摘要
本文用超滤法从菠萝果实中提取高纯度菠萝蛋白酶，其活力达50-140×10~4u／g。用化学分析、红外光谱和热分析研究了光、热、pH值、水分、添加剂对菠萝蛋白酶活力稳定性的影响，得到了菠萝蛋白酶变性热焓、变性温度以及不同水分含量时的变性温度曲线，并求出了菠萝蛋白酶热失活动力学参数。结果表明：光对菠萝蛋白酶贮存过程中的稳定性影响明显，室温贮存10天后，避光和无避光酶活力保留率相差10％。菠萝蛋白酶在pH4.0-5.5时，酶活力较稳定，pH5.0时酶活稳定性最好。随着贮存温度的升高，菠萝蛋白酶活力保留率明显下降。菠萝蛋白酶液在30-60℃之间，活力保留率(R)与贮存温度(T)呈线性关系R=121.6-1.493T。粉状菠萝蛋白酶在70℃前加热活力下降幅度较小，在70℃以上加热，酶活力急剧下降。DSC研究结果表明，菠萝蛋白酶的平衡热变性温度为85.2℃，变性热焓为71.1J／g，水分含量对菠萝蛋白酶热变性温度影响明显，当水分含量为0至20％时，变性温度由84.09℃降为30.48℃，水分含量为20-60％，变性温度为30-35℃。红外光谱(IR)研究表明，菠萝蛋白酶在80℃下加热后，其图谱无大变化，说明其一级结构没有改变，变性可能是由于二级或三级结构变化等原因所致。研究了半胱氨酸与维生素C对菠萝蛋白酶的保护作用，结果表明半胱氨酸的稳定化作用与保护作用都优于维生素C。苯甲酸钠是菠萝蛋白酶的防腐剂，加入量为0.05％。EDTA可以消除有害金属影响，使菠萝蛋白酶活力保持率得到显著改善。β-环糊精和B-1都是高分子型菠萝蛋白酶稳定剂，能明显提高酶活力保留率。由半胱氨酸、β-环糊精和EDTA组成的复合稳定剂对菠萝蛋白酶稳
定性的影响相当明显。菠萝蛋白酶热失活动力学研究结果表明，在30oC下的热失
活速率常数K。，。＝0．2897八，酶的热失活半衰期T。，；＝2．39h；45OC时K：；；。＝0．5787／h，
T．；。＝1．20h 60 oC时 K”。＝1．245／h，T。。＝0．557h。菠萝蛋白酶的热失活活化能AE．
（30－45oC）为36．gkJ／mol，频率因子A，＝8．39x10‘／h；A巳（45－60oC）为22．5
kJ／mol，A。＝4．23xl0’／h。菠萝蛋白酶力入 B－1稳定齐后，酶失活速率常数变刁，
半衰期延长，活化能增加，从而使酶的稳定性提高。
The high purity Bromelain with the activity of 50-140×104u/g were extracted from the pineapple fruit by means of ultra-fine filter in this paper. The effects of photo, heat, pH, and the additives on the stability of Bromelain were studied by using chemical analyses, infrared spectroscopy and thermal analyses. The thermal inactivation point, the denatural enthalpy and the thermal inactivation curves of the bromelain of different water contents were obtained and the kinetic parameters of deactivation of Bromelain were also calculated. The results indicate that during the storage the effect of photo on the stability of Bromelain is obvious. The difference between the activity retention indexes of the bromelain stored for 10 days with and without light blocking is up to 10%. The Bromelain shows a relatively high stability at pH 5.0. With the rising of storage temperature, the activity retention index reduced obviously. When the liquid bromelain is incubated at the temperature ranging from 30 to 60℃, the
Bromelain activity retention(R)and the storage temperature (T) follow to the relation of R=121.6-1.493T, When heating at 70℃, the descent in activity of powdered Bromelain is small, whereas heating above 70℃, the activity is decreased abruptly. The DSC results indicate that the equilibrium thermal denaturation point of Bromelain is about 85.2℃ and the denaturation enthalpy is 71.1J/g. The effect of moist on the equilibrium inactivation temperature is obvious. When the water content is ranging from 0 to 20%, the equilibrium denaturation point is decreased from 84.09 to 30.48℃ and is rebounded slowly. The IR spectrum of Bromelain heating at 80℃ shows no obvious difference comparing with that of unheated Bromelain, indicating that the primary structure of Bromelain is not changeable and the denaturation of Bromelain should be assigned to the variations in the secondary and tertiary structures. Therefore,
the improvements of Half-cystine and Vitamin C on the stability of Bromelain were investigated and the results indicated that both the stabilization and protection of cysteine on Bromelain are superior to those of Vitamin C. The sodium benzoate can be used as the preservative for Bromelain and 0.05% dosage of sodium benzoate shows a certain protection on Bromelain. EDTA used to eliminate the effect of hazardous metals can improve the activity retention of Bromelain obviously. The macromolecular stabilizing agents such as p-cyclodextrin and B-1 can increase the protease activity retention index of Bromelain. A composite stabilizing system including cysteine, b-cyclodextrin and EDTA can improve the activity stability of bromelain obviously. The studies of the deactivation kinetics of Bromelain indicate that the thermal inactivation rate constant(K303) and deactivation half-life valve( r 303) of Bromelain at 30℃ are 0.2897/h and 2.39h,and those at 45℃ and 60℃ are K3180.5787/h, r 318=1.20h and KM =1.245/h, r 3
33=0.557h; respectively. The activation energies and frequency factors for 30-45℃ and 45-60℃ are as follows, AE, (30-45 ℃) =36.9 kJ/mol, A,=8. 39×10Yh ; AE2 (45-60℃) =22. 5 kJ/mol, A2=4. 23×103/h. By adding of B-1, the inactivation rate constant of Bromelain becomes small, the half-life is prolonged and the activation energy increases, indicating that the stability of Bromelain can be improved by B-1.
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