Enzyme production mechanism of anaerobic fungus Orpinomyces sp. YF3 in yak rumen induced by different carbon source / 生物工程学报

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, β-glucan metabolic process, cellulase activity, endo-1,4-β-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.

Process optimization for enhanced production of cellulases form locally isolated fungal strain by submerged fermentation

Cellulase has myriad applications in various sectors like pharmaceuticals, textile, detergents, animal feed and bioethanol production, etc. The current study focuses on the isolation, screening and optimization of fungal strain through one factor at a time technique for enhanced cellulase production. In current study sixteen different fungal cultures were isolated and the culture which quantitatively exhibits higher titers of cellulase activity was identified both morphologically and molecularly by 18S rDNA and designated as Aspergillus niger ABT11. Different parameters like fermentation medium, volume, temperature, pH and nutritional components were optimized. The highest CMCase and FPase activities was achieved in 100ml of M5 medium in the presence of 1% lactose and sodium nitrate at 30 oC, pH5 after 72 hours. The result revealed A. niger can be a potential candidate for scale up studies.

Progress in the production of lignocellulolytic enzyme systems using Penicillium species / 生物工程学报

The efficient production of lignocellulolytic enzyme systems is an important support for large-scale biorefinery of plant biomass. On-site production of lignocellulolytic enzymes could increase the economic benefits of the process by lowering the cost of enzyme usage. Penicillium species are commonly found lignocellulose-degrading fungi in nature, and have been used for industrial production of cellulase preparations due to their abilities to secrete complete and well-balanced lignocellulolytic enzyme systems. Here, we introduce the reported Penicillium species for cellulase production, summarize the characteristics of their enzymes, and describe the strategies of strain engineering for improving the production and performance of lignocellulolytic enzymes. We also review the progress in fermentation process optimization regarding the on-site production of lignocellulolytic enzymes using Penicillium species, and suggest prospect of future work from the perspective of building a "sugar platform" for the biorefinery of lignocellulosic biomass.

Cultivable cellulolytic fungi isolated from the gut of Amazonian aquatic insects

Fungos filamentosos tem sido alvo de estudos de bioprospecção devido à sua grande eficiencia em produzir enzimas extracelulares, as quais tem grande potencial para uso em bioindústrias. Neste estudo, fungos filamentosos foram isolados do intestino de larvas de insetos aquáticos da Amazônia, para avaliar sua atividade celulolítica. Foram coletadas 69 larvas de insetos aquáticos fragmentadores de três gêneros: Phylloicus (Trichoptera: Calamoceratidae), Triplectides (Trichoptera:Leptoceridae) e Stenochironomus (Diptera: Chironomidae) em dez igarapés de uma área protegida na Amazônia central brasileira. O crescimento dos fungos isolados foi feito em meio de cultura Ágar Batata Dextrose (BDA). Os isolados fúngicos foram transferidos para o meio sintético com Carboximetil celulose e utilizou-se vermelho Congo para determinar o índice enzimático. O halo de hidrólise, indicando a produção de celulases, foi observado em 175 isolados fúngicos (70% do total), dos quais 25 tiveram um índice enzimático ≥ 2,0 e pertencem a sete gêneros fúngicos. Os táxons fúngicos Cladosporium, Gliocephalotrichum, Penicillium, Pestalotiopsis, Talaromyces, Trichoderma e Umbelopsis foram isolados dos intestinos das larvas de Phylloicus, Triplectides e Stenochironomus e são tradicionalmente utilizados em aplicações biotecnológicas. Os resultados indicam um potencial celulolítco destes fungos associados ao intestino de insetos aquáticos amazônicos. (AU)

Screening of cellulose degradation bacteria from Min pigs and optimization of its cellulase production

