CN116256326A - A method for detecting enzyme activity of maltase and its application - Google Patents
A method for detecting enzyme activity of maltase and its applicationDownload PDFInfo
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Abstract
Translated fromChinese
Description
Translated fromChinese技术领域Technical Field
本发明属于酶制剂检测技术领域，具体涉及一种麦芽糖酶的酶活力检测方法及其应用。The invention belongs to the technical field of enzyme preparation detection, and specifically relates to a method for detecting the enzyme activity of maltase and an application thereof.
背景技术Background Art
麦芽糖酶为淀粉水解酶类中的一种，能够专一性地催化麦芽糖生成葡萄糖，是一种新型饲用酶制剂，能够提高饲料利用率，促进动物生长。幼龄动物由于消化系统发育不成熟，各种内源酶分泌不足，限制了淀粉的消化吸收。随着动物日粮配方富营养化、饲养条件应激和环境污染问题越来越突出，成年健康动物添加"外源性营养消化酶"的作用也越来越明显，意义也越来越大。通过添加外源性麦芽糖酶来提高饲料淀粉的消化吸收，对于提高动物生产性能、节约饲料资源意义重大。Maltase is a type of starch hydrolyzing enzyme that can specifically catalyze maltose to produce glucose. It is a new type of feed enzyme preparation that can improve feed utilization and promote animal growth. Due to the immature development of the digestive system of young animals, the secretion of various endogenous enzymes is insufficient, which limits the digestion and absorption of starch. With the increasing eutrophication of animal diets, stressful feeding conditions and environmental pollution, the role of adding "exogenous nutritional digestive enzymes" to adult healthy animals is becoming more and more obvious and of greater significance. Improving the digestion and absorption of feed starch by adding exogenous maltase is of great significance for improving animal production performance and saving feed resources.
鉴于麦芽糖酶能够取代抗生素作用的特性，使用市场前景广阔。目前，我国尚缺乏麦芽糖酶的产品标准及检测方法标准。大部分企业使用自己的标准，主要是通过生成的过氧化氢和还原型邻联茴香胺在过氧化氢酶的作用下生成水和红色的氧化型邻联茴香胺。该方法中邻联茴香胺等试剂属于可致癌的化合物，对人体有一定的危害。或者通过麦芽糖酶将淀粉水解成还原糖，淀粉对碘呈蓝紫色的特异性反应逐渐消失、呈红棕色，其颜色消失的速度与酶活相关，故可通过固定反应后的吸光度计算其酶活力，存在操作繁琐等问题。In view of the fact that maltase can replace the action of antibiotics, it has a broad market prospect. At present, my country still lacks product standards and detection method standards for maltase. Most companies use their own standards, mainly through the generated hydrogen peroxide and reduced o-dianisidine to generate water and red oxidized o-dianisidine under the action of catalase. In this method, o-dianisidine and other reagents are carcinogenic compounds and have certain harm to the human body. Alternatively, starch is hydrolyzed into reducing sugars by maltase, and the specific reaction of starch to iodine, which is blue-purple, gradually disappears and becomes reddish brown. The speed at which its color disappears is related to the enzyme activity, so the enzyme activity can be calculated by the absorbance after the fixed reaction, which has problems such as cumbersome operation.
发明内容Summary of the invention
为了克服现有技术中麦芽糖酶检测标准的缺乏、试剂毒害性大、操作繁琐等问题，本发明提供一种麦芽糖酶的酶活力检测方法。该方法利用麦芽糖酶催化分解麦芽糖转化为葡萄糖，葡萄糖再被葡萄糖氧化酶催化氧化生成葡萄糖酸和过氧化氢以及过氧化氢在紫外区240nm处有较强吸收的原理，能够简单快速、稳定、准确、安全地测定麦芽糖酶的酶活力。In order to overcome the problems of lack of maltase detection standards, high reagent toxicity, and cumbersome operation in the prior art, the present invention provides a method for detecting the enzyme activity of maltase. The method utilizes the principle that maltase catalyzes the decomposition of maltose into glucose, glucose is then catalyzed by glucose oxidase to generate gluconic acid and hydrogen peroxide, and hydrogen peroxide has a strong absorption at 240nm in the ultraviolet region, and can simply, quickly, stably, accurately, and safely determine the enzyme activity of maltase.
