Enzyme Cocktail Research Articles

Deciphering the efficient cellulose degradation by the thermophilic fungus Myceliophthora thermophila focused on the synergistic action of glycoside hydrolases and lytic polysaccharide monooxygenases

The thermophilic fungus Myceliophthora thermophila as an efficient decomposer secretes various glycoside hydrolases and auxiliary oxidation enzymes to deconstruct cellulose. However, the core enzymes critical for efficient cellulose degradation and their interactions with other cellulolytic enzymes remain unclear. Herein, the transcriptomic analysis of M. thermophila grown on Avicel exhibited that cellulases from GH5_5, GH6 and GH7, and lytic polysaccharide monooxygenases (LPMOs) from AA9 contributed to cellulose degradation. Moreover, the peptide mass fingerprinting analysis of major extracellular proteins and corresponding gene-knockout strains studies revealed that MtCel7A and MtCel5A were the core cellulolytic enzymes. Furthermore, synergistic experiments found that hydrolytic efficiencies of MtCel7A and MtCel5A were both improved by mixture C1/C4 oxidizing MtLPMO9H, but inhibited by C1 oxidizing MtLPMO9E and C4 oxidizing MtLPMO9J respectively. These results demonstrated the potential application of C1/C4 oxidizing LPMOs for future designing novel cellulolytic enzyme cocktails on the efficient conversion of cellulose into biofuels and biochemicals.

Enhancement of an efficient enzyme cocktail from Penicillium consortium on biodegradation of pretreated poplar

Lignocellulosic biomass is the most abundant feedstock in production of bio-based fuels and materials. In order to achieve high conversion efficiency, enzyme cocktail has been assessed by numerous studies as a powerful tool to allocate diverse specific activities in one matrix. This study reported an efficient cellulase cocktail produced by co-cultivation of several Penicillium strains with lytic polysaccharide monooxygenases (LPMOs) producing strains. The optimal inoculum proportion was screened with hydrolysis yield as the indicator and through ANOVA analysis. When blending the consortium-derived enzyme cocktail with commercial cellulase preparation, the highest hydrolysis yield reached 88.7%, equivalent to 32% increase compared to using commercial cellulase alone. The behind structure-effect relationship between the substrate and enzymatic degradation was illustrated. Comparative analysis of the enzyme components, activities and the strains transcriptome provided a comprehensive evaluation and insights of the proposed Penicillium consortium, indicating it as a potent cellulase cocktail producer for enhancing lignocellulose biodegradation.

A sustainable process for co-production of xylooligosaccharides and ethanol from alkali treated sugarcane bagasse: A strategy towards waste management

Present study aims at sustainable utilization of sugarcane bagasse (SCB) for production of valuable prebiotic xylooligosaccharides (XOS) along with second generation ethanol. Fractionation of SCB into hemicellulose rich liquid fraction and cellulose rich solid residue was achieved using alkaline treatment. Carbohydrate rich precipitate obtained from liquid fraction was utilized for XOS production using inhouse produced endoxylanase. XOS production from SCB xylan was optimized by employing response surface methodology. Under optimized conditions, maximum XOS yield was 227.72 mg/g of carbohydrate rich precipitates. The solid residue obtained after alkaline pretreatment was used for ethanol fermentation by prehydrolysis and simultaneous saccharification and fermentation (P-SSF) process using cellulolytic enzyme cocktail and Saccharomyces cerevisiae SM1. Maximum ethanol concentration, productivity and yield were 79.76 ± 0.16 g/L, 0.83 g/L/h and 69.38%, respectively by employing P-SSF process. Based on the experimental data it can be predicted that bioconversion of 100 g raw SCB can yield 6.26 g of XOS (DP 2-DP 5), 15.95 g ethanol and 1.44 g of xylitol. Present investigation reports an integrated process for effective bioconversion of SCB into value added products by maximum utilization of cellulosic and hemicellulosic fractions simultaneously using indigenously produced fungal enzymes.

