摘要
酶法脱胶是油脂精炼过程中的一种生化脱胶技术，相比传统脱胶方法，具有高效、绿色环保等优点。目前市场上使用的磷脂酶存在反应条件要求严格，不能回收利用等问题，限制了磷脂酶在工业生产中的应用。
本研究以实验室保存的Bacillus cereus sp. AF-1为出发菌株，以提高发酵液磷脂酶活力为目的，优化发酵工艺得到较高酶活力的粗酶液，通过分离纯化得到磷脂酶并对其性质进行分析评价；采用化学修饰、固定化等方法来提高磷脂酶的稳定性和重复使用性，以期为磷脂酶的开发利用探索新的途径。主要研究内容与结果如下：
1.采用NaOH滴定法测定磷脂酶活力，通过单因素实验和响应面实验优化，确定磷脂酶活力测定的最优条件为：底物质量浓度4g/100mL、反应终点pH值9.0、酶液稀释倍数100倍、反应pH值5.1、反应温度53℃、反应时间8min。结果表明NaOH滴定法是一种准确、快捷的测定磷脂酶活力的方法。
2.以Bacillus cereus sp. AF-1为出发菌株，采用发酵法得到粗酶液，通过对培养基组成和发酵工艺条件的优化，并从摇瓶水平扩大到15L发酵罐水平，得到的最佳培养基为：葡萄糖浓度为2%，蛋白胨浓度为3%，无机盐的组成为：Na_2HPO_4·12H_2O1.5%，KH_2PO_4·2H_2O0.3%，MgSO_4·7H_2O0.06%，CaCl_20.01%；15L发酵罐最佳发酵条件为：发酵时间为30h、发酵温度为35℃、发酵液pH为7.5、接种量为10%、通气量为0.3m3/h。此条件下得到的粗酶液酶活力为41.57U/mL。
3.粗酶液通过硫酸铵沉淀及透析、DEAE-52离子交换层析、葡聚糖凝胶G-75凝胶层析等方法进行分离纯化，得到的磷脂酶类型为PLA1，其分子量为20kD，酶活力为1195.24U/mg，磷脂酶A1的Km和Vmax分别为11.11mg/mL和1.302mmol/L mg；最佳作用温度和pH值分别为35℃和7.5；4℃下贮藏5周，磷脂酶的酶活力没有变化，贮藏8周，能保持90%以上的酶活力。
4.采用化学修饰剂CC-mPEG对磷脂酶进行修饰，当修饰率为14%，酶活力回收率为88.75%时，得到的修饰酶MPLA1分子量约为25kD，MPLA1的热稳定性、pH稳定性、对底物的亲和力以及催化效率都得到了提高。酶在337nm处最大荧光强度的降低和圆二色谱β-折叠的降低和回转的升高，从MPLA1结构上进一步说明了修饰酶比PLA1具有更好的稳定性。
5.采用聚乙烯醇-海藻酸钠为载体，以固定化酶的活力，酶活力回收率和稳定性等参数为主要评价指标，对磷脂酶进行固定化，确定最佳工艺条件为：载体聚乙烯醇和海藻酸钠的浓度分别为10%和2%，交联剂硼酸和氯化钙的浓度分别为4%和2%，固定化时间为30min，酶的添加量为10mg/100g，固定化酶(IPLA1)的粒径为4mm；相比游离酶，IPLA1的最佳作用温度和pH值以及热稳定性和pH稳定性较游离酶得到提高；IPLA1重复使用8次，剩余酶活力仍然超过50%；在4℃下贮藏9周，IPLA1能保持90%以上的酶活力。
6.粗酶、纯化后的磷脂酶（PLA1）、修饰酶（MPLA1）和固定化酶（IPLA1）用于菜籽毛油脱胶，以毛油中磷含量为依据，对四种酶的脱胶效果进行比较，研究结果表明：粗酶能将毛油中磷含量降低到10mg/kg以下，但酶的使用量和作用时间均高于其他三种酶；当添加量为480U/kg，处理3h时，PLA1和IPLA1将毛油中磷的含量降低到10mg/kg以下，MPLA1将毛油中磷的含量降低到5mg/kg以下。PLA1经化学修饰和固定化后得到的MPLA1和IPLA1具有较好的脱胶效果。
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, NaH_2PO_4·12H_2O1.5, KH_2PO_4·2H_2O0.3, MgSO_4·7H_2O0.06, CaCl_20.01, 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.
引文
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