具体地，本发明采用如下技术方案来实现上述目的：Specifically, the present invention adopts the following technical solutions to achieve the above objectives:
一种麦芽糖酶的酶活力检测方法，包括以下步骤：A method for detecting the enzyme activity of maltase comprises the following steps:
S1、绘制过氧化氢含量与过氧化氢在240nm处的吸光度值之间关系的标准曲线，得到标准曲线斜率a；称取质量为m的待测麦芽糖酶，加入缓冲液提取，将提取液稀释n倍得到待测麦芽糖酶溶液；S1. Draw a standard curve of the relationship between the hydrogen peroxide content and the absorbance value of hydrogen peroxide at 240 nm to obtain the slope a of the standard curve; weigh the mass m of maltase to be tested, add buffer to extract, and dilute the extract n times to obtain a maltase solution to be tested;
S2、实验组取所述待测麦芽糖酶溶液预热，向其中加入麦芽糖底物溶液和葡萄糖氧化酶，反应T分钟，反应结束后加入硫酸溶液混匀得到第一混合溶液，测定所述第一混合溶液在240nm处的吸光度值A1；对照组取所述待测麦芽糖酶溶液预热，向其中加入硫酸溶液混匀，反应T分钟，加入麦芽糖底物溶液和葡萄糖氧化酶，得到第二混合溶液，测定所述第二混合溶液在240nm处的吸光度值A0；所述实验组和所述对照组的反应体系的总体积均为V；S2, the experimental group took the maltase solution to be tested and preheated it, added maltose substrate solution and glucose oxidase thereto, reacted for T minutes, added sulfuric acid solution after the reaction to obtain a first mixed solution, and measured the absorbance valueA1 of the first mixed solution at 240nm; the control group took the maltase solution to be tested and preheated it, added sulfuric acid solution thereto to mix, reacted for T minutes, added maltose substrate solution and glucose oxidase to obtain a second mixed solution, and measured the absorbance valueA0 of the second mixed solution at 240nm; the total volume of the reaction system of the experimental group and the control group is V;
S3、根据以下公式计算待测麦芽糖酶的比酶活X：S3. Calculate the specific enzyme activity X of the maltase to be tested according to the following formula:
优选的，所述缓冲液使所述待测麦芽糖酶溶液的pH＝4.0～7.0。Preferably, the buffer solution makes the pH of the maltase solution to be tested to be 4.0-7.0.
进一步优选的，所述缓冲液使所述待测麦芽糖酶溶液的pH＝4.0～6.5。More preferably, the buffer solution makes the pH of the maltase solution to be tested be 4.0-6.5.
再进一步优选的，所述缓冲液使所述待测麦芽糖酶溶液的pH＝4.3～6.4。Still more preferably, the buffer solution makes the pH of the maltase solution to be tested be 4.3-6.4.
更进一步优选的，所述缓冲液使所述待测麦芽糖酶溶液的pH＝4.5～6.0。More preferably, the buffer solution makes the pH of the maltase solution to be tested be 4.5-6.0.
最优选的，所述缓冲液使所述待测麦芽糖酶溶液的pH＝5.0～5.5。Most preferably, the buffer solution makes the pH of the maltase solution to be tested be 5.0-5.5.
优选的，所述待测麦芽糖酶溶液中麦芽糖酶的酶活力范围以控制步骤S3的公式中A1-A0＝0.2～0.5为准。Preferably, the enzyme activity range of the maltase in the maltase solution to be tested is based on the formula in step S3 where A1 -A0 = 0.2 to 0.5.
优选的，步骤S1中绘制标准曲线的步骤具体为：取不同浓度的过氧化氢溶液，分别各自加入所述缓冲液、葡萄糖氧化酶和硫酸溶液，充分混匀后在240nm下测定吸光度值，以吸光度值为横坐标，以过氧化氢含量为纵坐标绘制标准曲线。Preferably, the step of drawing a standard curve in step S1 is specifically as follows: taking hydrogen peroxide solutions of different concentrations, adding the buffer solution, glucose oxidase and sulfuric acid solution respectively, mixing them thoroughly, measuring the absorbance at 240 nm, and drawing a standard curve with the absorbance as the abscissa and the hydrogen peroxide content as the ordinate.