Improving saccharification of ramie stalks by synergistic effect of in-house cellulolytic enzymes consortium

The high cost of cellulase is one of the main obstacles hindering the large-scale biorefining of lignocellulosic biomass. Therefore, developing efficient method for preparation of cellulase is promising. In the present study, the production of cellulase by Trichoderma reesei, Trichoderma harzianum, and Aspergillus niger was optimized, and the synergistic effect of these cellulase on enzymatic hydrolysis of pretreated ramie stalks was also evaluated. The maximum CMCase (Carboxymethyl Cellulase) and filter paper activity (FPA) produced by T. reesei reached to 3.12 IU/mL and 0.13 IU/mL, respectively. The maximum activities of CMCase (3.68 IU/mL), FPA (0.04 IU/mL) and β-glucosidase (8.44 IU/mL) were obtained from A. niger. The results also showed that under the premise of the same FPA activity, the contribution of β-glucosidase activity to yield of reducing sugar was greater than that of CMCase. Besides, cellulase produced by T. reesei and A. niger had the best synergistic effect on enzymatic hydrolysis of pretreated ramie stalks. The highest reducing sugars yield (417 mg/g dry substrate) was achieved when enzyme cocktail was prepared at the ratio of 1:1, which was 1.36–3.35 folds higher than that of different single enzymes. The present research has provided a novel method for efficient preparation of enzymes consortium for enzymatic hydrolysis of ramie stalks.Graphical

Effect of Azolla cristata with or without enzyme supplementation on blood biochemistry and intestinal histomorphology of broiler chicken

A study was undertaken to determine the effect of partially replacing soybean protein with Azolla (AZ) with or without enzyme on blood biochemistry and intestinal histomorphology of broiler chicken. The experiment was performed on 210 Cobb broiler chicks randomly assigned to 7 treatment groups each replicated 3 times with 10 birds in each replicate. Basal diet was offered to the control (T1) group, whereas soybean protein was replaced with 5 (T2), 10 (T3) and 15% (T4) AZ and the other three groups, i.e. T5, T6 and T7 contained the respective inclusion levels of AZ, but were supplemented with cocktail enzyme. The results revealed that there was no statistical difference on blood glucose, cholesterol, protein, SGPT and SGOT levels among various treatment groups including control. Also there was a no significant effect in villus height (V), crypt depth (C) and V/C ratio among various treatment groups when compared to control. Highest villus height was observed in the control group (T1) and there was reduction in villus height in all the groups wherein AZ was included in the diet either alone or with enzyme supplementation. It is thus concluded that dietary inclusion of AZ had no adverse effect on the blood biochemistry and intestinal histomorphology when provided alone or in combination with enzyme supplementation, indicating that AZ could be included up to 15% level in the diet of broiler chicken without any adverse effect.

Shapeshifting in the Venus flytrap (Dionaea muscipula): Morphological and biomechanical adaptations and the potential costs of a failed hunting cycle

The evolutionary roots of carnivory in the Venus flytrap (Dionaea muscipula) stem from a defense response to plant injury caused by, e.g., herbivores. Dionaea muscipula aka. Darwin’s most wonderful plant underwent extensive modification of leaves into snap-traps specialized for prey capture. Even the tiny seedlings of the Venus flytrap already produce fully functional, millimeter-sized traps. The trap size increases as the plant matures, enabling capture of larger prey. The movement of snap-traps is very fast (~100–300 ms) and is actuated by a combination of changes in the hydrostatic pressure of the leaf tissue with the release of prestress (embedded energy), triggering a snap-through of the trap lobes. This instability phenomenon is facilitated by the double curvature of the trap lobes. In contrast, trap reopening is a slower process dependent on trap size and morphology, heavily reliant on turgor and/or cell growth. Once a prey item is caught, the trap reconfigures its shape, seals itself off and forms a digestive cavity allowing the plant to release an enzymatic cocktail to draw nutrition from its captive. Interestingly, a failed attempt to capture prey can come at a heavy cost: the trap can break during reopening, thus losing its functionality. In this mini-review, we provide a detailed account of morphological adaptations and biomechanical processes involved in the trap movement during D. muscipula hunting cycle, and discuss possible reasons for and consequences of trap breakage. We also provide a brief introduction to the biological aspects underlying plant motion and their evolutionary background.

