Optimized microplate β-1,3-glucanase assay system for Trichoderma spp. screening

被引:30
作者
Soller Ramada, Marcelo Henrique [1 ]
Cardoso Lopes, Fabyano Alvares [1 ]
Ulhoa, Cirano Jose [1 ]
Silva, Roberto do Nascimento [1 ]
机构
[1] Univ Fed Goias ICB, Lab Enzimol, Dept Bioquim & Biol Mol, BR-74001970 Goiania, Go, Brazil
关键词
Biocontrol; Laminarin; Microplate assay; Trichoderma; beta-1,3-glucanase; BIOCHEMICAL-CHARACTERIZATION; RHIZOCTONIA-SOLANI; CELL-WALL; HARZIANUM; EXO-BETA-1,3-GLUCANASE; MYCOPARASITISM; PURIFICATION; GLUCANASES; ASPERELLUM; MECHANISMS;
D O I
10.1016/j.mimet.2010.01.010
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
beta-1,3-glucanase is an important cell wall-degrading enzyme involved in mycoparasitism by Trichoderma spp. during antagonism against phytopathogenic fungi. A simple microplate-based method to assay beta-1,3-glucanase activity is described here as an alternative to an expensive tube-assay method. The reaction ;volume of the micro-assay was reduced to 130 mu l from the 1150 mu l used in the standard beta-1,3-glucanase macro-assay. Statistical analyses showed significant difference in sensitivity between the micro- and the macro-assay. The micro-method was optimized using the Response Surface Quadratic Model. The sensitivity of the optimized micro-method was shown to be four-fold greater than the macro-assay and two-fold higher than the micro-assay. The optimized micro-assay was significantly more sensitive in all of the twenty examined isolates during Trichoderma spp. beta-1,3-glucanase screening. We conclude that this modified and optimized method is more convenient, faster, cheaper and more reproducible than the traditional tube-assay. (C) 2010 Elsevier B.V. All rights reserved.
引用
收藏
页码:6 / 10
页数:5
相关论文
共 25 条
[1]   Fungal cell wall chitinases and glucanases [J].
Adams, DJ .
MICROBIOLOGY-SGM, 2004, 150 :2029-2035
[2]   Purification and characterization of an exo-β-1,3-glucanase produced by Trichoderma asperellum [J].
Bara, MTF ;
Lima, AL ;
Ulhoa, CJ .
FEMS MICROBIOLOGY LETTERS, 2003, 219 (01) :81-85
[3]   Pathogenesis-related functions of plant beta-1,3-glucanases investigated by antisense transformation - A review [J].
Beffa, R ;
Meins, F .
GENE, 1996, 179 (01) :97-103
[4]  
Benítez T, 2004, INT MICROBIOL, V7, P249
[5]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P680
[6]   Mycoparasitism studies of Trichoderma harzianum strains against Rhizoctonia solani:: evaluation of coiling and hydrolytic enzyme production [J].
Bruno dos Reis Almeida, Fausto ;
Cerqueira, Fernanda Menezes ;
Silva, Roberto do Nascimento ;
Ulhoa, Cirano Jose ;
Lima, Adilson Lopes .
BIOTECHNOLOGY LETTERS, 2007, 29 (08) :1189-1193
[7]   Characterization of a tissue-specific and developmentally regulated β-1,3-glucanase gene in pea (Pisum sativum) [J].
Buchner, P ;
Rochat, C ;
Wuillème, S ;
Boutin, JP .
PLANT MOLECULAR BIOLOGY, 2002, 49 (02) :171-186
[8]   Molecular characterization of a novel β-1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum [J].
Cohen-Kupiec, R ;
Broglie, KE ;
Friesem, D ;
Broglie, RM ;
Chet, I .
GENE, 1999, 226 (02) :147-154
[9]  
CUADRADO ABM, 2008, FUNGAL GENET BIOL, V45, P542
[10]   The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-β-1,3-glucanase [J].
Genta, Fernando A. ;
Dumont, Alexandra F. ;
Marana, Sandro R. ;
Terra, Walter R. ;
Ferreira, Clelia .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, 2007, 1774 (09) :1079-1091