Caatinga, Amazon and Atlantic Forest as natural sources for microbial lignocellulolytic enzymes.

Brazilian biomes are important sources for environmental microorganisms, including efficient metabolic machineries, like actinomycetes. These bacteria are known for their abilities to produce many bioactive compounds, including enzymes with multiple industrial applications. The present work aimed to evaluate lignocellulolytic abilities of actinomycetes isolated from soil and rhizosphere samples collected at Caatinga, Atlantic and Amazon Forest. Laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and cellulase were evaluated for their efficiency. These enzymes have an essential role in lignin decomposition, through oxidation of phenolic and non-phenolic compounds, as well as enzymatic hydrolysis of vegetal biomass. In this sense, a total of 173 actinomycetes were investigated. Eleven (11) of them were selected by their enzymatic performance. The actinomycete AC166 displayed some activity in all analysed scenarios in terms of Lac, MnP and LiP activity, while AC171 was selected as the most promising strain, showing the following activities: 29.7 U.L-1 for Lac; 2.5 U.L-1 for LiP and 23 U.L-1 for MnP. Cellulolytic activities were evaluated at two pH conditions, 4.8 and 7.4, obtaining the following results: 25 U.L-1 and 71 U.L-1, respectively. Thermostability (4, 30 and 60 o C) and salinity concentrations (0 to 4 M) and pH variation (2.0 to 9.0) stabilities of the obtained LiP and Lac enzymatic extracts were also verified. The actinomycete strain AC171 displayed an adaptable response in distinct pH and salt profiles, indicating that bacterial LiP was some halophilic type. Additionally, the strain AC149 produced an alkali and extreme halophilic lignin peroxidase, which are promising profiles for their future application under lignocellulosic biomass at bioethanol biorefineries.

Functional annotation and comparative modeling of ligninolytic enzymes from Trametes villosa (SW.) Kreisel for biotechnological applications.

Functional annotation of Trametes villosa genome was performed to search Class II peroxidase proteins in this white-rot fungus, which can be valuable for several biotechnological processes. After sequence identification and manual curation, five proteins were selected to build 3 D models by comparative modeling. Analysis of sequential and structural sequences from selected targets revealed the presence of two putative Lignin Peroxidase and three putative Manganese Peroxidase on this fungal genome. All 3 D models had a similar folding pattern from selected 3 D structure templates. After minimization and validation steps, the best 3 D models were subjected to docking studies and molecular dynamics to identify structural requirements and the interactions required for molecular recognition. Two reliable 3 D models of Class II peroxidases, with typical catalytic site and architecture, and its protein sequences are indicated to recombinant production in biotechnological applications, such as bioenergy.Communicated by Ramaswamy H. Sarma.

Lignocellulolytic Enzyme Production from Wood Rot Fungi Collected in Chiapas, Mexico, and Their Growth on Lignocellulosic Material.

Wood-decay fungi are characterized by ligninolytic and hydrolytic enzymes that act through non-specific oxidation and hydrolytic reactions. The objective of this work was to evaluate the production of lignocellulolytic enzymes from collected fungi and to analyze their growth on lignocellulosic material. The study considered 18 species isolated from collections made in the state of Chiapas, Mexico, identified by taxonomic and molecular techniques, finding 11 different families. The growth rates of each isolate were obtained in culture media with African palm husk (PH), coffee husk (CH), pine sawdust (PS), and glucose as control, measuring daily growth with images analyzed in ImageJ software, finding the highest growth rate in the CH medium. The potency index (PI) of cellulase, xylanase, and manganese peroxidase (MnP) activities was determined, as well as the quantification of lignin peroxidase (LiP), with the strains Phlebiopsis flavidoalba TecNM-ITTG L20-19 and Phanerochaete sordida TecNM-ITTG L32-1-19 being the ones with the highest PI of hydrolase activities with 2.01 and 1.83 cellulase PI and 1.95 and 2.24 xylanase PI, respectively, while Phlebiopsis flavidoalba TecNM-ITTG L20-19 and Trametes sanguinea TecNM-ITTG L14-19 with 7115 U/L LiP activity had the highest oxidase activities, indicating their ability to oxidize complex molecules such as lignin.

Decolorization of azo and anthraquinone dyes by crude laccase produced by Lentinus crinitus in solid state cultivation.