Reutilization of waste fungal biomass for concomitant production of proteochitinolytic enzymes and their catalytic products by Alcaligenes faecalis SK10

Fungal biomass, being organic waste, could be an excellent source of protein, carbohydrate and minerals. However, it has not been exploited fully until now. Efficient management of this waste can not only address the environmental impact on its disposal but also yield value-added metabolites. In the present study, in order to explore its potential, we subjected dead fungal biomass of Aspergillus niger SKN1 as substrate for both fermentative and enzymatic biodegradation, respectively by potent proteo-chitinolytic bacteria Alcaligenes faecalis SK10 and its enzyme cocktail. The results revealed that reasonable amount of protease and chitinase could be biosynthesized by the fermentative mode of utilization, while a mixture of amino acid, peptides and low-molecular weight amino-sugar (mono and oligomeric form of N-acetylglucosamine) could be generated through enzymatic hydrolysis. The physicochemical condition of both the bioprocess was subsequently optimized through statistical approach. The projected utilization of waste zero-valued fungal biomass offer a sustainable and environmentally sound method for production of microbial metabolites and large scale execution of the same could be proficient and in tune with the principle of circular economy.

Customized optimization of lignocellulolytic enzyme cocktails for efficient conversion of pectin-rich biomass residues

Pectin is a major component in many agricultural feedstocks. Despite the wide use in industrial production of cellulases and hemicellulases, the fungus Trichoderma reesei lacks a complete enzyme set for pectin degradation. In this study, three representative pectinolytic enzymes were expressed and screened for their abilities to improve the efficiency of T. reesei enzymes on the conversion of different agricultural residues. By replacing 5 % of the T. reesei proteins, endopolygalacturonase and pectin lyase remarkably increased the release of sugars from inferior tobacco leaves. In contrast, pectin methylesterase showed the strongest improving effect (by 31.1 %) on the hydrolysis of beetroot residue. The pectin in beetroot residue was only mildly degraded with the supplementation of pectin methylesterase, which allowed the extraction of pectin keeping the original emulsifying activity with a 51.1 % higher yield. The results provide a basis for precise optimization of lignocellulolytic enzyme systems for targeted valorization of pectin-rich agricultural residues.

Ultrasound-aided enzyme-assisted efficient extraction of bioactive compounds from Gymnema sylvestre and optimization as per response surface methodology

The herb Gymnema sylvestre is known for its bioactivities. Though several studies have reported extraction of medicinal compounds from it, extraction efficiency is still an issue. The goal of the current project was, therefore, to explore an efficient and green method for the extraction of medicinal biomolecules from the plant with extraction optimization. Ultrasound-aided enzyme-assisted extraction (UAE-EAE) was carried out as per RSM (response surface methodology) with four factors, time, temperature, pH, and amount of enzyme, and the responses were % yield, TPC, TFC, and antioxidant activity. For the highest desirability, time, temperature, pH, and amount of enzyme cocktail were 150 min, 64.80 °C, 5.64, and 7.49 mL, respectively. On optimized conditions, % yield, TPC, TFC and antioxidant activity were 32.35%, 108.79 mg/g gallic acid equivalents, 54.20 mg/g rutin equivalents and 68.15%, respectively. The EAE-UAE proved to be highly effective as it was almost 3 times more efficient than the conventional maceration carried out as the control. Morphological studies conducted by scanning electron microscopy (SEM) confirmed the formation of large cavities in plant cell walls (30.6–56.3 μm) as compared to the control (6.2–11.4 μm). The Energy Dispersive X-ray (EDX) analysis of the sample showed the presence of various elements (Zn, Al, K, Ca, Se, Mg, Na, Mo, S, P, and Cl) in it. Validation studies had very low error rates (1.47–4.08%) in the responses indicating high fitting of the predicted models. The discovered method was not only efficient but also green and relatively cost-effective and has the potential to be explored for application on an industrial scale.

Differential Activity of the Extracellular Phenoloxidases in Different Strains of the Phytopathogenic Fungus, Microdochium nivale.

