CN1181182C - 快速测量酶活性的装置 - Google Patents
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Abstract
本发明涉及用于快速测量固体饲料中酶活性的装置，包括(i)用于接受被试验样品的容器；(ii)对需要测量活性的酶有特异性的试剂；和(iii)使酶溶解的缓冲液。
Description
本发明涉及快速测量固体饲料中酶活性的装置，包括(i)用于容纳试验样品的容器，(ii)对需要测量的酶有特异性的试剂和(iii)溶解酶的缓冲液。
饲料优选的是测量前不作处理的固体饲料。
用于畜牧业的饲料通常补充有各种酶，其作用主要是改进所给予饲料的可消化性。这些酶通常以液体形式喷洒在饲料上，EP 0,789,291作了特别的介绍。酶也可以粉末形式加入饲料。
这样引起了两个问题，第一个是检查加入饲料的酶分布的均匀性，第二个问题是快速并方便地评估加入饲料的酶的活性。饲料制造者和希望检查他们给予动物的饲料的质量的饲养者特别提出了这些问题。迄今为止，可以在实验室里测量酶活性，这样造成了后勤紧张和延误，在马上需要结果时这些限制是真正的阻碍。
本发明通过提供用于测量喂养动物的任何富酶饲料的酶活性的装置来解决该问题。该装置的测量基于比色反应，可以定性测定和半定量测量试验样品的酶活性。
图1代表本发明用于测量酶活性装置的实施方案，它为柱的形式。
下面结合上述附图进行描述。
实施本发明的装置包括用于容纳试验样品的容器、对需要测量其活性的酶有特异性的试剂和用于溶解所述酶的缓冲液。
该装置的容器可为，但不限于，由标有刻度的窄的底部(11)和用于向柱里引入各种试剂并在搅拌时对其混合的宽的漏斗状顶部(12)构成的柱(图1)。柱也可装有防漏的开口和关闭系统(13)，如塞子，它通过突片(tab)(131)连接到柱体上。
容器也可由单用途的试管构成(图2)。
容器可由诸如单用途塑料的合成材料构成。
容器优选地在其基部包括可分离的突出物(14)，让其内含物的液体部分流出。有利的是突出物上备有留住饲料中固体小块收缩部(141)。
酶活性的测量基于Azo方法的显色反应。Azo方法的显色反应原理基于与发色团连接的酶的特征底物的酶水解。反应产生了可溶的低聚物，它使介质变蓝。在590纳米处测量介质的吸收度。
用于该装置的试剂是由与发色团连接的酶催化反应的底物。因此，酶水解反应释放了发色底物。
该装置也包括用于溶解喷洒在饲料上的酶并使酶保持在其最佳pH值的缓冲液。
可提及(仅供例举，不作限定)用于测量木聚糖酶活性的装置。
为了测量木聚糖酶的活性，所用的试剂是“Oat spelt Xylan RemazolBrilliant Blue R”或“Xylazyme AX”(Megazyme公司出售，由与染料连接的燕麦或小麦阿拉伯木聚糖构成)。
所用的缓冲液选自乙酸/乙酸钠；甘氨酸盐酸盐/甘氨酸；乌头酸/氢氧化钠；甲酸/甲酸钠缓冲液。
也可提及用于测量β-葡聚糖酶活性的装置，该测量也是基于Azo方法的显色反应。
可使用的底物由1，3：1，4-β-D-葡聚糖构成，具有Remazol Brilliant BlueR和BetaGlucazyme(由Megazyme公司出售)，包含与天青精(azurine)结合的β-葡聚糖。
所用的缓冲液选自乙酸/乙酸钠；甘氨酸盐酸盐/甘氨酸；乌头酸/氢氧化钠；甲酸/甲酸钠缓冲液。
为了测量纤维素酶的活性，所用的底物为Cellazyme锭(Megazyme公司出售)。这些锭由与天青精染料聚合的纤维素和/或纤维素和木葡聚糖的底物构成。
在本发明一个优选的实施方案中，试剂是固体形式。
有利的是，为了便于酶溶解，可向含有发色团试剂的底物中加入表面活性剂。该表面活性剂特别选自月桂基硫酸钠和十二烷基硫酸钠。
根据本发明较佳的实施方案，按下列四个步骤进行测量：
-向容器(1)中引入1毫升需要测量其酶活性的样品，-对于固体样品，应当将固体填充到容器的10毫升刻度标记处；
-引入固体珠形式的试剂；
-引入特定的缓冲液到20毫升刻度标记处；
-用塞子关闭柱后，将柱剧烈振摇数次。
可任选地加入(通过离心或过滤)分离液相和固相的附加步骤，以回收液相，并通过分光光度计测量颜色强度或简单地通过与色标(colour scale)比较来测量颜色强度。
反应4-8小时后出现蓝色证明有活性酶存在，颜色强度与存在于样品中的酶的活性成正比。
本发明另一个优点是对酶活性能进行半定量测量。柱中有色的液相可通过关掉可分离的突出物从柱中回收。其颜色强度可与光密度(O.D.)校准曲线比较。
除了快速外，测量方法也很简单，装置可用于任何地方而不需要特殊的设备。例如，一旦制得了饲料，制造者或饲养者可进行控制测量。
本发明将借助于下列实施例作更详细的阐述，这些实施例并非用于限定本发明。
实施例
对Rovabio木聚糖LC(木聚糖酶和β-葡聚糖酶混合物，来自绳状青霉(Penicillium funiculosum))和对Rovabio木聚糖酶TRLC(来自木霉Trichoderma reesei的木聚糖酶)进行两个系列的试验，它们的木聚糖酶活性为350-550uAXC/毫升。估计在饲料上喷洒液体组合物处理得出的水平为70-110uAXC/千克饲料。
所用的缓冲液是乙酸缓冲液，以将pH保持在4.7。喷洒可在粉状或颗粒状的饲料上进行。
| 样品 | (调整前)活性 | 3小时后观察 | 4小时30分时，590nm下的O.D. | 8小时时，590nm下的O.D. | 8小时时观察 |
| 在颗粒上的木聚糖酶TRLC | 1336 | 蓝色：+++ | ＞3.0 | ＞3.0 | 蓝色：+++ |
| 在颗粒上的木聚糖酶TRLC | 886.7 | 蓝色：+ | 1.567 | 2.685 | 蓝色：++ |
| 在颗粒上的木聚糖酶TRLC | 1469.25 | 蓝色：+ | 1.429 | 2.652 | 蓝色：++ |
| 制粒前木聚糖酶粉末 | 631.4 | 无颜色 | 0.201 | 0.666 | 蓝色：+ |
| 在颗粒上的木聚糖酶LC | 1144 | 蓝色：+++ | 2.309 | 2.376 | 蓝色：+++ |
| 在颗粒上的木聚糖酶LC | 1386.7 | 蓝色：+ | 1.382 | 2.484 | 蓝色：+++ |
| 在颗粒上的木聚糖酶LC | 1450.5 | 蓝色：+++ | 2.872 | 2.85 | 蓝色：+++ |
