本DNS试剂严格依据国家标准GB/T23874《饲料添加剂木聚糖酶活力的测定》配制，试剂中3,5-二硝基水杨酸浓度为3.15g/L，适配饲料原料、饲料添加剂中木聚糖酶活力定量检测，试剂由氢氧化钠、3,5-二硝基水杨酸、酒石酸钾钠、酚等组分复配而成，是木聚糖酶DNS比色法检测专用显色试剂。
检测原理：木聚糖酶可将底物木聚糖降解为还原性寡糖与木糖（还原糖）；还原糖在碱性沸水浴环境下与DNS试剂发生氧化还原反应，3,5-二硝基水杨酸的硝基被还原为氨基，溶液呈现橙红色；在有效线性范围内，橙红色深浅与酶解生成还原糖浓度成正比，还原糖生成量与木聚糖酶活力正相关；采用分光光度计于540 nm波长测定吸光度，结合木糖标准曲线定量还原糖含量，换算得到样品木聚糖酶活力。本产品仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
1、试剂：蒸馏水、醋酸缓冲液(pH 5.3)、木聚糖底物溶液(1%)、木糖标准液(1 mg/mL)
2、耗材器具：剪刀、研钵/高速匀浆机、离心管、试管、容量瓶、移液管
3、仪器设备：恒温水浴锅、离心机、分光光度计、比色皿
操作步骤（仅供参考）：
1、酶液提取（固体/液体饲料添加剂通用）
① 称取饲料酶制剂样品0.5-3 g，剪碎研磨，加入pH 5.3醋酸缓冲液约3 mL充分匀浆，转移至三角瓶，缓冲液多次冲洗研磨器具，洗液全部并入三角瓶；
② 50 ℃水浴浸提30 min，期间间断搅拌，充分溶出木聚糖酶；
③ 混合液转入50 mL离心管，4000 g离心5 min，收集上清；沉淀再加20 mL醋酸缓冲液混匀，同等条件二次离心；
④ 合并两次上清液，用pH 5.3醋酸缓冲液定容至100 mL，混匀，根据预估酶活适当稀释，作为待测酶液。
2、木糖标准曲线绘制
取洁净试管按下表加样，以0号管空白调零，540 nm测定吸光度，以吸光度为纵坐标、木糖标准浓度(mg/mL)为横坐标绘制标准曲线。
| 加入物质(mL) | 0 | 1 | 2 | 3 | 4 | 5 | | 木糖标准液(1 mg/mL) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 | | pH 5.3醋酸缓冲液 | 1 | 0.8 | 0.6 | 0.4 | 0.2 | 0 | | DNS试剂 | 2 | 2 | 2 | 2 | 2 | 2 | | 蒸馏水 | 9 | 9 | 9 | 9 | 9 | 9 | | 相当于木糖量 (mg) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 |
加入DNS试剂后沸水浴准确煮沸5 min，取出自来水冷却至室温。
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3、木聚糖酶活样品测定
每组同步设置样品管与空白对照管：
① 试管预先50 ℃水浴预热10 min；各管加入1 mL待测稀释酶液；空白管替换为等量醋酸缓冲液；
② 加入2 mL预热至50 ℃的1%木聚糖底物溶液，混匀，50 ℃精准水浴30 min；
③ 立即加入2 mL DNS试剂终止酶促反应，充分摇匀；空白管此时补加等量待测酶液；
④ 全部试管沸水浴准确煮沸5 min，流水冷却至室温，加9 mL蒸馏水混匀；
⑤ 分光光度计540 nm测定各管吸光度，对照标准曲线查出对应木糖质量。
计算：
木聚糖酶活力定义：在50 ℃、pH 5.3条件下，每分钟催化木聚糖水解生成1 μmol木糖所需酶量，定义为1个木聚糖酶活力单位U。
固体饲料添加剂酶活 U(U/g) = (C × VT × k) ÷ (m × VS × t × 0.1501)
液体饲料添加剂酶活 U(U/mL) = (C × VT × k) ÷ (VT × VS × t × 0.1501)
C ：标准曲线查得木糖质量 (mg)
VT ：酶提取液定容体积 (mL)
VS ：测定时取用酶液体积 (mL)
k ：待测酶液稀释倍数
t ：酶促反应时间 (min)
m ：称取固体样品质量 (g)
0.1501 ：木糖摩尔质量换算系数（1 μmol木糖 = 0.1501 mg）
注意事项：
1、DNS试剂避光室温保存，严禁冷冻、反复冻融，冻融后显色效率大幅下降，直接失效。
2、醋酸缓冲液、木聚糖底物现配现用，底物低温易析出，使用前充分水浴溶解。
3、酶促反应、沸水浴显色时间必须精准把控，温差、时长偏差会造成数值大幅漂移。
4、样品还原糖浓度过高时，需用醋酸缓冲液稀释酶液后重新检测，最终结果乘以稀释倍数。
5、待测酶提取液若无法当天检测，2-8 ℃冷藏可保存2天，超过2天需重新制备。
6、沸水浴操作时试管管口勿朝向人体，防止液体喷溅烫伤。
7、分光光度计波长严格设置490 nm，不可沿用植物还原糖检测540 nm参数，否则数据不满足国标判定要求。
8、试剂开封后3个月内用完，长期敞口存放易氧化失效。
9、为了您的安全和健康，请穿实验服并戴一次性手套操作。
This DNS reagent is strictly prepared in accordance with the national standard GB/T23874 "Determination of xylanase activity in feed additives". The concentration of 3,5-dinitrosalicylic acid in the reagent is 3.15 g/L, and it is suitable for quantitative determination of xylanase activity in feed materials and feed additives. The reagent is composed of sodium hydroxide, 3,5-dinitrosalicylic acid, potassium sodium tartrate, phenol and other components, and is a special chromogenic reagent for xylanase DNS colorimetric detection.
