Bioprospecting of novel and biologically active compounds and enzymes from a new fungal biobank for industrial biotechnology

Abstract

Here, we present methods for screening any newly isolated microbial biobank for enzymes, antioxidants, and additional secondary metabolites. Bioactive compounds and enzymes produced by fungi have many applications in industry as valuable leads for pharmaceuticals, nutraceuticals, and potential strains for industrial biotechnology. In this study, fungi collected from Irish habitats were purified and identified by sequencing Internal Transcribed Spacer (ITS) ribosomal DNA regions. Reducing sugars released because of enzyme hydrolysis was used to estimate the relative enzyme activity for carboxymethyl cellulase, pectinase, β-xylanase, arabinoxylanase, mannanase, and galacto-mannanase in the fungal extracts and was normalized against the crude protein content to express relative enzyme activity/milligram. The best enzyme producers were as follows: F1 Clonostachys rosea demonstrated 351.63 U/mg β-xylanase and 645.50 U/mg arabinoxylanase activity; F2 Penicillium expansum had 25 U/mg cellulase and 112.5 U/mg pectinase activity; F3 Fusarium avenaceum had 139.36 U/mg arabinoxylanase, 52.33 U/mg galacto-mannanase, and 45 U/mg mannanase activity; and F9 Trichoderma koningii had 372.15 U/mg β-xylanase and 655.18 U/mg arabinoxylanase activity. In addition to enzyme activity, these strains also exhibited antioxidant activity when tested in Oxygen radical absorbance capacity (ORAC), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric ion reducing antioxidant power (FRAP) assays, with activity expressed as μmol Trolox equivalents (TE) per gram of dried fungal extract. In results from the ORAC assay, F1 C. rosea reported 655,669 µmol TE/g; F2 P. expansum had 636,889 µmol TE/g; and F3 F. avenaceum had 4,488,035 µmol TE/g. High DPPH activity was observed for F1 C. rosea at 12,572 µmol TE/g, F3 F. avenaceum at 22,646 µmol TE/g, and F9 T. koningii at 22,558 µmol TE/g. F1 C. rosea, F3: F. avenaceum, and F9 T. koningii also reported high FRAP activity at 36,019 µmol TE/g, 69,325 µmol TE/g, and 25,812 µmol TE/g, respectively. In terms of secondary metabolites, the main compounds detected for the isolates were F1 C. rosea produced benzoic acid, 3-pyridinepropionic acid, and cyclo(-Gly-Phe); F2 P. expansum produced Cyclo(-Gly-Phe) and prenitremone A; F3 F. avenaceum produced cyclo(-Leu-Leu) and p-anisaldehyde; and F9 T. koningii produced cyclo(-Gly-Phe), canescin, citreoviridin X, and cyclopaldic acid. These selected isolates indicate that Irish fungi are a potential source of enzymes and novel biologically active compounds useful in industrial biotechnology and bioproduction supporting sustainability and responsible environmental management.