南极磷虾类胰蛋白酶分离纯化及性质鉴定

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南极磷虾类胰蛋白酶分离纯化及性质鉴

定

摘要

南极磷虾作为地球上最大的单种生物资源之一，被人们认为是地球上最有希望，

也是最后一个动物蛋白库，对于解决人口膨胀、食品短缺等问题具有美好的前景。然而

由于南极磷虾具有独特的消化系统及其体内功能强大的酶系统使得南极磷虾能够迅速

自溶，这就造成南极磷虾肉质软化和鲜度下降，南极磷虾死后即使在低温的条件下品

质下降也很迅速，这就给南极磷虾的商业化加工及利用增添了很多的困难。因此，研

究分析南极磷虾蛋白酶的性质具有十分重大的意义。

本实验选取南极磷虾为原料，分析研究了南极磷虾粗酶的性质，并对在南极磷虾

自溶过程中起主要作用的蛋白酶进行了分离纯化及部分性质研究。本课题的主要内容

和结论如下：

1. 南极磷虾粗酶性质的初步研究

南极磷虾粗酶主要含有 3种蛋白酶，最适 pH 值分别为 3.0、6.0、8.0；各最适 pH 值

条件下最适温度分别为 40、50、50℃。苯甲基磺酰氟(PMSF)和大豆胰蛋白酶抑制剂(STI)

对南极磷虾粗酶活性有较强的抑制作用，最高抑制率分别为 81%和75%，抑制范围分

别在 pH 3.0～9.0 和pH 6.0～10.5，EDTA 对其的抑制作用很小，约为 12%。南极磷虾

粗酶含有类胰蛋白酶并且可能不存在金属蛋白酶。Cu2+和Ba2+对粗酶活性的抑制作用较

强，抑制率分别为 74%和28%。Ca2+对粗酶活性几乎没有抑制作用，而 Mg2+对粗酶活

性有一定的激活作用。

2. 南极磷虾类胰蛋白酶分离纯化及部分性质研究

粗酶液经过硫酸铵分级沉淀，DEAE-Sepharose F. F.阴离子交换柱，Sephacryl S-

200 HR 凝胶层析后得到电泳纯级南极磷虾类胰蛋白酶，该酶比活力为 13.63U/mg，纯

化倍数为 45，产率为 17%；该酶的分子量大约为 28 kD。南极磷虾类胰蛋白酶的最适

pH 和最适温度分别为 8.0 和50℃；并且有较好的 pH 稳定性和热稳定性。

PMSF，TLCK，STI 对南极磷虾类胰蛋白酶有较强的抑制作用，抑制率分别为

86%，93%，95%；TPCK，EDTA 对该酶的活性基本没有影响。

3. STI 对南极磷虾类胰蛋白酶抑制动力学研究

探讨了大豆胰蛋白酶抑制剂（STI）对南极磷虾类胰蛋白酶的抑制动力学。实验结

果表明：南极磷虾类胰蛋白酶的 Km和Vmax 分别为 0.155 mmol/L 和8.44 μmol/(L·min)。

酶活力随着 STI 溶液浓度增大而减小，IC50 约为 2.8 μg/mL；低浓度的 STI 溶液对酶的

抑制作用为竞争性慢可逆抑制。正向微观速度常数 k+0 为0.184 mmol/(L·min)，逆向微

观速度常数 k-0 为0.0394 min-1，k+0 和k-0 代表了抑制反应过程中的正向微观抑制速度常

数和逆向微观抑制速度常数。k+0 越大，酶失活越快；k-0 越小，酶恢复活性越慢。k+0 比

k-0 大3.7 倍，酶和抑制剂结合速度远大于酶和抑制剂复合物的速度。STI 溶液浓度逐渐

增大，该酶最终将完全失活。

关键词：南极磷虾类胰蛋白酶分离纯化抑制动力学

ABSTRACT

As one of the largest fisheries resources of monophyletic species in the world,

Euphausia superba was consindered as the most promising and the last animal protein

resources in the world, which could solve the problems of human expansion and food

shortage. However, Euphausia superba had special digestion systems and strong

enzymatic functions in vivo, which made it autolyze fast and the qualities declined

quickly, even in the low temperature after its post mortem. So it made sense to analysis

the characterizations of protease from Antarctic krill.

With Euphausia superba as raw material, the characterizations of autolysis

enzymes from Antarctic krill were researched. Moreover, a protease, which plays a role

on autolysis of Antarctic krill, was purified from Antarctic krill and its partial

characterizations were studied. The main work and results of this research were as

follows:

1. Initial Research on crude enzymes from Antarctic Krill

Crude enzymes from Antarctic krill were studied systematically by optimum pH,

optimum temperature, effects of inhibitors and metal ions. The results showed that the

crude enzymes from Antarctic krill were mainly composed of three kinds of protease

and the optimum working pHs were 3.0, 6.0, and 8.0, respectively. The optimum

working temperatures were 40℃, 50℃ and 60℃, respectively. PMSF (phenylmethyl

sulfonyl fluoride) and STI (Soybean trypsin inhibitor) can obviously inhibit the enzyme,

and the pH, efficiently inhibit the activity of the enzyme, ranges from 3.0 to 9.0 and 6.0

to 10.5, respectively. Cu2+ and Ba2+ can also strongly inhibit the activity of the enzyme,

Ca2+ has little inhibition, but the enzyme can be activated by Mg2+.