White-rot basidiomycetes such as Lentinus crinitus produce laccases with potential use in dye biodegradation. However, high productivity and enzymes with specific properties are required in order to make viable laccase production. We aimed to produce laccase from Lentinus crinitus grown in sugarcane bagasse for dye decolorization. Solid state cultivation medium had sugarcane bagasse added with a nutrient solution of 10 g/L glucose, 1 g/L KH2PO4, 0.5 g/L MgSO4, 0.001 g/L FeSO4, 0.01 g/L ZnSO4, and 0.01 g/L MnSO4. The addition of different nitrogen sources (peptone, urea, or peptone plus urea) and different nitrogen concentrations (0, 0.4, 0.6, 0.8, 1.0, and 1.2 g/L) were evaluated. Enzymatic extract was used in the decolorization of azo dyes, reactive blue 220 (RB220) and reactive black 5 (RB5), and anthraquinone dye, Remazol brilliant blue R (RBBR). The greatest laccase activity (4800 U/g dry mass) occurred when the peptone and urea mixture was added to the solid state cultivation medium. When the nitrogen concentration was 1 g/L, the laccase activity increased to 6555 U/g dry mass. The laccase activity peak occurred on the 10th day, and the maximum decolorization within 24 h was observed with enzymatic extracts obtained on different cultivation days, i.e., 6th day for RB220, 10th day for RB5, and 9th day for RBBR. Manganese and lignin peroxidases were not produced when nitrogen was added to the cultivation medium. The crude enzymatic extract was more effective in the decolorization of azo dyes (RB220 and RB5), more than 90% of decolorization, than anthraquinone dye with 77% decolorization.

Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study.

Lignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzyme with its substrates is essential in the rational design of engineered peroxidases for biotechnological application. Here, we combine computational and experimental approaches to analyze the interaction of Phanerochaete chrysosporium LiP (isoenzyme H8) with VA and its radical cation (VA•+, resulting from substrate oxidation by the enzyme). Interaction energy calculations at semiempirical quantum mechanical level (SQM) between LiP and VA/VA•+ enabled to identify those residues at the acidic environment of catalytic Trp171 involved in the main interactions. Then, a battery of variants, with single and multiple mutations at these residues (Glu168, Asp165, Glu250, Asp264, and Phe267), was generated by directed mutagenesis, and their kinetics parameters were estimated on VA and two additional substrates. The experimental results show that Glu168 and Glu250 are crucial for the binding of VA, with Glu250 also contributing to the turnover of the enzyme. The experimental results were further rationalized through new calculations of interaction energies between VA/VA•+ and LiP with each of the single mutations. Finally, the delocalization of spin density was determined with quantum mechanics/molecular mechanics calculations (QM/MM), further supporting the contribution of Glu250 to VA oxidation at Trp171.

Pulp improvement of oil palm empty fruit bunches associated to solid-state biopulping and biobleaching with xylanase and lignin peroxidase cocktail produced by Aspergillus sp. LPB-5.

Oil palm empty fruit bunches is a lignocellulosic feedstock with biotechnological potential and thousands of tons are generated in the world each year. Filamentous fungi producing xylanases and ligninases in biopulping to obtain cellulose is a pulp improvement alternative. The enzymatic cocktail was produced in solid-state biopulping by Aspergillus sp. LPB-5 with 54.32 U/g xylanase, 13.41 U/g lignin peroxidase and low cellulase activity. Biological, thermal and chemical pretreatments were compared and enzymatic biobleaching was applied to pretreated pulps. Biopulping and biobleaching combination had 36.80% lignin loss, 26.27% hemicellulose reduction, 74.36% pulp yield with 36.56% digestibility. Alkaline and biobleaching combination removed 81.97% hemicellulose and 93.89% lignin with 73.59% digestibility. Enzymatic biobleaching increased the pulp digestibility in all pretreatments. Finally, the development of a bio-pretreatment to remove hemicellulose and alter the lignin-carbohydrate complex interface presented a soft process with great eco-friendly potential, where mild pre-treatments would reduce the use of aggressive agents.

On the Reaction Mechanism of the 3,4-Dimethoxybenzaldehyde Formation from 1-(3',4'-Dimethoxyphenyl)Propene.