Detection principle: Xylanase can degrade the substrate xylan into reducing oligosaccharides and xylose (reducing sugars); reducing sugars undergo redox reaction with DNS reagent in alkaline boiling water bath environment, the nitro groups of 3,5-dinitrosalicylic acid are reduced to amino groups, and the solution appears orange-red; within the effective linear range, the depth of orange-red color is proportional to the concentration of reducing sugars generated by enzymatic hydrolysis, and the amount of reducing sugars generated is positively correlated with xylanase activity; the absorbance is measured at 540 nm wavelength by spectrophotometer, and the reducing sugar content is quantified with xylose standard curve, and the xylanase activity of the sample is obtained by conversion. This product is only for research field, not suitable for clinical diagnosis or other purposes.
Materials required (self-provided):
1.Reagents: distilled water, acetate buffer (pH 5.3), xylan substrate solution (1%), xylose standard solution (1 mg/mL)
2.Consumables and tools: scissors, mortar/high-speed homogenizer, centrifuge tubes, test tubes, volumetric flasks, pipettes
3.Instruments and equipment: constant-temperature water bath, centrifuge, spectrophotometer, cuvettes
Procedure (for reference only):
1.Enzyme extraction (general for solid/liquid feed additives)
① Weigh 0.5-3 g of feed enzyme preparation sample, cut and grind, add about 3 mL of pH 5.3 acetate buffer for thorough homogenization, transfer to Erlenmeyer flask, rinse the grinding tools several times with buffer, and combine all washing solutions into the Erlenmeyer flask;
② Extract in water bath at 50 ℃ for 30 min, with intermittent stirring during the period, to fully dissolve xylanase;
③ Transfer the mixture to 50 mL centrifuge tube, centrifuge at 4000 g for 5 min, collect the supernatant; add 20 mL acetate buffer to the precipitate and mix well, centrifuge again under the same conditions;
④ Combine the two supernatants, dilute to 100 mL with pH 5.3 acetate buffer, mix well, dilute appropriately according to the estimated enzyme activity, and use as the enzyme solution to be tested.
