【作者】 张跃；

【导师】 麻建国；

【作者基本信息】 江南大学 ， 食品科学， 2007， 硕士

【摘要】 果胶酯酶（PE）广泛存在于植物、霉菌和细菌中。在一些水果和蔬菜的加工中,天然存在的果胶酯酶起保护果蔬质构的作用。高纯度的果胶酯酶（PE）酶制剂主要应用于果蔬保藏、制备低甲氧基果胶、研究酶的行为模式以及确定果胶结构和功能性质之间的基本关系等。本研究以商品果胶酶作为原料,探索一种制备高纯度果胶酯酶（PE）制剂的生产工艺。采用超滤和SP Sepharose（?）Fast Flow强阳离子交换凝胶色谱结合的方法复合果胶酶制剂Pc-3中的果胶酯酶和聚半乳糖醛酸酶进行分离。研究结果表明,超滤后,果胶酯酶活力由11.22U/ml上升至98.12U/ml,总酶活力由1402.5下降到1349.15,活力损失3.8%;聚半乳糖醛酸酶酶活由17.46U/ml上升至156.35U/ml,总活力由2182.5下降到2110.73,活力损失了3.3%;酶活比（PE:PG）没有发生改变,均为0.64。在pH4.2时,粗酶中果胶酯酶的吸附率为43.08%,聚半乳糖醛酸酶的吸附率为71.08%。在0.25ml/min的洗脱流速下得到最高的第一部分酶活比（PE:PG）:3.69,但是果胶酯酶的总得率仅为55.97%,而且整个洗脱结果也并未将两种酶完全分离。研究了PC-3中果胶酯酶（PE）和聚半乳糖醛酸酶（PG）的热稳定性,发现粗酶中果胶酯酶的最适温度为43℃,聚半乳糖醛酸酶有两个最适温度,分别为30℃和50℃。不同温度下两种酶的热失活研究表明,果胶酯酶是热不稳定的,聚半乳糖醛酸酶分为热稳定和热不稳定两个部分。在55℃加热10min后,有78.16%的果胶酯酶酶活力保留下来,聚半乳糖醛酸酶残留酶活为9.98%,两种酶的失活均为不可逆失活。说明通过适当的热处理可以改变粗酶制剂中果胶酯酶和聚半乳糖醛酸酶的酶活比例,从而得到高果胶酯酶酶活的酶制剂。研究了抑制剂对PC-3中果胶酯酶（PE）和聚半乳糖醛酸酶（PG）抑制效果,发现高氯化铁（FeCl₃）,七水合硫酸亚铁（FeSO₄·7H₂O）和L-谷氨酸钠对聚半乳糖醛酸酶酶活均具有明显的抑制作用,甘氨酸并不具有很好的抑制能力。同时上述几种试剂对果胶酯酶都没有抑制作用。而脲对两种酶均有抑制作用。从食品安全和抑制效果两个角度出发,七水合硫酸亚铁（FeSO₄·7H₂O）是一种较好的聚半乳糖醛酸酶酶活抑制剂。在其浓度为1mmol/L的时候,PG酶活迅速下降到原酶活的33.15%,在浓度为5mmol/L时,PE残留酶活仍高达92.7%。通过正交实验,得到制备高纯果胶酯酶制剂的最优工艺条件是:温度55℃,七水合硫酸亚铁（FeSO₄·7H₂O）浓度1mol/L,时间10min。在此条件下,果胶酯酶酶活残余为80.57%,而聚半乳糖醛酸酶的活力仅仅残留了8.29%。研究了自制果胶酯酶（PE）制剂对菠萝颗粒硬度的影响,并将实验结果与Rapidase（?）FP Super进行了比较。正交实验表明自制果胶酯酶制剂应用于菠萝果粒的最优条件是果胶酯酶溶液浓度为0.5%,乳酸钙浓度为0.3%,处理时间是25min。在此条件处理,放置一周后,菠萝颗粒硬度较未处理的样品增加了84.29%。在相同果胶酯酶活力的处理条件下,Rapidase（?）FP Super使菠萝颗粒的硬度增加了1.63倍。更多还原

【Abstract】 Pectinesterase（PE） exists in the plant,fungi and the germ widely.In some fruits and vegetable processing,the natural PE has the function of protecting the texture.The PE production of high pure degree is mainly applied to preserve the fruit and vegetable,product low methyl oxygen pectin,study the basic relationship of PE’s behavior pattern and the pectin’s structure and function property,and so on.This research investigated a kind of production craft that makes a high pure degree PE production from commercial pectinase preparation.The PE and Polygalacturonase（PG） of Pc-3 were isolated by ultrafiltration and SP Sepharose（?） Fast Flow—one sort of intense cationic gel filtration chromatography.After ultrafiltration,the PE activity was raised from 11.22U/ml to 98.12U/ml and the total activity was descended from 1402.5 to 1349.15.The PG activity was raised from 17.46U/ml to 156.35U/ml and the total activity was descended from 2182.5 to 2110.73.But the activity ratios（PE:PG） are both 0.64.At pH4.2,the adsorption ratio of PE in the crude enzyme solution was 43.08%and that of PG was 71.08%.The maximal activity ratio（PE:PG） was obtained at 0.25ml/min and the value was 3.69.But the yield of PE was only 55.97%.They were not separated completely.The thermal Characterization of PE and PG of Pc-3 was studied.The optimal temperature of PE is 43℃.PG has two optimal temperatures:30℃and 50℃.The study on stability of PE and PG at different temperatures declared that PE is thermal unstable and PG has two sorts:thermal stable and thermal unstable.After treated 10min at 55℃,PE activity retained 78.16%and PG activity remained only 9.98%.It proves we can obtain high PE activity production through proper thermal treatment.The inhibiting effect on PE and PG activity of commercial pectinase preparation by Iron（Ⅲ） chloride hexahydrate,Iron（Ⅱ） sulfate heptahydrate,glycine,L-Glutamic acid sodium salt and urea was studied. Iron（Ⅲ） chloride hexahydrate,Iron（Ⅱ） sulfate heptahydrate and L-Glutamic acid sodium salt have the obvious and similar inhibiting effect:their inhibiting abilities increased companied with their concentration in a certain range and kept unalterable out of the range.Glycine has unstable inhibiting ability.The above four reagents have no inhibiting effect on PE.Urea has the inhibiting effect on two enzymes and the effect on PG is stronger.Considering to safe,FeSO₄·7H₂O is the best inhibitor among them.When it’s concentration was 1mmol/L,the PG activity dropped off to 33.15%and When it’s concentration was 5mmol/L,the PE activity remained 92.7%.The optimum processing conditions of high PE activity production was studied and obtained as-following:temperature:55℃,FeSO₄·7H₂O concentration:1mol/L,time:10min.At this condition,the PE residual activity（%） was 80.57%and the PG residual activity（%） was 8.29%respectively。The influence on the hardness of pineapple grain by the self-made PE production was studied.Under the optimal condition（PE concentration:0.5%,the lactic acid calcium concentration:0.3%,time:25min.）, the hardness increased 84.29%after bestowed 1 week.Rapidase（?） FP Super made the hardness raised 163%with the equal PE activity.更多还原