Bioturbation analysis of microbial communities and flavor metabolism in a high-yielding cellulase Bacillus subtilis biofortified Daqu.

In this study, a fortified Daqu (FF Daqu) was prepared using high cellulase-producing Bacillus subtilis, and the effects of in situ fortification on the physicochemical properties, flavor, active microbial community and metabolism of Daqu were analyzed. The saccharification power, liquefaction power, and cellulase activity of the FF Daqu were significantly increased compared with that of the traditional Daqu (CT Daqu). The overall differences in flavor components and their contents were not significant, but the higher alcohols were lower in FF Daqu. The relative abundance of dominant active species in FF Daqu was 85.08% of the total active microbiota higher than 63.42% in CT Daqu, and the biomarkers were Paecilomyces variotii and Aspergillus cristatus, respectively. The enzymes related to starch and sucrose metabolic pathways were up-regulated and expressed in FF Daqu. In the laboratory level simulation of baijiu brewing, the yield of baijiu was increased by 3.36% using FF Daqu.

Modulating flavanone compound for reducing the bitterness and improving dietary fiber, physicochemical properties, and anti-adipogenesis of green yuzu powder by enzymatic hydrolysis.

Yuzu (Citrus junos Sieb.) is a peel-edible fruit with a pleasant aroma, but its bitter taste can impact consumer appeal. In this study, an efficient enzymatic method reduced bitterness in green yuzu powder (GYP). Cellulase KN and naringinase from Aspergillus oryzae NYO-2 significantly decreased naringin and neohesperidin content by over 87 %, while increasing total dietary fiber and soluble dietary fiber by up to 10 % and 51 %, respectively. Insoluble dietary fiber decreased by up to 22 %. Cellulose, hemicellulose, lignin, and pectin contents in enzyme-treated YP decreased by 1.15-2.00-fold, respectively. Enzyme-treated GYP exhibited improved physicochemical properties, including enhanced solubility, oil-holding capacity, and water swelling capacities. 3T3-L1 cells treated with cellulase-treated GYP and naringinase-treated GYP showed lower lipid accumulation and higher lipolysis capability than GYP, along with decreased fatty acid synthase contents. These findings suggest that enzyme-treated GYP holds potential as a functional ingredient in the food industry.

Small GTPase Rab7 is involved in stress adaptation to carbon starvation to ensure the induced cellulase biosynthesis in Trichoderma reesei.

BACKGROUND: The saprophytic filamentous fungus Trichoderma reesei represents one of the most prolific cellulase producers. The bulk production of lignocellulolytic enzymes by T. reesei not only relies on the efficient transcription of cellulase genes but also their efficient secretion after being translated. However, little attention has been paid to the functional roles of the involved secretory pathway in the high-level production of cellulases in T. reesei. Rab GTPases are key regulators in coordinating various vesicle trafficking associated with the eukaryotic secretory pathway. Specifically, Rab7 is a representative GTPase regulating the transition of the early endosome to the late endosome followed by its fusion to the vacuole as well as homotypic vacuole fusion. Although crosstalk between the endosomal/vacuolar pathway and the secretion pathway has been reported, the functional role of Rab7 in cellulase production in T. reesei remains unknown. RESULTS: A TrRab7 was identified and characterized in T. reesei. TrRab7 was shown to play important roles in T. reesei vegetative growth and vacuole morphology. Whereas knock-down of Trrab7 significantly compromised the induced production of T. reesei cellulases, overexpression of the key transcriptional activator, Xyr1, restored the production of cellulases in the Trrab7 knock-down strain (Ptcu-rab7KD) on glucose, indicating that the observed defective cellulase biosynthesis results from the compromised cellulase gene transcription. Down-regulation of Trrab7 was also found to make T. reesei more sensitive to various stresses including carbon starvation. Interestingly, overexpression of Snf1, a serine/threonine protein kinase known as an energetic sensor, partially restored the cellulase production of Ptcu-rab7KD on Avicel, implicating that TrRab7 is involved in an energetic adaptation to carbon starvation which contributes to the successful cellulase gene expression when T. reesei is transferred from glucose to cellulose. CONCLUSIONS: TrRab7 was shown to play important roles in T. reesei development and a stress response to carbon starvation resulting from nutrient shift. This adaptation may allow T. reesei to successfully initiate the inducing process leading to efficient cellulase production. The present study provides useful insights into the functional involvement of the endosomal/vacuolar pathway in T. reesei development and hydrolytic enzyme production.

