Current Biotechnology - Volume 6, Issue 4, 2017

Background: Microorganisms such as filamentous fungi are important sources of enzymes and secondary metabolites with wide application in biotechnological processes conducted in different industrial areas. Aspergillus, one of the most important genera of filamentous fungi for this purpose, has been used as a model to understand different biological processes. Despite the existence of some pathogenic Aspergillus strains, many others have been explored from a biotechnological perspective. Characteristics such as the presence of a secretory pathway, the possibility of genetic manipulation, and high productivity using different fermentative processes are advantageous and favorable for the use of Aspergillus spp. for different purposes. Production of both hydrolases and secondary metabolites has been reported for different Aspergillus species. Objective: This review describes the production, importance, and application of the main hydrolases (amylases, cellulases, fructofuranosidases, xylanases, proteases, tannases, and phytases) produced by Aspergillus spp. considering their importance in the global enzyme market. The production of secondary metabolites such as bioactive peptides and pigments is also presented. Conclusion: Different Aspergillus strains have demonstrated biotechnological potential for the production of hydrolases and secondary metabolites for application in food and feed, beverage, pharmaceutical, pulp, and bioremediation industries among others. In the future new species should be described and their biotechnological potential evaluated.

Background: Current studies about lipases production using agro-industrial residues and newly isolated microorganisms aimed at increasing economic attractiveness. Objective: The present study evaluated the lipase production from Aspergillus niger using canola cake as substrate and its extraction and characterization. Methods: Firstly, the microorganism was identified using sequencer ABI PRISM 3100 Genetic Analyzer. After that, experimental design technique was used to determine the variables that can influence the fermentation process, such as: nitrogen supplement and medium moisture; and for the extraction process, the influence of factors as temperature and pH. The lipase was characterized in terms of hydrolytic and esterification activity and specificity, and stability at 10 and -1°C. Results: The obtained results showed that the best condition for the lipase production is 60 wt% of moisture and 2 wt% of urea as supplemental source of nitrogen, and the most appropriate temperature for extraction is 50°C and pH 8.0. In terms of hydrolytic activity, the best conditions were obtained using pH 6.0, 35°C and 32 minutes. The coconut and soybean oil showed the best hydrolytic activity (90 U/g and 74 U/g, respectively), demonstrating that lipases act preferentially in triglycerides of medium and long chain. For the esterification reactions, the higher activity (837 U/g) was obtained using lauric acid as fatty acid and butanol as solvent. Conclusion: In the literature there are few reports about the lipase production from Aspergillus niger using canola cake as substrate. So, the study of the enzyme extraction and characterization after the fermentative process is of extreme relevance.

Background: Lipases are enzymes widely used in many industrial applications. However, its use can be increased by reducing costs of production, being important to this effort the screening of new microorganisms producing and the use of low cost culture mediums, such as agricultural residuals. Objective: To produce lipases using two new isolates of filamentous fungi in solid-state fermentation. Methods: The selection of significant variables on the lipase production was studied through a 27-3 Fractional Factorial Design. The studied variables were: the type of bran used as support in solid state fermentation (wheat or soy), nitrogen source (urea or nitrate), inducer (soy or olive oil), nitrogen source concentration (1 or 3%), inducer concentration (1 or 3%), pH (5 or 7) and filamentous fungal strain (Aspergillus O-4 or Aspergillus E-6). Also, a 24 Full Factorial Design was used in the sequence of optimization. Results: The first experimental design showed that the variables type of bran, nitrogen source, inducer, and pH were the most significant (p<0.05) on lipolytic activity, being the higher activity obtained of 45.95 U.g-1. These variables were studied in the second experimental design, in which high lipolytic activities were obtained (57 U) with soy bran, sodium nitrate as nitrogen source, olive oil as inducer and pH 5. Conclusion: The statistical methods of optimization enabled an increased lipase production in solidstate fermentation from Aspergillus niger (O-4).

Backgrond: The aqueous two-phase systems (ATPS) have been used as the first purification step in enzyme industrial isolation. Such systems allow the removal of large amounts and different types of contaminants by a simple and economic unit operation. Objective: The tannase extraction was conducted through ATPS composed of polyethylene glycol (PEG) and sodium citrate salt (PEG/citrate). Method: A factorial design model (24) was used to assess the influence of PEG molar mass (400-8000 g mol−1 - MPEG), PEG concentration (12.5-17.5%, w/w - CPEG), citrate concentration (15-25%, w/w, CCIT) and pH (6.0-8.0) on tannase extraction. Results: The tannase is preferably partitioned at the top phase. The highest purification factor (PF = 18.18) was achieved in ATPS using 25% (w/w) PEG 8000 (g mol−1) and 25% (w/w) CCIT at pH 8.0, in which a yield (Y) of 23.37% was obtained. Furthermore, pH was the independent variable that most significantly influenced the response variables. Tannase showed pH and optimum temperature of 5.0 and 60°C, respectively. It was stable in a very short pH range (3.0-6.0) and unstable in the studied temperature range (25-70°C). The tannase activity was stimulated by Ca2+, Co2+, Fe2+ and Mn2+. Organic solvents also increased enzyme activity, particularly in the following rates: 40% chloroform, 20% and 40%, glycerol and 20% and 40% toluene. Conclusion: The ATPS could be used as the first step in tannase extraction from a crude extract, and as a promising and low cost alternative for partial purification of tannase produced by P. rolfssi URM 6216.

