本DNS试剂严格依据国家标准 GB 20287‑2006《农用微生物菌剂》附录 D 配制，专为农用微生物菌剂纤维素酶活检测设计，试剂内含氢氧化钠、3,5‑二硝基水杨酸、酒石酸钾钠、酚等核心显色组分，适配国标规定 DNS 比色检测体系。
检测原理：以羧甲基纤维素钠盐 (CMC‑Na) 为反应底物，样品中纤维素酶在 60℃水浴条件下水解 CMC 生成还原糖；还原糖在碱性沸水浴环境下将 3,5‑二硝基水杨酸中的硝基还原为氨基，生成橙红色氨基化合物；在标准反应区间内，橙红色深浅与还原糖生成量呈正比，于 490nm 波长测定吸光度，结合葡萄糖标准曲线定量还原糖，最终换算得到样品纤维素酶活力单位。
本试剂仅适用于农用微生物菌剂、腐熟菌剂纤维素酶活科研与国标合规检测，不可用于临床诊断、食品出厂定量等其他用途。
自备材料
1、蒸馏水、柠檬酸缓冲液 (pH4.8)、1% CMC‑Na 底物溶液、葡萄糖标准液 (1 mg/mL)
2、三角瓶、玻璃珠、四层纱布、50 mL离心管、试管、天平、恒温水浴锅、分光光度计、比色皿、摇床、离心机
操作步骤：(仅供参考)
1、原样酶液制备 (固体 / 液体菌剂通用)
①称取菌剂样品 10.0 g (液体样品取 10 mL)，置于含玻璃珠的 250 mL 三角瓶，加入适量柠檬酸缓冲液定容至 100 mL；
②室温静置 20 min，200 r/min 振荡 30 min 充分浸提；
③四层纱布粗过滤，滤液转移离心管，3000 r/min 离心 10 min；
④取上清液，根据预估酶活力用缓冲液梯度稀释，得到待测原样酶液。
2、葡萄糖标准曲线绘制
取洁净试管按下表加样，以 0 号管调零，沸水浴显色冷却后 490 nm 测吸光度；以葡萄糖质量 (μg) 为横坐标、吸光度 OD 值为纵坐标绘制标准曲线。
| 加入物质 (mL) | 0 | 1 | 2 | 3 | 4 | 5 | | 葡萄糖标准液 (1mg/mL) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 | | 蒸馏水 | 1 | 0.8 | 0.6 | 0.4 | 0.2 | 0 | | DNS 试剂 | 3 | 3 | 3 | 3 | 3 | 3 | | 对应葡萄糖总量 (mg) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 |
加入DNS试剂后混匀，沸水浴准确加热 5min，流水冷却至室温。补蒸馏水至 20mL。 |
3、纤维素酶活样品测定
每组设置 1 支空白对照管、2 支平行样品管，严格控温计时：
①所有试管预先放入 60℃水浴预热 10 min；
②样品管各加入 1 mL 稀释后原样酶液，空白管加 1 mL 缓冲液；
③全部试管加入 4mL 预热至 60℃的 1% CMC‑Na 底物溶液，混匀；
④60℃水浴精准保温 20 min (酶促反应计时起点从加完底物开始)；
⑤立即向全部试管加入 3 mL DNS试剂 (GB 20287)，充分摇匀；
⑥空白对照管补加 1 mL原样酶液 (消除酶液本身还原糖干扰)；
⑦全部试管沸水浴准确显色 5 min，取出流水冷却至室温；
⑧蒸馏水定容至 20 mL，颠倒混匀，分光光度计 490 nm 测定 OD 值。
计算 (GB 20287 国标标准公式)：
纤维素酶活力定义：60℃条件下，1 mL 原样酶液每分钟水解 CMC 产生 1 μg 葡萄糖，定义为 1 个酶活力单位，符号 U。
液体菌剂酶活 (U /ml)= [k × (M₁ - M₀)] / 20
固体菌剂酶活 (U/g) = [k × V总 × (M₁ - M₀)] / (m × 20); × (M₁ - M₀)] / (m × 20)
k：酶液稀释倍数
U：样品纤维素酶活 (单位U/ml或U/g)
20：酶促反应保温时间 (min)
M₁：样品管由标准曲线查得葡萄糖质量 (μg)
V总：样品浸提定容体积 (mL)
M₀：空白对照管葡萄糖质量 (μg)
m：称取菌剂样品质量 (g)
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。
2、底物 CMC‑Na 溶液现配现用，60℃水浴搅拌溶解，低温易析出，影响检测结果。
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、配制缓冲液、底物时酸碱试剂操作注意防护，防止腐蚀灼伤。
7、试剂开封后尽快用完，长期敞口接触空气易氧化失效。
8、检测波长固定 540 nm，不可随意更改，严格遵循 GB/T 23874 检测波长要求。
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 Appendix D of the national standard GB 20287‑2006 Agricultural Microbial Inoculants, and is specifically designed for the determination of cellulase activity in agricultural microbial inoculants. The reagent contains core chromogenic components including sodium hydroxide, 3,5‑dinitrosalicylic acid, potassium sodium tartrate, and phenol, and is compatible with the DNS colorimetric detection system specified in the national standard.
Detection Principle: Using carboxymethyl cellulose sodium salt (CMC‑Na) as the reaction substrate, cellulase in the sample hydrolyzes CMC under water bath conditions at 60°C to generate reducing sugars. In an alkaline boiling water bath, the reducing sugars reduce the nitro groups in 3,5‑dinitrosalicylic acid to amino groups, producing an orange‑red amino compound. Within the standard reaction range, the intensity of the orange‑red color is proportional to the amount of reducing sugars produced. The absorbance is measured at 490 nm, and the reducing sugar content is quantified using a glucose standard curve, from which the cellulase activity of the sample is calculated.