优选的，所述待测麦芽糖酶溶液的制备方法为：称取质量为m的待测麦芽糖酶，加入所述缓冲液后，置于水浴摇床提取30min，静置10min，取上清液稀释n倍作为待测麦芽糖酶溶液备用。Preferably, the preparation method of the maltase solution to be tested is: weigh the maltase to be tested with a mass of m, add the buffer, extract it in a water bath shaker for 30 minutes, let it stand for 10 minutes, take the supernatant and dilute it n times as the maltase solution to be tested for later use.
优选的，步骤S2中所述麦芽糖底物溶液的浓度为100mg/mL，所述葡萄糖氧化酶的浓度为50U/mL，所述硫酸溶液的浓度为1mol/L。Preferably, in step S2, the concentration of the maltose substrate solution is 100 mg/mL, the concentration of the glucose oxidase is 50 U/mL, and the concentration of the sulfuric acid solution is 1 mol/L.
优选的，步骤S2中所述待测麦芽糖酶溶液、所述麦芽糖底物溶液、所述葡萄糖氧化酶、所述硫酸溶液的体积比为1:1:1:2。Preferably, in step S2, the volume ratio of the maltase solution to be tested, the maltose substrate solution, the glucose oxidase, and the sulfuric acid solution is 1:1:1:2.
优选的，步骤S2中反应T分钟是在35℃～65℃下保温5min。保温方式优选水浴保温。Preferably, the reaction time T in step S2 is 5 minutes at 35° C. to 65° C. The preferred insulation method is water bath insulation.
进一步优选的，步骤S2中反应T分钟是在37℃～65℃下保温5min。More preferably, the reaction time T minutes in step S2 is maintained at 37° C. to 65° C. for 5 minutes.
优选的，步骤S2中所述预热为在37℃水浴中保温5min。Preferably, the preheating in step S2 is to keep the mixture in a 37° C. water bath for 5 minutes.
本发明还涉及上述任一项所述的麦芽糖酶的酶活力检测方法在检测麦芽糖酶的酶活力中的应用。The present invention also relates to the use of any one of the above-mentioned methods for detecting the enzyme activity of maltase in detecting the enzyme activity of maltase.
与现有技术相比，本发明的有益效果是：Compared with the prior art, the present invention has the following beneficial effects:
本发明中利用麦芽糖被麦芽糖酶催化分解为葡萄糖，葡萄糖被催化氧化生成葡萄糖酸和过氧化氢(H2O2)，过氧化氢在紫外区240nm有较强的吸收的原理，首先绘制出过氧化氢含量与吸光度值之间关系的标准曲线，然后测定反应生成的过氧化氢的吸光度值，同时设置空白对照进行吸光度值校正，根据吸光度值计算出生成的过氧化氢的含量，进而根据过氧化氢的含量计算出麦芽糖酶的酶活力。该方法优化了麦芽糖酶的检测步骤，提高了麦芽糖酶活力检测的安全性，用于检测麦芽糖酶酶活力时具有简单、快速、稳定、准确的特点。The present invention utilizes the principle that maltose is catalytically decomposed into glucose by maltase, glucose is catalytically oxidized to generate gluconic acid andhydrogen peroxide (H2O2 ), and hydrogen peroxide has strong absorption at 240nm in the ultraviolet region. First, a standard curve of the relationship between the content of hydrogen peroxide and the absorbance value is drawn, and then the absorbance value of the hydrogen peroxide generated by the reaction is measured, and a blank control is set to correct the absorbance value. The content of the generated hydrogen peroxide is calculated according to the absorbance value, and then the enzyme activity of the maltase is calculated according to the content of hydrogen peroxide. The method optimizes the detection step of maltase, improves the safety of maltase activity detection, and has the characteristics of being simple, rapid, stable and accurate when used for detecting the enzyme activity of maltase.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为本发明实施例1中的过氧化氢含量与吸光度值关系的标准曲线图；FIG1 is a standard curve diagram showing the relationship between the hydrogen peroxide content and the absorbance value in Example 1 of the present invention;
图2为本发明实施例1中麦芽糖酶酶活力随pH值变化的曲线图；FIG2 is a graph showing the change in maltase activity with pH value in Example 1 of the present invention;
图3为本发明实施例1中麦芽糖酶酶活力随温度变化的曲线图。FIG3 is a curve diagram showing the change of maltase activity with temperature in Example 1 of the present invention.
