Combined effects of α-amylase, xylanase, and cellulase coproduced by Stachybotrys microspora on dough properties and bread quality as a bread improver.

This study aims to explore the feasibility of introducing, during the manufacture of bakery bread, an enzymatic cocktail coproduced by the fungus Stachybotrys microspora: α-amylases, xylanases and cellulases, using wheat bran as a nutrient source. Among the characteristics of the alveograph (dough tenacity "P" and dough extensibility "L"), the addition of a cocktail of enzymes at a concentration of 2 %, to weak wheat flour, has made it possible to significantly reduce its P/L ratio from 2.45 to 1.41. Furthermore, the use of enzyme cocktails at 2 %, 4 %, and 6 % concentrations increases the brown color of the bread crust. The great reduction in the rate of bread firmness, during storage over 5 days, was obtained in the presence of an enzyme cocktail in comparison with bread control (65.13 N for the control and 22.99 N, 23.24 N, and 18.24 N for bread enriched with enzyme cocktail at 2 %, 4 % and 6 % concentrations, respectively). In conclusion, the enzyme cocktail added can synergistically improve bread dough rheology and bread properties.

Efficient pathway-driven scyllo-inositol production from myo-inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.

Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo-inositol into scyllo-inositol at 30°C. In a scaled-up reaction, 10 g of myo-inositol was converted to 1.8 g of scyllo-inositol, which was further purified to yield 970 mg of pure powder. Notably, myo-inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. IMPORTANCE: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo-inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo-inositol, holding potential pharmaceutical significance as scyllo-inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems.

Visualization of Fagopyrum esculentum and Fagopyrum tataricum Chromosomes and Micronuclei.

This chapter presents the squash chromosome preparation technique for Fagopyrum esculentum and F. tataricum, using the root tips as the source of the material. Using an optimized version of this method, the chromosomes are free of cytoplasmic debris and are spread evenly on the glass slide. What comes of it is the possibility to make observations of the chromosome number and structure at the metaphase stage. This technique's modified version allows micronuclei analysis in interphase cells of buckwheats.

Simple one-step treatment for saccharification of mango peels using an optimized enzyme cocktail of Aspergillus niger ATCC 9642.

Developing efficient microbiological methods to convert polysaccharide-rich materials into fermentable sugars, particularly monosaccharides, is vital for advancing the bioeconomy and producing renewable chemicals and energy sources. This study focused on optimizing the production conditions of an enzyme cocktail from Aspergillus niger ATCC 9642 using solid-state fermentation (SSF) and assessing its effectiveness in saccharifying mango peels through a simple, rapid, and efficient one-step process. A rotatable central composite design was employed to determine optimal conditions of moisture, time, and pH for enzyme production in SSF medium. The optimized enzyme cocktail exhibited cellulase activity (CMCase) at 6.28 U/g, filter paper activity (FPase) at 3.29 U/g, and pectinase activity at 117.02 U/g. These optimal activities were achieved with an SSF duration of 81 h, pH of 4.66, and a moisture content of 59%. The optimized enzyme cocktail effectively saccharified the mango peels without the need for chemical agents. The maximum saccharification yield reached approximately 81%, indicating efficient conversion of mango peels into sugars. The enzyme cocktail displayed consistent thermal stability within the tested temperature range of 30-60°C. Notably, the highest sugar release occurred within 36 h, with glucose, arabinose, galactose, and xylose being the primary monosaccharides released during saccharification. This study highlights the potential application of Aspergillus niger ATCC 9642 and SSF for enzymatic production, offering a simple and high-performance process for monosaccharide production. The optimized enzyme cocktail obtained through solid-state fermentation demonstrated efficient saccharification of mango peels, suggesting its suitability for industrial-scale applications.

A new paradigm in lignocellulolytic enzyme cocktail optimization: Free from expert-level prior knowledge and experimental datasets.

