尖孢镰刀菌中几丁质酶活性的定量检测

Carmen Ruiz-Roldan; M. Isabel G. Roncero

doi:10.21769/BioProtoc.1882

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Peer-reviewed

Quantification of Chitinase Activity in Fusarium oxysporum

尖孢镰刀菌中几丁质酶活性的定量检测

Carmen Ruiz-Roldan email

M. Isabel G. Roncero

发布: 2016年08月05日第6卷第15期 DOI: 10.21769/BioProtoc.1882 浏览次数: 12930

评审: Zhaohui LiuClaudia CatalanottiAndreea Daraba

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Abstract

Fungal morphogenetic development requires modification and plasticity of the cell wall, which implies synthesis and remodelling of its components, including chitin and glucan. Thus chitinase and glucanase activities are crucial for cell-wall biogenesis and cell division. Quantification of chitinase activity might be useful to identify structural defects that could negatively influence growth and morphogenesis of some filamentous fungi like Fusarium oxysporum, which produces both intracellular and secreted chitinases. The chitinolytic enzymes are categorized based on their enzymatic action on chitin substrates. Endochitinases are defined as the enzymes catalyzing the random cleavage at internal points in the chitin chain. Exochitinases catalyze the progressive release of acetylchitobiose or N-acetylglucosamine from the non-reducing end of chitin, and thus, are referred to as chitobiosidase and β-N-acetylglucosaminidase, respectively. Here we describe a simple method to easily purify chitinases in order to compare both endo- and exo-chitinase activity of different F. oxysporum strains. The protocol can be adapted to any fungal species.

Keywords: Chitinase activity (池体娜涩的活动)

Fusarium (镰刀菌)

Extracellular enzyme (胞外酶)

Intracellular enzyme (细胞内的酶)

Materials and Reagents

Monodur nylon filters 15 µm diameter (Filtravibracion S.L., catalog number: Nylon-15 )
Sterile plastic funnels (80 mm diameter) (Tecnylab, catalog number: 45000150 )
Eppendorf tubes
Sterile spatula (Instrumentación Científica, S.L., catalog number: 2004NE8044 )
Microtiter plates (Thermo Fisher Scientific, catalog number: 2205 )
Dialysis membranes, cut-off 12 kDa (Sigma-Aldrich, catalog number: D9527-100FT )
Fusarium oxysporum f.sp. lycopersici microconida suspensions from wild type (J. Tello, Universidad de Almeria, Spain), the Δcon7-1 and the cΔcon7-1 mutant strains (Ruiz-Roldan et al., 2015)
Glycerol (Merck, catalog number: 104092 )
Potato dextrose broth medium (PDB) (Scharlau, catalog number: 01483 )
Chitin (Sigma-Aldrich, catalog number: C6137 )
Bio-Rad protein assay Dye reagent concentrate (Bio-Rad Laboratories, catalog number: 500-0006 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 10 735 078 001 )
Polyethylenglycol 35,000 (Sigma-Aldrich, catalog number: 81310 )
Na₂HPO₄ (Merck, catalog number: 1.06586.0500 )
NaH₂PO₄ monohydrate (Merck, catalog number: 567549 )
MgSO₄·7H₂O (Merck, catalog number: 1058865.5000 )
KH₂PO₄ (Merck, catalog number: 104873.1000 )
KCl (Merck, catalog number: 104933.0500 )
NH₄NO₃ (Merck, catalog number: 1.01187.1000 )
FeSO₄ (Merck, catalog number: 103965.0500 )
ZnSO₄·7H₂O (Merck, catalog number: 108883 )
MnSO₄ monohydrate (Merck, catalog number: 105941 )
Glucose (Coger SAS, catalog number: 24379.363 )
NaCl (Merck, catalog number: 1.06404.5000 )
Fluorimetric Chitinase Assay Kit (Sigma-Aldrich, catalog number: CS1030 ). Contents:
1. Assay Buffer (25 ml) (Sigma-Aldrich, catalog number: A8730 )
2. 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide (5 mg) (Sigma-Aldrich, catalog number: M2133 )
3. 4-Methylumbelliferyl β-D-N,N’-diacetylchitobioside hydrate (5 mg) (Sigma-Aldrich, catalog number: M9763 )
4. 4-Methylumbelliferyl β-D-N,N’,N’’-triacetylchitotriose (5 mg) (Sigma-Aldrich, catalog number: M5639 )
5. Chitinase from Trichoderma viride (1 mg) (Sigma-Aldrich, catalog number: C6242 )
6. 4-Methylumbelliferone Standard Solution, 50 mg/ml (1 ml) (Sigma-Aldrich, catalog number: M3570 )
7. Sodium carbonate (2 g) (Sigma-Aldrich, catalog number: S2127 )
8. Dimethyl sulfoxide (DMSO) (1 ml) (Sigma-Aldrich, catalog number: D8418 )
0.05 M phosphate buffer (see Recipes)
Synthetic medium (SM) (see Recipes)
Phosphate-buffered saline (PBS) (see Recipes)
Strate stock solutions (20 mg/ml) (see Recipes)
Strate working solutions (see Recipes)
Chitinase control enzyme (see Recipes)
Stop solution (sodium carbonate solution) (see Recipes)
4-Methylumbelliferone standard solution (see Recipes)

Equipment

Hemocytometer (Thoma) (Marienfeld, catalog number: 06 407 10 )
Mini-BeadBeater -16 homogenizer (BioSpec Products)
Mortar and pestle
Orbital incubator with shaking (Infors, model: Multitron Pro )
Microcentrifuge (Eppendorf MiniSpin plus, Fisher Scientific)
Fluorimeter (SpectraFluorPlus, TECAN, catalog number: F129005 )
Water bath (Selecta, catalog number: 6000138 )
Vortex (IKA, model: MS2 Minishaker )

Procedure

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如何引用

Ruiz-Roldan, C. and Roncero, M. I. G. (2016). Quantification of Chitinase Activity in Fusarium oxysporum. Bio-protocol 6(15): e1882. DOI: 10.21769/BioProtoc.1882.