Background: Manganese peroxidase (MnP) is a common extracellular ligninolytic peroxidase produced by Pleurotus spp. It catalyzes the H2O2-dependent oxidation of Mn2+ into highly reactive Mn3+, which in turn oxidizes phenolic and nonphenolic compounds including a large list of xenobiotics such as synthetic dyes. Objective: To purify and characterize the MnP of Pleurotus pulmonarius and to evaluate its capability to decolorize synthetic dyes. Methods: P. pulmonarius was cultured for 14 days under solid state conditions using pineapple waste as substrate. Under this condition, MnP was the main ligninolytic enzyme found in the culture filtrates. The enzyme was purified to apparent electrophoretic homogeneity through acetone precipitation and gel filtration using a Sephadex G-100 column. Results: MnP was purified 12.1-fold with a yield of 28.4% and a specific activity of 135.52 U/mg protein. The 42 kDa-monomeric protein was active over a large range of pH (4.0-6.0) and at a temperature of 50°C. The enzyme was stable at temperatures up to 40°C. At -20°C, it was stable for at least 6 months. The KM for Mn2+ and H2O2 at pH 4.5 and 40°C were 19.2 and 16.8 μM, respectively. The enzyme was strictly dependent on Mn2+ for oxidizing phenolic and nonphenolic compounds. It showed high activity and stability in the presence of organic solvents such as acetone, ethanol, isopropanol and acetonitrile, and was able to decolorize the anthraquinonic dye remazol brilliant blue R and the azo dye Congo red in the presence of 1 M Na2SO4 and NaCl. Conclusion: The properties of the P. pulmonarius MnP certainly make this enzyme a good agent for textile dye effluent treatment.

Background: Endophytic fungi are considered a source of novel activities, compounds and biotechnological processes with great and underexplored potential. The aims of this paper were: a) to evaluate the antibiotic activity of mangrove endophytic fungi against the human pathogens Staphylococcus aureus, Salmonela typhi and Escherichia coli and against the phytopathogen Xantomonas axonopodis citri; b) to elucidate the structure of the antibiotic produced by the endophytic fungus Hypocrea virens strain 89(3), using spectroscopic methods, mainly 1D and 2D NMR, as well as mass spectra and comparison with previously published data. Methods: The selection of the endophytic fungi producer of antibiotics was based on agar piece method. The pure substance (viridiol) was obtained by carrying out biological tests with the extract and its fractions against pathogenic bacteria (Escherichia coli), and this study was monitored by NMR spectroscopy. Viridiol was isolated from the mangrove endophytic fungus H. virens, which was obtained from branches of Avicennia nitida. The structure of the compound was elucidated by spectroscopic methods, mainly 1D and 2D NMR spectroscopy and confirmed by mass spectrometry. Results: In bioassays, compound viridiol showed antimicrobial activity against Escherichia coli, with an MIC of 64 μg/ml. The antibiotic-producing strain was identified as H. virens using internal transcribed spacer (ITS) sequence data. The spectroscopic methods employed were an efficient means of elucidating the structure of antibiotic viridiol. Conclusion: The isolation of a bioactive substance using 1H NMR monitoring was highly successful because it showed step by step the spectral signals concerning the bioactive substance from crude extract to pure substance. This is the first report of antimicrobial activity by viridiol against E. coli, a medically important pathogen.

Background: This review is intended to give an account of the knowledge about enzymatic reactions performed by brazilian marine-derived fungi. These biocatalysts can offer a range of enzymes capable to produce a large spectra of added value compounds. Screenings with varied species of marine-derived fungi presented active enzymes capable of performing the biotransformation of nitriles and epoxides, bioreduction of carbon-carbon double bonds, bioreduction of free or immobilized ketones and biohydroxylation of natural compounds. Results: Different marine-derived fungi presented active nitrilases that catalyzed the production of acetic acid derivatives, epoxide hydrolases that were responsible for the biotransformation of glycidyl ether derivatives, enoate reductases that catalyzed the chemoselective biohydrogenation of α, β- and α, β, γ, δ- unsaturated ketones, as well as enantioselective alcohol deydrogenases (ADHs) capable of producing chiral alcohols through different processes and active oxidoreductases efficient in the regioselective hydroxylation of natural products such as terpenes. Conclusion: These studies led to the production of compounds such as chiral alcohols, epoxides and terpene derivatives, which are of great interest due to their enantiomeric purity and applicability in different industry and academic sectors. The success of these reactions was possible due to the presence of secondary metabolites in marine-derived fungi as well the presence of different classes of active enzymes (nitrilases, epoxide hydrolases, alcoholdeydrogenases). These examples may inspire the scientific community to perform new marine-derived fungi screenings and produce enantiomerically pure compounds through the favorable environmental conditions given by these biocatalysts.

