CN108165605A - Culture medium and method a kind of while that screen protease and cellulase - Google Patents
Culture medium and method a kind of while that screen protease and cellulase Download PDFInfo
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- 108091005804 Peptidases Proteins 0.000 title claims abstract description 28
- 108010059892 Cellulase Proteins 0.000 title claims abstract description 27
- 229940106157 cellulase Drugs 0.000 title claims abstract description 27
- 239000004365 Protease Substances 0.000 title claims abstract description 26
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 title claims abstract description 16
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims abstract description 22
- 230000000694 effects Effects 0.000 claims abstract description 22
- 229910052740 iodine Inorganic materials 0.000 claims abstract description 22
- 239000011630 iodine Substances 0.000 claims abstract description 22
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 16
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 16
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims abstract description 16
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 claims abstract description 14
- 238000012216 screening Methods 0.000 claims abstract description 14
- 238000010186 staining Methods 0.000 claims abstract description 14
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims abstract description 13
- 235000010948 carboxy methyl cellulose Nutrition 0.000 claims abstract description 13
- 108010010803 Gelatin Proteins 0.000 claims abstract description 12
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims abstract description 10
- 239000008108 microcrystalline cellulose Substances 0.000 claims abstract description 10
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims abstract description 10
- 229940016286 microcrystalline cellulose Drugs 0.000 claims abstract description 10
- 239000008273 gelatin Substances 0.000 claims abstract description 9
- 229920000159 gelatin Polymers 0.000 claims abstract description 9
- 235000019322 gelatine Nutrition 0.000 claims abstract description 9
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 9
- 229920001817 Agar Polymers 0.000 claims abstract description 8
- 229920000936 Agarose Polymers 0.000 claims abstract description 8
- 239000008272 agar Substances 0.000 claims abstract description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 8
- 239000001103 potassium chloride Substances 0.000 claims abstract description 8
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 8
- 235000010344 sodium nitrate Nutrition 0.000 claims abstract description 8
- 239000004317 sodium nitrate Substances 0.000 claims abstract description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000012138 yeast extract Substances 0.000 claims abstract description 7
- 230000001580 bacterial effect Effects 0.000 claims abstract 5
- 229920002678 cellulose Polymers 0.000 claims description 4
- 239000001913 cellulose Substances 0.000 claims description 4
- 235000010980 cellulose Nutrition 0.000 claims description 4
- 239000000779 smoke Substances 0.000 claims description 2
- 235000019419 proteases Nutrition 0.000 claims 3
- 102000035195 Peptidases Human genes 0.000 claims 2
- 235000019833 protease Nutrition 0.000 claims 2
- 230000002255 enzymatic effect Effects 0.000 claims 1
- 239000012530 fluid Substances 0.000 claims 1
- 239000000499 gel Substances 0.000 claims 1
- 239000001768 carboxy methyl cellulose Substances 0.000 abstract description 10
- 239000008112 carboxymethyl-cellulose Substances 0.000 abstract description 10
- 239000000843 powder Substances 0.000 abstract description 9
- 238000004042 decolorization Methods 0.000 abstract description 7
- 238000003958 fumigation Methods 0.000 abstract description 6