Effectively pairing diverse lignocellulolytic enzyme cocktails with intricately structured lignocellulosic substrates is an enduring challenge for science and technology. To date, extensive trial-and-error remains the primary approach and no deep-learning methods were developed to address it due to limited experimental data and incomplete expert-level knowledge of enzyme-cocktail-substrate structure-dynamics-function relationships. Here, a novel model is developed to tackle this issue in efficient, cost-effective, and high-throughput manners. It needs no pre-labeled datasets, instead utilizing simple features, eliminating the reliance on expert-level prior knowledge of reaction mechanisms. Experimentally optimal combinations were found within predicted ranges of tailor-made combinations with precision of 91.98%, covering 80.00% of overall top-100. Practical tests demonstrated its effectiveness in narrowing down potential optimal combinations, speeding up targeted screening, and enabling efficient degradation of lignocellulosic biomass. The method has good applications in artificial proteins biosynthesis from low-value lignocellulosic straw, providing alternative solutions for biomass biorefining challenges in complex enzyme-cocktail-substrate interactions.

The effects of Moringa peregrina seed meal, autoclaving, and/or exogenous enzyme cocktail on performance, carcass traits, meat quality, and blood lipids of broilers.

The effects of Moringa peregrina seed meal (MPSM), autoclaving, and/or enzyme cocktail addition on performance, profitability, carcass traits, meat quality, and blood lipids of broilers between 1 and 35 d of age were investigated. Seven experimental diets were employed: the control 0% MPSM, 10% raw MPSM, 10% autoclaved MPSM (at a temperature of 120°C and 1 kg/cm2 pressure for 30 min), 10% raw MPSM supplemented with enzymes at 0.1 or 0.2 g/kg feed, and 10% autoclaved MPSM supplemented with the same previous enzymes and doses. Each diet was fed to 8 replicates with 5 broilers in each. At the end of the experiment, 3 broilers from each replicate were randomLy chosen to determine carcass traits, meat quality, and blood lipids. Findings at 35 d of age indicated that all 10% raw MPSM treatments with or without enzymes addition impaired growth, feed conversion (FCR), and profitability (p < 0.05), but increased feed intake (p < 0.05) and did not affect mortality when compared with the control group. The 10% autoclaved MPSM treatments with or without enzymes addition increased feed intake (p < 0.05) when compared with the control group, inducing growth equal to the control group (p > 0.05), and improving FCR and profitability. Enzymes addition to raw MPSM did not produce positive effects (p < 0.05), and no additive effect was observed when autoclaving and enzymes addition were combined (p > 0.05) as compared to the autoclaving group. Carcass traits, meat quality, and blood lipids were not significantly affected by MPSM, autoclaving, and enzymes addition. However, intestine, cecum, and gizzard percentages increased (p < 0.05) with all 10% raw MPSM treatments, while all 10% autoclaved MPSM treatments could return these values (p > 0.05) to the control group, except with gizzard, which exhibited less improvement. Additionally, all autoclaved groups had lower meat pH measured 24 h postmortem (p <0.05) compared to the control group. In conclusion, autoclaved MPSM can be included in broilers' diets at a 10% level without negative effects on performance, carcass traits, meat quality, and blood lipids. This indicates that autoclaving alone is adequate.

Distinct lignocellulolytic enzymes produced by Trichoderma harzianum in response to different pretreated substrates.

In order to optimize the composition of enzyme cocktail for improving the hydrolytic efficiency of lignocellulose, different substrates were tested as inducers for producing lignocellulolytic enzymes by Trichoderma harzianum EM0925 in this study. As results, ultrafine grinding or steam explosion pretreated substrates can induce T. harzianum EM0925 to secret holo lignocellulolytic enzymes; acid treated substrate can induce cellobiohydrolase; while alkali or sodium chlorite treated substrates can induce ß-xylosidase specifically. Furthermore, the combination of enzyme cocktails with different hydrolysis characteristics can further improve the hydrolysis efficiency, since 100% yields of glucose and xylose were obtained simultaneously from ultrafine grinding treated corn stover at low enzyme dosage (1.2 mg proteins/g substrate). This study for the first time demonstrated an effective solution that specific-pretreated substrates can be used as inducers for specific enzyme production by T. harzianum, which provided new idea and potential strategy for the construction of highly-efficient lignocellulolytic enzyme cocktails.

Production of glutathione from probiotic Bacillus amyloliquefaciens KMH10 using banana peel extract.

