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4.2 果胶酶活力测定
4.2.1 原理
果胶酶水解果胶,生成半乳糖醛酸。半乳糖醛酸具有还原性糖醛基,可用次亚碘酸法定量测定,以此来表示果胶酶的活性。
4.2.2 试剂和溶液
a.果胶粉(Sigma公司出品) 10g/L水溶液
称取果胶粉1.0000g,精确至0.0002g,加水溶解,煮沸,冷却。如有不溶物则需进行过滤。pH至3.5,用水定容至100ml,在冰箱中贮存备用。使用时间不超过三天;
b.硫代硫酸钠标准溶液c(Na2S2O3)=0.05mol/L 按GB 601配制与标定 0.1mol/L溶液。使用时准确稀释一倍;
c.碳酸钠溶液c(1/2Na2CO3)=1mol/L 按GB 601配制;
d.碘标准溶液c(1/2I2)=0.1mol/L 按GB 601配制与标定,贮存于棕色瓶中;
e.硫酸溶液c(1/2H2SO4)=2mol/L 取浓硫酸(d=1.84)5.6ml,缓慢加入适量水中,冷却后用水定容至100ml,摇匀;
f.可溶性淀粉指示液(10g/L) 按GB 603配制;
g.0.1mol/L柠檬酸—柠檬酸钠缓冲液(pH=3.5)
甲液 称取柠檬酸(C6H8O7·H2O)21.01g,用水溶解并定容至1000mL;
乙液 称取柠檬酸三钠(C6H5Na3O7·2H2O)29.41g,用水溶解并定容至1000ml;
取甲液140ml、乙液60ml,混匀,缓冲液的pH应为3.5(用pH计调试)。
4.2.3 仪器
a.比色管 25ml;
b.恒温水浴 50±0.2℃;
c.容量瓶 25ml、50ml、100ml、200ml、250ml;
d.碘量瓶 250ml;
e.吸管 1ml、5ml;
f.滴定管 25ml。
4.2.4 试验程序
4.2.4.1 制备酶液
a.固体酶 用已知重量的50ml小烧杯,称取样品1.0000g,精确至0.0002g,以少量柠檬酸—柠檬酸钠缓冲液(pH=3.5)溶解,并用玻璃棒捣研,将上清液小心倾入适当的容量瓶中,沉渣再加少量缓冲液,反复捣研3~4次,最后全部移入容量瓶,用缓冲液定容,摇匀,以四层纱布过滤,滤液供测试用;
b.液体酶 准确吸取浓缩酶液1.00ml于一定体积的容量瓶中,用柠檬酸—柠檬酸钠缓冲液(pH=3.5)稀释定容;
c.固体酶或浓缩酶液均须按附录A要求,准确稀释至一定倍数,酶液浓度应控制在消耗0.05mol/L硫代硫酸钠标准溶液(A-B)之差在0.5~1.0ml范围内。必要时可先做预备试验。
4.2.4.2 测定
a.于甲、乙两支比色管中,分别加入10g/L果胶溶液5ml,在50±0.2℃水浴中预热5~10min;
b.向甲管(空白)中加柠檬酸—柠檬酸钠缓冲液(pH=3.5)5ml;乙管(样品)中
加稀释酶液1ml、柠檬酸—柠檬酸钠缓冲液(pH=3.5)4ml,立刻摇匀,计时。在此温度下准确反应0.5h,立即取出,加热煮沸5min终止反应,冷却;
c.取上述甲、乙管反应液各5ml放入碘量瓶中,准确加入1mol/L碳酸钠溶液1ml、0.1mol/L碘液5ml,摇匀,于暗处放置20min;
d.取出,加入2mol/L硫酸溶液2ml,用0.05mol/L硫代硫酸钠标准溶液滴定至浅黄色,加淀粉指示液3滴,继续滴定至蓝色刚好消失为其终点,记录甲管(空白)、 乙管(样品)反应液消耗硫代硫酸钠标准溶液的体积。同时作平行样品测定。
4.2.5 试验结果的计算
1g酶粉或1ml酶液在50℃、pH3.5的条件下,1h分解果胶产生1mg半乳糖醛酸为一个酶活单位。
10
X=(A-B)×c×0.51×194.14×n×—————=(A-B)×c×n×396.05 .......(1)
5×1×0.5
式中X——样品的酶活力,u/g(u/ml);
A——空白消耗硫代硫酸钠标准溶液的体积,ml;
B——样品消耗硫代硫酸钠标准溶液的体积,ml;
c——硫代硫酸钠标准溶液的浓度,mol/L;
0.51——1毫摩尔硫代硫酸钠相当于0.51毫摩尔的游离半乳糖醛酸;
194.14——半乳糖醛酸的毫摩尔质量,mg;
n——酶液稀释倍数;
10——反应液总体积,ml;
5——滴定时取反应混合物的体积,ml;
1——反应时加入稀释酶液的体积,ml;
0.5——反应时间,h。
所得结果应表示至整数。
注:果胶暂定用Sigma公司产品为标准底物。若购不到,使用其他厂产品时, 必须作对照试验。
4.2.6 结果的允许差
平行试验,滴定时消耗硫代硫酸钠标准溶液的体积(毫升)不得超过0.05ml。
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