Search Results (103)

Reprocessing lignocellulosic waste to obtain new products for industrial purposes is a vital part of circular economy. This paper reports the cellulase production by newly isolated Bacillus methylotrophicus cultured on lignocellulosic agro-industrial by-products, out of which brewer’s spent grain (BSG) was selected as most beneficial. Plackett–Burman design was used for screening medium components, while Box–Behnken design was further applied to model the impact of the three most influential variables. The maximum approximated cellulase activity was 0.469 U/mL (1 U = 1 µmol of reducing sugars/1 min), at 48.6 g/L substrate, 5.3 g/L ammonium sulfate, pH 6.1. The partially purified cellulase was characterized, which demonstrated broad range of optimal pH (6.5–9.4), temperature (50–60 °C), and sensitivity to metals. Changes in lignin and pentosans content was demonstrated as a result of BSG hydrolysis with a cell-free cellulase preparation. The produced enzyme was used for hydrolysis of various chemically pretreated (NaOH and H₂SO₄) cellulosic substrates, where for reused alkali-pretreated BSG (after microbial enzyme production) the saccharification efficiency was at a level of 25%. The cellulolytic potential of the bacterial strain, along with its resistance to ethanol, present a beneficial combination, potentially applicable to aid saccharification of lignocellulosic by-products for biofuel production. Full article

Ultraviolet (UV)-induced mutagenesis is a cost-effective and straightforward technique for introducing random genetic variations without the use of chemical reagents or genetic engineering. It is commonly employed to enhance enzyme activity in industrial trains. In this study, Trichoderma sp. was exposed to UV radiation at varying distances (4, 9, and 13 cm) and durations (2, 4, 6, and 8 min) to induce mutations. The activities of endoglucanase (EG), β-glucosidase (BGL), and cellobiohydrolase (CBH) were assessed following treatment. The 4 cm exposure distance yielded the highest enhancement, with EG, BGL, and CBH activities increasing 1.5-, 1.3-, and 0.9-fold, respectively. When the distance was fixed at 4 cm, the optimal exposure time was identified as 4 min, yielding further enhancements of 1.9-, 1.6-, and 1.4-fold, respectively. The resulting mutant, designated Mut-4, was scaled up in a 10-L bioreactor to assess its industrial applicability. Mut-4 retained its enhanced performance, achieving 1.9-, 2.0-, and 1.4-fold enhancements in EG, BGL, and CBH activities, respectively, compared with the original strain. These findings indicate that combining UV-induced mutagenesis with basic screening is an effective strategy for enhancing cellulolytic enzyme production, representing a promising approach for lignocellulosic biomass conversion. Full article

Annually, a considerable amount of agricultural waste is produced leading to serious environmental pollution if not managed effectively. A wide range of bio-decomposers, including fungi, bacteria, and actinomycetes may break down the complex agro-residues in an eco-friendly way through secreting many cellulolytic and amylolytic enzymes. The present study aimed at exploring the ability of Frankia to degrade cellulose and starch and identifying the cellulase and α-amylase genes in Frankia genomes for potential agricultural waste degradation. Frankia alni ACN14a and Frankia casuarinae CcI₃ produced clear zones around growing hyphae on carboxymethyl cellulose (CMC) and starch substrates. The hydrolytic index (HI) ranged from 1 to 2.14 reflecting variation in their degradation efficacy. Quantification of CMCase (carboxymethyl cellulase) production in strain ACN14a presented the maximum activity (0.504 U/mL) under 1% CMC after 16 days whereas strain CcI₃ produced a weak activity after 6 days from incubation. Besides, amylase activity in strain ACN14a reached the highest value (3.215 U/mL) after 4 days of growing with 1% starch, while strain CcI₃ had the superior production (3.04 U/mL) after 12 days from 1% starch condition. Data mining and genome blasting led to the identification of multiple genes related to cellulose and starch degradation. Two endoglucanases (celA1, FRAAL4955 and celA2, FRAAL4956), two glycosyl hydrolase family 16 (FRAAL6120 and FRAAL2663), and one glycosyl hydrolase family 16 (Francci3_3843) were predicted in the two genomes. Likewise, the α-amylase genes (FRAAL5900) from Frankia alni ACN14a and (Francci3_3679) from strain CcI₃ were identified. The gene expression of endo-1, 4-beta-glucanase (celA2, FRAAL4956) revealed the maximum increment in its mRNA abundance under 0.25% CMC exposure and showed a 3.3-fold increase. Frankia capability to degrade cellulose and starch represents a critical process in nutrient cycling and environment protection. Full article