BACKGROUND: Cellulose as a potential feed resource hinders its utilization because of its complex structure, and cellulase is the key to its biological effective utilization. Animal endogenous probiotics are more susceptible to colonization in the intestinal tract, and their digestive enzymes are more conducive to the digestion and absorption of feed in young animals. Min pigs are potential sources of cellulase probiotics because of the high proportion of dietary fiber in their feed. In this study, the cellulolytic bacteria in the feces of Min pigs were isolated and screened. The characteristics of enzymes and cellulase production were studied, which provided a theoretical basis for the rational utilization of cellulase and high-fiber food in animal production. RESULTS: In our study, 10 strains of cellulase producing strains were isolated from Min pig manure, among which the M2 strain had the best enzyme producing ability and was identified as Bacillus velezensis. The optimum production conditions of cellulase from strain M2 were: 2% inoculum, the temperature of 35°C, the pH of 5.0, and the liquid loading volume of 50 mL. The optimum temperature, pH and time for the reaction of cellulase produced by strain M2 were 55°C, 4.5 and 5 min, respectively. CONCLUSIONS: Min pigs can be used as a source of cellulase producing strains. The M2 strain isolated from feces was identified as Bacillus velezensis. The cellulase from M2 strain had a good activity and the potential to be used as feed additive for piglets.

Moringa straw as cellulase production inducer and cellulolytic fungi source / Residuo de moringa como inductor de la producción de celulasas y como fuente de hongos celulolíticos

Abstract Currently, the valorization of agroindustrial waste is of great interest. Moringa oleifera is a multipurpose tree whose softwood residues could be used as raw material for low-cost cellulase production. The aim of this study was to isolate, identify, and characterize microorganisms with cellulolytic activity in different carbon sources. We isolated and puri-fied 42 microorganisms from M. oleifera biomass. Fungi presenting the largest hydrolytic halos in carboxymethylcellulose as a substrate were molecularly identified as Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) and Cladosporium cladosporioides (FC2). The ability of these fungal strains to break down cellulose was assessed in a submerged fermentation using either amorphous CMC or crystalline form (Avicel). P. funiculosum and C. cladosporioides displayed similar endoglucanase (606 U/l) and exoglucanase (205 U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of p-glucosidase activity (664 U/l) in the carboxymethylcellulose medium. In addition, the effect of three culture media (A, B, and C) on cellulase production was evaluated in P. funiculosum using moringa straw as a carbon source. The results showed a volumetric productivity improvement of cellulases that was 2.77-, 8.26-, and 2.30-fold higher for endoglucanase, exoglucanase and p-glucosidase, respectively when medium C containing moringa straw was used as a carbon source. The enzymatic extracts produced by these fungi have biotechnological potential especially for second-generation bioethanol production (2G) from moringa straw. This is the first report on the use of M. oleifera biomass to induce the production of various cellulases in P. funiculosum. © 2019 Asociación Argentina de Microbiología. Published by Elsevier Espana, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).

Resumen Actualmente, la valorización de los residuos agroindustriales es de gran interés. En este trabajo se emplearon residuos de madera blanda de Moringa oleifera para la producción de celulasas de bajo costo. El objetivo fue aislar, identificar y caracterizar microorganismos con actividad celulolítica en diferentes fuentes de carbono. A partir de la biomasa de M. oleifera, se aislaron e identificaron 42 microorganismos productores de celulasas. Los hongos que presentaron los mayores halos de hidrólisis en carboximetilcelulosa como sustrato fueron identificados molecularmente como Penicillium funiculosum (FG1), Fusarium verticillioides (FG3) y Cladosporium cladosporioides (FC2). Mediante fermentación sumergida, se evaluó la capacidad de estas cepas en la producción de celulasas utilizando celulosa cristalina (Avicel) y amorfa (CMC) como fuentes de carbono. P. funiculosum y C. cladosporioides presentaron las mayores actividades de endoglucanasa (606 U/l) y exoglucanasa (205 U/l) en medio Avicel, mientras que F. verticillioides mostró la mayor actividad de p-glucosidasa (664 U/l) en medio CMC. Además, se evaluó el efecto de tres medios de cultivo (A, B y C) sobre la producción de celulasas en P. funiculosum empleando residuos de moringa como fuente de carbono. Los resultados mostraron que en el medio C, la productividad volumétrica de celulasas se incrementó en 2,77; 8,26 y 2,30 veces para las actividades de endoglucanasa, exoglucanasa y p-glucosidasa, respectivamente. Los extractos enzimáticos producidos tienen gran potencial para su utilización biotecnológica, especialmente en la sacarificación de residuos de moringa y la producción de bioetanol de segunda generación. Este es el primer estudio del uso de la biomasa de M. oleifera para inducir la producción de diversas celulasas en P. funiculosum.

