Luiz Alberto Beraldo Moraes

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60°C and 4.0, respectively. The enzyme was stable for 1 h at 55°C, showing a t₅₀ of 53 min at 60°C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).

Microcystin production by a freshwater spring cyanobacterium of the genus Fischerella.

We investigated the production of a hepatotoxic, cyclic heptapeptide, microcystin, by a filamentous branched cyanobacterium belonging to the order Stigonematales, genus Fischerella. The freshwater Fischerella sp. strain CENA161 was isolated from spring water in a small concrete dam in Piracicaba, São Paulo State, Brazil, and identified by combining a morphological description with 16S rRNA gene sequencing and phylogenetic analysis. Microcystin (MCYST) analysis performed using an ELISA assay on cultured cells gave positive results. High performance liquid chromatography-mass spectrometry (HPLC-MS) analysis detected 33.6microg MCYST-LR per gram dry weight of cyanobacterial cells. Microcystin profile revealed by quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) analysis confirmed the production of MCYST-LR. Furthermore, genomic DNA was analyzed by PCR for sequences similar to the ketosynthase (KS) domain of the type I polyketide synthase gene, which is involved in microcystin biosynthesis. This revealed the presence of a KS nucleotide fragment similar to the mcyD and ndaD genes of the microcystin and nodularin synthetase complexes. Phylogenetic analysis grouped the Fischerella KS sequence together with mcyD sequences of the three known microcystin synthetase operon (Microcystis, Planktothrix and Anabaena) and ndaD of the nodularin synthetase operon, with 100% bootstrap support. Our findings demonstrate that Fischerella sp. CENA161 produces MYCST-LR and for the first time identify a nucleotide sequence putatively involved in microcystin synthesis in this genus.

Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity

Cyanobacterial strains isolated from terrestrial and freshwater habitats in Brazil were evaluated for their antimicrobial and siderophore activities. Metabolites of fifty isolates were extracted from the supernatant culture media and cells using ethyl acetate and methanol, respectively. The extracts of 24 isolates showed antimicrobial activity against several pathogenic bacteria and one yeast. These active extracts were characterized by Q-TOF/MS. The cyanobacterial strains Cylindrospermopsis raciborskii 339-T3, Synechococcus elongatus PCC7942, Microcystis aeruginosa NPCD-1, M. panniformis SCP702 and Fischerella sp. CENA19 provided the most active extracts. The 50 cyanobacterial strains were also screened for the presence of non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes and microcystin production. Putative fragment genes coding for NRPS adenylation domains and PKS keto-synthase domains were successfully PCR amplified from 92% and 80% of cyanobacterial strains, respectively. The potential therapeutical compounds siderophores were detected in five cyanobacterial isolates. Microcystin production was detected by ELISA test in 26% of the isolates. Further a protease inhibitor substance was detected by LC-MS/MS in the M. aeruginosa NPLJ-4 extract and the presence of aeruginosin and cyanopeptolin was confirmed by PCR amplification using specific primers, and sequenced. This screening study showed that Brazilian cyanobacterial isolates are a rich source of natural products with potential for pharmacological and biotechnological applications.

Antifungal compound produced by the cassava endophyte Bacillus pumilus MAIIIM4a

In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the diversity of endophytic bacteria from ethnovarieties of cassava cultivated by Brazilian Amazon Indian tribes and also to study the secondary metabolites produced by a Bacillus pumilus strain. Sixty seven cassava endophytic bacteria were subjected to 16S rRNA sequencing and FAME analysis. The bacterial profile revealed that 25% of all endophytic isolates belonged to the genus Bacillus. The isolate B. pumilus MAIIIM4a showed a strong inhibitory activity against the fungi Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii. Secondary metabolites of this strain were extracted using hexane, dichloromethane and ethyl acetate. Extracts were subjected to bioautography and LC/MS analysis, which allowed the identification of pumilacidin, an antifungal compound produced by B. pumilus MAIIIM4a. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy.