| 在颗粒上的木聚糖酶LC | 1330.5 | 蓝色：++ | 1.233 | 2.096 | 蓝色：+++ |
Claims (10)
1.一种用于测量固体饲料样品酶活性的装置，其特征在于，它包括用于容纳试验样品的容器，对需要测量活性的酶有特异性的试剂和用于溶解所述酶的缓冲液。
2.根据权利要求1所述的装置，其特征在于，试验样品是固体饲料。
3.根据权利要求2所述的装置，其特征在于，所述的固体饲料是未作处理的固体饲料。
4.根据权利要求1或2所述的装置，其特征在于，所述的容器是单一用途的刻度柱或管，具有防漏开口和封闭系统。
5.根据权利要求4所述的装置，其特征在于，所述的容器包括在其基部的可分离的突出物，让其内容物的液体部分流出。
6.根据权利要求1所述的装置，其特征在于，试剂是与发色团连接的酶的底物。
7.根据权利要求1所述的装置，其特征在于，试剂是固体形式或液体形式。
8.根据权利要求1所述的装置，其特征在于，用于测量酶活性的缓冲剂选自乙酸/乙酸钠；甘氨酸盐酸盐/甘氨酸；乌头酸/氢氧化钠；甲酸/甲酸钠缓冲液。
9.根据权利要求1所述的装置在定量测量酶活性中的应用，其特征在于，所得的颜色与标准曲线比较。
10.一种测量饲料中酶活性的方法，其特征在于，将需要测量酶活性的样品10毫升引入权利要求1-6任一所述的装置，引入固体珠形式的试剂；引入特定的缓冲液到达20毫升刻度标记处；用塞子关闭柱子，剧烈振摇数次；液相从固相中分离出来，回收液相，通过与色标比较来测量颜色强度。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR9810533A FR2782523B1 (fr) | 1998-08-19 | 1998-08-19 | Dispositif de mesure rapide de l'activite enzymatique |
| FR98/10533 | 1998-08-19 |
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| Publication Number | Publication Date |
|---|---|
| CN1312854A CN1312854A (zh) | 2001-09-12 |
| CN1181182C true CN1181182C (zh) | 2004-12-22 |
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| CNB998097853A Expired - Lifetime CN1181182C (zh) | 1998-08-19 | 1999-08-16 | 快速测量酶活性的装置 |
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| Country | Link |
|---|---|
| US (1) | US6821722B1 (zh) |
| EP (1) | EP1105456B1 (zh) |
| JP (1) | JP4531258B2 (zh) |
| KR (1) | KR20010072751A (zh) |
| CN (1) | CN1181182C (zh) |
| AT (1) | ATE254169T1 (zh) |
| AU (1) | AU752174B2 (zh) |
| BR (1) | BR9914294B1 (zh) |
| CA (1) | CA2341581C (zh) |
| DE (1) | DE69912806T2 (zh) |
| DK (1) | DK1105456T3 (zh) |
| EA (1) | EA002734B1 (zh) |
| ES (1) | ES2209474T3 (zh) |
| FR (1) | FR2782523B1 (zh) |
| ID (1) | ID28921A (zh) |
| MX (1) | MXPA01001628A (zh) |
| PT (1) | PT1105456E (zh) |
| SA (1) | SA00200919B1 (zh) |
| WO (1) | WO2000011136A1 (zh) |
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| AU2004263844A1 (en) * | 2003-07-07 | 2005-02-17 | Syngenta Participations Ag | Reagents, methods and kits for detecting feed enzymes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| FR2341865A1 (fr) * | 1976-02-19 | 1977-09-16 | Api Labor | Support pour le dosage d'activites enzymatiques et procede de dosage a l'aide de ce support |
| IT8505253A0 (it) * | 1985-11-15 | 1985-11-15 | Copan Srl | Tampone con serbatoio di terreno di trasporto per il prelievo e il trasferimento in provetta di campioni di sostenze da analizzare. |
| ATE97450T1 (de) * | 1989-02-02 | 1993-12-15 | Abbott Lab | Verfahren und vorrichtung zur quantitativen chromatographierung. |
| JPH04229168A (ja) * | 1990-12-26 | 1992-08-18 | Hoechst Japan Ltd | 酵素の活性と酵素反応阻害物質の活性を測定するための器具及び方法 |
| GB2301103B (en) * | 1995-05-23 | 1999-12-22 | Danisco | An enzyme system comprising ferulic acid esterase |