Glutathione, a tri-peptide (glutamate-cysteine-glycine) with the thiol group (-SH), is most efficient antioxidative agent in eukaryotic cells. The present study aimed to isolate an efficient probiotic bacterium having the potential to produce glutathione. The isolated strain Bacillus amyloliquefaciens KMH10 showed antioxidative activity (77.7 ± 2.56) and several other essential probiotic attributes. Banana peel, a waste of banana fruit, is chiefly composed of hemicellulose with various minerals and amino acids. A consortium of lignocellulolytic enzyme was used for the saccharifying banana peel to produce 65.71 g/L sugar to support the optimal glutathione production of 181 ± 4.56 mg/L; i.e., 1.6 folds higher than the control. So, the studied probiotic bacteria could be an effective resource for glutathione; therefore, the stain could be used as natural therapeutics for the prevention/treatment of different inflammation-related gastric ailments and as an effective producer of glutathione using valorized banana waste that has excellent industrial relevance.

Improvement of the Nutraceutical Profile of Brewer's Spent Grain after Treatment with Trametes versicolor.

Brewer's spent grain (BSG) is an important secondary raw material that provides a readily available natural source of nutraceuticals. It finds its largest application as animal feed and part of the human diet, while the future perspective predicts an application in the production of value-added products. In order to investigate a sustainable BSG treatment method, two BSG samples (BSG1 and BSG2) were evaluated as substrates for the production of hydrolytic (xylanase, ß-glucosidase and cellulase) and lignolytic enzymes (laccase, manganese peroxidase and lignin peroxidase) by solid-state fermentation (SSF) with Trametes versicolor while improving BSG nutritional value. The biological treatment was successful for the production of all hydrolytic enzymes and laccase and manganese peroxidase, while it was unsuccessful for the production of lignin peroxidase. Because the two BSGs were chemically different, the Trametes versicolor enzymes were synthesized at different fermentation times and had different activities. Consequently, the chemical composition of the two BSG samples at the end of fermentation was also different. The biological treatment had a positive effect on the increase in protein content, ash content, polyphenolic compounds, and sugars in BSG1. In BSG2, there was a decrease in the content of reducing sugars. Cellulose, hemicellulose, and lignin were degraded in BSG1, whereas only cellulose was degraded in BSG2, and the content of hemicellulose and lignin increased. The fat content decreased in both samples. The safety-related correctness analysis showed that the biologically treated sample did not contain any harmful components and was therefore safe for use in nutritionally enriched animal feed.

Development of an economically competitive Trichoderma-based platform for enzyme production: Bioprocess optimization, pilot plant scale-up, techno-economic analysis and life cycle assessment.

Despite decades of research and industrial applications of Trichoderma reesei, the development of industrially relevant strains for enzyme production including a low-cost and scalable bioprocess remains elusive. Herein, bioprocess optimization, pilot plant scale-up, techno-economic analysis and life-cycle assessment for enzyme production by an engineered T. reesei strain are reported. The developed bioprocess increased in âˆ¼ 2-fold protein productivity (0.39 g.L-1.h-1) and 1.6-fold FPase activity (196 FPU.L-1.h-1), reducing the fermentation in 4 days. Cultivation in a 65-L pilot plant bioreactor resulted in 54 g.L-1 protein in 7 days, highlighting the robustness and scalability of this bioprocess. Techno-economic analysis indicates an enzyme cost of âˆ¼ 3.2 USD.kg-1, which is below to the target proposed (4.24 USD.kg-1) in the NREL/TP-5100-47764 report, while life-cycle assessment shows a carbon footprint reduction of approximately 50% compared to a typical commercial enzyme. This study provides the fundamental knowledge for the design of economically competitive Trichoderma technologies for industrial use.

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 ß-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 ß-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.

Effect of Exogenous Enzymes Cocktail on Performance, Carcass Traits, Biochemical Metabolites, Intestinal Morphology, and Nutrient Digestibility of Broilers Fed Normal and Low-Energy Corn-Soybean Diets.

