Nitrogen-doped carbon quantum dots fabricated from cellulolytic enzyme lignin and its application to the determination of cytochrome c and trypsin.

A sensitive and effective strategy for the detection of cytochrome c (Cyt c) and trypsin was developed using biomass nitrogen-doped carbon quantum dots (N-CQDs) as the fluorescence probe. N-CQDs were synthesized through a one-pot hydrothermal method by utilizing cellulolytic enzyme lignin as the carbon source and ammonia as the solvent and nitrogen source. The obtained N-CQDs had good water solubility and stable optical properties. The introduction of nitrogen increased fluorescence quantum yield (QY) to 8.23%, which was almost four times as high as that before nitrogen doping. The N-CQDs were fabricated as a label-free biosensor to detect Cyt c and trypsin. The fluorescence of N-CQDs was quenched with positively charged Cyt c due to electrostatic induction aggregation and static quenching. However, Cyt c tended to be hydrolyzed into small peptides in the presence of trypsin, which caused fluorescence recovery of the N-CQDs/Cyt c complex. A wide linear response range was achieved for Cyt c within 1-50 µM and the developed N-CQDs/Cyt c complex displayed a linear response for trypsin within 0.09-5.4 U/mL. The detection limits were 0.29 µM for Cyt c and 0.013 U/mL for trypsin, respectively. Furthermore, this assay had been applied to Cyt c and trypsin detection in serum samples with the recoveries in the range of 94.6-98.5% and 95.5-102.0%, respectively. The established method was sensitive, selective, easy to operate, and low cost, which proved its potential application in clinical diagnosis. The synthesis and fluorescence mechanism of N-CQDs and the strategy for Cyt c and trypsin detection.

A novel amylolytic/xylanolytic/cellulolytic multienzyme complex from Clostridium manihotivorum that hydrolyzes polysaccharides in cassava pulp.

Some anaerobic bacteria, particularly Clostridium species, produce extracellular cellulolytic and xylanolytic enzymes as multienzyme complexes (MECs). However, an amylolytic/xylanolytic/cellulolytic multienzyme complex (AXC-MEC) from anaerobic bacteria is rarely found. In this work, the glycoprotein AXC-MEC, composed of subunits of amylolytic, xylanolytic, and cellulolytic enzymes, was isolated from crude extracellular enzyme of the mesophilic anaerobic bacterium Clostridium manihotivorum CT4, grown on cassava pulp, using a milled cassava pulp column and Sephacryl S-500 gel filtration chromatography. The isolated AXC-MEC showed a single band upon native-polyacrylamide gel electrophoresis (native-PAGE). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed at least eight protein bands of the multienzyme complex which predominantly exhibited amylolytic enzyme activity, followed by xylanolytic and cellulolytic enzyme activities. The AXC-MEC is highly capable of degrading starch and non-starch polysaccharides present in cassava pulp into glucose and oligosaccharides, without conventional pretreatment. Base on the genomic analysis of C. manihotivorum CT4, we found no evidence of the known structural components of the well-known multienzyme complexes from Clostridium species, cellulosomes such as scaffoldin, cohesin, and dockerin, indicating that AXC-MEC from strain CT4 exhibit a different manner of assembly from the cellulosomes. These results suggest that AXC-MEC from C. manihotivorum CT4 is a new MEC capable of hydrolyzing cassava pulp into value-added products, which will benefit the starch industry. KEY POINTS: • Glycoprotein AXC-MEC was first reported in Clostridium manihotivorum. • Unlike cellulosomes, AXC-MEC consists of amylase, xylanase, and cellulase. • Glucose and oligosaccharides were hydrolysis products from cassava pulp by AXC-MEC.

Enzyme production and oil palm empty fruit bunch bioconversion to ethanol using a hybrid yeast strain.

Oil palm empty fruit bunch (OPEFB) is a lignocellulosic biomass generated in palm oil mills. It is a sustainable resource for fuels and chemicals. In this study, OPEFB was converted to ethanol by an integrative OPEFB conversion process including dilute alkaline pretreatment, cellulolytic enzyme production, separate OPEFB hydrolysis, and cofermentation using a hybrid xylose-fermenting yeast. OPEFB was pretreated using 1% (w/v) NaOH solution followed by 1% (v/v) H2 O2 . Further, cellulolytic enzymes were produced by submerged fermentation using Trichoderma reesei Rut C30 and used for OPEFB hydrolysis. The filter paper cellulase activity of the crude cellulolytic enzymes was 15.1 IU/mL, which was higher than those obtained by reported Trichoderma strains under laboratory conditions. Glucose and xylose yields reached 66.9% and 74.2%, respectively, at 30 filter paper unit (FPU)/g-biomass enzyme dosage and 10% (w/v) biomass loading. The hybrid yeast strain ScF2 was previously constructed through recursive genome shuffling of Pichia stipitis and Saccharomyces cerevisiae and was used in OPEFB hydrolysate fermentation. About 16.9 g/L ethanol was produced with an ethanol yield of 0.34 g/g sugars, which was 67% of theoretical ethanol yield.

