CN107446897A - 一种鱼肝脏粗酶液冻干粉及其制备方法 - Google Patents

一种鱼肝脏粗酶液冻干粉及其制备方法 Download PDF

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CN107446897A
CN107446897A CN201710675790.7A CN201710675790A CN107446897A CN 107446897 A CN107446897 A CN 107446897A CN 201710675790 A CN201710675790 A CN 201710675790A CN 107446897 A CN107446897 A CN 107446897A
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王海燕
王碧燕
韩大雄
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Third Institute of Oceanography SOA
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Abstract

本发明公开了鱼肝脏粗酶液冻干粉及其制备方法和应用。所述鱼肝脏粗酶液冻干粉为鱼肝脏粗酶液的干粉制品。本发明同时公开了该鱼肝脏粗酶液冻干粉的制备方法，通过冷冻干燥机将新制的鱼肝脏粗酶液制备成可长期保存的鱼肝脏粗酶液冻干粉。本发明的鱼肝脏粗酶液冻干粉可以应用于7‑乙氧基‑3‑异吩噁唑酮脱乙基酶、谷胱甘肽硫转移酶、超氧化物歧化酶等活性的检测以及还原型谷胱甘肽等含量的测定。

Description

一种鱼肝脏粗酶液冻干粉及其制备方法
技术领域
本发明属于生物化学领域，具体涉及一种鱼肝脏粗酶液冻干粉及其制备方法和应用。
背景技术
微粒体是细胞被匀浆破碎时，内膜系统的膜结构破裂后自己重新封闭起来的小囊泡(主要是内质网),这些小囊泡的直径大约100nm左右，是异质性的集合体，将它们称为微粒体。微粒体主要含有细胞色素P450酶系(CYP)和抗氧化系统蛋白。鱼肝脏粗酶液为鱼肝脏匀浆破碎离心后得到的上清液，主要由微粒体组成，含有多种生物代谢酶和蛋白，如7-乙氧基-3-异吩噁唑酮脱乙基酶(7-ethoxyresorufin-o-deethylase,EROD)、谷胱甘肽硫-转移酶(glutathione S-transferase,GST)、超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽(glutathione,GSH)等。研究发现细胞色素P450(如：EROD等)和抗氧化系统酶系(如：GST、SOD等)及抗氧化物质(如：GSH等)对环境中的污染物(如重金属、溴代阻燃剂、多环芳烃、多氯联苯等)有一定的响应关系。已有很多学者通过检测鱼肝脏粗酶液中的EROD、GST、SOD等酶的活性和GSH等的含量的变化来指示环境状况，但鱼肝脏粗酶液为液体状态，不方便储存与定量，且随着保存时间的延长，容易失活，而以鱼肝脏粗酶液冻干粉作为酶源进行酶活性及相关蛋白含量测定的研究尚未见报道。
发明内容
本发明的目的在于克服现有技术的不足之处，提供了鱼肝脏粗酶液冻干粉及其制备方法和应用。
本发明解决其技术问题所采用的技术方案之一是：
鱼肝脏粗酶液冻干粉为鱼肝脏粗酶液的冷冻干燥后得到的干粉制品。
其中，所述鱼肝脏粗酶液冻干粉为新制鱼肝脏粗酶液冷冻干燥后得到的干粉制品。
本发明解决其技术问题所采用的技术方案之二是：
上述鱼肝脏粗酶液冻干粉的制备方法，包括：
1)剖鱼取出肝脏后(避免弄破鱼胆)，用预冷的0.10～0.30％的KCl溶液洗涤，滤纸吸掉血渍。称重，按1:5(W/V)的比例加入预冷的PBS(pH 7.0～8.0),冰浴下匀浆。匀浆液在0～8℃下，8000～12000g离心10～30min，取上清液分装。
2)将制备得到的鱼肝脏粗酶液在-80～-40℃冰箱或液氮冻存24～72小时，至粗酶液完全冻结。
3)将冷冻的鱼肝脏粗酶液用冷冻干燥机在-80～-40℃及真空条件下干燥24～72小时至鱼肝脏粗酶液变为干燥的干粉状，于-20～-4℃保存。
本发明解决其技术问题所采用的技术方案之三是：
上述鱼肝脏粗酶液冻干粉在7-乙氧基-3-异吩噁唑酮脱乙基酶、谷胱甘肽硫-转移酶、超氧化物歧化酶等活性的检测以及谷胱甘肽等含量的测定上的应用。
本技术方案与背景技术相比，它具有如下优点：
1.本发明提供了鱼肝脏粗酶液冻干粉及其制备方法。