2.Preparation of xylose standard curve
Take clean test tubes and add samples according to the table below, use tube No. 0 as blank for zero adjustment, measure absorbance at 540 nm, plot the standard curve with absorbance as the ordinate and xylose standard concentration (mg/mL) as the abscissa.
| Added substance (mL) | 0 | 1 | 2 | 3 | 4 | 5 | | Xylose standard solution (1 mg/mL) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 | | pH 5.3 acetate buffer | 1 | 0.8 | 0.6 | 0.4 | 0.2 | 0 | | DNS reagent | 2 | 2 | 2 | 2 | 2 | 2 | | Distilled water | 9 | 9 | 9 | 9 | 9 | 9 | | Equivalent xylose amount (mg) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 |
After adding DNS reagent, accurately boil in boiling water bath for 5 min, then cool to room temperature under running water.
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3.Determination of xylanase activity in samples
Set up sample tubes and blank control tubes simultaneously for each group:
① Pre-incubate the test tubes in water bath at 50 ℃ for 10 min; add 1 mL of the diluted enzyme solution to be tested to each tube; replace the blank tube with equal amount of acetate buffer;
② Add 2 mL of 1% xylan substrate solution pre-warmed to 50 ℃, mix well, incubate precisely in water bath at 50 ℃ for 30 min;
③ Immediately add 2 mL of DNS reagent to terminate the enzymatic reaction, shake thoroughly; at this time, add equal amount of the enzyme solution to be tested to the blank tube;
④ Boil all test tubes in boiling water bath for exactly 5 min, cool to room temperature under running water, add 9 mL of distilled water and mix well;
⑤ Measure the absorbance of each tube at 540 nm with spectrophotometer, and determine the corresponding xylose mass from the standard curve.
Calculation:
Definition of xylanase activity: Under the conditions of 50 ℃ and pH 5.3, the amount of enzyme required to catalyze the hydrolysis of xylan to produce 1 μmol of xylose per minute is defined as 1 unit of xylanase activity, symbol U.
Solid feed additive enzyme activity U(U/g) = (C × VT × k) ÷ (m × VS × t × 0.1501)
Liquid feed additive enzyme activity U(U/mL) = (C × VT × k) ÷ (VT ×VS × t × 0.1501)
C : xylose mass determined from standard curve, mg
VT : constant volume of enzyme extract, mL
VS : volume of enzyme solution taken for determination, mL
k : dilution factor of enzyme solution to be tested
t : enzymatic reaction time, min
m : mass of solid sample weighed, g
0.1501 : conversion coefficient of xylose molar mass (1 μmol xylose = 0.1501 mg)
Precautions:
1.Store DNS reagent at room temperature in the dark, do not freeze or subject to repeated freeze-thaw cycles, as the color development efficiency will be significantly reduced or directly lost after freeze-thaw.
2.Acetate buffer and xylan substrate should be prepared fresh and used immediately. The substrate is prone to precipitation at low temperatures, fully dissolve in water bath before use.
3.The timing of enzymatic reaction and boiling water bath color development must be precisely controlled. Deviations in temperature or time will cause significant drift in values.
4.When the reducing sugar concentration of the sample is too high, dilute the enzyme solution with acetate buffer and re-test, and multiply the final result by the dilution factor.
5.If the enzyme extract to be tested cannot be tested on the same day, it can be stored at 2-8 ℃ for up to 2 days. If it exceeds 2 days, it needs to be freshly prepared.
6.During boiling water bath operation, do not point the test tube opening towards the body to prevent liquid splashing and scalding.
7.The spectrophotometer wavelength should be strictly set to 490 nm, do not use the 540 nm parameter used for plant reducing sugar detection, otherwise the data will not meet the requirements of national standard judgment.
8.Use the reagent within 3 months after opening. Long-term open storage may cause oxidation and failure.
9.For your safety and health, please wear a lab coat and disposable gloves during operation.
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