Effects of combined drying techniques and cellulase hydrolysis on the nutritional value and sensory properties of shiitake mushrooms (Lentinus edodes).

Dried shiitake mushrooms offer rich nutritional value and unique sensory properties, prompting further investigation. The effects of different drying techniques (hot air drying (HAD), infrared hot air drying (IRHAD), pulsed vacuum drying (PVD), vacuum freeze drying (VFD), and natural drying (ND)) combined with enzymatic hydrolysis on the release of flavor compounds and nutrients from shiitake mushrooms were explored. The combination of HAD with cellulase hydrolysis yielded notably high levels of umami amino acids (5.4723 ± 0.1501 mg/g) and 5'-nucleotides (4.0536 ± 0.0062 mg/g), and superior volatile flavors. Combined with cellulase hydrolysis, IRHAD achieved the highest level of total sugars (6.57 ± 0.34 mg/mL), VFD resulted in the greatest soluble protein content (153.21 ± 0.23 µg/mL), PVD yielded the highest total phenolics content (93.20 ± 0.41 µg GAE/mL), and ND produced the maximum reducing sugar content (5.79 ± 0.13 mg/mL). This study addresses crucial gap in the post-drying processing of shiitake mushrooms, offering valuable insights for further product development of shiitake mushrooms.

Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations.

BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.

Expression of cellobiose dehydrogenase gene in Aspergillus niger C112 and its effect on lignocellulose degrading enzymes.

Cellobiose dehydrogenase (CDH) is one of the cellulase auxiliary proteins, which is widely used in the field of biomass degradation. However, how to efficiently and cheaply apply it in industrial production still needs further research. Aspergillus niger C112 is a significant producer of cellulase and has a relatively complete lignocellulose degradation system, but its CDH activity was only 3.92 U. To obtain a recombinant strain of A. niger C112 with high cellulases activity, the CDH from the readily available white-rot fungus Grifola frondose had been heterologously expressed in A. niger C112, under the control of the gpdA promoter. After cultivation in the medium with alkali-pretreated poplar fiber as substrate, the enzyme activity of recombinant CDH reached 36.63 U/L. Compared with the original A. niger C112, the recombinant A. niger transformed with Grifola frondosa CDH showed stronger lignocellulase activity, the activities of cellulases, ß-1, 4-glucosidase and manganese peroxidase increased by 28.57, 35.07 and 121.69%, respectively. The result showed that the expression of the gcdh gene in A. niger C112 could improve the activity of some lignocellulose degrading enzymes. This work provides a theoretical basis for the further application of gcdh gene in improving biomass conversion efficiency.

Deciphering the complex interplay between gut microbiota and crop residue breakdown in forager and hive bees (Apis mellifera L.).

