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Xylanase (E.C. 3.2.1.8) degrades β-1, 4 xylan by cleaving β-1, 4 glycosidic linkages randomly, resulting in the generation of xylose and xylo-oligosaccharides. Xylanases are produced by organisms including fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans and insects. Xylanases present considerable industrial interest for their use in paper manufacturing, improvement of animal feed digestibility, and clarification of fruit juices. In addition, this enzyme is the component of cell wall-degrading enzymes (CWDEs) during plant–pathogen interaction. Thus, considering their various applications in plant defence and also in industry, the characterization of xylanase activity becomes an important aspect. Conventionally, xylanase activity is determined by radial gel diffusion assay using Congo red staining (Emami and Hack, 2001) and by DNSA assay which is a colorimetric method for xylanase activity (McLauchlan et al., 1999; Kutasi et al., 2001). Comparatively, radial gel diffusion assay using Congo red staining is a qualitative assay whereas DNSA method is a quantitative assay. Moreover, Congo red is a chemical considered as hazardous category 1B (Carcinogenicity) and category 12 (Reproductive toxicity) by the 2012 OSHA Hazard Communication Standard (29 CFR 1910.1200). In the present study, the proposed method enables qualitative detection of xylanase activity using ethanol precipitation in the radial gel diffusion assay which is safer and simpler. The ethanol precipitation in agar plate has been adapted from the method for detecting xylanase activity in polyacrylamide gels (Royer and Nakas, 1990).

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An Improved and Simplified Radial Gel Diffusion Assay for the Detection of Xylanase Activity
一种径向凝胶扩散的简单改进实验用于检测木聚糖酶的活性

生物化学 > 蛋白质 > 活性
作者: Raviraj M. Kalunke
Raviraj M. KalunkeAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3295
Ilaria Moscetti
Ilaria MoscettiAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3296
Silvio Tundo
Silvio TundoAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
Bio-protocol author page: a3297
 and Renato D’Ovidio
Renato D’OvidioAffiliation: Dipartimento di Scienze Agrarie e Forestali (DAFNE), Università degli Studi della Tuscia, Viterbo, Italy
For correspondence: dovidio@unitus.it
Bio-protocol author page: a3298
Vol 6, Iss 13, 7/5/2016, 1497 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1863

[Abstract] Xylanase (E.C. 3.2.1.8) degrades β-1, 4 xylan by cleaving β-1, 4 glycosidic linkages randomly, resulting in the generation of xylose and xylo-oligosaccharides. Xylanases are produced by organisms including fungi, bacteria, yeast, marine algae, protozoans, snails, crustaceans and insects. Xylanases present considerable industrial interest for their use in paper manufacturing, improvement of animal feed digestibility, and clarification of fruit juices. In addition, this enzyme is the component of cell wall-degrading enzymes (CWDEs) during plant–pathogen interaction. Thus, considering their various applications in plant defence and also in industry, the characterization of xylanase activity becomes an important aspect. Conventionally, xylanase activity is determined by radial gel diffusion assay using Congo red staining (Emami and Hack, 2001) and by DNSA assay which is a colorimetric method for xylanase activity (McLauchlan et al., 1999; Kutasi et al., 2001). Comparatively, radial gel diffusion assay using Congo red staining is a qualitative assay whereas DNSA method is a quantitative assay. Moreover, Congo red is a chemical considered as hazardous category 1B (Carcinogenicity) and category 12 (Reproductive toxicity) by the 2012 OSHA Hazard Communication Standard (29 CFR 1910.1200). In the present study, the proposed method enables qualitative detection of xylanase activity using ethanol precipitation in the radial gel diffusion assay which is safer and simpler. The ethanol precipitation in agar plate has been adapted from the method for detecting xylanase activity in polyacrylamide gels (Royer and Nakas, 1990).
Keywords: Xylanase(木聚糖酶), Radial gel diffusion(径向凝胶扩散), Xylan(木聚糖)

[Abstract]