Lignin peroxidase (LiP) is an important enzyme for degrading aromatic hydrocarbons not only in nature but also in industry. In the presence of H2O2, this enzyme can easily decompose lignin and analogue compounds under mild conditions. In this reaction mechanism, LiP catalyzes the C-C cleavage of a propenyl side chain, being able to produce veratraldehyde (VAD) from 1-(3',4'-dimethoxyphenyl) propene (DMPP). One of the few and complete proposed mechanisms includes several non-enzymatic reactions. In this study, we performed a computational study to gain insight about the non-enzymatic steps involved in the reaction mechanism of VAD formation from DMPP using LiP as a catalyst. A kinetic characterization of the reaction using the reaction force and the reaction force constant concepts within the density functional theory (DFT) framework is proposed. All theoretical calculations for the reaction pathway were performed using the Minnesota Global Hybrid functional M06-2X and a 6-31++G(d,p) basis set. The complete reaction comprises seven steps (five steps not including LiP as a catalyst), which include radical species formation, bond transformation, water and oxygen addition, atom reordering, and deacetylation. The overall mechanism is an endothermic process with mixed activation energies depending on the four transition states. These results are the first attempt to fully understand the catalytic role of LiP in the degradation of lignin and its aromatic derivative compounds in terms of the electronic structure methods and future hybrid calculation approaches that we have recently been performing.

Requalification of a Brazilian Trichoderma Collection and Screening of Its Capability to Decolourise Real Textile Effluent.

Water contamination with large amounts of industrial textile coloured effluents is an environmental concern. For the treatment of textile effluents, white-rot fungi have received extensive attention due to their powerful capability to produce oxidative (e.g., ligninolytic) enzymes. In addition, other groups of fungi, such as species of Aspergillus and Trichoderma, have also been used for textile effluents treatment. The main aim of the present study was to requalify a Brazilian Trichoderma culture collection of 51 Trichoderma strains, isolated from different sources in Brazil and preserved in the oldest Latin-American Fungal Service Culture Collection, The Micoteca URM WDCM 804 (Recife, Brazil). Fungal isolates were re-identified through a polyphasic approach including macro- and micro-morphology and molecular biology, and screened for their capability to decolourise real effluents collected directly from storage tanks of a textile manufacture. Trichoderma atroviride URM 4950 presented the best performance on the dye decolourisation in real textile effluent and can be considered in a scale-up process at industrial level. Overall, the potential of Trichoderma strains in decolourising real textile dye present in textile effluent and the production of the oxidative enzymes Lac, LiP and MnP was demonstrated. Fungal strains are available in the collection e-catalogue to be further explored from the biotechnological point of view.

Industrial and biotechnological applications of ligninolytic enzymes of the basidiomycota: a review

Ligninolytic enzymes of the basidiomycetes play a crucial role in the global carbon cycle. The demand for application of ligninolytic enzymes complexes of white-rot fungi in industry and biotechnology is ever increasing due to their use in a variety of processes. Ligninolytic enzymes have potential applications in a large number of fields, including the chemical, fuel, food, agricultural, paper, textile, cosmetic industrial sectors and more. This ligninolytic system of white-rot fungi is also directly involved in the degradation of various xenobiotic compounds and dyes. Their capacities to remove xenobiotic substances and produce polymeric products make them a useful tool for bioremediation purposes. This paper reviews the applications of ligninolytic enzymes of basidiomycetes within different industrial and biotechnological area.

Overview of parameters influencing biomass and bioreactor performance used for extracellular ligninase production from Phanerochaete chrysosporium

The production of extracellular enzymes is gaining momentum as commercial interests seek alternative ways to improve the productivity in the biotechnology and pharmaceutical industries. Early research studies looked at improving batch bioreactor operational challenges; however, the use of continuous cultures was indicated to be favourable. This led to a new approach developed to produce extracellular enzymes continuously using fixed-film bioreactors from biofilms immobilised on polymeric and inorganic membranes. In this review, the performance of P. chrysosporium biomass, evaluated in terms of ligninase production using different bioreactor operation conditions, is highlighted. Furthermore, the limitations related to the implementation of optimised batch culture conditions to continuous fixed-film bioreactors are discussed. DO transportation, trace element toxicity and lipid peroxidation effects on P. chrysosporium biomass in fixed-film bioreactors operated for elongated periods, are also discussed.

