Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Thermo-resistant enzyme-producing microorganisms isolated from composting

Abstract Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Resumo Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Endoglucanase production by endophytic fungi isolated from vitis labrusca l. with peanut hull and sawdust as substrates / Produção de endoglucanase por fungos endofíticos isolados de Vitis labrusca L. utilizando casca de amendoim e serragem como substratos

Endoglucanases are enzymes widely employed in different industrial fields, albeit with high production costs. Studies on new microbial sources and low-cost substrates are highly relevant, including those on agro-industrial. Current analysis evaluates peanut hull (PH) and sawdust (SD) as substrates for submerged cultures of 14 endophytic fungi isolated from grapevine (Vitis labrusca L.) cultivars Bordô and Concord. Endophytes were grown on a carboxymethylcellulose (CMC) medium and the cup plate assay showed that eight strains (belonging to genera Cochliobolus, Diaporthe, Fusarium and Phoma) had positive results: enzymatic halos ranged from 10.8±0.02to 15.5±0.07 mm in diameter. Diaporthe sp. strains (GenBank accession codes KM362392, KM362368 and KM362378) and Fusariumculmorum KM362384 were highlighted as the most promising sources. Further, PH and SD as substrates for the fermentation of these fungi were evaluated by the cup plate assay and endoglucanase activity assay. Highest halo diameters were obtained for Diaporthe sp. KM362392: 16.1±0.01 mm (CMC), 14.5±0.01 mm (PH) and 14.7±0.03 mm (SD). The fungus also presented the highest levels of endoglucanase activity: analysis of variance revealed that CMC (3.52±0.98 µmol/min), PH (2.93±0.23 µmol/min) and SD (3.26±0.38 µmol/min) were similarly efficient as substrates. Results deepen knowledge on V. labrusca endophytes that may be endoglucanase sources, eventhough further optimizations in submerged cultures with PH and SD should be undertaken to increase theenzymatic production from these wastes.

Endoglucanases são enzimas amplamente empregadas em diferentes setores industriais; embora sua produção apresente custos elevados. Estudos sobre novas fontes microbianas e substratos mais baratos são de grande importância, incluindo os resíduos agroindustriais. Nesse estudo, casca de amendoim (CA) e serragem (SE) foram testadas como substratos para o cultivo submerso de 14 fungos endofíticos isolados das cultivares Bordô e Concord de videira (Vitis labrusca L.) Os endófitos foram crescidos em meio contendo carboximetilcelulose (CMC) e o ensaio cup plate mostrou resultados positivos para oito fungos (pertencentes aos gêneros Cochliobolus, Diaporthe, Fusarium and Phoma); os halos enzimáticos variaram entre 10,8±0,02 e 15,5±0,07 mm de diâmetro. Linhagens de Diaporthe sp. (códigos de acesso no GenBank KM362392, KM362368 e KM362378) e Fusariumculmorum KM362384 se destacaram como produtores mais promissores. Então, o uso de CA e SE como substratos para a fermentação desses fungos foi avaliado pelo ensaio cup plate e pela quantificação da atividade de endoglucanase. Os maiores halos enzimáticos foram obtidos para Diaporthe sp. KM362392: 16,1±0,01 mm (CMC), 14,5±0,01 mm (CA) e 14,7±0,03 mm (SE). Esse fungo também apresentou os maiores níveis de endoglucanase: a análise de variância revelou que CMC (3,52±0,98 µmol/min), CA (2,93±0,23 µmol/min) e SE (3,26±0,38 µmol/min) foram substratos similarmente eficientes. Esses resultados expandem o conhecimento sobre endófitos de V. labrusca que são fontes de endoglucanases; futuras otimizações quanto ao cultivo submerso com CA e SE podem ser utilizadas para aumentar a produção enzimática a partir do uso desses resíduos.

