Bioprocess optimization for food-grade cellulolytic enzyme production from sorghum waste in a novel solid-state fermentation bioreactor for enhanced apple juice clarification.

Transforming global agricultural waste into eco-friendly products like industrial enzymes through bioconversion can help address sustainability challenges aligning with the United Nations' Sustainable Development Goals. Present study explored the production of high-yield food-grade cellulolytic enzymes from Trichoderma reesei MTCC 4876, using a novel media formulation with a combination of waste sorghum grass and cottonseed oil cake (3:1). Optimization of physical and environmental parameters, along with the screening and optimization of media components, led to an upscaled process in a novel 6-L solid-state fermentation (SSF)-packed bed reactor (PBR) with a substrate loading of 200 g. Saturated forced aeration proved crucial, resulting in high fungal biomass (31.15 ± 0.63 mg glucosamine/gm dry fermented substrate) and high yield cellulase (20.64 ± 0.36 FPU/g-ds) and xylanase (16,186 ± 912 IU/g-ds) production at an optimal airflow rate of 0.75 LPM. The PBR exhibited higher productivity than shake flasks for all the enzyme systems. Microfiltration and ultrafiltration of the crude cellulolytic extract achieved 94% and 71% recovery, respectively, with 13.54 FPU/mL activity in the cellulolytic enzyme concentrate. The concentrate displayed stability across wide pH and temperature ranges, with a half-life of 24.5-h at 50 °C. The cellulase concentrate, validated for food-grade safety, complies with permissible limits for potential pathogens, heavy metals, mycotoxins, and pesticide residue. It significantly improved apple juice clarity (94.37 T%) by reducing turbidity (21%) and viscosity (99%) while increasing total reducing sugar release by 63% compared with untreated juice. The study also highlighted the potential use of lignin-rich fermented end residue for fuel pellets within permissible SOx emission limits, offering sustainable biorefinery prospects. Utilizing agro wastes in a controlled bioreactor environment underscores the potential for efficient large-scale cellulase production, enabling integration into food-grade applications and presenting economic benefits to fruit juice industries.

Co-production of schizophyllan and cellulolytic enzymes from bagasse by Schizophyllum commune.

Schizophyllum commune is a mushroom-forming fungus well-known for its ability to degrade lignocellulosic materials and production of schizophyllan, a high added-value product for cosmeceutical, pharmaceutical, and biomaterial industries. Conventionally, schizophyllan is produced by submerged fermentation using glucose as a carbon source. In this work, we demonstrate that alkaline pretreated bagasse can be used by Schizophyllum commune as an alternative carbon source for the production of schizophyllan. The influence of different factors was investigated including cultivation time, biomass loading, and culturing media component and a co-product correlation model was proposed. In this lab-scale study, a yield of 4.4 g/L of schizophyllan containing 89% glucose was achieved. In addition to schizophyllan, the cellulolytic enzymes co-produced during this process were isolated and characterized and could find applications in a range of industrial processes. This demonstrates the potential of using agricultural waste as a cheaper alternative feedstock for this biorefinery process.

Nanocellulose-Based Polymer Composites Functionalized with New Gemini Ionic Liquids.

The manuscript discusses the application of dimeric imidazolium ionic liquids with an aliphatic linker of different lengths, constituting a new class of compounds called gemini, for the modification of renewable materials. This innovative functionalization with the use of ionic liquids made it possible to obtain polymer composite nanomaterials with renewable fillers, which will reduce the consumption of petroleum-based raw materials and also be directly related to the reduction of energy intensity. Renewable filler in the form of nanocellulose modified with ionic liquids, as well as polymer composites with such filler obtained by extrusion and injection molding techniques, were subjected to detailed characterization using techniques like: X-ray diffraction (XRD), Fourier transform spectroscopy (FTIR), dispersion studies (DLS), morphological analysis (SEM), differential scanning calorimetry (DSC), hot-stage polarized light microscopy and characterization of mechanical properties. The use of innovative dimeric ionic liquids proved to be an effective method to carry out efficient functionalization of cellulose. This provided a stable space structure between polysaccharide particles, limiting aggregate formation. It was shown that chemical modification with ionic liquids has a significant effect on the nucleation activity of cellulose fillers and the formation of the supermolecular structure of the polymer matrix, which consequently allowed to obtain polymer composites with excellent strength characteristics and increased flexibility, which will allow to increase their application potential. Innovative ionic liquids have contributed to obtaining green nanomaterials with excellent functional properties, which have not been described in the literature so far.