To cause plant diseases, phytopathogenic fungi use numerous extracellular enzymes, among which, the phenoloxidases (POs) seem underestimated for the pathogens of non-woody plants. Our study aimed to (1) compare extracellular PO activities (lignin peroxidase, Mn peroxidase, laccase, and tyrosinase) in differentially virulent strains (inhabiting winter rye in a single field) of the phytopathogenic species, Microdochium nivale; (2) check whether these activities are responsive to host plant metabolites; and (3) search for correlations between the activities, lignin-decomposing capacity, and virulence. All strains displayed all four enzymatic activities, but their levels and dynamics depended on the particular strain. The activities displayed the hallmarks of co-regulation and responsiveness to the host plant extract. No relationships between the virulence of strains and levels of their extracellular PO activities or lignin-degrading capacity were revealed. We consider that different strains may rely on different POs for plant colonization, and that different POs contribute to the “uniqueness” of the enzymatic cocktails that are delivered into host plant tissues by different virulent strains of M. nivale. Our study supports the hypothesis of the differential behavior of closely related M. nivale strains, and discusses an important role of POs in the interactions of phytopathogens with herbaceous plants.

Efficient saccharification of Lycium barbarum leaf biomass by using enzyme cocktails produced by a novel fungus Aspergillus costaricensis LS18

The utilization of agro-industrial residues is an interesting issue contributing to sustainable development and environmental protection. Lycium barbarum leaves (LBL) are agro-industrial residues of the L. barabrum berry cultivation and seriously underutilized, leading to resource waste and environmental pollution. In this study, we prepared cost-effective enzyme cocktails with high xylanase activity from a novel soil-derived fungal strain Aspergillus costaricensis LS18. The xylanase activity of these on-site produced enzyme cocktails was 3.49 ± 0.55 U/mL. Through the hydrolysis using the enzyme cocktails with 6% substrate loading at 45 °C for 12 h, 86.57 ± 1.81% of total reducing sugars (RS) from LBL was released. The concentration of RS in the hydrolysates reached 8.17 ± 0.33 mg/mL. In this study, LBL were added values by two mutually independent bioprocess ways. On the one hand, LBL were used as the only nutrients in the medium for the on-site production of enzyme cocktails by fermentation. On the other hand, through hydrolysis using this enzyme cocktail, LBL biomass was efficiently hydrolyzed and fermentable monosugars were gained. This study could benefit to the exploitation of LBL resources and provide the references for utilization of other agro-industrial residues.

Bacterial species metabolic interaction network for deciphering the lignocellulolytic system in fungal cultivating termite gut microbiota

Fungus-cultivating termite Odontotermes badius developed a mutualistic association with Termitomyces fungi for the plant material decomposition and providing a food source for the host survival. The mutualistic relationship sifted the microbiome composition of the termite gut and Termitomyces fungal comb. Symbiotic bacterial communities in the O. badius gut and fungal comb have been studied extensively to identify abundant bacteria and their lignocellulose degradation capabilities. Despite several metagenomic studies, the species-wide metabolic interaction patterns of bacterial communities in termite gut and fungal comb remains unclear. The bacterial species metabolic interaction network (BSMIN) has been constructed with 230 bacteria identified from the O. badius gut and fungal comb microbiota. The network portrayed the metabolic map of the entire microbiota and highlighted several inter-species biochemical interactions like cross-feeding, metabolic interdependency, and competition. Further, the reconstruction and analysis of the bacterial influence network (BIN) quantified the positive and negative pairwise influences in the termite gut and fungal comb microbial communities. Several key macromolecule degraders and fermentative microbial entities have been identified by analyzing the BIN. The mechanistic interplay between these influential microbial groups and the crucial glycoside hydrolases (GH) enzymes produced by the macromolecule degraders execute the community-wide functionality of lignocellulose degradation and subsequent fermentation. The metabolic interaction pattern between the nine influential microbial species has been determined by considering them growing in a synthetic microbial community. Competition (30%), parasitism (47%), and mutualism (17%) were predicted to be the major mode of metabolic interaction in this synthetic microbial community. Further, the antagonistic metabolic effect was found to be very high in the metabolic-deprived condition, which may disrupt the community functionality. Thus, metabolic interactions of the crucial bacterial species and their GH enzyme cocktail identified from the O. badius gut and fungal comb microbiota may provide essential knowledge for developing a synthetic microcosm with efficient lignocellulolytic machinery.