- 229940041514 candida albicans extract Drugs 0.000 abstract description 5
- 239000002609 medium Substances 0.000 abstract description 5
- 239000011543 agarose gel Substances 0.000 abstract description 4
- 238000000855 fermentation Methods 0.000 abstract description 4
- 230000004151 fermentation Effects 0.000 abstract description 4
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 238000007598 dipping method Methods 0.000 description 4
- 230000007812 deficiency Effects 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 239000003517 fume Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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Abstract
本发明公开了一种同时筛选蛋白酶和纤维素酶的培养基及方法。本发明培养基包括以下组分：1~3g硝酸钠、0.8~1.2g磷酸氢二钾、0.4~0.6g硫酸镁、0.4~0.6g氯化钾、1~3g羧甲基纤维素或微晶纤维素、8~12g明胶、1~3g酵母浸粉、16~18g琼脂或琼脂糖，加水定容至1000ml。本发明方法包括以下步骤：（1）将培养基制成琼脂糖凝胶平板，选择两个平板并接种相同的菌株或其发酵液培养24h；对一平板先进行碘熏后观察记录结果，碘熏颜色褪去后再进行考马斯亮蓝染色脱色观察记录结果；对另一平板直接进行考马斯亮蓝染色脱色后观察记录结果；根据观察记录结果判断出所具蛋白酶活性、纤维素酶活性的菌株。本发明环境友好，能同时检测和筛选纤维素酶和蛋白酶活性菌株，操作简单高效。The invention discloses a medium and a method for simultaneously screening protease and cellulase. The culture medium of the present invention comprises the following components: 1~3g sodium nitrate, 0.8~1.2g dipotassium hydrogen phosphate, 0.4~0.6g magnesium sulfate, 0.4~0.6g potassium chloride, 1~3g carboxymethyl cellulose or microcrystalline Cellulose, 8~12g gelatin, 1~3g yeast extract powder, 16~18g agar or agarose, add water to make up to 1000ml. The method of the present invention comprises the following steps: (1) making the culture medium into an agarose gel plate, selecting two plates and inoculating the same bacterial strain or its fermentation broth for 24 hours; first carrying out iodine fumigation on one plate and then observing and recording the results. Coomassie Brilliant Blue staining and decolorization was performed to observe and record the results after the smoked color faded; the results were observed and recorded after direct Coomassie Brilliant Blue staining and decolorization on another plate; the strains with protease activity and cellulase activity were judged according to the observation and record results. The invention is environmentally friendly, can simultaneously detect and screen cellulase and protease activity strains, and is simple and efficient in operation.
Description
技术领域technical field
本发明属于生物技术领域，尤其涉及一种同时筛选蛋白酶和纤维素酶的培养基及方法。The invention belongs to the field of biotechnology, in particular to a culture medium and method for simultaneously screening protease and cellulase.
背景技术Background technique
现今筛选蛋白酶或者纤维素酶活性都是使用单一的平板，配制可以筛选不同活性酶的平板，要么筛选蛋白酶，要么筛选纤维素酶等。此外，蛋白酶筛选用的较多的是脱脂奶粉，直接可以看出透明圈，但是如果和其他方法结合起来的话，容易造成干扰。而筛选纤维素酶活性，通常用的是底物羧甲基纤维素或者微晶纤维素，然后通过刚果红或碘液浸染的方法得到结果，碘液浸染比刚果红灵敏，但是需要消耗大量的碘液且对环境有一定影响。Nowadays, a single plate is used to screen protease or cellulase activity, and a plate that can screen different active enzymes is prepared, either for protease or cellulase. In addition, skimmed milk powder is mostly used for protease screening, and the transparent circle can be seen directly, but if combined with other methods, it is easy to cause interference. And screen cellulase activity, what usually use is substrate carboxymethyl cellulose or microcrystalline cellulose, then obtain the result by Congo red or iodine solution dipping method, iodine solution dipping is more sensitive than Congo red, but needs to consume a large amount of iodine solution and has a certain impact on the environment.
发明内容Contents of the invention
本发明的目的在于提供一种同时筛选蛋白酶和纤维素酶的培养基及方法，旨在解决蛋白酶和纤维素酶活性需要分开筛选、且各筛选方法均存在不足的问题。The object of the present invention is to provide a culture medium and method for simultaneously screening protease and cellulase, aiming at solving the problem that protease and cellulase activities need to be screened separately, and each screening method has deficiencies.