This study investigates A. mellifera gut microbiota diversity and enzymatic activities, aiming to utilize identified isolates for practical applications in sustainable crop residue management and soil health enhancement. This study sampled honey bees, analyzed gut bacterial diversity via 16S rRNA gene, and screened isolates for cellulolytic, hemicellulolytic, and pectinolytic activities, with subsequent assessment of enzymatic potential. The study reveals that cellulolytic and hemicellulolytic bacterial isolates, mainly from γ-Proteobacteria, Actinobacteria, and Firmicutes, have significant potential for crop residue management. Some genera, like Aneurinibacillus, Bacillus, Clostridium, Enterobacter, Serratia, Stenotrophomonas, Apilactobacillus, Lysinibacillus, and Pseudomonas, are very good at breaking down cellulose and hemicellulase. Notable cellulose-degrading genera include Cedecea (1.390 ± 0.57), Clostridium (1.360 ± 0.86 U/mg), Enterobacter (1.493 ± 1.10 U/mg), Klebsiella (1.380 ± 2.03 U/mg), and Serratia (1.402 ± 0.31 U/mg), while Aneurinibacillus (1.213 ± 1.12 U/mg), Bacillus (3.119 ± 0.55 U/mg), Enterobacter (1.042 ± 0.14 U/mg), Serratia (1.589 ± 0.05 U/mg), and Xanthomonas (1.156 ± 0.08 U/mg) excel in hemicellulase activity. Specific isolates with high cellulolytic and hemicellulolytic activities are identified, highlighting their potential for crop residue management. The research explores gut bacterial compartmentalization in A. mellifera, emphasising gut physiology's role in cellulose and hemicellulose digestion. Pectinolytic activity is observed, particularly in the Bacillaceae clade (3.229 ± 0.02), contributing to understanding the honey bee gut microbiome. The findings offer insights into microbiome diversity and enzymatic capabilities, with implications for biotechnological applications in sustainable crop residue management. The study concludes by emphasizing the need for ongoing research to uncover underlying mechanisms and ecological factors influencing gut microbiota, impacting honey bee health, colony dynamics, and advancements in crop residue management.

Enhanced crystalline cellulose degradation by a novel metagenome-derived cellulase enzyme.

Metagenomics has revolutionized access to genomic information of microorganisms inhabiting the gut of herbivorous animals, circumventing the need for their isolation and cultivation. Exploring these microorganisms for novel hydrolytic enzymes becomes unattainable without utilizing metagenome sequencing. In this study, we harnessed a suite of bioinformatic analyses to discover a novel cellulase-degrading enzyme from the camel rumen metagenome. Among the protein-coding sequences containing cellulase-encoding domains, we identified and subsequently cloned and purified a promising candidate cellulase enzyme, Celcm05-2, to a state of homogeneity. The enzyme belonged to GH5 subfamily 4 and exhibited robust enzymatic activity under acidic pH conditions. It maintained hydrolytic activity under various environmental conditions, including the presence of metal ions, non-ionic surfactant Triton X-100, organic solvents, and varying temperatures. With an optimal temperature of 40 °C, Celcm05-2 showcased remarkable efficiency when deployed on crystalline cellulose (> 3.6 IU/mL), specifically Avicel, thereby positioning it as an attractive candidate for a myriad of biotechnological applications spanning biofuel production, paper and pulp processing, and textile manufacturing. Efficient biodegradation of waste paper pulp residues and the evidence of biopolishing suggested that Celcm05-2 can be used in the bioprocessing of cellulosic craft fabrics in the textile industry. Our findings suggest that the camel rumen microbiome can be mined for novel cellulase enzymes that can find potential applications across diverse biotechnological processes.

Green ecofriendly enhancement of cellulase productivity using agricultural wastes by Aspergillus terreus MN901491: statistical designs and detergent ability on cotton fabrics.

BACKGROUND: Cellulase is considered a group member of the hydrolytic enzymes, responsible for catalyzing the hydrolysis of cellulose and has various industrial applications. Agricultural wastes are used as an inexpensive source for several utilizable products throughout the world. So, searching for cellulase enzymes from fungal strains capable of utilizing agricultural wastes to increase productivity, reduce costs and overcome waste accumulation in the environment is very important to evaluate its potency as a bio-additive to detergent agents. RESULTS: In the current study, the previously identified fungal strain Aspergillus terreus MN901491 was screened and selected for cellulase production. Medium parameters were optimized using one-factor-at-a-time (OFAT) and multi-factorial (Plackett-Burman and Box-Behnken) design methods. OFAT showed the ability of the fungal strain to utilize agricultural wastes (corn cob and rice straw) as a substrate. Also, yeast extract was the best nitrogen source for enhancing cellulase productivity. The most significant variables were determined by Plackett-Burman Design (PBD) and their concentrations were optimized by Response Surface Methodology (RSM) using Box-Behnken Design (BBD). Among eleven independent variables screened by PBD, malt extract, (NH4)2SO4, and KCl were the most significant ones followed by rice straw which affected cellulase production positively. The ANOVA results particularly the R2-value of PBD (0.9879) and BBD (0.9883) confirmed the model efficiency and provided a good interpretation of the experiments. PBD and BBD improved cellulase productivity by 6.1-fold greater than that obtained from OFAT. Medium optimization using OFAT and statistical models increased cellulase production from A. terreus MN901491 by 9.3-fold compared to the non-optimized medium. Moreover, the efficiency of cellulase activity on cotton fabrics as a bio-additive detergent was evaluated and estimated using whiteness and scanning electron microscope (SEM) that affirmed its potential effect and remarkable detergent ability to improve whiteness by 200% in comparison with non-washed fabric and by 190% in comparison with fabric washed by water. CONCLUSION: The presented work was stabilized as a multi-efficiency in which wastes were used to produce cellulase enzyme from the fungal strain, Aspergillus terreus MN901491 as a bio-additive to detergent applications that involved ecofriendly and green processes.