Materials and Reagents

  1. 90 x 15 mm petri plates (SARSTEDT AG, catalog number: 82.1473001 )
  2. 0.5-cm-diameter drinking straw
  3. 150 ml Erlenmeyer flask
  4. Aspergillus niger xylanase (Xylanase M4) (Megazyme) or Trichoderma longibrachiatum xylanase (Xylanase M3) (Megazyme)
  5. Disodium hydrogen phosphate (Sigma-Aldrich, catalog number: S5136 )
  6. Citric acid (Sigma-Aldrich, catalog number: C1909 )
  7. Birch wood xylan (Sigma-Aldrich, catalog number: X0502 )
  8. Agarose (AppliChem GmbH, catalog number: A8963.0500 )
  9. Absolute ethanol (VWR International, catalog number: 20821.321 )
  10. McIlvaine’s buffer (pH 5.0) (see Recipes)
  11. 95% (v/v) ethanol (see Recipes)

Equipment

  1. Microwave
  2. Hallow flat end with 0.5-cm-diameter drinking straw
  3. 30 °C incubator (Mini BATT 805 ) (230 V, 50 Hz, 270 W) (Asal Srl, model: 805)
  4. Scanner camera (Epson, model: perfection V30 )

Procedure

  1. Mix 1.0% (w/v) birch wood xylan with 1.0% (w/v) agarose in McIlvaine’s buffer (15 ml/petri dish) and boil in 150 ml Erlenmeyer flask (approximately 10 min) until the birch wood xylan is dissolved completely; mix thoroughly and then dispense it in a petri plate (44 mm base diameter x 12 mm depth).
  2. Allow the birch wood xylan–agarose solution to solidify in the petri dish and then prepare a 0.5-cm-diameter well on it. The hole in agarose is poked using a hallow flat end of a 0.5-cm-diameter drinking straw.
  3. Place 30 µl of A. niger or T. longibrachiatum xylanase solution (total volume to be adjusted with McIlvaine’s buffer) in 0.5-cm-diameter wells of birch wood xylan-agarose petri plate.
  4. Incubate the petri plate at 30 °C for 16 h.
  5. Overlay the plate with 95% ethanol and keep the plate at room temperature for 30 min to reveal the halo representing the degradative activity of xylanase.
  6. Measure the diameter of the halo using a scale.
  7. Take the image using a digital camera or scan the petri plate using a scanner to keep the record of the results (Figure 1).


    Figure 1. Example of agarose diffusion assays for detection of xylanase activity. Haloes represent xylanase activity. 1: Buffer (negative control); 2: Aspergillus niger xylanase M4 (0.006U); 3: A. niger xylanase M4 (0.012U); 4: A. niger xylanase M4 (0.018U); 5: A. niger xylanase M4 (0.024U).

Recipes

  1. McIlvaine’s buffer (pH 5.0)
    0.2 M disodium hydrogen phosphate
    0.1 M citric acid
  2. 95% (v/v) absolute ethanol
    Mix 95 ml absolute ethanol with 5% (v/v) distilled water

Acknowledgments

This protocol has been adapted from the previously published by Kalunke et al. (2013). This protocol was designed to determine xylanase inhibition for wheat transgenic plants overexpressingthe xylanase inhibitor TAXI‐III.
Research was supported by the Italian Ministry of University and Research (PRIN 2010-2011) to Renato D’Ovidio.

References

  1. Emami, K., Hack, E. (2001). Characterisation of a xylanase gene from Cochliobolussativus and its expression. Mycol. Res. 105 (3): 352-359.
  2. Kalunke, R. M., Janni, M., Benedettelli, S. and D’Ovidio, R. (2013). Using biolistics and hybridization to combine multiple glycosidase inhibitor transgenes in wheat. Euphytica 194(3): 443-457.
  3. Kutasi, J., Bata, A., Brydl, E., Rafai, P. and Jurkovich, V. (2001). Characterisation and effects of a xylanase enzyme preparation extracted from Thermomyces lanuginosus cultures. Acta Vet Hung 49(2): 175-184. 
  4. McLauchlan, W. R., Garcia-Conesa, M. T., Williamson, G., Roza, M., Ravestein, P. and Maat, J. (1999). A novel class of protein from wheat which inhibits xylanases. Biochem J 338 (Pt 2): 441-446.
  5. Royer, J. C. and Nakas, J. P. (1990). Simple, sensitive zymogram technique for detection of xylanase activity in polyacrylamide gels. Appl Environ Microbiol 56(6): 1516-1517.