Ligninases production by Basidiomycetes strains on lignocellulosic agricultural residues and their application in the decolorization of synthetic dyes / Produção de ligninases por linhagens de fungos Basidiomicetos usando resíduos agrícolas lignocelulósicos e aplicação das enzimas na descoloração de corantes sintéticos

Wood rotting Basidiomycetes collected in the ôEstação Ecológica do Noroeste Paulistaõ, São José do Rio Preto, São Paulo State, Brazil, concerning Aphyllophorales order and identified as Coriolopsis byrsina SXS16, Lentinus strigellus SXS355, Lentinus sp SXS48, Picnoporus sanguineus SXS 43 and Phellinus rimosus SXS47 were tested for ligninases production by solid state fermentation (SSF) using wheat branor rice straw as culture media. C. byrsina produced the highest laccase (200 U mL-1) and Lentinus sp produced the highest activities of manganese peroxidase (MnP) and lignin peroxidase (LiP) (7 and 8 U mL-1, respectively), when cultivated on wheat bran. The effect of N addition on enzyme production was studied in medium containing rice straw and the data showed an increase of 3 up to 4-fold in the laccase production compared to that obtained in SSF on wheat bran. The laccases presented optimum pH at 3.0-3.5 and were stable at neutral pH values. Optimum pH for MnP and LiP activities was at 3.5 and between 4.5 and 6.0, respectively. All the strains produced laccase with optimum activities between 55-60ºC while the peroxidases presented maximum activity at temperatures of 30 to 55ºC. The crude enzymes promoted decolorization of chemically different dyes with around 70% of decolorization of RBBR and cybacron blue 3GA in 6h oftreatment. The data indicated that enzymes from these basidiomycetes strains are able to decolorize synthetic dyes.

Fungos decompositores de madeira, do grupo Basidiomicetes, coletados na ôEstação Ecológica do NoroestePaulistaõ, São José do Rio Preto, São Paulo, Brasil, pertencentes a ordem Aphyllophorales e identificados como Coriolopsis byrsina SXS16, Lentinus strigellus SXS355, Lentinus sp. SXS48,Picnoporus sanguineus SXS 43 e Phellinus rimosus SXS47 foram estudados para a produção de ligninases por FES (fermentação em estado sólido) usando farelo de trigo ou palha de arroz como meio de cultura. A espécie C. byrsina produziu a maior quantidade de lacase (200 U mL-1) enquanto que Lentinus sp. foi o melhor produtor de manganês peroxidase (MnP) e lignina peroxidase (LiP) (7 e 8 U mL-1, respectivamente), quando cultivados em meio composto por farelo de trigo. A avaliação do efeito da suplementação de nitrogênio do substrato sólido lignocelulósico (palha de arroz) indicou um aumento de 3 a 4 vezes na produção de lacase. A caracterização das enzimasmostrou que as lacases apresentaram atividade ótima em pH 3,0-3,5 e foram estáveis em pH de neutro a alcalino. O pH ótimo para atividade de MnP e LiP foi de 3,5 e entre 4,5 e 6,0, respectivamente. Todas as linhagens produziram lacase com atividade ótima a 55-60ºC, enquanto as peroxidases apresentaram atividades máximas entre temperaturas de 30 e 55ºC. A aplicaçãodas soluções enzimáticas brutas, obtidas pelo cultivo das linhagens em meio de farelo de trigo, em testes de descoloração de corantes sintéticos de diferentes grupos químicos levou amais 70% de perda de cor dos corantes RBBR e de cybacron blue 3GA, em 6h de tratamento. Os dados obtidos indicaramque as soluções enzimáticas contendo ligninases produzidas pelas linhagens de basidiomicetos estudadas promoveram adescoloração de corantes sintéticos.

Ligninases production by Basidiomycetes strains on lignocellulosic agricultural residues and their application in the decolorization of synthetic dyes.