Construction of an engineered Saccharomyces cerevisiae expressing endoglucanase efficiently / 生物工程学报

Endoglucanase (EG) is an important component of cellulases and play an important role in cellulose degradation. However, its application is limited due to the low yield of endoglucanase from natural microorganisms. Efficient heterologous expression of endoglucanase is an effective way to solve this problem. To obtain the engineered Saccharomyces cerevisiae for high-yield endoglucanase, endoglucanase gene was cloned from Clostridium cellulovorans, with a total length of 1 996 bp, encoding 440 amino acids, and the complete expression cassette (PαEGC) was constructed with the PGK promoter sequence from Saccharomyces cerevisiae, α-signal peptide sequence from pPIC9K plasmid and CYC1 terminator sequence from pSH65 plasmid by gene splicing by overlap extension PCR (SOE PCR), and the expression vector of endoglucanase in Saccharomyces cerevisiae was constructed by rDNA integration. The relationship between copy number and protein expression was explored. Random multicopy expression of endoglucanase was performed in Saccharomyces cerevisiae. The copy number of endoglucanase was identified by Droplet Digital PCR and explore the relationship between copy number and protein expression.The engineered Saccharomyces cerevisiae of endoglucanase with copy numbers of 1, 3, 4, 7, 9, 11, 15, 16, 19, 21, 22 and 23 were obtained by rDNA integration, respectively. The results showed that when the copy number was 15, the enzyme activity was the highest, namely 351 U/mL. The engineered strain of Saccharomyces cerevisiae for endoglucanase was successfully constructed, which can provide reference for the heterologous expression of other industrial enzymes.

Progress in improving the properties of dissolving pulp by enzymes / 生物工程学报

Dissolving pulp consists of high purity cellulose and is widely used to as raw materials for the production of regenerated cellulose fiber, cellulose ester and cellulose ether. The characteristic of dissolving pulp affects greatly the production and processing performance of subsequent products. The α-cellulose content, hemicellulose content, pulp viscosity, ash, transition metal ion content, fiber morphology, molecular weight distribution of cellulose and the reactivity are the important properties. Because of its green, mild and high efficiency, the application of enzymes in improving the properties of dissolving pulp has a promising application prospect and has been researched significantly. In this review, the main properties of dissolving pulp are presented first, followed by a recommendation of the enzymes to improve these properties. The application and current research of cellulase and xylanase in improving the properties of dissolving pulp are emphasized. The main problems and the future research areas in improving the properties of dissolving pulp by enzymes are revealed. Finally, the technology prospects in this field are proposed.

Fermentation of cellulase with multiple types of Salvia miltiorrhiza residues and other solid wastes from Chinese materia medica industrialization / 中国中药杂志

The solid wastes of Chinese materia dedica industrialization represented by Salvia miltiorrhiza residues have a strong small-molecule bio-recalcitrance in the process of high-value utilization of biotransformation. Highly tolerant strains were bred to break bio-recalcitrance of Salvia miltiorrhiza residues and produce high-value added cellulose, which has a significant significance for recycling and industrial utilization of solid waste. In this study, a strain of fungus, Penicillium expansum SZ13, was found with small-molecule antibacterial substance tanshinone contained in Salvia miltiorrhiza residues by a biological method. The optimal enzyme production process and peak period of SZ13 were determined. It was found that SZ13 could maintain peak enzyme production for 5 days by degrading residues under the conditions of temperature 35 ℃, rotation speed 180 r·min~(-1), 5% of residues addition, and 5% seed solution addition. Meanwhile, the ability of SZ13 to degrade the enzyme production of multiple types of residues was explored. The results showed a high enzyme activity and stable enzyme production of SZ13 in the process of degrading residues. SZ13 could efficiently utilize various types of Chinese medicine residues, such as Salvia miltiorrhiza residues, to realize the high-value utilization of cellulose in multiple types of residues.

Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆ zface1 Strain

Abstract The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

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.