Creating a novel genetic diversity of Trichoderma afroharzianum by γ-radiation for xylanase-cellulase production.

Creating novel sources of a microbial strain using induced mutation can increase enzyme production for industrial use. According to this, we have developed a mutant strain of Trichoderma afroharzianum by Co60 gamma irradiation. Trichoderma mutants were isolated from an optimum dose of 250 Gy. The qualitative and quantitative screening were used for evaluating their enzyme production and the DNA barcoding method was used to identify the best Trichoderma mutant isolates. The highest cellulase (exo-glucanase, endoglucanase, ß-glucosidase, and total cellulase) and xylanase activities were observed in superior mutant isolates of Trichoderma afroharzianum NAS107-M44 and Trichoderma afroharzianum NAS107-M82, which is approximately 1.6-2.5 times higher than its parent strain, respectively. The electrophoretic pattern of proteins showed that the exo-glucanase I, endo-glucanase III, and the xylanase I enzymes hydrolyzed the corn bran, synergistically. Overall, gamma irradiation-induced mutation could be an expedient technique to access such superior mutants for the bioconversion of corn bran wastes.

Simultaneously efficient dissolution and structural modification of chrysanthemum pectin: Targeting at proliferation of Bacteroides.

Pectic polysaccharide is a bioactive ingredient in Chrysanthemum morifolium Ramat. 'Hangbaiju' (CMH), but the high proportion of HG domain limited its use as a prebiotic. In this study, hot water, cellulase-assisted, medium-temperature alkali, and deep eutectic solvent extraction strategies were firstly used to extract pectin from CMH (CMHP). CMHP obtained by cellulase-assisted extraction had high purity and strong ability to promote the proliferation of Bacteroides and mixed probiotics. However, 4 extraction strategies led to general high proportion of HG domain in CMHPs. To further enhance the dissolution and prebiotic potential of CMHP, pectinase was used alone and combined with cellulase. The key factor for the optimal extraction was enzymolysis by cellulase and pectinase in a mass ratio of 3:1 at 1 % (w/w) dosage. The optimal CMHP had high yield (15.15 %), high content of total sugar, and Bacteroides proliferative activity superior to inulin, which was probably due to the cooperation of complex enzyme on the destruction of cell wall and pectin structural modification for raised RG-I domain (80.30 %) with relatively high degree of branching and moderate HG domain. This study provided a green strategy for extraction of RG-I enriched prebiotic pectin from plants.

Crop residue heterogeneity: Decomposition by potential indigenous ligno-cellulolytic microbes and enzymatic profiling.