Ross 308 broilers in a randomized complete block design with a 2 × 2 factorial treatment arrangement (four treatments with 12 replications of six chicks each) were fed corn and SBMbased diets with two concentrations of metabolizable energy (ME) (normal (positive control, PC) and low (negative control, NC)) and two amounts of enzyme cocktail (EC) (0% and 0.005%) for 35 days. Performance, carcass traits, serum metabolites, ileal histology, and apparent nutrient digestibility were evaluated. Compared with the non-supplemented diet, the use of EC improved feed conversion ratio (FCR) over 26−35 and 0−35 days (p < 0.01), European performance efficiency factor (EPEF) over 26−35 days (p < 0.05), dressing yield (p < 0.01), villus height (p < 0.05), nitrogen-corrected apparent ME (AMEn) (p < 0.01), and serum glucose (p < 0.05). Compared with the NC diet, feeding the PC diet improved FCR over all experimental periods (p < 0.01, p < 0.05, p < 0.05, and p < 0.01, respectively), EPEF over 0−10 days (p < 0.05), and AMEn retention (p < 0.01). To conclude, the AMEn of broilers fed corn and SBM diets could be improved by adequately adjusting dietary ME and using a cocktail of non-starch polysaccharide-degrading enzymes, improving commercial benefits to producers.

Non-starch polysaccharide degradation in the gastrointestinal tract of broiler chickens fed commercial-type diets supplemented with either a single dose of xylanase, a double dose of xylanase, or a cocktail of non-starch polysaccharide-degrading enzymes.

The aim of this study was to examine non-starch polysaccharide (NSP) degradation in the gastrointestinal tract of chickens fed a range of commercial-type diets supplemented with a commercial dose of xylanase, a double dose of xylanase or a cocktail of NSP - degrading enzymes. Cobb 500 broilers (n = 1,080) were fed 12 dietary treatments; 4 diets with differing primary grain sources (barley, corn, sorghum, and wheat) and three different enzyme treatments (commercial recommended dose of xylanase (16,000 BXU/kg), a double dose of xylanase (32,000 BXU/kg) or an NSP-degrading enzyme cocktail (xylanase, ß-glucanase, cellulase, pectinase, mannanase, galactanase, and arabinofuranosidase at recommended commercial levels). There were 108 pens, approximately 10 birds per pen, 9 replicates per dietary treatment. The diets were fed as 3 phases, starter (d 0-12), grower (d 12-23), and finisher (d 23-35). On bird age d 12, 23, and 35, performance (total pen body weight, feed intake, and feed conversion ratio corrected for mortality [cFCR]), litter and excreta dry matter content, and ileal and total tract soluble and insoluble NSP degradability and free oligosaccharide digestibility was determined. On d 35, the quantity of NSP in the gizzard, jejunum, ileum and excreta was determined. Results from this study showed that the double xylanase dose and NSP-ase cocktail had positive impacts on starter phase performance in birds fed the corn- and wheat-based diets. In the grower phase in birds fed the barley-based diet, these enzyme treatments improved cFCR and increased litter dry matter content. The NSP-ase cocktail had a negative impact on finisher phase cFCR in birds fed the sorghum-based diet. The double xylanase dose induced a positive impact on NSP degradability and free oligosaccharide digestibility. In conclusion, there appears to be advantages to feeding broilers a double xylanase dose, but lack of consistency when using an NSP-ase cocktail containing many enzymes.

Recent advancements in prebiotic oligomers synthesis via enzymatic hydrolysis of lignocellulosic biomass.

Interest in functional food, such as non-digestible prebiotic oligosaccharides is increasing day by day and their production is shifting toward sustainable manufacturing. Due to the presence of high carbohydrate content, lignocellulosic biomass (LCB) is the most-potential, cost-effective and sustainable substrate for production of many useful products, including lignocellulose-derived prebiotic oligosaccharides (LDOs). These have the same worthwhile properties as other common oligosaccharides, such as short chain carbohydrates digestible to the gut flora but not to humans mainly due to their resistance to the low pH and high temperature and their demand is constantly increasing mainly due to increased awareness about their potential health benefits. Despite several advantages over the thermo-chemical route of synthesis, comprehensive and updated information on the conversion of lignocellulosic biomass to prebiotic oligomers via controlled enzymatic saccharification is not available in the literature. Thus, the main objective of this review is to highlight recent advancements in enzymatic synthesis of LDOs, current challenges, and future prospects of sustainably producing prebiotic oligomers via enzymatic hydrolysis of LCB substrates. Enzyme reaction engineering practices, custom-made enzyme preparations, controlled enzymatic hydrolysis, and protein engineering approaches have been discussed with regard to their applications in sustainable synthesis of lignocellulose-derived oligosaccharide prebiotics. An overview of scale-up aspects and market potential of LDOs has also been provided.

Covalent Immobilization of Chondrostereum purpureum Endopolygalacturonase on Ferromagnetic Nanoparticles: Catalytic Properties and Biotechnological Application.