The valorization of plant biomass is one of the main strategies for sustainable development. However, its use as energy, biofuels, fertilizers, value-added products, or even food is severely affected by the complexity of the plant cell wall. Therefore, the evaluation of fungi with high production of lignocellulolytic enzymes capable of efficiently degrading these substrates constitutes a viable, clean, and eco-friendly solution, allowing, for example, an increase in the digestibility and nutritional quality of alternative animal feed sources. For these reasons, the present study evaluated the ability of the mutant strain Trichodema viride M5-2 to improve the nutritional composition of the forage legumes Lablab purpureus and Mucuna pruriens through solid-state fermentation. Endo- and exoglucanase cellulolytic activity was assessed, as well as the effect of fermentation on the fiber’s physical properties and chemical composition. Molecular changes in the structure of plant fiber were analyzed using infrared spectroscopy. Increased production of the cellulolytic complex of the enzymes endoglucanase (3.29 IU/mL) and exoglucanase (0.64 IU/mL) was achieved in M. pruriens. The chemical composition showed an increase in true protein and a decrease in neutral fiber, hemicellulose, and cellulose, with a consequent improvement in nutritional quality. Fiber degradation was evident in the infrared spectrum with a significant decrease in the signals associated with cellulose and, to a lesser extent, with lignin. It can be concluded that the mutant strain T. viride M5-2 produced chemical, physical, and molecular changes in the fibrous and protein fractions of L. purpureus and M. pruriens through SSF, which improved their nutritional value as an alternative feed for animal nutrition. By promoting the use of this fungus, the nutritional quality of this source is increased through an effective and eco-friendly process, which contributes to mitigating the environmental impact of food production, in accordance with sustainability objectives and the need for more responsible agricultural practices. Full article

Fungal species secrete various enzymes and are considered the primary sources of industrially important cellulases. Cellulases are essential natural factors for cellulose degradation and have attracted significant interest for multiple applications. However, reducing the cost and enhancing cellulase production remains a significant challenge. Mutagenesis has opened a new window for enhancing enzyme secretion by modifying the organism’s genome. In this study, cellulases from Alternaria citri were produced and characterized, and the optimization for ideal fermentation conditions was performed for three types of cellulases (endoglucanase, exoglucanase, and β-glucosidase) by a wild-type (A. citri) and a mutant strain (A. citri 305). Ethyl methanesulfonate, a chemical mutagen, was used to enhance cellulase production by A. citri. The results demonstrate the improved cellulolytic ability of the mutant strain A. citri 305 utilizing lignocellulosic waste substances, particularly, orange-peel powder, wheat straw, sugarcane bagasse, and sawdust, making this study economically valuable. This evokes the potential for multi-dimensional applications in enzyme production, waste degradation, and biofuel generation. This study highlights that the activity of cellulases to hydrolyze various lignocellulosic substrates is enhanced after mutagenesis. Full article

This study investigates the enzyme-assisted extraction (EAE) of bioactive compounds from the pseudo-fruit of the wild rose (Rosa canina L.), also known as rosehip, using a commercial cellulolytic enzyme preparation, Cellic^® CTec3 HS. The effects of extraction time, solid to liquid ratio, and enzyme loading on total phenolic content (TPC) and total flavonoid content (TFC) were evaluated. The highest yields of TPC (168.3 ± 1.1 GAE/g DM) and TFC (72.3 ± 0.8 mg CAE/g DM) were obtained at 360 min, using 1% (v/v) enzyme loading and a 6% (w/v) solid to liquid ratio. Kinetic modeling of the extraction process was performed using first-order, second-order, Peleg’s, and power law models. The power law model best described the extraction dynamics. The obtained extracts were assessed for their biological activities including antioxidant, antimicrobial, anti-aging, and anti-diabetic properties. The extract obtained under optimal extraction conditions exhibited potent tyrosinase inhibition (80%) and moderate to low inhibition of α-glucosidase (15%) and α-amylase (20%) activities. The IC₅₀ for DPPH radical scavenging was 0.44 μL extract/mL while the extract exhibited significant antibacterial activity against Escherichia coli growth (79% inhibition). These findings suggest that the extract, obtained through EAE, has promising biological properties with potential applications in the food, pharmaceutical, and cosmetic industries. Full article

