摘要
双水相萃取技术是近年来发展起来的一种新型的生化分离技术，该技术具有生物相容性高，分离条件温和，分离步骤简单，易于放大，且不存在有机溶剂残留等优点。已广泛用于各类酶、核酸、生长激素及各种植物中有效成分的提纯。
本文首先研究了构造双水相体系的成相组分及不同操作条件对菠萝蛋白酶活性的影响。实验表明，不同分子量的聚乙二醇(PEG)以及在双水相成相浓度范围内PE61000对菠萝蛋白酶活性影响不大；在选定的浓度范围，(NH_4)_2SO_4、阴离子型表面活性剂十二烷基硫酸钠(SDS)和阳离子型表面活性剂十六烷基三甲基溴化铵(CTAB)对菠萝蛋白酶活性无显著影响；菠萝蛋白酶活性适合的温度范围为20℃～50℃；适合的pH范围为4.0～10.0；对于加入一定浓度的金属盐，NaCl和NaBr对菠萝蛋白酶活性影响不大，而加入MgCl_2和CaCl_2明显使菠萝蛋白酶活性降低。
本文研究菠萝蛋白酶在PEG／(NH_4)_2SO_4双水相体系，阴、阳离子表面活性剂双水相体系和PEG／羟丙基变性淀粉双水相体系中的分配行为及影响菠萝蛋白酶分配的因素。
在PEG／(NH_4)_2SO_4双水相中研究了PEG分子量、PEG浓度、(NH_4)_2SO_4浓度、NaCl等因素对菠萝蛋白酶在两相中的分配系数、酶活性回收率
的影响。实验结果表明，在以下的实验条件下获得了较高的酶活性回
收率:PEG平均分子量1 000，浓度17%，(NH4)2504浓度14%，pH为7.0，
室温的情况下，其分配系数达到3.9，酶活性回收率可达到引%。
通过实验测定了SDS和CTA日水溶液混合体系双水相的相图，考察
了温度和外加盐NaBr对双水相形成区域的影响。在构造的阴、阳离子
表面活性剂双水相双水相体系中，研究了温度、pH等因素对菠萝蛋白
酶分配行为的影响。在选定的实验条件下，SDS/CTAB/H 20双水相体系
对菠萝蛋白酶有很好的萃取作用，在成相的两个区间，酶活性回收率
均可以达到80%以上。
研究了以木薯淀粉为原料，用环氧丙烷为醚化剂制备轻丙基淀粉
过程，以轻丙基淀粉的取代度为考察指标，考察了工艺条件和操作条
件对制备经丙基淀粉过程的影响。用制备的轻丙基淀粉与pEG构造双
水相体系，测定了该体系的部分相图，并对菠萝蛋白酶在该双水相中
的分配行为进行了初步研究。在轻丙基淀粉的质量分率为11%，P〔Gl oco
质量分率为10.5%，NaCI的浓度0.15mol/L的条件下，菠萝蛋白酶的
分配系数为3.23，菠萝蛋白酶活性回收率最高达到71%。
对三个双水相体系用于菠萝蛋白酶分离的优点及存在的问题进行
了分析，并提出了解决所存在问题的一些建议。本项目获得广西青年
科学基金(桂科青01 35029)和广西教育厅科研基金资助。
Aqueous two-phase extraction is a new bioseparation technique developed in recent years. The bioseparation technique has many advantages such as higher biocompatibility, less separation procedures, milder operating condition, easier engineering amplification, less residue of organic solvents in the product and so on. It has been widely used to purify different enzymes, nucleic acids, growth hormone and bioactive component in plants.
In this experiment, the effects of several factors on bromelain activity were investigated. These factors included the components of aqueous two-phase system and different operating conditions. The experiment results show that the different molecular masses of polyethylene glycol (PEG) and the condition of PEG1000 in the experiment range have little effect to bromelain activity. In the selected concentration range, (NH4)2SO4, anionic surfactant sodium dodecyl sulfonate (SDS) and cationic surfactant cetyl trimethyl amine bromide(CTAB) all have little effect to bromelain activity. The experiment results also show that the suitable temperature range of bromelain activity is from 20C to 50C and the suitable pH value range of reaction is from 4.0 to 10.0. In the range of experiment, NaCl and NaBr have no obviously effect on enzyme activity, but MgCl2 and CaCl2 all inhibited the bromelain activity.
It was investigated that the distribution behaviour and influence factors on distribution of bromelain in the PEG/(NH4)2SO4, SDS/CTAB, PEG/ hydroxypropyl starch aqueous two-phase systems.
In the PEG/(NH4)2SO4 aqueous two-phase system, the effects of the PEG
with different molecular masses, concentrations of PEG, concentrations of (NH4)2SO4i and NaCl on the distribution factor of bromelain in two phase, the volume ratio of the top phase to the bottom phase and the recovery of bromelain were investigated. Under the selected separation conditions, a high recovery of bromelain was obtained from the mixed fluid. The experiment results show that the suitable extraction conditions are 17% PEG1000, 14% (NH4)2SO4, pH 7.0, and the distribution coefficient of K =3.9 and the yield of bromelain can reach 91% at room temperature.
Through the experiment, the phase diagrams of SDS and CTAB aqueous mixtures were discussed. The influence of temperature and added salt sodium bromide(NaBr) on the aqueous two-phase regions have been observed. Besides, bromelain was extracted under the condition of different temperatures and pH values and added salt. The experiment results show that the yield of bromelain can reach 80% in the system of SDS/CTAB/H2O.
Finally, the synthetis method of hydroxypropyl starch using maniocca as raw material and the effect of technological conditions on the substitution were investigated. Using the hydroxypropyl starch and PEG construct aqueous two-phase system, the phase diagrams and distribution behaviour of bromelain in this aqueous two-phase system was determined. The experiment results show that the suitable extraction conditions were 11% PEG1000, 10.5% hydroxypropyl starch, pH 7.0, 0.15 mol/L NaCl, and the distribution coefficient of K =3.23 and the yield of bromelain can reach 71% at room temperature.
In this paper, the advantages and the disadvantages of using three aqueous two-phase systems to extract bromelain were analyzed and some suggests to existing questions were put in forward. The project is subsidized by the Young People Fund of Guangxi Science and Technology Department (No 0135029) and the Scientific Research Fund of Guangxi Education Department.
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