The continuous depletion of fossil resources, energy-crisis and environmental pollution has gained popularity for careful selection of suitable microbial consortium to efficiently decompose crop residue and facilitate nutrient cycling. While crop residue is commonly incorporated into soil, the impact of the heterogeneity of residue on decomposition and biological mechanisms involved in extracellular carbon (C) cycle related enzyme activities remain not fully understood. To address this problem, an incubation study was conducted on chemical heterogeneity of straw and root residue with indigenous ligno-cellulolytic microbial consortium on extracellular enzymes as their activity is crucial for making in-situ residue management decisions under field condition. The activity of extracellular enzymes in different substrates showed differential variation with the type of enzyme and ranged from 16.9 to 77.6 µg mL-1, 135.7 to 410.8 µg mL-1, 66.9 to 177.1 µg mL-1 and 42.1 to 160.9 µg mL-1 for cellulase, xylanase, laccase and lignin peroxidase, respectively. Extracellular enzyme activities were sensitive to heterogeneity of biochemical constituent's present in straw and root residues and enhanced the decomposition processes with indigenous ligno-cellulolytic microbial consortium (Bacillus altitudinis, Streptomyces flavomacrosporus and Aspergillus terreus). Correlation matrix elucidated A. terreus and B. altitudinis as potential indigenous ligno-cellulolytic microbial inoculant influencing soil enzymatic activity (p < 0.001). This research work demonstrates a substantial impact of chemically diverse crop residues on the decomposition of both straw and root. It also highlights the pivotal role played by key indigenous decomposers and interactions between different microorganisms in governing the decomposition of straw and root primarily through release of extracellular enzyme. Consequently, it is novel bio-emerging strategy suggested that incorporation of the crop residues under field conditions should be carried out in conjunction with the potential indigenous ligno-cellulolytic microbial consortium for efficient decomposition in the short period of time under sustainable agriculture system.

UCST-Type Soluble Immobilized Cellulase: A New Strategy for the Efficient Degradation and Improved Recycling Performance of Wastepaper Cellulose.

This paper reports an innovative study that aims to address key issues in the efficient recycling of wastepaper cellulose. The research team utilized the temperature-responsive upper critical solution temperature (UCST) polymer P(NAGA-b-DMA) in combination with the LytA label's affinity for choline analogs. This innovative approach enabled them to successfully develop a novel soluble immobilized enzyme, P(NAGA-b-DMA)-cellulase. This new enzyme has proven highly effective, significantly enhancing the degradation of wastepaper cellulose while demonstrating exceptional stability. Compared with the traditional insoluble immobilized cellulase, the enzyme showed a significant improvement in the pH, temperature stability, recycling ability, and storage stability. A kinetic parameter calculation showed that the enzymatic effectiveness of the soluble immobilized enzyme was much better than that of the traditional insoluble immobilized cellulase. After the immobilization reaction, the Michaelis constant of the immobilized enzyme was only increased by 11.5%. In the actual wastepaper degradation experiment, the immobilized enzyme was effectively used, and it was found that the degradation efficiency of wastepaper cellulose reached 80% of that observed in laboratory conditions. This novel, thermosensitive soluble immobilized cellulase can efficiently catalyze the conversion of wastepaper cellulose into glucose under suitable conditions, so as to further ferment into environmentally friendly biofuel ethanol, which provides a solution to solve the shortage of raw materials and environmental protection problems in the paper products industry.

Use of Bacillus spp. as beneficial fermentation microbes in baking.

The development of minimally processed baked goods is dependent on new "clean label" functional ingredients that allow substitution of additives without compromising quality. We investigated the use of fermentation with Bacillus spp. as a novel approach to improve bread quality. Bacillus velezensis FUA2155 and Bacillus amyloliquefaciens Fad WE ferments were prepared using white wheat flour, wheat bran or buckwheat, and were added at a level of 2.5-20 % to bread dough. Ropy spoilage of bread was controlled by sourdough addition at a level of 10 or 20 %. The volume of white wheat bread and wheat bran bread increased by 47.4 and 62.5 % respectively with 2.5 % Bacillus ferments. Bread shelf-life was prolonged by the Bacillus ferment only at higher dosages that also reduced bread volume. The use of unfermented or sourdough fermented buckwheat improved bread volume and delayed mould spoilage. The characterization of water-soluble polysaccharides from sourdoughs and Bacillus ferments revealed that solubilization of arabinoxylans contributed to the increase in volume after fermentation of wheat but not after fermentation of buckwheat. In conclusion, Bacillus fermentation can be used to improve bread quality, adding to the diversity of microbes that are suitable for baking applications.

Specific activity and utility of recombinant cellobiohydrolase II (Cel6A) produced in maize endosperm.