Lingonberry pomace (LP) is a by-product rich in valuable bioactive compounds and can be used in the food industry after various treatments and property characterization. This study aimed to evaluate the impact of commercially available enzymes (Viscozyme^® L, Pectinex^® Ultra Tropical, and Celluclast^® 1.5 L) and supercritical carbon dioxide (SFE-CO₂) extraction technology on the chemical composition and technological properties of treated LP products. The Megazyme kit was used to determine the soluble dietary fiber (SDS) and insoluble dietary fiber (IDF) contents, while the changes in mono-, disaccharide, and oligosaccharides were analyzed by applying high-pressure liquid chromatography with a refractive index detector. The analyzed properties were as follows: the water swelling capacity (WSC), water retention capacity (WRC), water solubility index (WSI), oil retention capacity (ORC), bulk density (BD), and emulsion stability of modified LP. The tested LP contained 8.49 g/100 g of SDF and 65.36 g/100 g of IDF (in dry matter). The partial separation of lipophilic substances during SFE-CO₂ extraction did not significantly affect the enzymatic hydrolysis efficiency. The amount of oligosaccharides in the LP increased using enzymes with pectinolytic activity (Viscozyme^® L and Pectinex^® Ultra Tropical), while cellulolytic enzymes (Celluclast^® 1.5 L) increased the amount of SDF and improved the IDF/SDF ratio. Enzymatic hydrolysis increased the SI, WRC, and ORC of LP powder. Emulsions with LP hydrolyzed with Pectinex^® Ultra Tropical demonstrated the highest stability during storage. This study demonstrates that the modification of LP powders provides diverse technological properties, which could expand the application of such products for further food production. Full article

The aim of this study was to evaluate the effect of an extremely low-frequency oscillating magnetic field on the enzymatic activities of common airborne Aspergillus sp. strains that were isolated from indoor environments. A D-optimal experimental design with three factors was applied: magnetic field density (0.5 to 2 mT), exposure time (0.5 to 2 h), and Aspergillus sp. strains (A. ellipticus, A. japonicus, A. flavus, and A. fumigatus). The response variables were exoenzymatic indexes (cellulolytic, amylolytic, proteolytic, lipolytic, and hemolytic) and pH, as a measure of organic acid production. A. ellipticus was the highest producer of organic acids, and A. japonicus was as pathogenic as A. fumigatus. Different magnetobiological effects were observed: on enzyme secretion in the remaining strains, we detected no appreciable effect (I_lip and I_prot of A. flavus), inhibition (I_lip of A. ellipticus; I_cel and I_amil of A. japonicus; I_amil and I_prot of A. fumigatus), and stimulation. Predictive quadratic models were obtained, and 2 mT for 2 h was the magnetic treatment regime that influenced the fungal enzymatic activity. These physiological changes following magnetobiological effects could be influenced during fungal sporulation and must thus be considered in aeromicrobiology studies. They can also be beneficial for obtaining industrial-use enzymes, but detrimental to the biodeterioration of different materials and human health. Full article

The Fusarium oxysporum species commonly found in soil include plant and human pathogens, and nonpathogenic species. F. oxysporum grown on lignocellulosic substrates under submerged conditions produces an extracellular enzyme profile with hemicellulolytic and cellulolytic activities. Our aim was to produce an extract of Fusarium oxysporum f. sp. melonis with β-1,4-xylanase activity after fermentation on a Brewers’ spent grain (BSG)-containing substrate. We prepared the BSG substrate, with or without sonication, for the submerged fermentation of Fusarium oxysporum previously isolated from local soil and preserved at 4 °C. First, an enriched inoculum was prepared, and later, the production of β-1,4-xylanase using the BSG substrates was monitored for up to 6 or 10 days in the enriched inoculum or in the enzyme extract, respectively. An activity of β-1,4-xylanase 12.0 U/mL (day 3) was obtained in the enriched inoculum with the untreated BSG, remaining constant for 3 days. A significant increase in the activity of this enzyme was observed (day 6), especially in the extract obtained using the sonicated BSG substrate (39 U/mL). Applying ultrasound to the BSG before its use in a submerged fermentation with Fusarium oxysporum f. sp. melonis could be an alternative for producing β-1,4-xylanase. Full article