Enhancement of Versatile Extracellular Cellulolytic and Hemicellulolytic Enzyme Productions by Lactobacillus plantarum RI 11 Isolated from Malaysian Food Using Renewable Natural Polymers.

Lactobacillus plantarum RI 11 was reported recently to be a potential lignocellulosic biomass degrader since it has the capability of producing versatile extracellular cellulolytic and hemicellulolytic enzymes. Thus, this study was conducted to evaluate further the effects of various renewable natural polymers on the growth and production of extracellular cellulolytic and hemicellulolytic enzymes by this novel isolate. Basal medium supplemented with molasses and yeast extract produced the highest cell biomass (log 10.51 CFU/mL) and extracellular endoglucanase (11.70 µg/min/mg), exoglucanase (9.99 µg/min/mg), ß-glucosidase (10.43 nmol/min/mg), and mannanase (8.03 µg/min/mg), respectively. Subsequently, a statistical optimization approach was employed for the enhancement of cell biomass, and cellulolytic and hemicellulolytic enzyme productions. Basal medium that supplemented with glucose, molasses and soybean pulp (F5 medium) or with rice straw, yeast extract and soybean pulp (F6 medium) produced the highest cell population of log 11.76 CFU/mL, respectively. However, formulated F12 medium supplemented with glucose, molasses and palm kernel cake enhanced extracellular endoglucanase (4 folds), exoglucanase (2.6 folds) and mannanase (2.6 folds) specific activities significantly, indicating that the F12 medium could induce the highest production of extracellular cellulolytic and hemicellulolytic enzymes concomitantly. In conclusion, L. plantarum RI 11 is a promising and versatile bio-transformation agent for lignocellulolytic biomass.

Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes.

Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/ß-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii ß-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment.IMPORTANCE Ongoing research is focused on improving "green" pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/ß-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment. It demonstrates efficient synergism with endoxylanases PcXyn10C and PcXyn11A to depolymerize xylan in untreated rice straw and enhanced the xylose production and improved cellulose hydrolysis. Therefore, it can be considered an enzymatic pretreatment. Furthermore, the studies here show that glucose yield released from steam- and xylanolytic enzyme-treated rice straw by the combination of CtCel9R and TbCglT was higher than the glucose yield obtained from ammonia-treated rice straw saccharification. This work presents a novel environment-friendly xylanolytic enzyme pretreatment not only as a green pretreatment but also as an economically feasible biorefinery method.

Reactivity improvement of cellulolytic enzyme lignin via mild hydrothermal modification.

Isolated by the cellulolytic enzyme lignin (CEL) process, water-alcohol (1:1, v/v) was introduced as co-solvent in the process of the hydrothermal treatment. The modification parameters such as reaction temperature and time, solid-to-liquid ratio, and catalysts (NaOH and NaOAlO2) have been investigated in terms of the specific lignin properties, such as the phenolic hydroxyl content (OHphen), DPPH free radical scavenging rate, and formaldehyde value. The CELs were also characterized by GPC, FT-IR and 1H NMR spectroscopy, and Py-GC/MS. The key data are under optimal lignin modification conditions (solid-to-liquid ratio of 1:10 (w/v) and a temperature of 250°C for 60min) are: OHphen content: 2.50mmol/g; half maximal inhibitory concentration (IC50) towards DPPH free radicals: 88.2mg/L; formaldehyde value: 446.9g/kg). Both base catalysts decrease the residue rate, but phenol reactivities of the products were also detracted. Py-GC/MS results revealed that modified lignin had a higher phenolic composition than the CEL did, especially the modified lignin without catalyst (ML), which represented 74.51% phenolic content.

Investigation of Marine-Derived Fungal Diversity and Their Exploitable Biological Activities.

Marine fungi are potential producers of bioactive compounds that may have pharmacological and medicinal applications. Fungi were cultured from marine brown algae and identified using multiple target genes to confirm phylogenetic placement. These target genes included the internal transcribed spacer (ITS), the nuclear large subunit (LSU), and the ß-tubulin region. Various biological activities of marine-derived fungi were evaluated, including their antifungal, antioxidant and cellulolytic enzyme activities. As a result, a total of 50 fungi was isolated from the brown algae Sargassum sp. Among the 50 isolated fungi, Corollospora angusta was the dominant species in this study. The genus Arthrinium showed a relatively strong antifungal activity to all of the target plant pathogenic fungi. In particular, Arthrinium saccharicola KUC21221 showed high radical scavenging activity and the highest activities in terms of filter paper units (0.39 U/mL), endoglucanase activity (0.38 U/mL), and ß-glucosidase activity (1.04 U/mL).