Cellobiohydrolase II (CBH II) is an exo-glucanase that is part of a fungal mixture of enzymes from a wood-rot fungus, Trichoderma reesei. It is therefore difficult to purify and to establish a specific activity assay. The gene for this enzyme, driven by the rice Os glutelin promoter, was transformed into High II tissue culture competent corn, and the enzyme accumulated in the endosperm of the seed. The transgenic line recovered from tissue culture was bred into male and female elite Stine inbred corn lines, stiff stalk 16083-025 (female) and Lancaster MSO411 (male), for future production in their hybrid. The enzyme increases its accumulation throughout its 6 generations of back crosses, 27-266-fold between T1 and T2, and 2-10-fold between T2 and T3 generations with lesser increases in T4-T6. The germplasm of the inbred lines replaces the tissue culture corn variety germplasm with each generation, with the ultimate goal of producing a high-yielding hybrid with the transgene. The CBH II enzyme was purified from T5 inbred male grain 10-fold to homogeneity with 47.5% recovery. The specific activity was determined to be 1.544 units per µg protein. The corn-derived CBH II works in biopolishing of cotton by removing surface fibers to improve dyeability and increasing glucose from corn flour for increasing ethanol yield from starch-based first-generation processes.

Metabolomics-based study of chemical compositions in cellulase additives derived from a tobacco-origin Bacillus subtilis and their impact on tobacco sensory attributes.

To enhance the quality of tobacco leaves and optimize the smoking experience, diverse strains of functional bacteria and their associated metabolites have been used in tobacco aging. Exogenous cellulase additives are frequently employed to facilitate the degradation of cellulose and other macromolecular matrices and enhance the quality of the tobacco product. However, little is known about how microbial metabolites present in exogenous enzyme additives affect tobacco quality. In this study, crude cellulase solutions, produced by a tobacco-originating bacterium Bacillus subtilis FX-1 were employed on flue-cured tobacco. The incorporation of cellulase solutions resulted in the reduction of cellulose crystallinity in tobacco and the enhancement of the overall sensory quality of tobacco. Notably, tobacco treated with cellulase obtained from laboratory flask fermentation demonstrated superior scent and flavor attributes in comparison to tobacco treated with enzymes derived from industrial bioreactor fermentation. The targeted and untargeted metabolomic analysis revealed the presence of diverse flavor-related precursors and components in the cellulase additives, encompassing sugars, alcohols, amino acids, organic acids, and others. The majority of these metabolites exhibited significantly higher levels in the flask group compared to the bioreactor group, probably contributing to a pronounced enhancement in the sensory quality of tobacco. Our findings suggest that the utilization of metabolic products derived from B. subtilis FX-1 as additives in flue-cured tobacco holds promise as a viable approach for enhancing sensory attributes, establishing a solid theoretical foundation for the potential development of innovative tobacco aging additives.

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.

Enhancement of cellulolytic enzyme production from intrageneric protoplast fusion of Aspergillus species and evaluating the hydrolysate scavenging activity.

BACKGROUND: Lignocellulosic biomass provides a great starting point for the production of energy, chemicals, and fuels. The major component of lignocellulosic biomass is cellulose, the employment of highly effective enzymatic cocktails, which can be produced by a variety of microorganisms including species of the genus Aspergillus, is necessary for its utilization in a more productive manner. In this regard, molecular biology techniques should be utilized to promote the economics of enzyme production, whereas strategies like protoplast fusion could be employed to improve the efficacy of the hydrolytic process. RESULTS: The current study focuses on cellulase production in Aspergillus species using intrageneric protoplast fusion, statistical optimization of growth parameters, and determination of antioxidant activity of fermentation hydrolysate. Protoplast fusion was conducted between A. flavus X A. terreus (PFFT), A. nidulans X A. tamarii (PFNT) and A. oryzae X A. tubingensis (PFOT), and the resultant fusant PFNT revealed higher activity level compared with the other fusants. Thus, this study aimed to optimize lignocellulosic wastes-based medium for cellulase production by Aspergillus spp. fusant (PFNT) and studying the antioxidant effect of fermentation hydrolysate. The experimental strategy Plackett-Burman (PBD) was used to assess how culture conditions affected cellulase output, the best level of the three major variables namely, SCB, pH, and incubation temperature were then determined using Box-Behnken design (BBD). Consequently, by utilizing an optimized medium instead of a basal medium, cellulase activity increased from 3.11 U/ml to 7.689 U/ml CMCase. The following medium composition was thought to be ideal based on this optimization: sugarcane bagasse (SCB), 6.82 gm; wheat bran (WB), 4; Moisture, 80%; pH, 4; inoculum size, (3 × 106 spores/ml); and incubation Temp. 31.8 °C for 4 days and the fermentation hydrolysate has 28.13% scavenging activities. CONCLUSION: The results obtained in this study demonstrated the significant activity of the selected fusant and the higher sugar yield from cellulose hydrolysis over its parental strains, suggesting the possibility of enhancing cellulase activity by protoplast fusion using an experimental strategy and the fermentation hydrolysate showed antioxidant activity.