具体实施方式DETAILED DESCRIPTION
以下结合实施例对本发明技术方案进行清楚、完整地描述。显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明的实施例，本领域技术人员在没有做出创造性劳动的前提下，依据以下实施方式所作的任何等效变换或替代，均属于本发明的保护范围之内。The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, any equivalent transformation or substitution made by those skilled in the art according to the following implementation methods without creative work shall fall within the protection scope of the present invention.
以下实施例中未注明具体条件的方法，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明型号或生产厂商者，均为可以通过市售购买获得的常规产品。In the following examples, the methods without specific conditions were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without specifying the model or manufacturer were all conventional products that can be purchased commercially.
以下实施例中所需的试剂的配制方法如下：The preparation methods of the reagents required in the following examples are as follows:
高锰酸钾标准滴定溶液(c(KMnO4)＝0.1mol/L)的配制和标定参照GB/T601-2016《化学试剂标准滴定溶液的制备》中4.12节的方法。The preparation and calibration of potassium permanganate standard titration solution (c(KMnO4 )=0.1 mol/L) refer to the method in Section 4.12 of GB/T601-2016 “Preparation of Standard Titration Solutions for Chemical Reagents”.
0.2mol/L H2O2溶液的配制及标定：取2mL 30％ H2O2用蒸馏水定容至100mL，再用高锰酸钾标准滴定溶液标定该溶液，获得过氧化氢溶液的实际浓度C(根据实际情况，调整配制的溶液中H2O2的含量，控制C在0.2±0.02mol/L)。Preparation and calibration of 0.2mol/LH2O2solution : Take 2mL of 30%H2O2 and dilute it to 100mL with distilledwater , then calibrate the solution with potassium permanganate standard titration solution to obtain the actual concentration C of the hydrogen peroxide solution (according to the actual situation, adjusttheH2O2 content in the prepared solution to control C at 0.2±0.02mol/L).
实施例1～2中0.1mol/L乙酸缓冲液：称取23.14g三水乙酸钠，加入1.70mL冰乙酸，再加水溶解，定容至2000mL，调节pH至5.50。0.1 mol/L acetic acid buffer in Examples 1-2: weigh 23.14 g of sodium acetate trihydrate, add 1.70 mL of glacial acetic acid, then add water to dissolve, make the volume to 2000 mL, and adjust the pH to 5.50.
100mg/mL麦芽糖底物：称取10.0g D-(+)-麦芽糖一水合物(C12H22O11·H2O，CAS：6363-53-7，纯度≥99.0％)，加0.1mol/L的乙酸缓冲液溶解并稀释至100mL，混匀。100 mg/mL maltose substrate: weigh 10.0 g of D-(+)-maltose monohydrate (C12 H22 O11 ·H2 O, CAS: 6363-53-7, purity ≥ 99.0%), add 0.1 mol/L acetic acid buffer to dissolve and dilute to 100 mL, and mix well.
50U/mL葡萄糖氧化酶：将购买自Sigma公司的葡萄糖氧化酶(CAS：9001-37-0)标注的酶活力，用0.1mol/L乙酸缓冲液稀释到50U/mL。50 U/mL glucose oxidase: The glucose oxidase (CAS: 9001-37-0) purchased from Sigma was diluted to 50 U/mL with 0.1 mol/L acetic acid buffer.
1mol/L硫酸浓度：量取30mL 98％浓硫酸，溶于水，并用水定容至500mL。1 mol/L sulfuric acid concentration: Measure 30 mL of 98% concentrated sulfuric acid, dissolve it in water, and make up to 500 mL with water.
