Phospholipase A₁-catalyzed Hydrolysis Of Soybean Phospholipids

Phospholipids are integral components of biomembranes.Intensive studies have shown that functions of phospholipids are multifold,ubiquitous and fundamental for life.Being natural surfactants of amphiphilic molecules and physiological functions,commercial phospholipids are widely used in foods,pharmaceutical,cosmetic,agriculture and other industrial fields.Rapid progress in lipid biochemistry,pathology,pharmacology and nutriology has brought more and more attention to phospholipids modification.It is believed that enzymatic modification of phospholipids will be a potential and promising choice because of its unique advantages such as more specific reaction,milder reaction condition, less by-products and less environmental pollution.Preparation of lysophospholipids by phospholipase A₁-catalyzed hydrolysis of soybean phospholipids was investigated.Reaction system was investigated including homogenizing rate,stillness or non- stillness,aqueous phase or n-hexane and Ca²⁺concentration.It was found that acid value was remarkably different with different homogenizing rate;acid value increased obviously in non- stillness system compare with stillness system;reaction rate was faster and acid value was a little higher in n-hexane,but simple technics and safe product was obtained with a satisfying acid value in aqueous phase;phospholipase A₁ was independent of Ca²⁺with higher Ca²⁺concentration resulted in lower acid value.Reaction system was chose as homogenizing rate 1000prm,non- stillness and aqueous phase without Ca²⁺.Effects of enzyme dosage,substrate ratio(water/phospholipids,ml/g),reaction temperature and reaction time on acid value were investigated in single factor experiment. Satisfying acid value was obtained under enzyme dosage 400-600μl,substrate ratio 10-14, reaction temperature 35-40℃,reaction time 9-11h.Phospholipase A₁-catalyzed hydrolysis of soybean phospholipids was optimized using response surface methodology with response of acid value based on single factor experiment. A three-level four-factor Box-Behnken design was adopted.The second-order regression model was:AV=127.1+1.975X₁+2.075X₂+0.808334X₃+1.375X₄-1.09167X₁²-4.79166X₂²-5.26667X₃²-2.04166X₄²+0.449989X₁X₂+0.699998X₁X₃-0.125X₁X₄+ 0.299998X₂X₃-0.824999X₂X₄-0.275003X₃X₄.Substrate ratio and reaction temperature were the most remarkable factors.Hydrolysis was influenced by these factors in the following order:substrate ratio >reaction temperature>reaction time>enzyme dosage.The interaction of every two factors on acid value was also investigated.Optimal conditions were recommended as enzyme dosage 590μl,substrate ratio 12.4,reaction temperature 40.4℃,and reaction time 10.3h.At these experimental conditions,the estimated acid value of product was 128.9 mgKOH/g.Acid value of 128.5 mgKOH/g was obtained under these conditions in validation experiment which proved that the model fitted phospholipase A₁-catalyzed hydrolysis of soybean phospholipids well.