Pectinases are widely used in a variety of industrial processes. However, their application is limited by low catalytic processivity, reduced stability, high cost, and poor re-use compatibility. These drawbacks may be overcome by enzyme immobilization with ferromagnetic nanoparticles, which are easily recovered by a magnetic field. In this work, an endopolygalacturonase from Chondrostereum purpureum (EndoPGCp) expressed in Pichia pastoris was immobilized on glutaraldehyde-activated chitosan ferromagnetic nanoparticles (EndoPGCp-MNP) and used to supplement a commercial enzyme cocktail. No significant differences in biochemical and kinetic properties were observed between EndoPGCp-MNP and EndoPGCp, although the EndoPGCp-MNP showed slightly increased thermostability. Cocktail supplementation with EndoPGCp-MNP increased reducing sugar release from orange wastes by 1.8-fold and showed a synergistic effect as compared to the free enzyme. Furthermore, EndoPGCp-MNP retained 65% of the initial activity after 7 cycles of re-use. These properties suggest that EndoPGCp-MNP may find applications in the processing of pectin-rich agroindustrial residues.

Wild Halophytic Phragmites karka Biomass Saccharification by Bacterial Enzyme Cocktail.

Biofuel derived from halophytic biomass is getting attention owing to the concerns of energy versus food crisis. The disadvantages associated with edible bioenergy resources necessitate the need to explore new feedstocks for sustainable biofuel production. In this study, biomass from locally available abundant halophytes (Panicum antidotale, Phragmites karka, Halopyrum mucronatum, and Desmostachya bipinnata) was screened for saccharification by an enzyme cocktail composed of cellulase, xylanase, and pectinase from Brevibacillus borstelensis UE10 and UE27, Bacillus aestuarii UE25, Aneurinibacillus thermoaerophilus UE1, and Bacillus vallismortis MH 1. Two types of pretreatment, i.e., with dilute acid and freeze-thaw, were independently applied to the halophytic biomass. Saccharification of acid-pretreated P. karka biomass yielded maximum reducing sugars (9 mg g-1) as compared to other plants. Thus, the factors (temperature, pH, substrate concentration, and enzyme units) affecting its saccharification were optimized using central composite design. This statistical model predicted 49.8 mg g-1 of reducing sugars that was comparable to the experimental value (40 mg g-1). Scanning electron microscopy and Fourier-transform infrared spectroscopy showed significant structural changes after pretreatment and saccharification. Therefore, halophytes growing in saline, arid, and semi-arid regions can be promising alternative sources for bioenergy production.

Bioconversion of Lignocellulosic Materials with the Contribution of a Multifunctional GH78 Glycoside Hydrolase from Xylaria polymorpha to Release Aromatic Fragments and Carbohydrates.

A bifunctional glycoside hydrolase GH78 from the ascomycete Xylaria polymorpha (XpoGH78) possesses catalytic versatility towards both glycosides and esters, which may be advantageous for the efficient degradation of the plant cell-wall complex that contains both diverse sugar residues and esterified structures. The contribution of XpoGH78 to the conversion of lignocellulosic materials without any chemical pretreatment to release the water-soluble aromatic fragments, carbohydrates, and methanol was studied. The disintegrating effect of enzymatic lignocellulose treatment can be significantly improved by using different kinds of hydrolases and phenoloxidases. The considerable changes in low (3 kDa), medium (30 kDa), and high (> 200 kDa) aromatic fragments were observed after the treatment with XpoGH78 alone or with this potent cocktail. Synergistic conversion of rape straw also resulted in a release of 17.3 mg of total carbohydrates (e.g., arabinose, galactose, glucose, mannose, xylose) per gram of substrate after incubating for 72 h. Moreover, the treatment of rape straw with XpoGH78 led to a marginal methanol release of approximately 17 µg/g and improved to 270 µg/g by cooperation with the above accessory enzymes. In the case of beech wood conversion, the combined catalysis by XpoGH78 and laccase caused an effect comparable with that of fungal strain X. polymorpha in woody cultures concerning the liberation of aromatic lignocellulose fragments.

Agarase cocktail from agar polysaccharide utilization loci converts homogenized Gelidium amansii into neoagarooligosaccharides.