以下实施例中麦芽糖酶的酶活力的定义为：在37℃和pH值为5.5的条件下，每分钟分解麦芽糖生成葡萄糖且葡萄糖被葡萄糖氧化酶催化氧化生成葡萄糖酸和H2O2时，产生1μmol过氧化氢所需的麦芽糖酶的量，即为一个麦芽糖酶活力单位，以U表示。The enzyme activity of maltase in the following examples is defined as: the amount of maltase required to produce 1 μmol of hydrogen peroxide when maltose is decomposed into glucose per minute and glucose is oxidized by glucose oxidase to produce gluconic acid and H2 O2 at 37°C and pH 5.5, which is one maltase activity unit, expressed as U.
实施例1Example 1
本实施例提供了一种麦芽糖酶的酶活力的检测方法，包括以下步骤：This embodiment provides a method for detecting the enzyme activity of maltase, comprising the following steps:
S1、绘制过氧化氢含量与吸光度值的关系的标准曲线和配制待测麦芽糖酶溶液S1. Draw a standard curve of the relationship between hydrogen peroxide content and absorbance value and prepare the maltase solution to be tested
S1-1、绘制过氧化氢含量与吸光度值之间关系的标准曲线S1-1. Draw a standard curve of the relationship between hydrogen peroxide content and absorbance value
取0.2mol/L H2O2溶液，按照表1中的组成配制成不同浓度的H2O2溶液。室温条件下，在240nm波长下用石英比色皿测定各种浓度的H2O2溶液的吸光度值x，以测得的吸光度值x为横坐标，溶液中H2O2浓度y为纵坐标绘制标准曲线，得到的标准曲线如图1所示。该标准曲线方程为y＝32.059x(R2＝1)，斜率a＝32.059。Take 0.2mol/LH2 O2 solution and prepare H2 O2 solutions of different concentrations according to the composition in Table 1. Under room temperature, use a quartz cuvette to measure the absorbance value x of H2 O2 solutions of various concentrations at a wavelength of 240nm, and draw a standard curve with the measured absorbance value x as the abscissa and the H2 O2 concentration y in the solution as the ordinate. The obtained standard curve is shown in Figure 1. The equation of the standard curve is y=32.059x (R2 =1), and the slope a=32.059.
表1不同浓度的过氧化氢溶液Table 1 Hydrogen peroxide solutions of different concentrations
S1-2、配制待测麦芽糖酶溶液S1-2. Prepare the maltase solution to be tested
称取多份质量为m(见表2)的待测麦芽糖酶，精确至0.0001g。将每份麦芽糖酶置于三角瓶中，加入25mL 0.1mol/L pH＝5.5的乙酸缓冲液，水浴摇床提取30min，静置10min后，取上清用0.1mol/L pH＝5.5的乙酸缓冲液稀释n倍(稀释倍数见表2)，作为待测麦芽糖酶溶液。Weigh multiple portions of maltase to be tested with a mass of m (see Table 2) to an accuracy of 0.0001 g. Place each portion of maltase in a conical flask, add 25 mL of 0.1 mol/L pH=5.5 acetic acid buffer, extract for 30 min on a water bath shaker, let stand for 10 min, take the supernatant and dilute it n times with 0.1 mol/L pH=5.5 acetic acid buffer (see Table 2 for the dilution multiple) as the maltase solution to be tested.
步骤S1-1和S1-2不分先后顺序。There is no particular order for steps S1-1 and S1-2.
S2、测定待测麦芽糖酶溶液的吸光度值S2. Determine the absorbance value of the maltase solution to be tested
S2-1、测定实验组待测麦芽糖酶溶液的吸光度值S2-1. Determine the absorbance value of the maltase solution to be tested in the experimental group
实验组：取1mL 100mg/mL麦芽糖底物于37℃水浴锅中预热5min，加入1mL待测麦芽糖酶溶液，再加入1mL 50U/mL葡萄糖氧化酶，在37℃下准确反应5min，加入2mL 1mol/L硫酸溶液终止反应，得到第一混合溶液。测定第一混合溶液在240nm处的吸光度值A1。Experimental group: Take 1mL of 100mg/mL maltose substrate and preheat it in a 37℃ water bath for 5min, add 1mL of maltase solution to be tested, then add 1mL of 50U/mL glucose oxidase, react at 37℃ for 5min, add 2mL of 1mol/L sulfuric acid solution to terminate the reaction, and obtain the first mixed solution. Determine the absorbance value A1 of the first mixed solution at 240nm.