2.本发明通过冷冻干燥机将新制鱼肝脏粗酶液在-80～-40℃及真空条件下干燥24～72小时至鱼肝脏粗酶液变为干燥的干粉状，相比传统的鱼肝脏粗酶液而言，其干粉的形态比液体的形态更方便储存、定量，且稳定性较好，避免了传统冷冻保存的鱼肝脏粗酶液使用时冻融过程对酶活性的影响。
3.本发明的鱼肝脏粗酶液冻干粉可以用于7-乙氧基-3-异吩噁唑酮脱乙基酶、谷胱甘肽硫转移酶、超氧化物歧化酶等活性检测以及谷胱甘肽等含量的测定。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为实施例中得到的鱼肝脏粗酶液冻干粉样品图。
图2为实验例中得到的不同批次的鱼肝脏粗酶液与鱼肝脏粗酶液冻干粉的EROD酶的活性比较，其中横坐标为鱼肝脏酶源批次，纵坐标为EROD酶的活性。
具体实施方式
下面通过实施例具体说明本发明的内容。
实施例：制备鱼肝脏粗酶液冻干粉
剖鱼(罗非鱼)取出肝脏后(避免弄破鱼胆)，用预冷的0.15％的KCl溶液洗涤，滤纸吸掉血渍。称重，为13.8g，剪碎，按1:5(W/V)的比例加入预冷的PBS(pH 7.4)69.0mL,冰浴下匀浆3min。匀浆液在4℃下，10000g离心20min，取上清液分装。将制备得到的鱼肝脏粗酶液置于液氮冻存24小时。将冷冻的鱼肝脏粗酶液用冷冻干燥机在-80℃及真空条件下干燥36小时，取出干燥粉末，即为鱼肝脏粗酶液冻干粉，置于-20℃保存。
制备得到的鱼肝脏粗酶液冻干粉样品图如图1所示。
上述实施例均能实现下述实验例之技术效果：
实验例1：鱼肝脏粗酶液冻干粉的EROD酶活性测定
(1)样品准备：取10mg鱼肝脏粗酶液冻干粉，用200μL pH 7.5的PBS溶解成50mg/mL的粗酶液。
(2)活性测试：室温下，于96孔板中加入粗酶液20μL，0.01mol/L的PBS(pH 7.5)160μL，0.5mmol/L的7-乙氧基-3-异吩噁唑酮(ERF)溶液5μL，最后加入0.5mmol/L的NADPH溶液40μL，混匀.立即以532nm为激发波长，以580nm为发射波长，测定荧光强度。每30s测定一次荧光值，连续测定25min。
测得的7-乙氧基-3-异吩噁唑酮脱乙基酶活性为：0.59pmol/min/mg pro
实验例2：不同批次鱼肝脏粗酶液与鱼肝脏粗酶液冻干粉的EROD酶活性比较
(1)不同批次鱼肝脏粗酶液中的EROD酶活性测定：操作参照实验例1步骤(2)。
(2)不同批次鱼肝脏粗酶液冻干粉中的EROD酶活性测定：操作参照实验例1步骤(1)、(2)。
不同批次鱼肝脏粗酶液与鱼肝脏粗酶液冻干粉的EROD酶活性测定的实验结果如图2所示。
上述实验结果显示：各批次制得的鱼肝脏粗酶液冻干粉的EROD酶活性均不比粗酶液的活性低，基本上冻干粉活性为粗酶液活性的77-120％，说明冻干粉作为粗酶液新的储存形式具有可行性。
实验例3：鱼肝脏粗酶液冻干粉的GST酶活性测定
取10mg鱼肝脏粗酶液冻干粉，用200μL pH 6.5的PBS溶解成50mg/mL的粗酶液。室温下，于96孔板中加入粗酶液10μL，0.01mol/L的PBS(pH 6.5)170μL，30mmol/L的GSH溶液10μL，最后加入30mmol/L的CDNB溶液10μL，混匀。非酶促反应不加酶液，立即在340nm下每30s测定一次吸光值，连续测定5min。
测得的GST酶活性为：15U/mL
实验例4：鱼肝脏粗酶液冻干粉的SOD酶活性测定
取10mg鱼肝脏粗酶液冻干粉，用200μL pH 8.2的PBS溶解成50mg/mL的粗酶液。室温下，于96孔板中加入0.01mol/L的Tris-HCl溶液(pH 8.2)100μL，超纯水90μL，最后加入4.5mmol/L的邻苯三酚溶液10μL,混匀.立即在325nm下每30s测定一次吸光值，测定5min。计算邻苯三酚自氧化速率，按以上步骤加入上清液10μL，使自氧化速率抑制约50％。
测得的SOD酶活性为：110U/mL
实验例5：鱼肝脏粗酶液冻干粉的GSH含量测定
取10mg鱼肝脏粗酶液冻干粉，用200μL pH 8.0的PBS溶解成50mg/mL的粗酶液。室温下，于96孔板中加入10μL粗酶液，180μL 0.01mol/L的磷酸缓冲溶液(pH 8.0)和10μL邻苯二甲醛的乙醇溶液(1mg/mL)，充分混匀，室温下放置20min.以340nm为激发波长，在430nm下测定荧光强度。
测得的GSH的含量为：0.8mmol/L
以上所述，仅为本发明较佳实例而已，故不能依此限定本发明实施的范围，即依本发明专利范围及说明书内容所作的等效变化与修饰，皆应仍属于本发明涵盖的范围内。

