Enzymatic Bioprospecting of Fungi Isolated from a Tropical Rainforest in Mexico

The humid tropical environment provides an ideal place for developing a high diversity of plants; this is why it is an interesting site for the enzymatic bioprospecting of fungi that are responsible for the recycling of organic matter in an efficient and accelerated way and whose enzymes could have multiple biotechnological applications. For this study, 1250 isolates of macroscopic and microscopic fungal morphotypes were collected from soil, leaf litter, and wood. One hundred and fifty strains (50 from each source) were selected for the enzymatic screening. From the first phase, 51 strains with positive activity for laccase, protease, amylase, xylanase, and lipase enzymes were evaluated, of which 20 were isolated from leaf litter, 18 from the soil, and 13 from wood. The 10 best strains were selected for the enzymatic quantification, considering the potency index and the production of at least two enzymes. High laccase activity was detected for Trametes villosa FE35 and Marasmius sp. CE25 (1179 and 710.66 U/mg, respectively), while Daedalea flavida PE47 showed laccase (521.85 U/mg) and protease activities (80.66 U/mg). Fusarium spp. PH79 and FS400 strains had amylase (14.0 U/mg, 49.23 U/mg) and xylanase activities (40.05 U/mg, 36.03 U/mg) respectively. These results confirm the enzymatic potential of fungi that inhabit little-explored tropical rainforests with applications in industry.

LIGNOCELLULOSE WASTE VALORIZATION BY AN ENZYMATIC COCKTAIL OF P. ERYNGII AND P. PULMONARIUS

The present study aimed to characterize Pleurotus eryngii and P. pulmonarius ligninolytic enzymes and to determine their potential for polymer degradation in common agroforestry residues. The peak of laccase activity (36052.33 U L-1) was observed after P. pulmonarius cultivation on oak sawdust. The maximal Mn-dependent peroxidase activity was reached by P. eryngii (2511.36 U L-1), while the highest level of versatile peroxidase activity was noted in P. pulmonarius (3053.03 U L-1), after fermentation of corn stalks. The highest level of lignin loss (46.28%) was achieved after cultivation of P. pulmonarius on corn stalks, but the most selective degradation of lignocellulose polymers was observed after P. eryngii cultivation on wheat straw. The obtained results lead to the conclusion that the studied P. eryngii and P. pulmonarius strains are good producers of ligninolytic enzymes and effective and selective depolymerizers of agroforestry residues, and therefore their use would be beneficial in numerous environmentally friendly technologies.

The selection of laccase producing polypores fungi and inducer addition on the degradation of batik waste

Research on the selection of laccase producing polypores fungi and inducer addition on the degradation of batik waste has been carried out. The objectives of this study were to obtain isolates of polypores fungi with high laccase activity and data on the inducer effect in the batik waste degradation by selected isolate. Batik waste is potential pollution to the environment. It has been found that the Polyporaceae fungus reduce water pollution caused by batik waste. Fungal fruiting bodies were collected at the Cibinong Science Center and forest park IPB Campus Darmaga, Bogor. The inducers used included 15 g/L sucrose, 200 μM CuSO4, and 1.5% sorghum waste. Seven isolates of polypores fungi were obtained. Isolate J6 with high laccase activity was obtained and identified as Coriolopsis sp. The addition of sorghum waste to the growth medium increased the laccase activity of Coriolopsis sp. J6 by 1428 U/mL. Coriolopsis sp. J6 was able to reduce the color of Poly R-478 by 83.73% after the addition of sucrose and incubation for 10 days. This fungus was able to reduce the color of batik waste by 37.17% after the addition of 1.5% sorghum waste and incubation for 10 days.

Biological pretreatment of rice husks with the white-rot fungus pleurotus ostreatus: the role of laccase activity

The main objective of this study was to determine the influence of the composition of the culture medium and lignocellulosic compounds on the secretion of laccase enzymes by P. ostreatus in submerged cultures. These studies were done using a statistical and systematic approach that allowed the control of the culture media composition. The optimal nutritional conditions were found that simultaneously increased fungal growth and laccase activity in the absence and presence of copper sulfate, a recognized inducer of laccase. Under these conditions, the biochemical aspects of transcripts in P. ostreatus related to laccase secretion were evaluated , which revealed the participation of membrane transporters with high affinity for copper (CTRs) as intermediate candidates for the regulation of three laccase genes, lacc2, lacc6, and lacc10. Moreover, the evaluation of the results of the culture media composition suggests that the regulation of these transporters is closely linked to sufficient nutritional conditions in carbon and nitrogen, with central participation of the metabolism of organic nitrogen in this process. With these findings, it was possible to obtain more profound knowledge of the pretreatment of lignocellulosic biomass by P. ostreatus in a submerged culture that was oriented to determine the role of laccase activity in the biological pretreatment of rice husks.