The diversity and resource potential of macroscopic fungi in tropical regions remain understudied. Vietnam, being in a biodiversity hotspot, has a large number of new fungal species that are of interest for biotechnology and medicine. The presence of a large number of protected areas in Vietnam creates favorable opportunities for the study and ex situ conservation of tropical biodiversity. From 2012 to 2023, 785 strains of macrofungi from National Parks of Vietnam were preserved in the LE-BIN collection, 327 of which were barcoded with the sequences deposited in the NCBI GenBank. A taxonomic analysis demonstrated that many of the preserved isolates are potentially new or poorly studied species, representing a useful resource for taxonomical studies and a search for new medicinal mushrooms. More than 180 strains were studied for the first time for growth rate and enzymatic activities. Of these, 53 strains showed high growth rate, 43—high cellulolytic activity, 73—high oxidative enzymes activity, and 27 showed high proteolytic activity, making them promising candidates for biotechnological and medical applications and opening new opportunities for sustainable biomass management, discovery of new enzymes and bioactive substances, development of new drugs and efficient plant waste treatment technologies. The results confirm the importance of the ex situ conservation of fungal diversity in tropical regions as a valuable source for scientific and commercial applications and suggest certain new active strains for biotechnological study. Full article

The filamentous fungus Penicillium verruculosum (anamorph Talaromyces verruculosus) has been shown to be an efficient producer of secreted cellulases, used in biorefinery processes. Understanding the mechanisms of regulation of cellulase gene expression in the fungus P. verruculosum is a current task in industrial biotechnology, since it allows for targeted changes in the composition of the complex secreted by the fungus. Expression of cellulase genes in fungi is regulated mainly at the level of transcription via pathway-specific transcription factors (TF), the majority of which belong to the Zn(II)2Cys6 family of zinc binuclear cluster proteins. Transcriptional regulation of cellulase genes may have a species-specific pattern and involves several transcription factors. In this study, we used a qPCR method and transcriptome analysis to investigate the effect of knockouts and constitutive expression of genes encoding homologues of the regulatory factors XlnR and ClrB from P. verruculosum on the transcription of cbh1, egl2, and bgl1 genes, encoding three key cellulases, cellobiohydrolase, endoglucanase, and β-glucosidase, in the presence of various inducers. We have shown that the transcription factor XlnR of the filamentous fungus P. verruculosum is strictly responsible for the transcription of the main cellulolytic genes (cbh1, egl2, and bgl1) in the presence of xylose and xylobiose, but not in the presence of cellobiose. ClrB/Clr-2, a homologue from P. verruculosum, does not represent the main transcription factor regulating transcription of cellulolytic genes in the presence of selected inducers, unlike in the cases of Aspergillus nidulans, Aspergillus niger, and Penicillium oxalicum; apparently, it has a different function in fungi from the genus Talaromyces. We have also shown that constitutive expression of the transcription factor XlnR resulted in 3.5- and 2-fold increases in the activity of xylanase and β-glucosidase in a B1-XlnR enzyme preparation, respectively. In a practical sense, the obtained result can be used for the production of enzyme preparations based on the P. verruculosum B1-XlnR strain used for the bioconversion of renewable cellulose-containing raw materials into technical sugars. Full article

The enzyme-assisted approaches for plant phenolics extraction are more eco-friendly methods compared to acid or alkaline hydrolysis. Carbohydrase enzymes can release free phenolics from plant materials by cleaving the glycosidic bonds between phenolic compounds and cell wall polymers. In this study, the efficiency of carbohydrase-assisted treatment approaches was evaluated to extract bioactive phenolics from hawthorn (Crataegus orientalis) fruit residues. Enzymatic treatment of the fruits was operated by using a crude cellulolytic enzyme cocktail from Rhizomucor miehei NRRL 5282 and a pectinase preparate from Aspergillus niger. Both cellulase and combined cellulase–pectinase treatments improved the total phenolic content (TPC) and antioxidant activity of extracts. The TPC increased to 1899 ± 27 mg gallic acid equivalents/100 g dry matter during the combined enzyme treatment, showing a strong correlation with the average antioxidant capacity determined by ferric-reducing antioxidant power (1.7-fold increment) and 2,2-diphenyl-1-picrylhydrazyl (1.15-fold increment) reagents. The major phenolics in enzyme-treated extracts were vanillic and ferulic acids, the concentrations of which increased 115.6-fold and 93.9-fold, respectively, during carbohydrase treatment. The planktonic growth of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Chromobacterium violaceum was slightly inhibited by the extracts with minimum inhibitory concentration values between 15.0 and 77.9 mg/mL, while the yeasts tested were quite resistant to the samples. B. subtilis and yeast biofilms were sensitive to the enzyme-treated extracts, which also showed quorum-sensing inhibitory effects against C. violaceum. The obtained bioactive hawthorn extracts hold potential as a natural source of antioxidants and antimicrobials. Full article