Production, concentration and partial characterization of an enzymatic extract produced by an Aspergillus niger mutant in solid state fermentation.

An enzymatic extract from Aspergillus niger 3T5B8 was produced by Solid State Fermentation (SSF) in aerated columns, using wheat bran as substrate. A combination of extracts produced using three different process conditions varying temperature, pH and aeration formed the final extract (Mixture). The Mixture was concentrated by an ultrafiltration process that partially purified and provided an efficient recovery of the enzymatic activities of xylanase (88.89%), polygalacturonase (89.3%), ß-glucosidase (93.15%), protease (98.68%) and carboxymethylcellulase (CMCase) (98.93%). SDS-PAGE analysis showed 15 visible protein bands in the crude and concentrated Mixture with molecular weights ranging from 15.1 to 104.6 kDa. Thin layer chromatography confirmed the effective action of ß-glucosidase and xylanase hydrolysis activities over cellobiose and xylan, respectively. A central composite design (CCD) with two variables and four replicates at the center points was used to determine the optimal temperature and pH for CMCase and ß-glucosidase. The optimal temperature was 78.9 °C and pH 3.8 for CMCase and 52.8 °C and pH 4.8 for ß-glucosidase, respectively.

Impacts of biofilms on the conversion of cellulose.

Lignocellulose is a widely available renewable carbon source and a promising feedstock for the production of various chemicals in biorefineries. However, its recalcitrant nature is a major hurdle that must be overcome to enable economic conversion processes. Deconstruction of lignocellulose is part of the global carbon cycle, and efficient microbial degradation systems have evolved that might serve as models to improve commercial conversion processes. Biofilms-matrix encased, spatially organized clusters of microbial cells and the predominating lifestyle in nature-have been recognized for their essential role in the degradation of cellulose in nature, e.g., in soils or in the digestive tracts of ruminant animals. Cellulolytic biofilms allow for a high concentration of enzymes at the boundary layer between the solid substrate and the liquid phase and the more complete capture of hydrolysis products directly at the hydrolysis site, which is energetically favorable. Furthermore, enhanced expression of genes for carbohydrate active enzymes as a response to the attachment on solid substrate has been demonstrated for cellulolytic aerobic fungi and anerobic bacteria. In natural multispecies biofilms, the vicinity of different microbial species allows the creation of efficient food webs and synergistic interactions thereby, e.g., avoiding the accumulation of inhibiting metabolites. In this review, these topics are discussed and attempts to realize the benefits of biofilms in targeted applications such as the consolidated bioprocessing of lignocellulose are highlighted. KEY POINTS: Multispecies biofilms enable efficient lignocellulose destruction in the biosphere. Cellulose degradation by anaerobic bacteria often occurs by monolayered biofilms. Fungal biofilms immobilize enzymes and substrates in an external digestion system. Surface attached cultures typically show higher expression of cellulolytic enzymes.

Moringa straw as cellulase production inducer and cellulolytic fungi source.

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 purified 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 (606U/l) and exoglucanase (205U/l) activities in the Avicel-containing medium, whereas F. verticillioides showed the highest level of ß-glucosidase activity (664U/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 ß-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.

Regulation of genes encoding cellulolytic enzymes by Pal-PacC signaling in Aspergillus nidulans.