本发明是这样实现的，一种同时筛选蛋白酶和纤维素酶的培养基，该培养基包括以下组分：1~3g 硝酸钠、0.8~1.2g 磷酸氢二钾、0.4~0.6g硫酸镁、0.4~0.6g氯化钾、1~3g羧甲基纤维素或微晶纤维素、8~12g明胶、1~3g酵母浸粉、16~18g琼脂或琼脂糖，加水定容至1000ml。The present invention is achieved in this way, a culture medium for simultaneously screening protease and cellulase, which comprises the following components: 1~3g sodium nitrate, 0.8~1.2g dipotassium hydrogen phosphate, 0.4~0.6g magnesium sulfate, 0.4~0.6g potassium chloride, 1~3g carboxymethyl cellulose or microcrystalline cellulose, 8~12g gelatin, 1~3g yeast extract powder, 16~18g agar or agarose, add water to make up to 1000ml.
优选地，该培养基包括以下组分：2g 硝酸钠、1g 磷酸氢二钾、0.5g硫酸镁、0.5g氯化钾、2g羧甲基纤维素或2g微晶纤维素、10g明胶、2g酵母浸粉、17g琼脂或琼脂糖，加水定容至1000ml。Preferably, the culture medium comprises the following components: 2 g sodium nitrate, 1 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate, 0.5 g potassium chloride, 2 g carboxymethyl cellulose or 2 g microcrystalline cellulose, 10 g gelatin, 2 g yeast Dip powder, 17g agar or agarose, add water to make up to 1000ml.
本发明进一步提供了一种同时筛选蛋白酶和纤维素酶的方法，该方法包括以下步骤：The present invention further provides a method for simultaneously screening protease and cellulase, the method comprising the following steps:
（1）将上述培养基制成琼脂糖凝胶平板，选择两个平板并接种相同的菌株或其发酵液培养24h；(1) Make the above culture medium into agarose gel plate, select two plates and inoculate the same strain or its fermentation broth for 24 hours;
（2）对一平板先进行碘熏5~10分钟，随后观察结果，拍照记录，碘熏颜色褪去后再进行考马斯亮蓝染色脱色观察结果，拍照记录结果；(2) Fume with iodine for 5 to 10 minutes on a plate, then observe the results, take pictures and record, after the color of iodine fumigation fades, perform Coomassie brilliant blue staining and decolorization to observe the results, and take pictures to record the results;
（3）对另一平板直接进行考马斯亮蓝染色脱色后观察结果、拍照记录；(3) Observe the results, take photos and record the results after direct Coomassie Brilliant Blue staining and decolorization on the other plate;
（4）根据步骤（2）、（3）所得的观察记录结果判断出具有蛋白酶活性的菌株、具有纤维素酶活性的菌株，或者同时具有蛋白酶活性和纤维素酶活性的菌株。(4) Judging the strains with protease activity, cellulase activity, or both protease activity and cellulase activity according to the observation and recording results obtained in steps (2) and (3).
本发明克服现有技术的不足，提供一种同时筛选蛋白酶和纤维素酶的培养基及方法，采用同时添加蛋白酶底物明胶蛋白和纤维素酶底物羧甲基纤维素的方式，制备培养基，结合先碘熏后考马斯亮蓝染色的方法，建立同时检测和筛选纤维素酶和蛋白酶活性菌株的快捷方法。蛋白酶筛选用的较多的是脱脂奶粉，直接可以看出透明圈，但是如果和其他方法结合起来的话，容易造成干扰，本发明选用了明胶蛋白，使用考马斯亮蓝染色，经过试验是可行的；而筛选纤维素酶活性，通常用的是底物羧甲基纤维素或者微晶纤维素，然后刚果红或碘液浸染的方法，碘液浸染比刚果红灵敏，但是需要消耗大量的碘液且对环境有一定影响，碘液浸染改为碘液熏染同样灵敏快速，而且碘液耗量减少，而且对环境污染少，所以在本发明中，综合起来用明胶蛋白和羧甲基纤维素结合，然后先碘熏后考马斯亮蓝的染色方法，两者结合起来可以筛选蛋白酶和纤维素酶，考虑到碘熏可能会对考马斯亮蓝染色造成结果，实验做了一下，两者没有干扰，故此方法是可行的，这也为后续再添加其他底物筛选其他活性奠定基础。