Agave syrup as a substrate for inulinase production by Kluyveromyces marxianus NRRL Y-7571 / Xarope de agave como substrato para produção de inulinase por Kluyveromyces marxianus NRRL Y-7571

The factorial planning was used to plan and optimize inulinase production by the yeast Kluyveromyces marxianus NRRL Y-7571. The experiments were conducted using a Central Composite Design (CCD) 22, at different concentrations of agave syrup (3.6 to 6.4%) and yeast extract (2.2 to 3.0%). After 96 hours of fermentation, the best condition for the inulinase production was 5% agave syrup and 2.5% yeast extract, which yielded an average of 129.21 U mL-1 of inulinase. Partial characterization of the crude enzyme showed that the optimal pH and temperature were 4.0 and 60°C, respectively. The enzyme showed thermal stability at 55°C for 4 hours.

O planejamento fatorial foi utilizado para planejar e otimizar a produção de inulinase pela levedura Kluyveromyces marxianus NRRL Y-7571. Os experimentos foram conduzidos por meio de Delineamento Composto Central Rotacional (DCCR) 22 em diferentes concentrações de xarope de agave (3,6 a 6,4%) e extrato de levedura (2,2 a 3,0%). Depois de 96 horas de fermentação, a melhor condição para produção de inulinase foi xarope de agave 5% e extrato de levedura 2,5%, com uma produção média de 129,21 U mL-1. A caracterização parcial do extrato enzimático bruto mostrou que a enzima apresenta pH e temperatura ótimos de 4,0 e 60oC, respectivamente. A enzima mostrou estabilidade térmica a 55oC durante 4 horas.

Characterization of a multi-tolerant tannin acyl hydrolase II from Aspergillus carbonarius produced under solid-state fermentation

Background Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20-60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application.

Production of beta-glucosidase on solid-state fermentation by Lichtheimia ramosa in agroindustrial residues: characterization and catalytic properties of the enzymatic extract

Background β-Glucosidases catalyze the hydrolysis of cellobiose and cellodextrins, releasing glucose as the main product. This enzyme is used in the food, pharmaceutical, and biofuel industries. The aim of this work is to improve the β-glucosidase production by the fungus Lichtheimia ramosa by solid-state fermentation (SSF) using various agroindustrial residues and to evaluate the catalytic properties of this enzyme. Results A high production of β-glucosidase, about 274 U/g of dry substrate (or 27.4 U/mL), was obtained by cultivating the fungus on wheat bran with 65% of initial substrate moisture, at 96 h of incubation at 35°C. The enzymatic extract also exhibited carboxymethylcellulase (CMCase), xylanase, and β-xylosidase activities. The optimal activity of β-glucosidase was observed at pH 5.5 and 65°C and was stable over a pH range of 3.5-10.5. The enzyme maintained its activity (about 98% residual activity) after 1 h at 55°C. The enzyme was subject to reversible competitive inhibition with glucose and showed high catalytic activity in solutions containing up to 10% of ethanol. Conclusions β-Glucosidase characteristics associated with its ability to hydrolyze cellobiose, underscore the utility of this enzyme in diverse industrial processes.

Fibrinolytic protease production by new Streptomyces sp: DPUA 1576 from Amazon lichens

Background Streptomyces sp. DPUA 1576 from Amazon lichens was studied to protease and fibrinolytic production. A 2² factorial experimental design was applied to optimize its protease enzyme production using two independent variables, namely soybean flour and glucose concentrations. Results The optimal conditions to obtain high protease production (83.42 U/mL) were 1.26% soybean flour and 1.23% glucose concentration. A polynomial model was fitted to correlate the relationship between the two variables and protease activity. In relation to fibrinolytic activity, the highest activity of 706.5 mm² was obtained at 1.7% soybean flour and 1.0% glucose concentration, which was 33% higher than plasmin. Fibrinolytic production was not optimized in the studied conditions. Conclusions These results show that the optimization of the culture medium can enhance protease production, thus becoming a good process for further research. In addition, Streptomyces sp. DPUA 1576, isolated from Amazon lichens, might be a potential strain for fibrinolytic protease production.