Investigation of Optimal Conditions for Production, Characterization, and Immobilization of Fructosyltransferase and β-Fructofuranosidase by Filamentous Fungi

This work's objective was the extracellular production, partial characterization, and immobilization of the enzymes fructosyltransferase (Ftase) and β-fructofuranosidadase (Ffase) by filamentous fungi. Aspergillus niger ATCC 9642 and Penicillium brasilianum were evaluated for the production of fructosyltransferase (Ftase) and β-fructofuranosidadase (FASE) enzymes. The A. niger presented the highest activity of FTase (24.86 µmol/min.mL) and FFase (28.68 µmol/min.mL) in medium composed of 20% sucrose, 0.5% yeast extract, 1% NaNO3, 0.05% MgSO4.7 H2O, 0.25% KH2PO4, 0.5% NH4Cl and 0.25% NaCl inoculated using 5x107spores/mL and incubated at 25°C, pH 5.5, 150 rpm for 48 h. Presenting optimum pH and temperature of 2.39 and 60°C. Thermal stability has shown that the enzyme FFase is more thermally stable when compared to FTase. Stability against different pHs showed similar behavior for FTase and FFase; the optimum pH being between 2.0 and 3.0. FTase and FFase showed storage stability in freezing and refrigeration temperature for approximately 400 h. The kinetic parameters, Km and Vmax, for the sucrose substrate were 24.60mM and 104.16 μmol/min.mL for FTase and 3.91mM and 20.24 μmol/min.mL for FFase. The immobilization process displayed a yield of 6744.66% for FFase and 3928.90% for FTase, with enzymatic activities of 364.79 U/g and 220.34 U/g, and 4 and 3 times reuse, respectively.

First report of Penicillium brasilianum Bat., P. cluniae Quintan., and P. echinulonalgiovense S. Abe ex Houbraken & R.N. Barbosa (Eurotiales, Aspergillaceae) as endophytes from a bromeliad in the Caatinga dry forest in Brazil

Penicillium brasilianum Bat., P. cluniae Quintan., and P. echinulonalgiovense S. Abe ex Houbraken & R.N. Barbosa are reported for the first time as endophytes from the leaves of an endemic bromeliad in the Caatinga dry forest in Brazil. For species determination, phenotypic features were analysed along with the sequencing of the β-tubulin and calmodulin genes. Penicillium Link isolates obtained in this study showed the typical morphology of species in the Lanata-Divaricata section. These results contributed to increase the knowledge of fungal diversity in dry environments in the word.

Synthesis and potential application of polygalacturonase from a Penicillium brasilianum isolate

The aim of this study was to evaluate the synthesis of pectinase from Penicillium brasilianum in shake flasks and address their potential for industrial applications. A Plackett-Burman design followed by a complete second order design were used for the screening of most important factors and to maximize the polygalacturonase activity, respectively. Maximum polygalacturonase activity was 52.8 U mL-1 at 48 hours of bioproduction. The kinetic evaluation for substrate consumption showed that 42% total organic carbon, 52 nitrogen, 23 magnesium, and 60% potassium were consumed. The crude enzyme complex was used on commercial mango juice clarification, and, at a 0.5% concentration (v v-1) reduced viscosity by 10%, turbidity by 12% and clarification by 23%. Therefore, the results presented in this study could provide valuable and beneficial information for the food and enzyme industries (juice) as well as being a new landmark to microbiology by providing essential knowledge on P. brasilianum growing needs.

The histone deacetylase clr3 regulates secondary metabolite production and growth under oxidative stress conditions in Penicillium brasilianum

ABSTRACTMost of the biosynthetic gene clusters (BGCs) found in filamentous fungi are silent under standard laboratory cultivation conditions due to the lack of expression triggering stimuli, representing a considerable drawback in drug discovery. To access the full biosynthetic potential of these microbes, studies towards the activation of cryptic BGCs are essential. Histone acetylation status is an important regulator of chromatin structure which impacts in cell physiology and, therefore, expression of biosynthetic gene clusters in filamentous fungi. Histone deacetylases (HDACs) and histone acetyl-transferases (HATs) are responsible for maintaining and controlling this process under different cell conditions. In this study, clr3, a gene encoding a histone deacetylase in Penicillium brasilianum was deleted and associated phenotypic and metabolic changes evaluated. Results indicate reduced growth under oxidative stress conditions in the Δclr3 knockout strain. Also, the production of several secondary metabolites including austin-related meroterpenoids, brasiliamides, mycotoxins such as verruculogen and penicillic acid, as well as cyclodepsipeptides was reduced in the Δclr3 strain when compared to wild-type strain. Accordingly, addition of epigenetic modulators responsible for HDAC inhibition such as suberoylanilide hydroxamic acid (SAHA) and nicotinamide (NAA) to P. brasilianum growth media also culminated in reduction of secondary metabolite production. Mass Spectrometry Imaging (MSI) was applied to compare metabolite production and spatial distribution on the colony. Results suggest that Clr3 plays an important role in secondary metabolite biosynthesis in P. brasilianum, thus offering new strategies for regulation of natural product synthesis by assessing chromatin modification in P. brasilianum.

Discovery of Bioactive Indole-Diketopiperazines from the Marine-Derived Fungus Penicillium brasilianum Aided by Genomic Information

Identification and analysis of the whole genome of the marine-derived fungus Penicillium brasilianum HBU-136 revealed the presence of an interesting biosynthetic gene cluster (BGC) for non-ribosomal peptide synthetases (NRPS), highly homologous to the BGCs of indole-diketopiperazine derivatives. With the aid of genomic analysis, eight indole-diketopiperazines (1−8), including three new compounds, spirotryprostatin G (1), and cyclotryprostatins F and G (2 and 3), were obtained by large-scale cultivation of the fungal strain HBU-136 using rice medium with 1.0% MgCl2. The absolute configurations of 1−3 were determined by comparison of their experimental electronic circular dichroism (ECD) with calculated ECD spectra. Selective cytotoxicities were observed for compounds 1 and 4 against HL-60 cell line with the IC50 values of 6.0 and 7.9 μM, respectively, whereas 2, 3, and 5 against MCF-7 cell line with the IC50 values of 7.6, 10.8, and 5.1 μM, respectively.