The present invention overcomes the deficiencies of the prior art and provides a culture medium and method for simultaneously screening protease and cellulase. The culture medium is prepared by simultaneously adding protease substrate gelatin protein and cellulase substrate carboxymethyl cellulose , combined with the method of coomassie brilliant blue staining after iodine fumigation, to establish a quick method for simultaneous detection and screening of strains with cellulase and protease activity. Skimmed milk powder is mostly used for protease screening, and the transparent circle can be seen directly, but if it is combined with other methods, it will easily cause interference. The present invention selects gelatin protein and uses Coomassie brilliant blue staining, which is feasible after testing; And screen cellulase activity, what usually used is substrate carboxymethyl cellulose or microcrystalline cellulose, then Congo red or iodine solution dipping method, iodine solution dipping is more sensitive than Congo red, but needs to consume a large amount of iodine solution and Environment is had certain influence, iodine solution dyeing is changed into iodine solution and is equally sensitive and quick, and iodine solution consumption reduces, and environmental pollution is few, so in the present invention, combine together with gelatin protein and carboxymethyl cellulose, Then the staining method of Coomassie Brilliant Blue after iodine fumigation, the combination of the two can screen protease and cellulase. Considering that iodine fumigation may cause results in Coomassie Brilliant Blue staining, the experiment was done, and there was no interference between the two, so this method It is feasible, and this also lays the foundation for the subsequent addition of other substrates to screen for other activities.
相比于现有技术的缺点和不足，本发明具有以下有益效果：本发明环境友好，能同时检测和筛选纤维素酶和蛋白酶活性菌株，操作简单高效。Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: the present invention is environmentally friendly, can simultaneously detect and screen cellulase and protease active strains, and is simple and efficient in operation.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.
实施例1Example 1
培养基配方如下：2g 硝酸钠 1g 磷酸氢二钾 0.5g硫酸镁 0.5g氯化钾 2g羧甲基纤维素或2g微晶纤维素 10g明胶 2g酵母浸粉 17g琼脂或琼脂糖，加水定容至1000ml。The medium formula is as follows: 2g sodium nitrate 1g dipotassium hydrogen phosphate 0.5g magnesium sulfate 0.5g potassium chloride 2g carboxymethyl cellulose or 2g microcrystalline cellulose 10g gelatin 2g yeast extract powder 17g agar or agarose, add water to volume 1000ml.
实施例2Example 2
培养基配方如下：1g 硝酸钠、0.8g 磷酸氢二钾、0.4g硫酸镁 0.6g氯化钾、3g羧甲基纤维素或3g微晶纤维素、8g明胶、1g酵母浸粉、18g琼脂或琼脂糖，加水定容至1000ml。The formula of the medium is as follows: 1g sodium nitrate, 0.8g dipotassium hydrogen phosphate, 0.4g magnesium sulfate, 0.6g potassium chloride, 3g carboxymethyl cellulose or 3g microcrystalline cellulose, 8g gelatin, 1g yeast extract powder, 18g agar or Agarose, add water to make up to 1000ml.
实施例3Example 3
培养基配方如下：3g 硝酸钠、1.2g 磷酸氢二钾、0.6g硫酸镁、0.6g氯化钾、1g羧甲基纤维素或1g微晶纤维素、12g明胶、1g酵母浸粉、16g琼脂或琼脂糖，加水定容至1000ml。The medium formula is as follows: 3g sodium nitrate, 1.2g dipotassium hydrogen phosphate, 0.6g magnesium sulfate, 0.6g potassium chloride, 1g carboxymethyl cellulose or 1g microcrystalline cellulose, 12g gelatin, 1g yeast extract powder, 16g agar Or agarose, add water to make up to 1000ml.