S2-2、测定对照组的吸光度值S2-2. Determine the absorbance value of the control group
对照组：取1mL 100mg/mL麦芽糖底物于37℃水浴锅中预热5min，加入2mL 1mol/L硫酸溶液，于37℃下保温5min，加入1mL待测麦芽糖酶溶液，再加入1mL 50U/mL葡萄糖氧化酶，得到第二混合溶液。测定第二混合溶液在240nm处的吸光度值A0。Control group: Take 1mL of 100mg/mL maltose substrate and preheat it in a 37℃ water bath for 5min, add 2mL of 1mol/L sulfuric acid solution, keep it at 37℃ for 5min, add 1mL of maltase solution to be tested, and then add 1mL of 50U/mL glucose oxidase to obtain a second mixed solution. Determine the absorbance value A0 of the second mixed solution at 240nm.
步骤S2-1和S2-2不分先后顺序。从图1上可以看出，当溶液中过氧化氢的浓度在0～24μmol/L范围内(对应的吸光度值为0～0.75)时，溶液的吸光度值与过氧化氢浓度之间呈现出优异的线性关系。为了使检测结果更准确，本实施例中将第一混合溶液中过氧化氢的浓度控制在与吸光度值为0.2～0.5时对应的浓度范围内，即待测麦芽糖酶溶液中麦芽糖酶的酶活力控制范围以使A1-A0＝0.2～0.5为准。Steps S2-1 and S2-2 are in no particular order. As can be seen from Figure 1, when the concentration of hydrogen peroxide in the solution is in the range of 0 to 24 μmol/L (corresponding to an absorbance value of 0 to 0.75), an excellent linear relationship is shown between the absorbance value of the solution and the concentration of hydrogen peroxide. In order to make the test result more accurate, in this embodiment, the concentration of hydrogen peroxide in the first mixed solution is controlled within the concentration range corresponding to the absorbance value of 0.2 to 0.5, that is, the enzyme activity control range of the maltase in the maltase solution to be tested is such that A1 -A0 = 0.2 to 0.5.
S3、根据以下公式计算待测麦芽糖酶的酶活力X：S3. Calculate the enzyme activity X of the maltase to be tested according to the following formula:
表2麦芽糖酶酶活力检测结果Table 2 Maltase enzyme activity test results
从表2中的检测结果可以看出，当待测麦芽糖酶的酶活力范围使A1-A0＝0.2～0.5时，通过本发明中的检测方法检测麦芽糖酶的酶活力结果重复性好，精密度高。尤其是当第一混合溶液中麦芽糖酶的酶活力为固定值(稀释倍数为75的6组实验)进行平行测定时，检测结果的重现性更好。It can be seen from the test results in Table 2 that when the enzyme activity range of the maltase to be tested is A1 -A0 = 0.2 to 0.5, the results of the enzyme activity of maltase detected by the detection method of the present invention have good repeatability and high precision. In particular, when the enzyme activity of maltase in the first mixed solution is a fixed value (6 groups of experiments with a dilution factor of 75) and parallel determination is performed, the reproducibility of the test results is better.