This reagent is intended solely for research and national standard compliance testing of cellulase activity in agricultural microbial inoculants and decomposition inoculants. It must not be used for clinical diagnosis, food release quantitative testing, or other purposes.
Materials Required (Self‑Provided)
1.Distilled water, citrate buffer (pH 4.8), 1% CMC‑Na substrate solution, glucose standard solution (1 mg/mL)
2.Erlenmeyer flasks, glass beads, four‑layer gauze, 50 mL centrifuge tubes, test tubes, balance, constant‑temperature water bath, spectrophotometer, cuvettes, shaker, centrifuge
Procedure (For Reference Only)
1. Preparation of Original Enzyme Solution (General for Solid/Liquid Inoculants)
① Weigh 10.0 g of the inoculant sample (or take 10 mL for liquid samples) into a 250 mL Erlenmeyer flask containing glass beads, and add an appropriate amount of citrate buffer to a final volume of 100 mL.
② Let stand at room temperature for 20 min, then shake at 200 r/min for 30 min for thorough extraction.
③ Filter roughly through four‑layer gauze, transfer the filtrate to centrifuge tubes, and centrifuge at 3000 r/min for 10 min.
④ Collect the supernatant and dilute stepwise with buffer according to the estimated enzyme activity to obtain the original enzyme solution for testing.
2. Preparation of Glucose Standard Curve
Take clean test tubes and add samples as shown in the table below. Use tube No. 0 as the blank for zero adjustment. After color development in a boiling water bath and cooling, measure the absorbance at 490 nm. Plot the standard curve with glucose mass (μg) on the x‑axis and absorbance (OD value) on the y‑axis.
| Reagent (mL) | 0 | 1 | 2 | 3 | 4 | 5 | | Glucose standard solution (1 mg/mL) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 | | Distilled water | 1 | 0.8 | 0.6 | 0.4 | 0.2 | 0 | | DNS Reagent | 3 | 3 | 3 | 3 | 3 | 3 | | Total glucose amount (mg) | 0 | 0.2 | 0.4 | 0.6 | 0.8 | 1 |
Mix thoroughly after adding DNS Reagent, heat accurately in a boiling‑water bath for 5 min, then cool to room temperature under running tap water. Add distilled water to make up to 20 mL. |
3. Determination of Cellulase Activity in Samples
Set up one blank control tube and two parallel sample tubes per group. Strictly control temperature and timing:
① Pre‑incubate all test tubes in a 60°C water bath for 10 min.
② Add 1 mL of the diluted original enzyme solution to each sample tube, and add 1 mL of buffer to the blank tube.
③ Add 4 mL of 1% CMC‑Na substrate solution (pre‑warmed to 60°C) to all tubes and mix well.
④ Incubate accurately at 60°C for 20 min (timing starts immediately after adding the substrate).
⑤ Immediately add 3 mL of DNS reagent (GB 20287) to all tubes and mix thoroughly.
⑥ Add 1 mL of the original enzyme solution to the blank control tube (to eliminate interference from reducing sugars originally present in the enzyme solution).
⑦ Place all tubes in a boiling water bath for exactly 5 min for color development, then cool to room temperature under running water.
⑧ Dilute to a final volume of 20 mL with distilled water, mix by inversion, and measure the OD value at 490 nm using a spectrophotometer.
Calculation (According to GB 20287 National Standard Formula)
Definition of cellulase activity: One unit of enzyme activity (U) is defined as the amount of enzyme that produces 1 μg of glucose per minute from CMC hydrolysis per 1 mL of the original enzyme solution at 60°C.
Liquid inoculant activity (U/mL) = [k × (M₁ – M₀)] / 20
Solid inoculant activity (U/g) = [k × Vtotal × (M₁ – M₀)] / (m × 20); × (M₁ – M₀)] / (m × 20)
k = dilution factor of the enzyme solution
U = cellulase activity of the sample, in U/mL or U/g
20 = enzymatic reaction incubation time, in minutes
M₁ = glucose mass (μg) of the sample tube determined from the standard curve
Vtotal = final volume (mL) of the sample extraction
M₀ = glucose mass (μg) of the blank control tube
m = mass (g) of the inoculant sample weighed
Precautions
1.Store the DNS reagent at room temperature in the dark. Do not freeze or subject to repeated freeze‑thaw cycles, as this will significantly reduce color development efficiency.
2.The CMC‑Na substrate solution should be prepared fresh and used immediately. Dissolve it in a 60°C water bath with stirring; low temperatures may cause precipitation and affect results.
3.The timing for both the enzymatic reaction and the boiling water bath color development must be precisely controlled. Deviations in temperature or duration may cause large fluctuations in the measured values.
4.If the reducing sugar concentration in the sample is too high, dilute the enzyme solution with acetate buffer and re‑test; multiply the final result by the dilution factor.
5.If the enzyme extract cannot be tested on the same day, it may be stored at 2‑8°C for up to 2 days. Beyond 2 days, a fresh preparation is required.
6.When preparing buffers and substrates, take protective measures against acid and alkali reagents to prevent corrosion and burns.
7.Use the reagent promptly after opening; prolonged exposure to air may cause oxidation and loss of activity.
8.The detection wavelength is fixed at 540 nm and must not be changed arbitrarily, strictly following the wavelength requirement specified in GB/T 23874.
9.For your safety and health, please wear a lab coat and disposable gloves during operation.
危险品化学品经营许可证（带存储）