Wood rotting Basidiomycetes collected in the "Estação Ecológica do Noroeste Paulista", São José do Rio Preto, São Paulo State, Brazil, concerning Aphyllophorales order and identified as Coriolopsis byrsina SXS16, Lentinus strigellus SXS355, Lentinus sp SXS48, Picnoporus sanguineus SXS 43 and Phellinus rimosus SXS47 were tested for ligninases production by solid state fermentation (SSF) using wheat bran or rice straw as culture media. C. byrsina produced the highest laccase (200 U mL(-1)) and Lentinus sp produced the highest activities of manganese peroxidase (MnP) and lignin peroxidase (LiP) (7 and 8 U mL(-1), respectively), when cultivated on wheat bran. The effect of N addition on enzyme production was studied in medium containing rice straw and the data showed an increase of 3 up to 4-fold in the laccase production compared to that obtained in SSF on wheat bran. The laccases presented optimum pH at 3.0-3.5 and were stable at neutral pH values. Optimum pH for MnP and LiP activities was at 3.5 and between 4.5 and 6.0, respectively. All the strains produced laccase with optimum activities between 55-60ºC while the peroxidases presented maximum activity at temperatures of 30 to 55ºC. The crude enzymes promoted decolorization of chemically different dyes with around 70% of decolorization of RBBR and cybacron blue 3GA in 6h of treatment. The data indicated that enzymes from these basidiomycetes strains are able to decolorize synthetic dyes.

Ligninases production by Basidiomycetes strains on lignocellulosic agricultural residues and their application in the decolorization of synthetic dyes

Wood rotting Basidiomycetes collected in the "Estação Ecológica do Noroeste Paulista", São José do Rio Preto, São Paulo State, Brazil, concerning Aphyllophorales order and identified as Coriolopsis byrsina SXS16, Lentinus strigellus SXS355, Lentinus sp SXS48, Picnoporus sanguineus SXS 43 and Phellinus rimosus SXS47 were tested for ligninases production by solid state fermentation (SSF) using wheat bran or rice straw as culture media. C. byrsina produced the highest laccase (200 U mL-1) and Lentinus sp produced the highest activities of manganese peroxidase (MnP) and lignin peroxidase (LiP) (7 and 8 U mL-1, respectively), when cultivated on wheat bran. The effect of N addition on enzyme production was studied in medium containing rice straw and the data showed an increase of 3 up to 4-fold in the laccase production compared to that obtained in SSF on wheat bran. The laccases presented optimum pH at 3.0-3.5 and were stable at neutral pH values. Optimum pH for MnP and LiP activities was at 3.5 and between 4.5 and 6.0, respectively. All the strains produced laccase with optimum activities between 55-60°C while the peroxidases presented maximum activity at temperatures of 30 to 55°C. The crude enzymes promoted decolorization of chemically different dyes with around 70% of decolorization of RBBR and cybacron blue 3GA in 6h of treatment. The data indicated that enzymes from these basidiomycetes strains are able to decolorize synthetic dyes.

Fungos decompositores de madeira, do grupo Basidiomicetes, coletados na "Estação Ecológica do Noroeste Paulista", São José do Rio Preto, São Paulo, Brasil, pertencentes a ordem Aphyllophorales e identificados como Coriolopsis byrsina SXS16, Lentinus strigellus SXS355, Lentinus sp. SXS48, Picnoporus sanguineus SXS 43 e Phellinus rimosus SXS47 foram estudados para a produção de ligninases por FES (fermentação em estado sólido) usando farelo de trigo ou palha de arroz como meio de cultura. A espécie C. byrsina produziu a maior quantidade de lacase (200 U mL-1) enquanto que Lentinus sp. foi o melhor produtor de manganês peroxidase (MnP) e lignina peroxidase (LiP) (7 e 8 U mL-1, respectivamente), quando cultivados em meio composto por farelo de trigo. A avaliação do efeito da suplementação de nitrogênio do substrato sólido lignocelulósico (palha de arroz) indicou um aumento de 3 a 4 vezes na produção de lacase. A caracterização das enzimas mostrou que as lacases apresentaram atividade ótima em pH 3,0-3,5 e foram estáveis em pH de neutro a alcalino. O pH ótimo para atividade de MnP e LiP foi de 3,5 e entre 4,5 e 6,0, respectivamente. Todas as linhagens produziram lacase com atividade ótima a 55-60°C, enquanto as peroxidases apresentaram atividades máximas entre temperaturas de 30 e 55°C. A aplicação das soluções enzimáticas brutas, obtidas pelo cultivo das linhagens em meio de farelo de trigo, em testes de descoloração de corantes sintéticos de diferentes grupos químicos levou a mais 70% de perda de cor dos corantes RBBR e de cybacron blue 3GA, em 6h de tratamento. Os dados obtidos indicaram que as soluções enzimáticas contendo ligninases produzidas pelas linhagens de basidiomicetos estudadas promoveram a descoloração de corantes sintéticos.