Neoagarooligosaccharides (NAOs) are drawing more and more attention because of their numerous bioactivities, yet limited number of agarases impedes NAOs production from red algae. In this study, predicted agar polysaccharide utilization loci (agar-PUL) were firstly used as inventory for agarase. 6 agarases were identified from agar-PULs and two of them were successfully expressed and analyzed. Then enzyme cocktail (GH16-1:GH16-2:Aga50D = 2:1:1) was proved to have highest synergistic effect. Finally homogenization was applied to G. amansii and proved to be an efficient way to release agar from tissues. When liquid-to-solid ratio was 9 g/150 mL, homogenization time was 20 min, and enzyme cocktail loading was 150 U/150 mL, maximum NAOs production (90.2 mg per 9 g wet G. amansii) could be achieved. Enzyme supported one-step process (ESOP) proposed in study is environment-friendly, time saving, cost saving and none-destructive, therefore has a potential industrial application in red algae utilization.

Sequential pretreatment of sugarcane bagasse by alkali and organosolv for improved delignification and cellulose saccharification by chimera and cellobiohydrolase for bioethanol production.

Sequential pretreatments for sugarcane bagasse (scb) by NaOH followed by organosolv under mild conditions were evaluated for cellulose recovery and dilignification. The best-optimized sequential pretreatment of scb was obtained at 10% (w/v) of raw scb loading at 1% (w/v) NaOH (50 °C, 2 h) followed by treatment with organosolv (85%, v/v phosphoric acid, 50 °C, 1 h) with chilled acetone. This sequentially pretreated scb showed cellulose recovery, 66.1% (w/w) and delignification, 83.2% (w/w). NaOH or organosolv pretreated scb showed lower cellulose recovery 47.4% (w/w) or 54.5% (w/w) with lower delignification, 61% (w/w) or 56% (w/w), respectively. Pretreated solid residue of sequentially pretreated scb was enzymatically saccharified by chimera (ß-glucosidase and endoglucanase, CtGH1-L1-CtGH5-F194A) and cellobiohydrolase (CtCBH5A) cloned from Clostridium thermocellum. Enzymatic hydrolysate of best sequentially pretreated scb gave total reducing sugar (TRS) yield, 230 mg/g and glucose yield, 137 mg/g pretreated scb. Only organosolv pretreated scb gave TRS yield, 112.5 mg/g and glucose yield, 72 mg/g of pretreated scb. Thus, sequentially pretreated scb resulted in 37% higher enzymatic digestibility than only orgnaosolv pretreated scb. Higher enzymatic digestibility was supported by higher crystallinity index CrI (45%) than those obtained with only organosolv pretreated (38%) or raw scb (25%). Field Emission Scanning Electron Microscope (FESEM) and Fourier-transform infrared (FT-IR) analyses showed enhanced cellulose exposure in sequentially pretreated scb. Preliminary investigation of bioethanol production at small scale by separate hydrolysis and fermentation (SHF) of enzymatic hydrolysate from best sequentially pretreated scb by Saccharomyces cerevisiae gave maximum ethanol yield of 0.42 g/g of glucose. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02600-y.

Immobilization of enzyme cocktails on dopamine functionalized magnetic cellulose nanocrystals to enhance sugar bioconversion: A biomass reusing loop.

A combined enzymatic treatment/acid hydrolysis technique was utilized to synthesize cellulose nanocrystals (CNCs) from sugar beet pulp. CNCs were functionalized with magnetite nanoparticles and dopamine making a versatile nano-carrier (DA/Fe3O4NPs@CNCs) for covalent enzyme immobilization. Oxygene/amine functionalities, high magnetization value, and specific surface area of DA/Fe3O4NPs@CNCs made it a reusable and green candidate for conjugation to hydrolytic enzyme cocktails (three cellulases, two hemicellulases, and their combinations) to prepare an innovative and practical nano-biocatalyst for biomass conversion. The conjugated enzymes showed an enhanced optimum temperature (∼ 10 °C), improved thermal stability, and shifted optimum pH toward alkaline pHs. Covalent attachment could successfully suppress the enzyme leaching and provide easy recovery/reuse of the nano-biocatalyst up to 10 cycles, with > 50% of initial activity. Application of the nano-biocatalyst in hydrolysis of rice straw and sugar beet pulp showed an increase (20-76%) in the yield of fermentable sugars compared to the free enzyme cocktails.