实施例2Example 2
本实施例中麦芽糖酶的酶活力的检测方法与实施例1基本相同，不同之处在于步骤S1-2中稀释倍数与实施例1中不同，步骤S2-1中反应温度与实施例1中不同。本实施例中具体的稀释倍数和反应温度见表3。The method for detecting the enzyme activity of maltase in this embodiment is basically the same as that in
表3各个实验组中麦芽糖酶溶液的稀释倍数和反应温度Table 3 Dilution multiple and reaction temperature of maltase solution in each experimental group
以温度为横坐标，以步骤S3中计算出的酶活力结果为纵坐标绘制曲线，得到如图2中所示的麦芽糖酶酶活力随温度变化的曲线图。从图2中可以看出，麦芽糖酶的酶活力在55℃时最强；反应温度低于55℃时，随着温度升高酶活力增强；反应温度高于55℃时，随着温度升高酶活力逐渐下降。当反应温度在35℃～65℃范围内，麦芽糖酶的酶活力存留率为60％以上；在37～65℃范围内，麦芽糖酶的酶活力存留率为70％以上；在40～60℃范围内，麦芽糖酶的酶活力存留率为80％以上；在45～57℃范围内，麦芽糖酶的酶活力存留率达到90％以上；在55℃时，麦芽糖酶的酶活力存留率达到100％。当麦芽糖酶作为动物饲料添加剂使用时，为了保持检测结果的准确性，选择与畜禽体内温度一致的37℃作为定义酶活力的温度。The temperature is used as the abscissa and the enzyme activity result calculated in step S3 is used as the ordinate to draw a curve, and a curve of the maltase enzyme activity changing with temperature as shown in Figure 2 is obtained. As can be seen from Figure 2, the enzyme activity of maltase is strongest at 55°C; when the reaction temperature is lower than 55°C, the enzyme activity increases with the increase of temperature; when the reaction temperature is higher than 55°C, the enzyme activity gradually decreases with the increase of temperature. When the reaction temperature is in the range of 35°C to 65°C, the enzyme activity retention rate of maltase is more than 60%; in the range of 37 to 65°C, the enzyme activity retention rate of maltase is more than 70%; in the range of 40 to 60°C, the enzyme activity retention rate of maltase is more than 80%; in the range of 45 to 57°C, the enzyme activity retention rate of maltase reaches more than 90%; at 55°C, the enzyme activity retention rate of maltase reaches 100%. When maltase is used as an animal feed additive, in order to maintain the accuracy of the test results, 37°C, which is consistent with the body temperature of livestock and poultry, is selected as the temperature for defining enzyme activity.
实施例3Example 3
本实施例中麦芽糖酶的酶活力的检测方法与实施例1基本相同，不同之处在于步骤S1-2中稀释倍数和0.1mol/L乙酸缓冲液的pH值与实施例1中不同，具体的稀释倍数和pH值见表4。The method for detecting the enzyme activity of maltase in this embodiment is basically the same as that in Example 1, except that the dilution multiple and the pH value of the 0.1 mol/L acetic acid buffer in step S1-2 are different from those in Example 1. The specific dilution multiple and pH value are shown in Table 4.
表4各实验组中麦芽糖酶溶液的稀释倍数和乙酸缓冲液的pH值Table 4 Dilution multiples of maltase solution and pH value of acetate buffer in each experimental group
以反应体系pH值为横坐标，以步骤S3中计算出的酶活力结果为纵坐标绘制曲线，得到如图3中所示的麦芽糖酶酶活力pH值变化的曲线图。从图3中可以看出，麦芽糖酶的酶活力在pH＝5.5时最强；pH值低于5.5时，随着pH值升高酶活力增强；pH值高于5.5时，随着pH值升高酶活力下降。当pH值为4.0～7.0时，麦芽糖酶的酶活力存留率为60％以上；当pH值为4.0～6.5时，麦芽糖酶的酶活力存留率为70％以上；当pH值为4.3～6.4时，麦芽糖酶的酶活力存留率为80％以上；当pH值为4.5～6.0时，麦芽糖酶的酶活力存留率达到90％以上；当pH值为5.0～5.5时，麦芽糖酶的酶活力存留率达到100％。因此，本发明中选择pH＝5.5作为麦芽糖酶酶活力检测的最佳pH值。The pH value of the reaction system is used as the horizontal axis, and the enzyme activity result calculated in step S3 is used as the vertical axis to draw a curve, and a curve of the pH value change of the maltase enzyme activity is obtained as shown in Figure 3. As can be seen from Figure 3, the enzyme activity of maltase is strongest at pH = 5.5; when the pH value is lower than 5.5, the enzyme activity increases with the increase of the pH value; when the pH value is higher than 5.5, the enzyme activity decreases with the increase of the pH value. When the pH value is 4.0-7.0, the enzyme activity retention rate of maltase is more than 60%; when the pH value is 4.0-6.5, the enzyme activity retention rate of maltase is more than 70%; when the pH value is 4.3-6.4, the enzyme activity retention rate of maltase is more than 80%; when the pH value is 4.5-6.0, the enzyme activity retention rate of maltase reaches more than 90%; when the pH value is 5.0-5.5, the enzyme activity retention rate of maltase reaches 100%. Therefore, pH=5.5 is selected as the optimal pH value for maltase enzyme activity detection in the present invention.