Identification of a novel strain, Streptomyces blastmyceticus JZB130180, and evaluation of its biocontrol efficacy against Monilinia fructicola / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Peach brown rot, caused by Monilinia fructicola, is one of the most serious peach diseases. A strain belonging to the Actinomycetales, named Streptomyces blastmyceticus JZB130180, was found to have a strong inhibitory effect on M. fructicola in confrontation culture. Following the inoculation of peaches in vitro, it was revealed that the fermentation broth of S. blastmyceticus JZB130180 had a significant inhibitory effect on disease development by M. fructicola. The fermentation broth of S. blastmyceticus JZB130180 had an EC50 (concentration for 50% of maximal effect) of 38.3 µg/mL against M. fructicola, as determined in an indoor toxicity test. Analysis of the physicochemical properties of the fermentation broth revealed that it was tolerant of acid and alkaline conditions, temperature, and ultraviolet radiation. In addition, chitinase, cellulase, and protease were also found to be secreted by the strain. The results of this study suggest that S. blastmyceticus JZB130180 may be used for the biocontrol of peach brown rot.

A simplified and miniaturized glucometer-based assay for the detection of β-glucosidase activity / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

β-Glucosidase activity assays constitute an important indicator for the early diagnosis of neonatal necrotizing enterocolitis and qualitative changes in medicinal plants. The drawbacks of the existing methods are high consumption of both time and reagents, complexity in operation, and requirement of expensive instruments and highly trained personnel. The present study provides a simplified, highly selective, and miniaturized glucometer-based strategy for the detection of β-glucosidase activity. Single-factor experiments showed that optimum β-glucosidase activity was exhibited at 50 °C and pH 5.0 in a citric acid-sodium citrate buffer when reacting with 0.03 g/mL salicin for 30 min. The procedure for detection was simplified without the need of a chromogenic reaction. Validation of the analytical method demonstrated that the accuracy, precision, repeatability, stability, and durability were good. The linear ranges of β-glucosidase in a buffer solution and rat serum were 0.0873-1.5498 U/mL and 0.4076-2.9019 U/mL, respectively. The proposed method was free from interference from β-dextranase, snailase, β-galactosidase, hemicellulase, and glucuronic acid released by baicalin. This demonstrated that the proposed assay had a higher selectivity than the conventional dinitrosalicylic acid (DNS) assay because of the specificity for salicin and unique recognition of glucose by a personal glucose meter. Miniaturization of the method resulted in a microassay for β-glucosidase activity. The easy-to-operate method was successfully used to detect a series of β-glucosidases extracted from bitter almonds and cultured by Aspergillus niger. In addition, the simplified and miniaturized glucometer-based assay has potential application in the point-of-care testing of β-glucosidase in many fields, including medical diagnostics, food safety, and environmental monitoring.

Fungal Diversity and Enzyme Activity Associated with the Macroalgae, Agarum clathratum

Agarum clathratum, a brown macroalgae species, has recently become a serious environmental problem on the coasts of Korea. In an effort to solve this problem, fungal diversity associated with decaying A. clathratum was investigated and related β-glucosidase and endoglucanase activities were described. A total of 233 fungal strains were isolated from A. clathratum at 15 sites and identified 89 species based on morphology and a multigene analysis using the internal transcribed spacer region (ITS) and protein-coding genes including actin (act), β-tubulin (benA), calmodulin (CaM), and translation elongation factor (tef1). Acremonium, Corollospora, and Penicillium were the dominant genera, and Acremonium fuci and Corollospora gracilis were the dominant species. Fifty-one species exhibited cellulase activity, with A. fuci, Alfaria terrestris, Hypoxylon perforatum, P. madriti, and Pleosporales sp. Five showing the highest enzyme activities. Further enzyme quantification confirmed that these species had higher cellulase activity than P. crysogenum, a fungal species described in previous studies. This study lays the groundwork for bioremediation using fungi to remove decaying seaweed from populated areas and provides important background for potential industrial applications of environmentally friendly processes.

