Acidophilic β-xylanases have attracted considerable attention due to their excellent activity under extreme acidic environments and potential industrial utilizations. In this study, a novel β-xylanase gene (Xyl11) of glycoside hydrolase family 11, was cloned from Trichoderma asperellum ND-1 and efficiently expressed in Pichia pastoris (a 2.0-fold increase). Xyl11 displayed a maximum activity of 121.99 U/ml at pH 3.0 and 50°C, and exhibited strict substrate specificity toward beechwood xylan (Km = 9.06 mg/ml, Vmax = 608.65 μmol/min/mg). The Xyl11 retained over 80% activity at pH 2.0–5.0 after pretreatment at 4°C for 1 h. Analysis of the hydrolytic pattern revealed that Xyl11 could rapidly convert xylan to xylobiose via hydrolysis activity as well as transglycosylation. Moreover, the results of site-directed mutagenesis suggested that the Xyl11 residues, Glu127, Glu164, and Glu216, are essential catalytic sites, with Asp138 having an auxiliary function. Additionally, a high degree of synergy (15.02) was observed when Xyl11 was used in association with commercial β-xylosidase. This study provided a novel acidophilic β-xylanase that exhibits excellent characteristics and can, therefore, be considered a suitable candidate for extensive applications, especially in food and animal feed industries.
In this study, an acidophilic GH5 β-mannanase (TaMan5) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 2.0-fold increase, 67.5 ± 1.95 U/mL). TaMan5 displayed the highest specificity toward locust bean gum (Km = 1.34 mg/mL, Vmax = 749.14 μmol/min/mg) at pH 4.0 and 65°C. Furthermore, TaMan5 displayed remarkable tolerance to acidic environments, retaining over 80% of its original activity at pH 3.0–5.0. The activity of TaMan5 was remarkably decreased by Cu2+, Mn2+, and SDS, while Fe2+/Fe3+ improved the enzyme activity. A thin-layer chromatography (TLC) analysis of the action model showed that TaMan5 could rapidly degrade mannan/MOS into mannobiose without mannose via hydrolysis action as well as transglycosylation. Site-directed mutagenesis results suggested that Glu205, Glu313, and Asp357 of TaMan5 are crucial catalytic residues, with Asp152 playing an auxiliary function. Additionally, TaMan5 and commercial α-galactosidase displayed a remarkable synergistic effect on the degradation of galactomannans. This study provided a novel β-mannanase with ideal characteristics and can be considered a potential candidate for the production of bioactive polysaccharide mannobiose.
Trichoderma sp. was isolated from the rhizosphere soil of a healthy banana plant grown in a Panama disease-contaminated field (suppressive soil). The selected strain, identified as Trichoderma asperellum , exhibited antifungal activity against four species of phytopathogenic fungi from the Fusarium genus ( F. oxysporum, F. fujikuroi, F. tricinctum, F. cantenulatum ). Antagonism related mechanisms associated with the T. asperellum isolate against four fungal phytopathogens were studied in vitro both indirectly by the observation of growth competition and directly by antibiosis and mycoparasitism . It was shown that T. asperellum could suppress growth of phytopathogens in the range of 65–74%, as well as inhibits spore germination by 30- 75%. Moreover, T. asperellum was demonstrated to secrete mycolytic enzymes (chitinase and β-1,3, glucanase), which are potentially able to degrade cell walls of the phytopathogens. The observed accumulation of respective transcripts and enzymatic activities of both chitinase and β-1,3, glucanase were remarkably higher in pathogen-induced cultures. The reduction of disease severity by 70% was observed in pot cultures of banana plantlets infected previously with F. oxysporum after 9 weeks of treatment with T. asperellum. Our findings demonstrate that T. asperellum as originated from banana wilt-contaminated plantations express anti-pathogenic effects based on various modes of action, including competitiveness, antibiosis, and mycoparasitism and might be potentially used as a biological control agent to cope with diseases caused by a broad spectrum of pathogenic fungi of Fusarium genus.
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