实施例4Example 4
本实施例中验证了本发明中检测麦芽糖酶酶活力的方法的准确度。检测方法与实施例1基本相同，不同之处在于，步骤S1-2中稀释倍数与实施例1中不同。This example verifies the accuracy of the method for detecting maltase activity in the present invention. The detection method is substantially the same as that in Example 1, except that the dilution factor in step S1-2 is different from that in Example 1.
将比酶活为500～3000U/g的市售麦芽糖酶(Novozymes公司生产)用0.1mol/L乙酸缓冲液稀释成不同酶活力的标准麦芽糖酶溶液，采用实施例1中的方法检测其酶活力，具体的稀释倍数和检测结果见表5。Commercially available maltase (produced by Novozymes) with a specific enzyme activity of 500 to 3000 U/g was diluted with 0.1 mol/L acetic acid buffer to prepare standard maltase solutions with different enzyme activities. The enzyme activities were detected using the method in Example 1. The specific dilution multiples and test results are shown in Table 5.
表5方法准确度验证Table 5 Method accuracy verification
从表5中可以看出，采用本发明中的方法检测比酶活为500～3000U/g的市售麦芽糖酶稀释至6.5～14.0U/g时，检测结果重现性好，精密度高。与市售麦芽糖酶的理论比酶活数据相比较，误差较小(相对误差＜5％)，说明本发明中检测麦芽糖酶酶活力的方法准确度很高。As can be seen from Table 5, when the commercial maltase with a specific enzyme activity of 500-3000 U/g is diluted to 6.5-14.0 U/g by the method of the present invention, the test results have good reproducibility and high precision. Compared with the theoretical specific enzyme activity data of the commercial maltase, the error is small (relative error <5%), indicating that the method of detecting the activity of maltase in the present invention is very accurate.
以上所述仅为本发明的优选实施例而已，并不用于限制本发明的保护范围。对于任何熟悉本领域的技术人员来说，本发明可以有各种更改和变化。任何依据本发明申请保护范围及说明书内容所作的简单的等效变化和修饰，均应包含在本发明的保护范围之内。The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. For any person skilled in the art, the present invention may have various changes and modifications. Any simple equivalent changes and modifications made according to the scope of protection of the present invention and the contents of the specification should be included in the scope of protection of the present invention.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3879263A (en)* | 1973-09-06 | 1975-04-22 | Du Pont | Method for the determination of amylase |
| JPS5697863A (en)* | 1980-01-07 | 1981-08-06 | Hitachi Ltd | Method and apparatus for analyzing amylase |
| JPS60168399A (en)* | 1984-02-09 | 1985-08-31 | Sanko Junyaku Kk | Determination of amylase |
| JPS6131954A (en)* | 1984-07-25 | 1986-02-14 | Hitachi Ltd | Maltose sensor |
| CN112301092A (en)* | 2020-10-30 | 2021-02-02 | 大连大学 | Colorimetric analysis method for determining activity of glucose oxidase |
- 2023
- 2023-03-26CNCN202310313538.7Apatent/CN116256326A/enactivePending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3879263A (en)* | 1973-09-06 | 1975-04-22 | Du Pont | Method for the determination of amylase |
| JPS5697863A (en)* | 1980-01-07 | 1981-08-06 | Hitachi Ltd | Method and apparatus for analyzing amylase |
| JPS60168399A (en)* | 1984-02-09 | 1985-08-31 | Sanko Junyaku Kk | Determination of amylase |
| JPS6131954A (en)* | 1984-07-25 | 1986-02-14 | Hitachi Ltd | Maltose sensor |
| CN112301092A (en)* | 2020-10-30 | 2021-02-02 | 大连大学 | Colorimetric analysis method for determining activity of glucose oxidase |
Non-Patent Citations (1)
| Title |
|---|
| 曹建康等: "果蔬采后生理生化实验指导", 30 September 2007, 中国轻工业出版社, pages: 120 - 122* |
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