Co-expression of lignocellulase from termite and their endosymbionts / 生物工程学报

Natural lignocellulosic materials contain cellulose, hemicellulose, and lignin. Cellulose hydrolysis to glucose requires a series of lignocellulases. Recently, the research on the synergistic effect of lignocellulases has become a new research focus. Here, four lignocellulase genes encoding β-glucosidase, endo-1,4-β-glucanase, xylanase and laccase from termite and their endosymbionts were cloned into pETDuet-1 and pRSFDuet-1 and expressed in Escherichia coli. After SDS-PAGE analysis, the corresponding protein bands consistent with the theoretical values were observed and all the proteins showed enzyme activities. We used phosphoric acid swollen cellulose (PASC) as substrate to measure the synergistic effect of crude extracts of co-expressing enzymes and the mixture of single enzyme. The co-expressed enzymes increased the degradation efficiency of PASC by 44% compared with the single enzyme mixture; while the degradation rate increased by 34% and 20%, respectively when using filter paper and corn cob pretreated with phosphoric acid as substrates. The degradation efficiency of the co-expressed enzymes was higher than the total efficiency of the single enzyme mixture.

Artificial zinc finger protein mediated cellulase production in Trichoderma reesei Rut-C30 / 生物工程学报

Trichoderma reesei Rut-C30 is widely used in industrial cellulase production, and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery. In this study, T. reesei Rut-C30 was engineered with an artificial zinc finger proteins (AZFPs) library. Two mutants T. reesei M1 and M2 with improved cellulase production were obtained. Compared to the parent strain, the filter paper activity (FPase) of T. reesei M1 and M2 increased 100% and 53%, respectively. In addition, the total amount of extracellular protein from the M1 mutant increased 69%, whereas the endo-β-glucanase (CMCase) activity of the M2 mutant is 64% higher compared to the parental strain. Furthermore, RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants, but different patterns were observed in the two mutants. On the other hand, the cellulase transcriptional repressor ace1 was down-regulated in both mutants, but the transcription level of the activator xyr1 was only up-regulated in the strain M1. These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T. reesei. Analysis of the target genes of AZFPs from T. reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T. reesei, but also enable development of cellulase hyper-producing strains by metabolic engineering.

Endophytic Actinomycetes as Potential Producers of Hemicellulases and Related Enzymes for Plant Biomass Degradation

Abstract Tailor made enzymatic preparation must be design to hydrolyze efficiently plant biomass, once that each plant biomass possesses a distinct cell wall composition. Most of actinomycetes used for plant cell wall degradation are focused on the cellulases and xylanases production. However, a wide range of enzymes must be produced for an efficient degradation of lignocellulose materials. During the last decade several unusual environments were studied to obtain strains that produce glycohydrolases with innovator characteristics. In this context, the present work concerned the selection of endophytic actinomycetes as producers of hemicellulases and related enzymes with different enzymatic profiles, for use in the deconstruction of lignocellulosic biomass. A total of 45 Brazilian actinomycetes previously isolated from plants (endophytics) and soil were prospected for hemicellulases and β-glucosidase production. Four strains highlighted for hemicellulase production (DR61, DR63, DR69 and DR66) and were selected for cultivation under other inductors substrates (xylan and pectin). All strains belong to Streptomyces genera and have their extracts tested for degradation of several hemicellulolytic substrates. The strains presented different glicohydrolyse enzymes profiles mainly for xylans and glucans that can be used for specific formulations of enzymes applied on the biomass deconstruction, principally on sugar cane bagasse.

Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.

Inducible cellulase production from an organic solvent tolerant Bacillus sp. SV1 and evolutionary divergence of endoglucanase in different species of the genus Bacillus

Abstract Bacteria are important sources of cellulases with various industrial and biotechnological applications. In view of this, a non-hemolytic bacterial strain, tolerant to various environmental pollutants (heavy metals and organic solvents), showing high cellulolytic index (7.89) was isolated from cattle shed soil and identified as Bacillus sp. SV1 (99.27% pairwise similarity with Bacillus korlensis). Extracellular cellulases showed the presence of endoglucanase, total cellulase and β-glucosidase activities. Cellulase production was induced in presence of cellulose (3.3 times CMCase, 2.9 times FPase and 2.1 times β-glucosidase), and enhanced (115.1% CMCase) by low-cost corn steep solids. An in silico investigation of endoglucanase (EC 3.2.1.4) protein sequences of three Bacillus spp. as query, revealed their similarities with members of nine bacterial phyla and to Eukaryota (represented by Arthropoda and Nematoda), and also highlighted of a convergent and divergent evolution from other enzymes of different substrate [(1,3)-linked beta-d-glucans, xylan and chitosan] specificities. Characteristic conserved signature indels were observed among members of Actinobacteria (7 aa insert) and Firmicutes (9 aa insert) that served as a potential tool in support of their relatedness in phylogenetic trees.