Characterization of a microcystin and detection of microcystin synthetase genes from a Brazilian isolate of Nostoc

A nostocalean nitrogen-fixing cyanobacterium isolated from an eutrophic freshwater reservoir located in Piracicaba, São Paulo, Brazil, was evaluated for the production of hepatotoxic cyclic heptapeptides, microcystins. Morphologically this new cyanobacterium strain appears closest to Nostoc, however, in the phylogenetic analysis of 16S rRNA gene it falls into a highly stable cluster distantly only related to the typical Nostoc cluster. Extracts of Nostoc sp. CENA88 cultured cells, investigated using ELISA assay, gave positive results and the microcystin profile revealed by ESI-Q-TOF/MS/MS analysis confirmed the production of [Dha(7)]MCYST-YR. Further, Nostoc sp. CENA88 genomic DNA was analyzed by PCR for sequences of mcyD, mcyE and mcyG genes of microcystin synthetase (mcy) cluster. The result revealed the presence of mcyD, mcyE and mcyG genes with similarities to those from mcy of Nostoc sp. strains 152 and IO-102-I and other cyanobacterial genera. The phylogenetic tree based on concatenated McyG, McyD and McyE amino acids clustered the sequences according to cyanobacterial genera, with exception of the Nostoc sp. CENA88 sequence, which was placed in a clade distantly related from other Nostoc strains, as previously observed also in the 16S rRNA phylogenetic analysis. The present study describes for the first time a Brazilian Nostoc microcystin producer and also the occurrence of demethyl MCYST-YR variant in this genus. The sequenced Nostoc genes involved in the microcystin synthesis can contribute to a better understanding of the toxigenicity and evolution of this cyanotoxin.

Immunomodulatory action of Copaifera spp oleoresins on cytokine production by human monocytes

Copaifera spp oleoresins have been used in folk medicine for centuries; nevertheless, its immunomodulatory action has not been investigated. Thus, the goal of this study was to characterize different oleoresins and to verify their action on human monocytes regarding pro- and anti-inflammatory cytokine production (TNF-α and IL-10, respectively). The chemical composition of Brazilian Copaifera reticulata, Copaifera duckey and Copaifera multijuga oleoresins was analyzed by HPLC-MS. Cell viability was assessed by MTT method after incubation of cells with Copaifera spp. Noncytotoxic concentrations of oleoresins were incubated with human monocytes from healthy donors, and cytokine production was determined by ELISA. HPLC-MS analysis for terpenes allowed the identification of six diterpene acids and one sesquiterpene acid. Oleoresins exerted no cytotoxic effects on human monocytes. All oleoresins had a similar profile: LPS-induced TNF-α production was maintained by oleoresins, while a significant inhibitory action on IL-10 production was seen. Copaifera oleoresins seemed to exert an activator profile on human monocytes without affecting cell viability. Such effect may be due to the presence of either diterpene or sesquiterpene acids; however, further studies are necessary to determine the involvement of such compounds in Copaifera immunomodulatory effects.

Simultaneous determination of amino acids and neurotransmitters in plasma samples from schizophrenic patients by hydrophilic interaction liquid chromatography with tandem mass spectrometry

A sensitive, reproducible, and rapid method was developed for the simultaneous determination of underivatized amino acids (aspartate, serine, glycine, alanine, methionine, leucine, tyrosine, and tryptophan) and neurotransmitters (glutamate and γ-aminobutyric acid) in plasma samples using hydrophilic interaction liquid chromatography coupled to triple quadrupole tandem mass spectrometry. The plasma concentrations of amino acids and neurotransmitters obtained from 35 schizophrenic patients in treatment with clozapine (27 patients) and olanzapine (eight patients) were compared with those obtained from 38 healthy volunteers to monitor the effectiveness of treatment. The chromatographic conditions separated ten target compounds within 3 min. This method presented linear ranges that varied from (lower limit of quantification: 9.7-13.3 nmol/mL) to (upper limit of quantification: 19.4-800 nmol/mL), intra- and interassay precision with coefficients of variation lower than 10%, and relative standard error values of the accuracy ranged from -2.1 to 9.9%. The proposed method appropriately determines amino acids and neurotransmitters in plasma from schizophrenic patients. Compared with the control group (healthy volunteers), the plasma levels of methionine in schizophrenic patients treated with olanzapine are statistically significantly higher. Moreover, schizophrenic patients treated with clozapine tend to have increased plasma levels of glutamate.