Cellulosic biomass represents a valuable potential substitute for fossil-based fuels. As such, there is a strong need to develop efficient biotechnological processes for the enzymatic hydrolysis of cellulosic biomass via the optimization of cellulase production by fungi. Ambient pH is an important factor affecting the industrial production of cellulase. In the present study, we demonstrate that several Aspergillus nidulans genes encoding cellulolytic enzymes are regulated by Pal-PacC-mediated pH signaling, as evidenced by the decreased cellulase productivity of the palC mutant and pacC deletants of A. nidulans. The deletion of pacC was observed to result in delayed induction and decreased expression of the cellulase genes based on time course expression analysis. The genome-wide identification of PacC-regulated genes under cellobiose-induced conditions demonstrated that genes expressed in a PacC-dependent manner included 82 % of ClrB (a transcriptional activator of the cellulase genes)-regulated genes, including orthologs of various transporter and ß-glucosidase genes considered to be involved in cellobiose uptake or production of stronger inducer molecules. Together with the significant overlap between ClrB- and PacC-regulated genes, the results suggest that PacC-mediated regulation of the cellulase genes involves not only direct regulation by binding to their promoter regions but also indirect regulation via modulation of the expression of genes involved in ClrB-dependent transcriptional activation. Our findings are expected to contribute to the development of more efficient industrial cellulase production methods.

Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (ß-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development.

Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation.

With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed ß-glucosidase had a positie effect on cell growth in agreement with the fact that no putative ß-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction.

Investigating process parameters to enhance (hemi)cellulolytic enzymes activity produced by Trichoderma reesei RUT-C30 using deoiled oil palm mesocarp fiber in solid-state fermentation.

This study investigates the synthesis of (hemi)cellulolytic enzymes, including endoglucanase (CMCase), xylanase, and ß-glucosidase, employing Trichoderma reesei RUT-C30 and deoiled oil palm mesocarp fiber (OPMF) through solid-state fermentation (SSF). The objective was to determine the optimal process conditions for achieving high enzyme activities through a one-factor-at-a-time approach. The study primarily focused on the impact of the solid-to-liquid ratio, incubation period, initial pH, and temperature on enzyme activity. The effects of OPMF pretreatment, particularly deoiling and fortification, were explored. This approach significantly improved enzyme activity levels compared to the initial conditions, with CMCase increasing by 111.6 %, xylanase by 665.2 %, and ß-Glucosidase by 1678.1 %. Xylanase and ß-glucosidase activities, peaking at 1346.75 and 9.89 IU per gram dry substrate (GDS), respectively, under optimized conditions (1:4 ratio, pH 7.5, 20 °C, 9-day incubation). With lower moisture levels, CMCase reached its maximum activity of 227.84 IU/GDS. The study highlights how important it is for agro-industrial byproducts to support environmentally sustainable practices in the palm oil industry. It also emphasizes how differently each enzyme reacts to changes in process parameters.

Facile pretreatment strategies to biotransform Kans grass into nanocatalyst, cellulolytic enzymes, and fermentable sugars towards sustainable biorefinery applications.

The present investigation is targeted towards the facile fabrication of a carbon-based nanocatalyst (CNCs) using Kans grass biomass (KGB) and its sustainable application in microbial cellulase enhancement for the alleviation of enzymatic hydrolysis for sugar production. Different pretreatments, including physical, KGB extract-mediated treatment, followed by KOH pretreatment, have been applied to produce CNCs using KGB. The presence of CNCs influences the pretreatment of KGB substrate, fungal cellulase production, stability, and sugar recovery in the enzymatic hydrolysis of KGB. Using 1.0% CNCs pretreated KGB-based solid-state fermentation, 33 U/gds FPA and 126 U/gds BGL were obtained at 72 h, followed by 107 U/gds EG at 48 h in the presence of 0.5% CNCs. Further, 42 °C has been identified as the optimum temperature for cellulase production, while the enzyme showed thermal stability at 50 °C up to 20 h and produced 38.4 g/L sugar in 24 h through enzymatic hydrolysis of KGB.