材料和试剂

  1. 90×15mm培养皿(SARSTEDT AG,目录号:82.1473001)
  2. 0.5厘米直径的吸管
  3. 150ml锥形瓶
  4. 木聚糖酶(木聚糖酶M4)(Megazyme)或木霉菌木霉木聚糖酶(木聚糖酶M3)(Megazyme)
  5. 磷酸氢二钠(Sigma-Aldrich,目录号:S5136)
  6. 柠檬酸(Sigma-Aldrich,目录号:C1909)
  7. 桦木木聚糖(Sigma-Aldrich,目录号:X0502)
  8. 琼脂糖(AppliChem GmbH,目录号:A8963.0500)
  9. 无水乙醇(VWR International,目录号:20821.321)
  10. McIlvaine的缓冲液(pH 5.0)(参见配方)
  11. 95%(v/v)乙醇(见配方)

设备

  1. 微波
  2. 半圆平头,直径为0.5厘米的吸管
  3. 30℃培养箱(Mini BATT 805)(230V,50Hz,270W)(Asal Srl,型号:805)
  4. 扫描相机(爱普生,型号:完美V30)

程序

  1. 将1.0%(w/v)桦木木聚糖与在McIlvaine缓冲液(15ml /培养皿)中的1.0%(w/v)琼脂糖混合,并在150ml锥形瓶中煮沸(约10分钟),直到桦木木聚糖完全溶解;充分混合,然后将其分配在培养皿(44mm底径×12mm深)中
  2. 使桦木木聚糖琼脂糖溶液在培养皿中固化,然后在其上制备0.5cm直径的孔。使用0.5cm直径的吸管的中空平端戳刺琼脂糖中的孔
  3. 放置30微升的。尼日尔或 T。在用桦木木木聚糖 - 琼脂糖培养皿的0.5-cm直径的孔中培养长木霉素木聚糖酶溶液(用McIlvaine缓冲液调节的总体积)。
  4. 孵育培养皿在30℃下16小时。
  5. 用95%乙醇覆盖板,并将板在室温下保持30分钟以显示代表木聚糖酶的降解活性的晕。
  6. 使用标尺测量光晕的直径。
  7. 使用数码相机拍摄图像或使用扫描仪扫描培养皿以保留结果记录(图1)。


    图1.用于检测木聚糖酶活性的琼脂糖扩散测定的实施例。卤素代表木聚糖酶活性。 1:缓冲液(阴性对照); 2:黑曲霉木聚糖酶M4(0.006U); 3:
    。木霉聚糖酶M4(0.012U); 4:。木霉聚糖酶M4(0.018U); 5:

    A。木霉聚糖酶M4(0.024U)。

食谱

  1. McIlvaine的缓冲液(pH 5.0)
    0.2M磷酸氢二钠
    0.1M柠檬酸
  2. 95%(v/v)无水乙醇 将95ml无水乙醇与5%(v/v)蒸馏水混合

致谢

该协议已经从先前由Kalunke等人(2013)发表的。该方案设计用于确定过表达木聚糖酶抑制剂TAXI-III的小麦转基因植物的木聚糖酶抑制。
研究由意大利大学和研究部(PRIN 2010-2011)支持Renato D'Ovidio。

参考文献

  1. Emami,K.,Hack,E。(2001)。  来自Cochliobolussativus的木聚糖酶基因及其表达。 Res。 105(3):352-359
  2. Kalunke,RM,Janni,M.,Benedettelli,S.和D'Ovidio,R。(2013)。  使用生物射弹和杂交以在小麦中组合多种糖苷酶抑制剂转基因。 Euphytica 194(3):443-457。
  3. Kutasi,J.,Bata,A.,Brydl,E.,Rafai,P.and Jurkovich,V。(2001)。  从Thermomyces lanuginosus培养物提取的木聚糖酶制剂的表征和作用。 49(2):175 -184。 
  4. McLauchlan,WR,Garcia-Conesa,MT,Williamson,G.,Roza,M.,Ravestein,P.and Maat,J.(1999)。  来自小麦的抑制木聚糖酶的一类新型蛋白质。生物化学杂志338(Pt 2) :441-446。
  5. Royer,JC和Nakas,JP(1990)。  简单用于检测聚丙烯酰胺凝胶中木聚糖酶活性的灵敏酶谱技术。 Appl Environ Microbiol 56(6):1516-1517。
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How to cite this protocol: Kalunke, R. M., Moscetti, I., Tundo, S. and D’Ovidio, R. (2016). An Improved and Simplified Radial Gel Diffusion Assay for the Detection of Xylanase Activity. Bio-protocol 6(13): e1863. DOI: 10.21769/BioProtoc.1863; Full Text



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