| US6001587A (en) * | 1997-04-08 | 1999-12-14 | The United States Of America As Represented By The Secretary Of The Navy | Chemically specific patterning on solid surfaces using surface immobilized enzymes |
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- 1998-08-19 FR FR9810533A patent/FR2782523B1/fr not_active Expired - Fee Related
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- 1999-08-16 AU AU51727/99A patent/AU752174B2/en not_active Expired
- 1999-08-16 ES ES99936736T patent/ES2209474T3/es not_active Expired - Lifetime
- 1999-08-16 KR KR1020017002077A patent/KR20010072751A/ko not_active Application Discontinuation
- 1999-08-16 AT AT99936736T patent/ATE254169T1/de active
- 1999-08-16 JP JP2000566393A patent/JP4531258B2/ja not_active Expired - Fee Related
- 1999-08-16 EP EP99936736A patent/EP1105456B1/fr not_active Expired - Lifetime
- 1999-08-16 EA EA200100247A patent/EA002734B1/ru not_active IP Right Cessation
- 1999-08-16 MX MXPA01001628A patent/MXPA01001628A/es active IP Right Grant
- 1999-08-16 US US09/763,018 patent/US6821722B1/en not_active Expired - Lifetime
- 1999-08-16 WO PCT/FR1999/001990 patent/WO2000011136A1/fr not_active Application Discontinuation
- 1999-08-16 CN CNB998097853A patent/CN1181182C/zh not_active Expired - Lifetime
- 1999-08-16 BR BRPI9914294-5A patent/BR9914294B1/pt not_active IP Right Cessation
- 1999-08-16 DE DE69912806T patent/DE69912806T2/de not_active Expired - Lifetime
- 1999-08-16 PT PT99936736T patent/PT1105456E/pt unknown
- 1999-10-16 ID IDW20010613A patent/ID28921A/id unknown
-
2000
- 2000-02-01 SA SA00200919A patent/SA00200919B1/ar unknown
Also Published As
| Publication number | Publication date |
|---|---|
| FR2782523B1 (fr) | 2000-10-06 |
| KR20010072751A (ko) | 2001-07-31 |
| SA00200919B1 (ar) | 2006-06-10 |
| EA200100247A1 (ru) | 2001-08-27 |
| JP4531258B2 (ja) | 2010-08-25 |
| US6821722B1 (en) | 2004-11-23 |
| ATE254169T1 (de) | 2003-11-15 |
| MXPA01001628A (es) | 2002-04-08 |
| DE69912806T2 (de) | 2004-09-23 |
| FR2782523A1 (fr) | 2000-02-25 |
| EA002734B1 (ru) | 2002-08-29 |
| EP1105456A1 (fr) | 2001-06-13 |
| BR9914294B1 (pt) | 2011-01-25 |
| WO2000011136A1 (fr) | 2000-03-02 |
| AU5172799A (en) | 2000-03-14 |
| JP2002523038A (ja) | 2002-07-30 |
| BR9914294A (pt) | 2001-11-06 |
| CA2341581C (fr) | 2010-10-12 |
| CN1312854A (zh) | 2001-09-12 |
| PT1105456E (pt) | 2004-04-30 |
| CA2341581A1 (fr) | 2000-03-02 |
| DE69912806D1 (de) | 2003-12-18 |
| ID28921A (id) | 2001-07-12 |
| EP1105456B1 (fr) | 2003-11-12 |
| AU752174B2 (en) | 2002-09-05 |
| ES2209474T3 (es) | 2004-06-16 |
| DK1105456T3 (da) | 2004-02-23 |
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