The gut microbiota of insecticide-resistant insects houses insecticide-degrading bacteria: A potential source for biotechnological exploitation

The exploration of new niches for microorganisms capable of degrading recalcitrant molecules is still required. We hypothesized the gut microbiota associated with insect-resistant lines carry pesticide degrading bacteria, and predicted they carry bacteria selected to degrade pesticides they were resistant to. We isolated and accessed the pesticide-degrading capacity of gut bacteria from the gut of fifth instars of Spodoptera frugiperda strains resistant to lambda-cyhalothrin, deltamethrin, chlorpyrifos ethyl, spinosad and lufenuron, using insecticide-selective media. Sixteen isolates belonging to 10 phylotypes were obtained, from which four were also associated with the susceptible strain. However, growth of gut bacteria associated with larvae from the susceptible strain was not obtained in any of the insecticide-based selective media tested. Growth of isolates was affected by the concentration of insecticides in the media, and all grew well up to 40 μg/ml. The insecticide-degrading capacity of selected isolates was assessed by GC or LC-MS/MS analyses. In conclusion, resistant strains of S. frugiperda are an excellent reservoir of insecticide-degrading bacteria with bioremediation potential. Moreover, gut-associated bacteria are subjected to the selection pressure imposed by insecticides on their hosts and may influence the metabolization of pesticides in insects.

Streptomyces araujoniae Produces a Multiantibiotic Complex with Ionophoric Properties to Control Botrytis cinerea

A recently described actinomycete species (Streptomyces araujoniae ASBV-1(T)) is effective against many phytopathogenic fungi. In this study, we evaluated the capacity of this species to inhibit Botrytis cinerea development in strawberry pseudofruit, and we identified the chemical structures of its bioactive compounds. An ethyl acetate crude extract (0.1 mg ml(-1)) of ASBV-1(T) fermentation broth completely inhibited fungus growth in strawberry pseudofruit under storage conditions. The crude extract was fractionated by preparative high-performance liquid chromatography; the active fraction was further evaluated by tandem mass spectrometry. ASBV-1(T) produced a multiantibiotic complex with ionophoric properties. This complex contained members of the macrotetralides class (including monactin, dinactin, trinactin, and tetranactin) and the cyclodepsipeptide valinomycin, all of which were active against B. cinerea. Furthermore, the addition of 2 mM MgSO4 and 1 mM ZnSO4 enhanced macrotetralide and valinomycin production, respectively, in the culture broth. These compounds are considered to be the main active molecules that S. araujoniae produces to control B. cinerea. Their low to moderate toxicity to humans and the environment justifies the application of ASBV-1(T) in biological control programs that aim to mitigate the damage caused by this phytopathogen.

Dereplication of Streptomyces sp. AMC 23 polyether ionophore antibiotics by accurate-mass electrospray tandem mass spectrometry.

Actinomycetes, especially those belonging to the genus Streptomyces, are economically important from a biotechnological standpoint: they produce antibiotics, anticancer compounds and a variety of bioactive substances that are potentially applicable in the agrochemical and pharmaceutical industries. This paper combined accurate-mass electrospray tandem mass spectrometry in the full scan and product ion scan modes with compounds library data to identify the major compounds in the crude extract produced by Streptomyces sp. AMC 23; it also investigated how sodiated nonactin ([M + Na](+)) fragmented. Most product ions resulted from elimination of 184 mass units due to consecutive McLafferty-type rearrangements. The data allowed identification of four macrotetrolides homologous to nonactin (monactin, isodinactin, isotrinactin/trinactin and tetranactin) as well as three related linear dimer compounds (nonactyl nonactoate, nonactyl homononactoate and homononactyl homononactoate). The major product ions of the sodiated molecules of these compounds also originated from elimination of 184 and 198 mass units. UPLC-MS/MS in the neutral loss scan mode helped to identify these compounds on the basis of the elimination of 184 and 198 mass units. This method aided monitoring of the relative production of these compounds for 32 days and revealed that the biosynthetic process began with increased production of linear dimers as compared with macrotetrolides. These data could facilitate dereplication and identification of these compounds in other microbial crude extracts.