Trichoderma species have been reported as masters in producing cellulolytic enzymes for the biodegradation of lignocellulolytic biomass and biocontrol agents against plant pathogens and pests. In our previous study, a novel Trichoderma strain LZ117, which shows potent capability in cellulase production, was isolated. Herein, we conducted multilocus phylogenetic analyses based on DNA barcodes and performed time-scaled phylogenomic analyses using the whole genome sequences of the strain, annotated by integrating transcriptome data. Our results suggest that this strain represents a new species closely related to T. atrobrunneum (Harzianum clade). Genes encoding carbohydrate-active enzymes (CAZymes), transporters, and secondary metabolites were annotated and predicted secretome in Trichoderma sp. LZ117 was also presented. Furthermore, genetic manipulation of this strain was successfully achieved using PEG-mediated protoplast transformation. A putative transporter gene encoding maltose permease (Mal1) was overexpressed, which proved that this transporter does not affect cellulase production. Moreover, overexpressing the native Cre1 homolog in LZ117 demonstrated a more pronounced impact of glucose-caused carbon catabolite repression (CCR), suggesting the importance of Cre1-mediated CCR in cellulase production of Trichoderma sp. LZ117. The results of this study will benefit further exploration of the strain LZ117 and related species for their applications in bioproduction. Full article

Species belonging to the genus Bacillus produce many advantageous extracellular enzymes that have tremendous applications on a commercial scale for the textile, detergent, feed, food, and beverage industries. This study aimed to isolate potent thermo-tolerant amylolytic and cellulolytic bacterium from the local environment. Using the Box–Behnken design of response surface methodology, we further optimized the amylase and cellulase activity. The isolate was identified by 16S rRNA gene sequencing as Bacillus subtilis QY4. This study utilized potato peel waste (PPW) as the biomaterial, which is excessively being dumped in an open environment. Nutritional status of the dried PPW was determined by proximate analysis. All experimental runs were carried out in 250 mL Erlenmeyer flasks containing acid treated PPW as a substrate by the thermos-tolerant Bacillus subtilis QY4 incubated at 37 °C for 72 h of submerged fermentation. Results revealed that the dilute H₂SO₄ assisted autoclaved treatment favored more amylase production (0.601 IU/mL/min) compared to the acid treatment whereas high cellulase production (1.269 IU/mL/min) was observed in the dilute acid treatment and was found to be very effective compared to the acid assisted autoclaved treatment. The p-value, F-value, and coefficient of determination proved the significance of the model. These results suggest that PPW could be sustainably used to produce enzymes, which offer tremendous applications in various industrial arrays, particularly in biofuel production. Full article

Enzyme-production microorganisms typically occupy a dominant position in composting, where cellulolytic microorganisms actively engage in the breakdown of lignocellulose. Exploring strains with high yields of cellulose-degrading enzymes holds substantial significance for the industrial production of related enzymes and the advancement of clean bioenergy. This study was inclined to screen cellulolytic bacteria, conduct genome analysis, mine cellulase-related genes, and optimize cellulase production. The potential carboxymethylcellulose-hydrolyzing bacterial strain Z2.6 was isolated from the maturation phase of pig manure-based compost with algae residuals as the feedstock and identified as Bacillus velezensis. In the draft genome of strain Z2.6, 31 related cellulolytic genes were annotated by the CAZy database, and further validation by cloning documented the existence of an endo-1,4-β-D-glucanase (EC 3.2.1.4) belonging to the GH5 family and a β-glucosidase (EC 3.2.1.21) belonging to the GH1 family, which are predominant types of cellulases. Through the exploration of ten factors in fermentation medium with Plackett–Burman and Box–Behnken design methodologies, maximum cellulase activity was predicted to reach 2.98 U/mL theoretically. The optimal conditions achieving this response were determined as 1.09% CMC-Na, 2.30% salinity, and 1.23% tryptone. Validation under these specified conditions yielded a cellulose activity of 3.02 U/mL, demonstrating a 3.43-fold degree of optimization. In conclusion, this comprehensive study underscored the significant capabilities of strain Z2.6 in lignocellulolytic saccharification and its potentialities for future in-depth exploration in biomass conversion. Full article