Optimization and Implementation of Fed-Batch Strategy to Produce Ligninolytic Enzyme from the White-Rot Basidiomycete Pycnoporus sanguineus in Bubble Column Reactor

The current work evaluates the production of ligninolytic enzyme optimization via response surface methodology using different inducers: acid cellulignin (CA); MnSO4 (Mn2+); CuSO4·5H2O (Cu2+); veratryl (3, 4-dimethoxybenzyl); alcohol (VA); Tween 80% (T80); and the carbon-to-nitrogen ratio (C/N). A further goal was implementing a fed-batch strategy to produce ligninolytic enzyme extracts from P. sanguineus 2512 using a bubble column reactor (BCR). The best optimized experimental condition in the shake flasks was a 7.5 C/N ratio, 0.025 g/L Cu2+, 1.5 mM Mn2+, 3.0 mM VA and 0.025 mM T80, resulting in 64,580, 9.10 and 80.72 U/L for Laccase (Lac), Manganese (MnP) and Lignin peroxidase (LiP) activities, respectively. In the BCR, three feedings were performed at 24 h intervals on the 6th, 7th and 8th days with a significant increase in Lac (99,600 U/L) and MnP (47.53 U/L) activities on the 8th day and a reduction on the 9th day of cultivation. The LiP activity peak was achieved on the 5th day (416 U/L) of cultivation, decreasing thereafter. Enzyme cocktails concentrated in hollow fiber in the third cultivation batch showed contents of 4 × 105 U/L, 220 U/L and 2.5 g/L for Lac, MnP and total proteins, respectively. The enzymatic cocktail with the highest LiP activity (1200 U/L) was obtained in the first batch. The results showed that the optimization of the biosynthesis of the ligninolytic enzymes provided satisfactory improvement in terms of Lac and MnP production per run.

Statistical optimization of pretreatment of municipal solid waste by response surface methodology for fermentable sugar production

AbstractThis research aims to use low‐cost and easily available chemicals for chemical pretreatment to achieve a high yield of fermentable sugars using biodegradable municipal solid waste as a substrate. Biodegradable municipal solid waste was pretreated with locally available toilet cleaner (acidic in nature) and washing detergent (basic in nature) and to achieve maximum yield of reducing sugars, two variables; chemical concentration and time were selected for optimization using a central composite design. In Central composite design 13 runs for toilet cleaner (acidic in nature) and washing detergent (basic in nature) pretreatment models were created while ranges for two variables were selected as 1, 2, 3 days for time and 1%, 3%, 5% for chemical concentration. In the toilet cleaner (acidic in nature) pretreatment, optimum conditions were 5% (vol/vol) of toilet cleaner (acidic in nature) for 3 days. At optimal conditions, the model for toilet cleaner (acidic in nature) pretreatment released 167.755 g/L of reducing sugars and 2.684 MJ/L energy while the model for washing detergent (basic in nature) pretreatment released 159.141 g/L of reducing sugars and 2.546 MJ/L energy. An enzyme cocktail hydrolyzed the toilet cleaner (acidic in nature) pretreated sample, and the highest value of fermentable sugar was achieved at 36 h of 237.83 ± 11.028 g/L. Thus, the chemical pretreatment using toilet cleaner and washing detergent has a great potential for fermentable sugar production and effectively be used in industrial and household digesters for the conversion of waste into useful products.

Ursodeoxycholic acid production by Gibberella zeae mutants

Ursodeoxycholic acid (UDCA) is a highly demanded pharmaceutical steroid widely used in medicine. An ascomycete Gibberella zeae VKM F-2600 is capable of producing UDCA by 7β-hydroxylation of lithocholic acid (LCA). The present study is aimed at the improvement of the fungus productivity. The original procedures for the protoplast obtaining followed by UV mutagenesis and screening of ketoconazole-resistant mutant clones have been applied. The highest yield of G. zeae protoplasts was obtained when using the mycelium in the active growth phase, ammonium chloride as an osmotic stabilizer and treatment of the fungal cells by the lytic enzymes cocktail from Trichoderma hurzanium. The conditions for effective protoplast regeneration and the UV-mutagenesis were found to provide 6–12% survival rate of the protoplasts with superior number of possible mutations. Three of 27 ketoconazole-resistant mutant clones obtained have been selected due to their increased biocatalytic activity towards LCA. The mutant G. zeae M23 produced 26% more UDCA even at relatively high LCA concentration (4 g/L) as compared with parent fungal strain, and the conversion reached 88% (w/w). The yield of UDCA reached in this study prefers those ever reported. The results contribute to the knowledge on ascomycete mutagenesis, and are of importance for biotechnological production of value added cholic acids.Graphical

Alkalization of Kraft Pulps from Pine and Eucalyptus and Its Effect on Enzymatic Saccharification and Viscosity Control of Cellulose.

