Assessments on the catalytic and kinetic properties of beta-glucosidase isolated from a highly efficient antagonistic fungus Trichoderma harzianum

Elham Khalili,Kalaivani Batumalaie,Roswanira Abdul Wahab,Muhammad Arshad Javed,Fahrul Huyop,Naji Arafat Mahat

doi:10.14393/bj-v34n1a2018-39384

Abstract

Due to the toxicity and inefficiency of chemical fungicides to control infestation of Macrophomina phaseolina (Tassi) Goid which causes charcoal rot in plants, a biotechnological approach using β-glucosidase (EC.3.2.1) as the alternative bioactive ingredient in fungicide is hereby, proposed. The extracellular enzyme was isolated from a highly efficient fungal antagonist, Trichoderma harzianum T12. The highly similar molecular masses obtained using SDS-PAGE (96 kDa) and MALDI-TOF mass spectrometry (98.3 kDa) affirmed that the β-glucosidase was purified to homogeneity. Consequently, optimum catalytic parameters that rendered the highest enzyme activity were found to be: 45˚C, pH 7, inoculum size of 10 % (w/v), supplementation with metal ions Zn2+ and Mn2+ ions, and Tween 80. Addition of wheat bran and (NH4)2SO4 as carbon and nitrogen sources also improved enzyme activity. BLASTn showed the sequence of β-glucosidase T12 was highly identical to other β-glucosidases viz. T. harzianum strain IOC-3844 (99%), T. gamsii and T. virens bgl1 (86 %) as well as T. reesei strain SJVTR and T. viride strain AS 3.3711 (84 %). Kinetic assessment showed that β-glucosidase T12 catalyzes hydrolytic activity is characterized by a Km of 0.79 mM and Vmax of 8.45 mM min-1 mg-1 protein, with a corresponding kcat of 10.69 s-1.

Highlights

Charcoal rot is a disease prevalent in commercial crops caused by a pathogenic root fungus Macrophomina phaseolina (Tassi) Goid which led to substantial losses in crop yields such as corn (Zea mays L.), sorghum (Sorghum bicolor), sesame (Sesamum indicum L.) and soybean (Glycine max L.) (RAYATPANAH et al, 2012; KHALILI et al, 2016) worldwide
The alternative technique suggested here focuses on the use of an enzyme, βglucosidase (EC.3.2.1) naturally secreted by the antagonistic fungal isolate, T. harzianum, as a bioactive ingredient in a fungicide. β-glucosidase belongs to a group of enzymes called hydrolases which hydrolyse O- and S-glycosyl compounds (EXPASY., 2017)
SDS-PAGE Results show that purified β-glucosidase appeared to be homogeneous in the F3 and F4 on SDS-PAGE (Figure 1) as estimated from the obtained 96 kDa molecular mass, indicating that the 60 and 80% saturation fractions of ammonium sulfate contained pure β-glucosidase, respectively

Summary

Introduction

Charcoal rot is a disease prevalent in commercial crops caused by a pathogenic root fungus Macrophomina phaseolina (Tassi) Goid which led to substantial losses in crop yields such as corn (Zea mays L.), sorghum (Sorghum bicolor), sesame (Sesamum indicum L.) and soybean (Glycine max L.) (RAYATPANAH et al, 2012; KHALILI et al, 2016) worldwide. Utilization of chemical fungicides against M. phaseolina is inefficient due to the soil-borne nature of the fungus (ANIS et al, 2010). In view of the shortcomings in current pest management strategies, the study hereby suggests a greener approach to better control charcoal rot caused by M. phaseolina. The alternative technique suggested here focuses on the use of an enzyme, βglucosidase (EC.3.2.1) naturally secreted by the antagonistic fungal isolate, T. harzianum, as a bioactive ingredient in a fungicide. T. harzianum has evolved the capability to secrete a type of β-glucosidase that digests the ordered layers of amorphic β-1, 3-glucan (GAJERA et al, 2010; KHALILI et al, 2017) in M. phaseolina. The hydrolytic activity of this βglucosidase targets the sugar-based polymer that

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