Optimized microplate beta-1,3-glucanase assay system for Trichoderma spp. screening.
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 microl from the 1150 microl 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.
Ramada
Marcelo Henrique Soller
MH
Laboratório de Enzimologia, Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Goiás (ICB), Goiânia, GO, CEP 74001-970, Brazil.
Lopes
Fabyano Alvares Cardoso
FA
Ulhoa
Cirano José
CJ
Silva
Roberto do Nascimento
Rdo N
D000818:Animals; D019411:Clinical Laboratory Techniques / Q000379:methods*; D043326:Glucan 1,3-beta-Glucosidase / Q000378:metabolism*; D008403:Mass Screening / Q000379:methods*; D009172:Mycology / Q000379:methods*; D012680:Sensitivity and Specificity; D014242:Trichoderma / Q000201:enzymology* / Q000302:isolation & purification*
D043326:Glucan 1,3-beta-Glucosidase