Coconut waste valorization to produce biochar catalyst and its application in cellulose-degrading enzymes production via SSF.

Solid waste management and waste valorization are key concerns and challenges around the globe. Solid wastes generated by food industries are found in a diverse variety, are key sources of enormously valuable compounds, and can be effectively transformed into useful products for broad industrial applications. Biomass-based catalysts, industrial enzymes, and biofuels are some of the very prominent and sustainable products that are developed using these solid wastes. The aims of the current study are therefore centered on the multiple valorizations of coconut waste (CWs) to develop biochar as a catalyst and its application in fungal enzyme production in solid-state fermentation (SSF). Biochar as a catalyst using CWs has been prepared via a calcination process lasting 1 h at 500 °C and characterized through X-ray diffraction, Fourier-transformed infrared spectroscopy, and scanning electron microscope techniques. The produced biochar has been implemented for boosting enzyme production through SSF. In addition, studies have been performed on enzyme production with varying time and temperature, and it is found that the maximum 92 IU/gds BGL enzyme could be produced at a 2.5 mg concentration of biochar-catalyst at 40 °C in 72 h.

Prospects of soil microbiome application for lignocellulosic biomass degradation: An overview.

Sustainable and practically viable biofuels production technology using lignocellulosic biomass is still seeking its way of implementation owing to some major issues involved therein. Unavailability of efficient microbial sources for the degradation of cellulosic biomass is one of the major roadblocks in biomass to biofuels production technology. In this context, utilization of microbiomes to degrade lignocellulaosic biomass is emerging as a rapid and effective approach that can fulfill the requirements of biomass based biofuels production technology. Therefore, the present review is targeted to explore soil metagenomic approach to improve the lignocellulosic biomass degradation processing for the cost-effective and eco-friendly application. Soil microbiomes consist of rich microbial community along with high probability of cellulolytic microbes, and can be identified by culture independent metagenomics method which can be structurally and functionally explored via genomic library. Therefore, in depth analysis and discussion have also been made via structural & functional metagenomics tools along with their contribution to genomic library. Additionally, the present review highlights currently existing bottlenecks along with their feasible solutions. This review will help to understand the basic research as well as industrial concept for the process improvement based on soil microbiome mediated lignocellulosic biomass degradation, and this may likely to implement for the low-cost commercial biofuels production technology.

Evaluation of Blend Production of Cellulases and Xylanases Using Pretreated and Recycled Carnauba Straw.

Carnauba (Copernicia prunifera) is a Brazilian palm tree used for wax production, which usually generates a large amount of waste. This work evaluated the carnauba waste for cellulase and xylanase production using Trichoderma reesei CCT2768 through a solid-state fermentation (SSF). Carnauba waste was used in its crude form (C-IN), pretreated (C-P) with alkaline hydrogen peroxide (AHP), and also recycled after the SSF process (C-PR). C-IN, C-P, and C-PR were characterized by XRD, FTIR, and SEM. Cellulase and xylanase production was performed by SSF for 72 h, and the enzymatic extracts obtained were mixed each other in different concentrations. FPase, CMCase, and xylanase activities were determined. Trichoderma reesei CCT-2768 has shown high performance to produce cellulases and xylanases. Total cellulase, CMCase, and ß-glycosidase presented a highest activity when C-PPR1 (25% of C-PR and 75% of C-P) was used as a carbon source, with yield of 2.85 U/g, 41.21 U/g, and 2.80 U/g, respectively. The highest xylanase production was achieved when only the pretreated carnauba waste (C-P) was used, with an enzyme activity of 224.93 U/g. Carnauba has shown a promising carbon source capacity to induce the production of cellulolytic and xylanolytic enzymes by using T. reesei CCT2768, promoting the circular and ecofriendly economy, as well as a cost reduction, of the production process of these enzymes.

Enhancement of the enzymatic hydrolysis efficiency of wheat bran using the Bacillus strains and their consortium.