2. Partial purification and characterization of trypsin-like from Antarctic krill

(Euphausia Superba)

A trypsin-like, which plays a role on autolysis of Antarctic krill, was purified from

Antarctic krill and its major physical and chemical characteristics were studied. The

purified enzyme was obtained after 30%~70% ammonium sulfate grading precipitation,

DEAE-Sepharose Fast Flow anion-exchange chromatography and Sephacryl S-200 HR

chromatography. The enzyme was confirmed by a single band on SDS-PAGE and was

purified by 45-fold with the yield of 17%. The relative molecular mass of the enzyme

was determined to be about 28 kD. The characterization of the enzyme was made by

using casein as substrate to measure activity. The optimum pH and temperature of the

enzyme were 8.0 and 50℃, respectively. The enzyme was stable at pH ranging from 7.0

to 9.5 and the temperature blow 45℃; PMSF (phenylmethyl sulfonyl fluoride), STI

(strontium bismuth titanate) and TLCK (N-α-Tosyl-L-Lysine Chloromethyl Ketone) can

obviously inhibit the enzyme.

3. Kinetics of inhibition of trypsin-like from Antarctic krill by STI

The kinetics of inhibition of trypsin-like from Antarctic krill by STI has been

studied. The results showed that the value of Km and Vmax were 0.155 mM and 8.44

μM·min-1, respectively. As the concentration of STI increased, the activity of trypsin-

like was decreased with IC50 of 2.8μg/mL. STI could slowly, reversibly and

competitively inhibit the enzyme activity at low concentration. The rate constant of the

forward inactivation (k+0), which is 0.184 mM·min-1, is about five times as much as that

of the reverse reactivation (k−0), which is 0.0394 min-1. Therefore, when the STI

concentration was sufficiently large, the enzyme would be completely inactivated.

Key word: Antarctic krill, Trypsin-like, Purification, Inhibition

kinetics

目  录
中文摘要
ABSTRACT
第一章  绪  论...............................................................................................................1
§1.1  南极磷虾简介.....................................................................................................1
§1.1.1 南极磷虾的生命周期...................................................................................1
§1.1.2  南极磷虾氟含量分析..................................................................................2
§1.1.3  南极磷虾营养成分分析..............................................................................3
§1.1.4 南极磷虾自溶...............................................................................................4
§1.1.5  南极磷虾酶及其应用..................................................................................4
§1.2  胰蛋白酶研究状况分析.....................................................................................6
§1.2.1  胰蛋白酶的分布..........................................................................................6
§1.2.2  胰蛋白酶分离纯化......................................................................................6
§1.2.3  分离纯化方法..............................................................................................6
§1.2.4  虾类胰蛋白酶的生化特性..........................................................................9
§1.3  立题背景及意义.................................................................................................10
§1.4  本课题研究内容.................................................................................................11
第二章  南极磷虾粗酶性质的初步研究.....................................................................12
§2.1  材料与方法.........................................................................................................12
§2.1.1  材料与试剂..................................................................................................12
§2.1.2  仪器与设备..................................................................................................12
§2.2   实验方法............................................................................................................13
§2.2.1   粗酶液制备.................................................................................................13
§2.2.2  酶活力测定..................................................................................................13
§2.2.3  粗酶最适 pH 值测定....................................................................................13
§2.2.4  粗酶最适温度测定......................................................................................13
§2.2.5  抑制剂与金属离子对粗酶活性的影响......................................................13
§2.3  结果与分析.........................................................................................................14
§2.3.1  pH 值对南极磷虾粗酶活性的影响............................................................14
§2.3.2  温度对南极磷虾粗酶活性的影响..............................................................15
§2.3.3  抑制剂在不同pH 值条件下对南极磷虾粗酶活性的影响........................15
§2.3.4  金属离子对南极磷虾粗酶活性的影响......................................................17
§2.4  本章小结.............................................................................................................17
第三章  南极磷虾类胰蛋白酶分离纯化及部分性质研究.........................................18
§3.1  材料与方法.........................................................................................................18
§3.1.1  原料..............................................................................................................18
§3.1.2  实验试剂与设备..........................................................................................18

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摘要：

南极磷虾类胰蛋白酶分离纯化及性质鉴定摘要南极磷虾作为地球上最大的单种生物资源之一，被人们认为是地球上最有希望，也是最后一个动物蛋白库，对于解决人口膨胀、食品短缺等问题具有美好的前景。然而由于南极磷虾具有独特的消化系统及其体内功能强大的酶系统使得南极磷虾能够迅速自溶，这就造成南极磷虾肉质软化和鲜度下降，南极磷虾死后即使在低温的条件下品质下降也很迅速，这就给南极磷虾的商业化加工及利用增添了很多的困难。因此，研究分析南极磷虾蛋白酶的性质具有十分重大的意义。本实验选取南极磷虾为原料，分析研究了南极磷虾粗酶的性质，并对在南极磷虾自溶过程中起主要作用的蛋白酶进行了分离纯化及部分性质研究。本课题的主要内容和...

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