Study On Production, Purification And Catalytic Characterization Of Phospholipase A1

Enzymatic degumming is a biochemical degumming technology in oil refining process whichwas much more effective and environmentally friendly than traditional methods. At present,commercial phospholipases have strict requirement for reaction condition and can’t be recycled.All these limit phospholipase application in industrial production.Phospholipase produced from Bacillus cereus sp. AF-1which perserved in the laboratory.The purpose of this study is to improve activity of phospholipase by optimizing fermentationprocess. The phospholipase was purified and the characteristics of the purified enzyme have beenstudied. In order to explore new method to improve stability and reusability, chemicalmodification and immobilization of phospholipase were applied. The main results and conclusionsobtained are as follows.1. The activity of phospholipase was messured by liberated fatty acids titrated with NaOH.The optimal condition was obtained through single factor experiments and response surfacemethodology. The optimal conditions for determining phospholipase activity required substrateconcentration of4g/100mL, final pH9.0, enzyme dilution factor of100, reaction pH5.1, reactiontemperature of53℃and reaction time of8min.The results revealed that titration is a accurateand rapid method for assay activity of phospholipase.2. Medium component and fermentation process were optimized for production ofphospholipase from Bacillus cereus sp. AF-1. Fermentation parameters were further optimized infermentor（15L）. The results showed that the culture medium were as the following （%）: peptone3,glucose2, NaH₂PO₄·12H₂O1.5, KH₂PO₄·2H₂O0.3, MgSO₄·7H₂O0.06, CaCl₂0.01, pH7.2. Thefermentation parameters were as follow: cultured at35°C for30h, initial pH was adjusted to7.0,at inoculation of10%, at ventilatory volume of0.3m3/h. In such condition, the activity ofphospholipase can reach41.57U/mL.3. The enzyme was purified with precipitated by （NH4）2SO4, eluted in DEAE-52and G-75columns. The enzyme was identified as phospholipase A1. The purified enzyme has a molecularweight of20kDa with specific activity1195.24U/mg. The enzyme showed maximum activity（Vmax） of1.302mmol/L mg-1proteins with its corresponding Km value of11.11mg/mL. Theoptimal temperature and pH for phospholipase A1were35℃and7.5. The enzyme biocatalystretained100%and90%of the initial activity after storing5and8weeks at4℃, respectively.4. PLA1was modified with CC-mPEG. The optimum modification degree and recovery ofthe enzyme activity were14%and88.75%, respectively. The purified MPLA1has a molecularweight of25kDa. Compared with PLA1, the thermal stability, pH stability, affinity towards thesubstrate and catalytic efficiency value of MPLA1have been improved. The decrease of thefluorescence intensity at337nm and the changes of β-turns, rotation structure which could explainthe better stability of MPLA1.5. Polyvinyl alcohol （PVA）–alginate beads was used as a carrier for immobilized enzymes.Effects of PVA–alginate on phospholipase A1immobilization was evaluated by testing someparameters, such as immobilized phospholipase activity, immobilization yield and stability. Theresults showed that the optimal condition were as follow: beads prepared with10%PVA and2%sodium alginate,4%boric acid and2%calcium chloride solution, process time in boric acid solution was30minutes,10mg enzyme per100g PVA–alginate matrix, diameter of bead is4mm. The pH and temperature optimum for the PVA–alginate immobilized phospholipase A1werehigher than free phospholipase A1. The PVA-alginate beads showed better thermal stability andpH stability than free phospholipase A1. The enzyme immobilized in the beads remained morethan50%of the initial activity in the eighth cycle. The enzyme biocatalyst immobilized in thebeads retained more than90%of the initial activity after storing9weeks at4℃.6. Crude phospholipase, PLA1, MPLA1and IPLA1were used for rapeseed oil degumming.Effects of4enzymes were evaluated by phosphorus content of oil. The result revealed that theother three enzymes were more effective than crude phospholipase in oil degumming. The residualphosphorus content decreases steadily to less than10mg/kg, when the optimum degummingconditions are determined as time3h and PLA1（or IPLA1） dosage480U/kg. The residualphosphorus content even decreases under5mg/kg with3h and MPLA1dosage480U/kg. MPLA1and IPLA1have better effect of degumming after chemical modify and immobilization.