Bleached kraft pulps from eucalyptus and pine were subjected to cold caustic extraction (CCE) with NaOH (5, 10, 17.5, and 35%) for hemicelluloses removal and to increase cellulose accessibility. The effect of these changes was evaluated in enzymatic saccharification with the multicomponent Cellic CTec3 enzyme cocktail, and in viscosity reduction of pulps with the monocomponent Trichoderma reesei endoglucanase (EG). After CCE with 10% NaOH (CCE10) and 17.5% NaOH (CCE17.5), hemicellulose content lower than 1% was achieved in eucalyptus and pine pulps, respectively. At these concentrations, cellulose I started to be converted into cellulose II. NaOH concentrations higher than 17.5% decreased the intrinsic viscosity (from 730 to 420 mL/g in eucalyptus and from 510 to 410 mL/g in pine). Cellulose crystallinity was reduced from 60% to 44% in eucalyptus and from 71% to 44% in pine, as the NaOH concentration increased. Enzymatic multicomponent saccharification showed higher glucose yields in all CCE-treated eucalyptus samples (up to 93%) while only CCE17.5 and CCE35 pine pulps achieved 90% after 40 h of incubation. Untreated bleached pulps of both species presented saccharification yields lower than 70%. When monocomponent EG was used to treat the same pulps, depending on enzyme charge and incubation time, a wide range of intrinsic viscosity reduction was obtained (up to 74%). Results showed that eucalyptus pulps are more accessible and easier to hydrolyze by enzymes than pine pulps and that the conversion of cellulose I to cellulose II hydrate only has the effect of increasing saccharification of CCE pine samples. Viscosity reduction of CCE pulps and EG treated pulps were obtained in a wide range indicating that pulps presented characteristics suitable for cellulose derivatives production.

Aspergillus welwitschiae inulinase enzyme cocktails obtained on agro-material inducers for the purpose of fructooligosaccharides production

Production of fructooligosaccharides (FOS) is a trending topic due to their prebiotic effect becoming increasingly important for the modern human diet. The most suitable process for FOS production is the one using fungal inulinases. Introduction of new fungal inulinase producers and their implementation in production of inulinase enzymes is therefore gaining interest. This study provides a new approach to FOS synthesis by fungal enzyme complex without prior separation of any specific enzyme. Inulinase enzyme complexes could be used for the synthesis of FOS in two possible ways – hydrolysis of inulin (FOSh) and transfructosylation process of sucrose (FOSs), as demonstrated here. Depending on the fungal growth inducing substrate, a variety of inulinase enzyme complexes was obtained – one of which was most successful in production of FOSh and another one of FOSs. Substrates derived from crops: triticale, wheat bran, Jerusalem artichoke and Aspergillus welwitschiae isolate, previously proven as safe for use in food, were utilized for production of inulinase enzyme cocktails. The highest FOSs production was obtained by enzyme complex rich in β-fructofuranosidase, while the highest FOSh production was obtained by enzyme complex rich in endoinulinase. Both FOSh and FOSs showed antioxidant potential according to ABTS and ORAC, which classifies them as a suitable additive in functional food. Simultaneous zymographic detection of inulinase enzymes, which could contribute to expansion of the knowledge on fungal enzymes, was developed and applied here. It demonstrated the presence of different inulinase isoforms depending on fungal growth substrate. These findings, which rely on the innate ability of fungi to co-produce all inulinases from a cocktail, could be useful as a new, easy approach to FOS production by fungal enzymes without their separation and purification, contributing to cheaper and faster production processes.