Effects of heterologous expression and N-glycosylation on the hyperthermostable endoglucanase of Pyrococcus furiosus.

Hyperthermostable endoglucanases of glycoside hydrolase family 12 from the archaeon Pyrococcus furiosus (EGPf) catalyze the hydrolysis of ß-1,4-glucosidic linkages in cellulose and ß-glucan structures that contain ß-1,3- and ß-1,4-mixed linkages. In this study, EGPf was heterologously expressed with Aspergillus niger and the recombinant enzyme was characterized. The successful expression of EGPf resulted as N-glycosylated protein in its secretion into the culture medium. The glycosylation of the recombinant EGPf positively impacted the kinetic characterization of EGPf, thereby enhancing its catalytic efficiency. Moreover, glycosylation significantly boosted the thermostability of EGPf, allowing it to retain over 80% of its activity even after exposure to 100 °C for 5 h, with the optimal temperature being above 120 °C. Glycosylation did not affect the pH stability or salt tolerance of EGPf, although the glycosylated compound exhibited a high tolerance to ionic liquids. EGPf displayed the highest specific activity in the presence of 20% (v/v) 1-butyl-3-methylimidazolium chloride ([Bmim]Cl), reaching approximately 2.4 times greater activity than that in the absence of [Bmim]Cl. The specific activity was comparable to that without the ionic liquid even in the presence of 40% (v/v) [Bmim]Cl. Glycosylated EGPf has potential as an enzyme for saccharifying cellulose under high-temperature conditions or with ionic liquid treatment due to its exceptional thermostability and ionic liquid tolerance. These results underscore the potential of N-glycosylation as an effective strategy to further enhance both the thermostability of highly thermostable archaeal enzymes and the hydrolysis of barley cellulose in the presence of [Bmim]Cl.

Unveiling the Role of Hydrolytic Enzymes from Soil Biocontrol Bacteria in Sustainable Phytopathogen Management.

BACKGROUND: Phytopathogens, encompassing fungi, bacteria, viruses, and nematodes, pose a significant threat to the agricultural industry by causing substantial economic losses through severe plant diseases. The excessive use of synthetic fungicides to combat phytopathogens has raised environmental and human health concerns. RESULTS: Consequently, there is an increasing demand for safe and environmentally friendly biopesticides to align with consumer preferences for uncontaminated food. One particularly promising alternative to synthetic fungicides involves harnessing biocontrol bacteria that produce extracellular hydrolytic enzymes. These enzymes serve to effectively manage phytopathogens while concurrently fostering sustainable plant protection. Among the pivotal hydrolytic enzymes generated by biocontrol bacteria are chitinase, cellulase, protease, lipase, glucanase, and amylase. These enzymes exert their influence by breaking down the cell wall, proteins, and DNA of phytopathogens, thereby establishing a dependable method of biocontrol. CONCLUSIONS: Recognizing the critical role of these hydrolytic enzymes in sustainable biocontrol, this review seeks to delve into their primary functions, contribution to sustainable plant protection, and mechanisms of action. Through an exploration of the potential presented by biocontrol bacteria and their enzymatic mechanisms, we can discern effective and environmentally conscious strategies for managing phytopathogens in agriculture.