In the downstream process, the bioconversion of lignocellulosic biomass can be improved by applying a biological pretreatment procedure using microorganisms to produce hydrolytic enzymes to modify the recalcitrant structure of lignocellulose. In this study, various Bacillus strains (B. subtilis B.01162 and B.01212, B. coagulans B.01123 and B.01139, B. cereus B.00076 and B.01718, B. licheniformis B.01223 and B.01231) were evaluated for the degrading capacity of wheat bran in the submerged medium using enzymatic activities, reducing sugars and weight loss as indicators. The obtained results revealed that the B. subtilis B.01162, B. coagulans B.01123 and B. cereus B.00076 could be promising degraders for the wheat bran pretreatment. Besides, the application of their consortium (the combination of 2-3 Bacillus species) showed the positive effects on cellulose bioconversion compared with monocultures. Among them, the mixture of B. subtilis B.01162 and B. coagulans B.01123 increased significantly the cellulase, endo-glucanase, and xylanase enzyme activity resulting in accelerating the lignocellulose degradation. Our results served a very good base for the development of microbial consortium for biological pretreatment of lignocellulosic raw materials.

From pomiculture waste to biotechnological raw material: efficient transformation using ligninosomes and cellulosomes from Pleurotus spp.

The goal of this study was to determine the capacity of Pleurotus spp. lignocellulosome to transform frequent pomiculture residues (grapevine-, plum-, and raspberry sawdust) into raw materials for biotechnological processes. All three lignocellulosics induced the synthesis of ligninolytic and cellulolytic enzymes in the tested species. Laccase was dominant in the ligninolytic cocktail, with a maximum activity of 40,494.88 U L-1 observed after the cultivation of P. pulmonarius on grapevine sawdust. Grapevine sawdust also proved to be the optimal substrate for the synthesis of versatile peroxidases especially in P. eryngii (1010.10 U L-1), while raspberry sawdust favored the production of Mn-dependent peroxidase in P. pulmonarius (479.17 U L-1). P. pulmonarius was the dominant cellulolytic agent and raspberry sawdust was optimal for the synthesis of xylanases, and endo- and exo-cellulases (15,746.35 U L-1, 9741.56 U L-1, and 836.62 U L-1), while grapevine sawdust mostly induced ß-glucosidase activity (166.11 U L-1). The degree of residues delignification was more substrate- than species-dependent, ranging between 6.44 and 23.72% after the fermentation of grapevine and raspberry sawdust with P. pulmonarius. On the other hand, the lowest level of cellulose consumption was also observed on raspberry sawdust after the cultivation of P. eryngii, which together with high delignification also induced the highest selectivity index (1.27). The obtained results show the exceptional lignocellulolytic potential of Pleurotus spp. enzyme cocktails which opens up many possibilities for their application in numerous biotechnological processes.

From simple and specific zymographic detections to the annotation of a fungus Daldinia caldariorum D263 that encodes a wide range of highly bioactive cellulolytic enzymes.

BACKGROUND: Lignocellulolytic enzymes are essential for agricultural waste disposal and production of renewable bioenergy. Many commercialized cellulase mixtures have been developed, mostly from saprophytic or endophytic fungal species. The cost of complete cellulose digestion is considerable because a wide range of cellulolytic enzymes is needed. However, most fungi can only produce limited range of highly bioactive cellulolytic enzymes. We aimed to investigate a simple yet specific method for discovering unique enzymes so that fungal species producing a diverse group of cellulolytic enzymes can be identified. RESULTS: The culture medium of an endophytic fungus, Daldinia caldariorum D263, contained a complete set of cellulolytic enzymes capable of effectively digesting cellulose residues into glucose. By taking advantage of the unique product inhibition property of ß-glucosidases, we have established an improved zymography method that can easily distinguish ß-glucosidase and exoglucanase activity. Our zymography method revealed that D263 can secrete a wide range of highly bioactive cellulases. Analyzing the assembled genome of D263, we found over 100 potential genes for cellulolytic enzymes that are distinct from those of the commercially used fungal species Trichoderma reesei and Aspergillus niger. We further identified several of these cellulolytic enzymes by mass spectrometry. CONCLUSIONS: The genome of Daldinia caldariorum D263 has been sequenced and annotated taking advantage of a simple yet specific zymography method followed by mass spectrometry analysis, and it appears to encode and secrete a wide range of bioactive cellulolytic enzymes. The genome and cellulolytic enzyme secretion of this unique endophytic fungus should be of value for identifying active cellulolytic enzymes that can facilitate conversion of agricultural wastes to fermentable sugars for the industrial production of biofuels.