Production of cellulose nanofibers and sugars using high dry matter feedstock

Abstract Sugars are a sustainable platform for producing chemicals and polymers, while cellulose nanofibers (CNF) are sustainable materials with high specific mechanical properties that have an important role to play in many applications. This study highlights the feasibility of producing sugars and CNF using high dry matter (DM) content of 20 %. A commercial enzyme cocktail at low dosage, 5 mg EP/g DM, and a high 15 mg EP/g DM, were used to hydrolyze the pulp cellulose to produce sugars and CNF. HPLC was used to evaluate the cellulose conversion rate and amount of sugar realized. The CNF were studied using TEM, the diameter of the CNF were measured and estimate the crystallinity. The cellulose conversion plateaued at about 48 h for both the low and high dosage, indicating inhibition due to higher sugar concentration and/or increased recalcitrance of the remaining CNF. The CNF obtained at low enzyme indicated the fibrils were insufficiently separated. At the high dosage, less clumps were observed and with smaller diameter than with the low enzyme dosage. The high dosage also produced CNF with significantly higher crystallinity index, confirming that amorphous cellulose portions readily underwent enzymatic hydrolysis and left more recalcitrant CNF.

Systematic study of single-cell isolation from musculoskeletal tissues for single-sell sequencing

BackgroundThe single-cell platform provided revolutionary way to study cellular biology. Technologically, a sophistic protocol of isolating qualified single cells would be key to deliver to single-cell platform, which requires high cell viability, high cell yield and low content of cell aggregates or doublets. For musculoskeletal tissues, like bone, cartilage, nucleus pulposus and tendons, as well as their pathological state, which are tense and dense, it’s full of challenge to efficiently and rapidly prepare qualified single-cell suspension. Conventionally, enzymatic dissociation methods were wildly used but lack of quality control. In the present study, we designed the rapid cycling enzymatic processing method using tissue-specific enzyme cocktail to treat different human pathological musculoskeletal tissues, including degenerated nucleus pulposus (NP), ossifying posterior longitudinal ligament (OPLL) and knee articular cartilage (AC) with osteoarthritis aiming to rapidly and efficiently harvest qualified single-cell suspensions for single-cell RNA-sequencing (scRNA-seq).ResultsWe harvested highly qualified single-cell suspensions from NP and OPLL with sufficient cell numbers and high cell viability using the rapid cycling enzymatic processing method, which significantly increased the cell viability compared with the conventional long-time continuous digestion group (P < 0.05). Bioanalyzer trace showed expected cDNA size distribution of the scRNA-seq library and a clear separation of cellular barcodes from background partitions were verified by the barcode-rank plot after sequencing. T-SNE visualization revealed highly heterogeneous cell subsets in NP and OPLL. Unfortunately, we failed to obtain eligible samples from articular cartilage due to low cell viability and excessive cell aggregates and doublets.ConclusionsIn conclusion, using the rapid cycling enzymatic processing method, we provided thorough protocols for preparing single-cell suspensions from human musculoskeletal tissues, which was timesaving, efficient and protective to cell viability. The strategy would greatly guarantee the cell heterogeneity, which is critical for scRNA-seq data analysis. The protocol to treat human OA articular cartilage should be further improved.

Enhanced enzymatic hydrolysis of corn stover using twin‐screw extrusion under mild conditions

AbstractThis paper aims to investigate the effect of extrusion at high solid loading on corn stover (CS) properties and its enzymatic hydrolysis. This biomass was extruded under different screw speeds and different solid loadings and the impact of these parameters on the physicochemical properties was evaluated. It was found that lignocellulosic components were not significantly affected by the pretreatment, while the surface area increased with solid loading and rotation speed. Different enzyme cocktails were used for the enzymatic hydrolysis of extruded and untreated CS. Overall, mild twin‐extrusion enhanced the enzymatic hydrolysis of CS through an increase in glucose and xylose yields by 134–212% and 214–294%, respectively, when using Trichoderma longibrachiatum cellulase. The highest sugar content was obtained from CS extruded under 400 g total solids per liter and 200 rpm. The energy efficiency of the pretreatment was also assessed and was found to be maximal at 400 g total solids per liter and 200 rpm. © 2022 Society of Chemical Industry and John Wiley &amp; Sons, Ltd.