Biodegradation Capacity and Activity Enzymatic of Bacillus subtilis against Low-Density Polyethylene

Aims: The objective of this work was to determine the degradation capacity of low-density polyethylene by the bacterium Bacillus subtilis and analyze the production of extracellular laccase activity. Methodology: The experiments was realized in 50 mL of culture medium, added with a fragment of known dry weight (1 cm2 colorless polyethylene bag squares), and were incubated at 28°C, pH 6.5, for 6 months under static conditions, determining the growth of the bacterium by dry weight (68, 75, and 91 mg), the production of extracellular protein (271, 234, and 326.1 mg/mL), and the degradation of the substrate by dry biodegraded (8.57%, 5.88%, and 11.76%). Results: The production of extracellular laccase enzyme was analyzed in presence of polyethylene, finding an enzymatic activity of laccase of 2.06, 1.49, and 2,03 U/mL, while in the control without substrate, no enzymatic activity was observed, which suggests that this enzyme may participate in the degradation of polyethylene. In addition, some characteristics of the extracellular enzymatic activities were analyzed, such as stability at 4oC and 28oC, optimal pH and temperature, the effect of protein and substrate concentration. Conclusion: The extracellular protein production and dry weight of the bacterium are higher in the presence of low-density polyethylene. The laccase activity is very stable at 4oC and 28oC, the most effective pH and temperature, were 4.5 and 28oC, and present an incubation time of 5 minutes, and this data suggest that this enzymatic activitiy may participate in the degradation of low density polyethylene.

Laccase Activity in Fungus Cryphonectria parasitica Is Affected by Growth Conditions and Fungal–Viral Genotypic Interactions

Laccase activity reduction in the chestnut blight fungus Cryphonectria parasitica usually accompanies the hypovirulence caused by the infection of fungus with Cryphonectria hypovirus 1 (CHV1). However, the different methods utilized for assessing this phenomenon has produced varied and often conflicting results. Furthermore, the majority of experimental setups included only one prototypic system, further confounding the results. Considering the diversity of fungal isolates, viral strains, and variability of their effects on the phytopathogenic process observed in nature, our goal was to ascertain if laccase activity variability is affected by (1) different C. parasitica isolates infected with several CHV1 strains, and (2) growth conditions. We have demonstrated that some CHV1 strains, contrary to previous assumptions, increase the activity of C. parasitica laccases. The specific fungal isolates used in the experiments and culture conditions also affected the results. Furthermore, we showed that two commonly used laccase substrates, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,4-dimethoxyphenol, cannot be used interchangeably in C. parasitica laccase activity measurements. Our results illustrate the importance of conducting this type of study in experimental systems and culture conditions that resemble natural conditions as much as possible to be able to infer the most relevant conclusions applicable to natural populations.

Improvement of laccase activity by silencing PacC in Ganoderma lucidum

Ganoderma lucidum is a representative white-rot fungus that has great potential to degrade lignocellulose biomass. Laccase is recognized as a class of the most important lignin-degrading enzymes in G. lucidum. However, the comprehensive regulatory mechanisms of laccase are still lacking. Based on the genome sequence of G. lucidum, 15 laccase genes were identified and their encoding proteins were analyzed in this study. All of the laccase proteins are predicted to be multicopper oxidases with conserved copper-binding domains. Most laccase proteins were secreted enzymes in addition to Lac14 in which the signal peptide could not be predicted. The activity of all laccases showed the highest level at pH 3.0 or pH 7.0, with total laccase activity of approximately 200 U/mg protein. Silencing PacC resulted in a 5.2 fold increase in laccase activity compared with WT. Five laccase genes (lac1, lac6, lac9, lac10 and lac14) showed an increased transcription levels (approximately 1.5-5.6 fold) in the PacC-silenced strains versus that in WT, while other laccase genes were downregulated or unchanged. The extracellular pH value was about 3.1, which was more acidic in the PacC-silenced strains than in the WT (pH 3.5). Moreover, maintaining the fermentation pH resulted in a downregulation of laccase activity which is induced by silencing PacC Our findings indicate that in addition to its function in acidification of environmental pH, PacC plays an important role in regulating laccase activity in fungi.

An Engineered Thermostable Laccase with Great Ability to Decolorize and Detoxify Malachite Green

Laccases can catalyze the remediation of hazardous synthetic dyes in an eco-friendly manner, and thermostable laccases are advantageous to treat high-temperature dyeing wastewater. A novel laccase from Geothermobacter hydrogeniphilus (Ghlac)was cloned and expressed in Escherichia coli. Ghlac containing 263 residues was characterized as a functional laccase of the DUF152 family. By structural and biochemical analyses, the conserved residues H78, C119, and H136 were identified to bind with one copper atom to fulfill the laccase activity. In order to make it more suitable for industrial use, Ghlac variant Mut2 with enhanced thermostability was designed. The half-lives of Mut2 at 50 °C and 60 °C were 80.6 h and 9.8 h, respectively. Mut2 was stable at pH values ranging from 4.0 to 8.0 and showed a high tolerance for organic solvents such as ethanol, acetone, and dimethyl sulfoxide. In addition, Mut2 decolorized approximately 100% of 100 mg/L of malachite green dye in 3 h at 70 °C. Furthermore, Mut2 eliminated the toxicity of malachite green to bacteria and Zea mays. In summary, the thermostable laccase Ghlac Mut2 could effectively decolorize and detoxify malachite green at high temperatures, showing great potential to remediate the dyeing wastewater.