Proteomic Profiling of the Secretome of Trichoderma reesei.

Trichoderma reesei (T. reesei) is the workhorse for the production of industrial cellulolytic enzyme cocktails for cellulose hydrolysis. However, the current industrial process using enzyme cocktails is not efficient enough for the cost-effective generation of cellulosic sugar. Here, we describe a protocol for the application of a state-of-the-art LC-MS/MS-based proteomics method for studying the T. reesei secretome. A protein-free minimal chemically defined cell culture medium must be used for a successful secretome analysis. A lignocellulose substrate can be added to this minimal medium to stimulate the fungal secretion of enzymes specific to that substrate. The secretory proteins in the conditioned medium can be purified for quantitative proteomics profiling. T. reesei secretes several hundred enzymes including cellulases, hemicellulases, pectinases, proteases, oxidoreductases, and many putative proteins when it is stimulated with lignocellulose. By combining an understanding of the basic biomass hydrolytic mechanisms with the discovery of novel enzymes, more effective enzyme cocktails can be designed for a sustainable biochemical-based biorefinery.

Biocompatible natural deep eutectic solvent-based extraction and cellulolytic enzyme-mediated transformation of Pueraria mirifica isoflavones: a sustainable approach for increasing health-bioactive constituents.

The presence of specific gut microflora limits the biotransformation of Pueraria mirifica isoflavone (PMI) glycosides into absorbable aglycones, thus limiting their health benefits. Cellulolytic enzyme-assisted extraction (CAE) potentially solves this issue; however, solvent extraction requires recovery of the hydrophobic products. Here, we established the simultaneous transformation and extraction of PMIs using cellulolytic enzymes and natural deep eutectic solvents (NADESs). The NADES compositions were optimized to allow the use of NADESs as CAE media, and the extraction parameters were optimized using response surface methodology (RSM). The optimal conditions were 14.7% (v/v) choline chloride:propylene glycol (1:2 mol ratio, ChCl:PG) at 56.1 °C for the cellulolytic enzyme (262 mU/mL) reaction in which daidzin and genistin were extracted and wholly transformed to their aglycones daidzein and genistein. The extraction of PMIs using ChCl:PG is more efficient than that using conventional solvents; additionally, biocompatible ChCl:PG enhances cellulolytic enzyme activity, catalyzing the transformation of PMIs into compounds with higher estrogenicity and absorbability.

Ecological Genomics and Evolution of Trichoderma reesei.

The filamentous fungus Trichoderma reesei (Hypocreales, Ascomycota) is an efficient industrial cell factory for the production of cellulolytic enzymes used for biofuel and other applications. Therefore, researches addressing T. reesei are relatively advanced compared to other Trichoderma spp. because of the significant bulk of available knowledge, multiple genomic data, and gene manipulation techniques. However, the established role of T. reesei in industry has resulted in a frequently biased understanding of the biology of this fungus. Thus, the recent studies unexpectedly show that the superior cellulolytic activity of T. reesei and other Trichoderma species evolved due to multiple lateral gene transfer events, while the innate ability to parasitize other fungi (mycoparasitism) was maintained in the genus, including T. reesei. In this chapter, we will follow the concept of ecological genomics and describe the ecology, distribution, and evolution of T. reesei, as well as critically discuss several common misconceptions that originate from the success of this species in applied sciences and industry.