addition amount 4.0 mL/g.Under this condition,the enzyme activity was 2 850U/g and the immobilization rate was 73.1%.The immobilized PLA₂ showed a better effect in terms of storage stability than the free PLA₂.At the same time,the immobilized PLA₂ was used for degumming of rapeseed oil.The optimum degumming reaction condition was obtained according to single factor and response surface test:immobilized PLA₂ addition amount 100.00 mg/kg,reaction pH 5.6,reaction temperature 55°C,degumming time 4.5 h.Under this condition,phosphorus content of the degummed oil was 2.520 mg/kg.The immobilized PLA₂ also had the advantage of reusing the enzyme.After 10 degumming cycles,the immobilized PLA₂ retains 51.1%of its initial activity.