Claims (4)

1.鱼肝脏粗酶液冻干粉，其特征在于：所述肝脏粗酶液冻干粉为鱼肝脏粗酶液冷冻干燥后的干粉制品。
2.根据权利要求1所述的鱼肝脏粗酶液冻干粉，其特征在于：所述鱼肝脏粗酶液冻干粉为新制鱼肝脏粗酶液冷冻干燥后得到的干粉制品。
3.根据权利要求1所述的鱼肝脏粗酶液冻干粉的制备方法，其特征在于：
1)剖鱼取出肝脏后，用预冷的0.10～0.30％的KCl溶液洗涤，滤纸吸掉血渍；称重，按1:5(W/V)的比例加入预冷的PBS(pH 7.0～8.0),冰浴下匀浆；匀浆液在0～8℃下，8000～12000g离心10～30min，取上清液分装；
2)将制备得到的鱼肝脏粗酶液在-80～-40℃冰箱或液氮冻存24～72小时，至粗酶液完全冻结；
3)将冷冻的鱼肝脏粗酶液用冷冻干燥机在-80～-40℃及真空条件下干燥24～72小时至鱼肝脏粗酶液变为干燥的干粉状，于-20～-4℃保存。
4.根据权利要求1所述的鱼肝脏粗酶液酶干粉在包括7-乙氧基-3-异吩噁唑酮脱乙基酶、谷胱甘肽硫转移酶、超氧化物歧化酶在内的酶活性的检测以及还原型谷胱甘肽含量的测定上的用途。
CN201710675790.7A 2017-08-09 2017-08-09 一种鱼肝脏粗酶液冻干粉及其制备方法 Pending CN107446897A (zh)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002062969A9 (en) * 2001-02-06 2002-10-17 Massachusetts Inst Technology Cellular reprogramming in peptide hydrogel and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002062969A9 (en) * 2001-02-06 2002-10-17 Massachusetts Inst Technology Cellular reprogramming in peptide hydrogel and uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
曾青兰 等: "《生物制药工艺》", 29 February 2012, 华中科技大学出版社 *
朱智 等: "EROD、GST测试法在评价污染物生态效应的研究", 《中国科技论文在线》 *

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Application publication date: 20171208