实施例4Example 4
（1）将实施例1培养基制成琼脂糖凝胶平板，选择两个平板并接种相同的菌株或其发酵液培养24h；(1) Make an agarose gel plate from the culture medium of Example 1, select two plates and inoculate the same strain or its fermentation broth for 24 hours;
（2）对一平板先进行碘熏5~10分钟（将该平板倒扣在装有革兰氏碘液的平皿盖子上碘熏5~10分钟，纤维素酶的筛选通常是采用以羧甲基纤维素（CMC）或微晶纤维素（Avicel）为底物制成的琼脂糖凝胶平板结合刚果红或碘液染色的方法），用于验证碘熏是否对后来的考马斯亮蓝染色造成影响，随后观察结果，拍照记录；碘熏颜色褪去后再进行考马斯亮蓝染色脱色观察结果，拍照记录结果。(2) Fume with iodine for 5-10 minutes on a plate first (put the plate upside down on the lid of a plate containing Gram's iodine solution for 5-10 minutes, cellulase is usually screened with carboxymethyl Agarose gel plates made of cellulose-based cellulose (CMC) or microcrystalline cellulose (Avicel) combined with Congo red or iodine solution staining) to verify whether iodine fumigation has any effect on the subsequent Coomassie brilliant blue staining Effect, then observe the results, take pictures and record; after the iodine smoke fades, carry out Coomassie Brilliant Blue staining and decolorization to observe the results, take pictures and record the results.
（3）对另一平板直接进行考马斯亮蓝染色脱色后观察结果、拍照记录；(3) Observe the results, take photos and record the results after direct Coomassie Brilliant Blue staining and decolorization on the other plate;
（4）根据步骤（2）、（3）所得的观察记录结果判断出具有蛋白酶活性的菌株、具有纤维素酶活性的菌株，或者同时具有蛋白酶活性和纤维素酶活性的菌株；其中，用明胶代替脱脂奶粉配筛选蛋白酶的培养基，接种菌株或其发酵液后，肉眼不能观察溶解圈，可以用考马斯亮蓝溶液对平板染色脱色，有溶解圈的周围没有蛋白，染色脱色后蓝色褪去，没有溶解圈的地方则有大量的蛋白就会染成蓝色。(4) Judging the strains with protease activity, cellulase activity, or both protease activity and cellulase activity according to the observation and recording results obtained in steps (2) and (3); among them, use gelatin Instead of skimmed milk powder, mix protease screening medium. After inoculating the strain or its fermentation broth, the dissolution circle cannot be observed with the naked eye, and the plate can be stained and decolorized with Coomassie brilliant blue solution. There is no protein around the dissolution circle, and the blue color fades after staining and decolorization. Where there is no dissolution zone, there is a large amount of protein and it will be stained blue.
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1387578A (en) * | 1999-09-03 | 2002-12-25 | 三共株式会社 | Novel compound F-15078 |
| EP3209132A1 (en) * | 2014-10-23 | 2017-08-30 | Futureco Bioscience, S.A. | Bacteria with nematicidal activity and the ability to promote plant growth |
-
2018
- 2018-01-12 CN CN201810032356.1A patent/CN108165605A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1387578A (en) * | 1999-09-03 | 2002-12-25 | 三共株式会社 | Novel compound F-15078 |
| EP3209132A1 (en) * | 2014-10-23 | 2017-08-30 | Futureco Bioscience, S.A. | Bacteria with nematicidal activity and the ability to promote plant growth |
Non-Patent Citations (3)
| Title |
|---|
| 刘丽莉等: "胶原蛋白酶产生菌的筛选及酶的分离纯化", 《生物工程学报》 * |
| 陈晨等: "一种筛选胞外纤维素酶产生菌的快捷、灵敏、有效的方法", 《微生物学通报》 * |
| 韩锐: "《抗癌药物研究与实验技术》", 30 April 1997, 北京医科大学 中国协和医科大学联合出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734954A (en) * | 2019-11-21 | 2020-01-31 | 河北省微生物研究所 | Rapid identification culture medium and application thereof in cellulase qualitative detection |
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