Background: Plants live in a dynamic environment subjected to frequent pathogenic and environmental stresses and have evolved diverse self-defense pathways for their survival. Many of these defense responses are energy-intensive and antagonistic against each other to enable prioritization of strong targeted responses. Viruses cause significant damage in the agricultural and natural world. They are obligate intracellular pathogens that depend on their host cell for replication and movement. Method: We searched bibliographic databases for peer-reviewed research literature on the topic of natural and engineered plant defense responses against plant viruses. Results: While programmed cell death is an appropriate initial defense response to isolate viruses, RNA silencing or the RNAi pathway presents a highly effective defense mechanism that plants employ against virus attack. But RNA silencing is also essential for normal gene regulation within the host. RNA silencing largely dominates the literature on plant virus defense, but there are a number of other effective defense mechanisms that will be discussed. Conclusion: This review first covers different viral replication strategies based on genome organization, then presents an overview of known virus defense mechanisms and pathways, and finally highlights recent work and new strategies to engineer viral resistance in crop plants.

Background: Biodegradation of lignocellulosic materials to monomeric sugars by the action of cellulolytic enzymes has recorded a significant value for industrial processes. These sugars can be converted to ethanol which is of great economic value to salvage the ever-increasing global dependence on fossil fuel and its gradual depletion. Exploitation of this cellulosic groundnut shell biomass possesses both significant industrial and environmental implications. Objective: To isolate microorganisms of soil origin capable of producing cellulase, investigate hydrolysis of groundnut shell biomass and characterize the enzyme. Methods: Bacteria were isolated and screened from saw mill soil and were identified morphologically and biochemically using analytical profile index kit and apiweb software. Enzyme extract was assayed using carboxyl methyl cellulase assay method and partially purified. Enzyme saccharification of the pretreated biomass, pH, temperature and thermal stability were studied. Results: Out of the twelve bacteria species isolated and screened for cellulase producing potential, eight showed positive on screening medium indicated by clear or halo zone around growing microbial colonies. However, Bacillus subtilis was selected based on zone of inhibition exhibited on screening medium (28 mm) and hydrolysis efficiency of 100%. Both the crude and partially purified cellulase exhibited similar optimum pH 4.0 and differing optimal temperature 50°C and 60°C and thermally stable at 60°C and 70°C retaining about 78% and 71% activity respectively. Conclusion: Cheap, unexploited, renewable and readily available agricultural cellulosic biomass has been effectively utilized as substrate for the production of a thermally stable enzyme as criteria for industrial application.

Background: β-Glucosidases are terminal enzymes in cellulose hydrlysis which hydrolyze glycosidic bonds between two or more carbohydrates. Not much information is available on purification and characterization of β-glucosidase from Penicillium janthinellum. Here we report the purification and characterization of thermostable β-glucosidase from P. janthinellum mutant EU2D-21 produced under submerged conditions. Methods: The thermostable β-glucosidase was purified to homogeneity by anion exchange chromatography followed by gel permeation chromatography. It was characterized for its physical and biochemical properties using variety of methods such as MALDI-TOF MS, SDS-PAGE, CD spectra, pH and temperature optima and stability. Results: The protocol followed for purification resulted in high recovery of purified enzyme with specific activity increased from 1.0 to 169.84 IU/mg. It is a glycoprotein with molecular mass of 120 kDa as estimated by SDS-Page and MALDI-TOF MS. The enzyme is active at pH 4.5 and temperature 65°C. The purified β-glucosidase possesses tryptophan and carboxyl residues and histidine at the active site. Tandem MS analysis and MASCOT based database interrogations revealed low homology with known beta-glucosidases including of P. decumbens. The absence of a protein match with any P. janthinellum entries indicate that this β-glucosidase sequence has not been previously reported. Conclusions: The study demonstrated the importance of this enzyme at commercial levels for the hydrolysis of lignocellulose to their monomers. Tandem MS analysis and MASCOT based database interrogations revealed low homology with known beta-glucosidases including of P. decumbens. The absence of a protein match with any P. janthinellum entries indicate that this β-glucosidase sequence has not been previously reported.