Isolation and molecular identification of a cellulotic bacterium from muncipal waste and investigation of its cellulase production / Isolamento e identificação molecular de uma bactéria celulolítica do lixo municipal e investigação de sua produção de celulase

Municipal waste is rich in lignocellulosic compounds which contain cellulose, lignin and hemicellulose. Microorganisms can break down such compounds and convert them into glucose and other carbohydrates. The current study was performed to isolate and identify cellulolytic bacteria in municipal waste. Municipal waste samples were collected and plated on Carboxymethyl cellulose (CMC) agar. Preliminary identification of the isolates was performed using standard biochemical assays. The activity of carboxymethyl cellulose (CMCase) was specified through measuring the release of reducing sugars from CMC. Different nitrogen sources at various concentrations and initial pH values were evaluated for their effect on enzyme production. Further the enzyme production was determined at different fermentation times. Molecular identification was then performed using bacterial 16s rRNA gene amplification and sequencing. A cellulolytic bacterium was isolated from municipal waste samples and identified based on morphological, physiological and biochemical characteristics along with 16S rRNA analysis. The isolated bacterium was identified as Bacillus subtilis (accession number: KU681044). Whose growth characteristics showed that its growth curve entered the logarithmic phase following 10­18 h with the stable growth phase ranging from 23 to 37 h. The optimal carbon source for fermentation was 1% rice hull, with the nitrogen source comprised of 2% peptone and yeast extract. The the minimum CMCase activity was observed at an initial medium pH of 4.0, while the maximum was observed at pH 7. The strain grew vigorously and the cellulase yield was high at 6­24 h fermentation time period. The isolated bacteria showed the degrading potential of cellulose which could be employed in local industrial process.

Resíduos urbanos são ricos em compostos lignocelulósicos que contêm celulose, lignina e hemicelulose. Microrganismos podem quebrar esses compostos e convertê-los em glicose e outros carboidratos. O presente estudo foi realizado para isolar e identificar bactérias celulolíticas em resíduos urbanos. Amostras de resíduos municipais foram coletadas e plaqueadas em ágar Carboximetilcelulose (CMC). A identificação preliminar dos isolados foi realizada utilizando ensaios bioquímicos padrão. A atividade da carboximetilcelulose (CMCase) foi especificada através da medição da liberação de açúcares redutores da CMC. Diferentes fontes de nitrogênio em várias concentrações e valores iniciais de pH foram avaliados quanto ao seu efeito na produção de enzimas. Além disso, a produção de enzima foi determinada em diferentes tempos de fermentação. A identificação molecular foi então realizada utilizando amplificação e sequenciamento do gene bacteriano 16s rRNA. Uma bactéria celulolítica foi isolada de amostras de resíduos urbanos e identificada com base em características morfológicas, fisiológicas e bioquímicas, juntamente com a análise 16S rRNA. A bactéria isolada foi identificada como Bacillus subtilis (número de acesso: KU681044). Cujas características de crescimento mostraram que sua curva de crescimento entrou na fase logarítmica após 10-18 h com a fase de crescimento estável variando de 23 a 37 h. A fonte de carbono ótima para a fermentação foi 1% de casca de arroz, com a fonte de nitrogênio composta de 2% de peptona e extrato de levedura. A atividade mínima de CMCase foi observada em um pH médio inicial de 4,0, enquanto a máxima foi observada em pH 7. A linhagem cresceu vigorosamente e o rendimento de celulase foi alto no período de 6 a 24 horas de fermentação. As bactérias isoladas mostraram o potencial de degradação da celulose que poderia ser empregada no processo industrial local.