Structural characterization of lignin from the green pretreatments for co-producing xylo-oligosaccharides and glucose: Toward full biomass utilization.

Three green non-enzymatic catalysis pretreatments (NECPs) including autohydrolysis, subcritical CO2-assisted seawater autohydrolysis, and inorganic salt catalysis were utilized to simultaneously produce xylo-oligosaccharides (XOS), glucose, and cellulolytic enzyme lignin (CEL) from sugarcane bagasse (SCB). The yield of XOS in all three NECPs was over 50 % with a competitive glucose yield of enzymatic hydrolysis. And the effects of different pretreatments on the chemical structure and composition of CEL samples were also investigated. The pretreatments significantly increased the thermal stability, yield, and purity of the CEL samples. Moreover, the net yield of lignin was 58.3 % with lignin purity was 98.9 % in the autohydrolysis system. Furthermore, there was a decrease in the molecular weight of CEL samples as the pretreatment intensity increased. And the original lignin structural units sustained less damage during the NECPs, due to the cleavage of the ß-O-4 bonds dominating lignin degradation. Meanwhile, these pretreatments increased the phenolic-OH in CEL samples, making the lignin more reactive, and enhancing its subsequent modification and utilization. Collectively, the described techniques have demonstrated practical significance for the coproduction of XOS and glucose, and lignin, providing a promising strategy for full utilization of biomass.

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities.

BACKGROUND: The polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear. RESULTS: An LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid). CONCLUSIONS: This study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

A novel multifunctional cellulolytic enzyme screened from metagenomic resources representing ruminal bacteria.

Metagenomic resources representing ruminal bacteria were screened for novel exocellulases using a robotic, high-throughput screening system, the novel CelEx-BR12 gene was identified and the predicted CelEx-BR12 protein was characterized. The CelEx-BR12 gene had an open reading frame (ORF) of 1140 base pairs that encoded a 380-amino-acid-protein with a predicted molecular mass of 41.8 kDa. The amino acid sequence was 83% identical to that of a family 5 glycosyl hydrolase from Prevotella ruminicola 23. Codon-optimized CelEx-BR12 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography. The Michaelis-Menten constant (Km value) and maximal reaction velocity (Vmax values) for exocellulase activity were 12.92 µM and 1.55 × 10(-)(4 )µmol min(-1), respectively, and the enzyme was optimally active at pH 5.0 and 37°C. Multifunctional activities were observed against fluorogenic and natural glycosides, such as 4-methylumbelliferyl-ß-d-cellobioside (0.3 U mg(-1)), CMC (105.9 U mg(-1)), birch wood xylan (132.3 U mg(-1)), oat spelt xylan (67.9 U mg(-1)), and 2-hydroxyethyl-cellulose (26.3 U mg(-1)). Based on these findings, we believe that CelEx-BR12 is an efficient multifunctional enzyme as endocellulase/exocellulase/xylanase activities that may prove useful for biotechnological applications.

Selecting Suitable Near-Native Lignins for Research.

There are several methods to isolate near-native lignins, including milled-wood lignin, enzymatic lignin, cellulolytic enzyme lignin, and enzymatic mild-acidolysis lignin. Which one is the most representative of the native lignin? Herein, near-native lignins were isolated from different plant groups and structurally analyzed to determine how well these lignins represented their native lignin counterparts. Analytical methods were applied to understand the molecular weight, monomer composition, and distribution of interunit linkages in the structure of the lignins. The results indicated that either enzymatic lignin or cellulolytic enzyme lignin may be used to represent native lignin in softwoods and hardwoods. None of the lignins, however, appeared to represent native lignins in grasses (monocot plants) because of substantial syringyl/guaiacyl differences. Complicating the understanding of grass lignin structure, large amounts of hydroxycinnamates acylate their polysaccharides and, when released, are often conflated with actual lignin monomers.

On-site enriched production of cellulase enzyme using rice straw waste and its hydrolytic performance evaluation through systematic dynamic modeling.

The application of on-site produced cellulolytic enzymes in place of commercial enzymes towards hydrolytic preparations of reducing sugars using inexpensive lignocellulosic wastes is considered the most efficient strategy to accomplish a cost-effective biofuel production process. Along with improved production, intrinsic and systematic performance evaluation of the produced enzyme during the hydrolysis process through kinetic intervention remains a crucial requirement for achieving the improved performance of the process. With this motivation, the present study primarily deals with the nutritionally optimized production strategy of cellulases from rice straw (RS) waste using Trichoderma reesei (MTCC 164). The highest cellulase production was obtained 8.09 ± 0.32 g/l in batch mode at optimized combinations of 3.5% (w/v) RS inducer, 3.0% (w/v) lactose, and 1.5% (w/v) peptone. Production was further improved through pH-regulated (pH 5.5 to 6.5) fed-batch fermentations. The enzyme produced at pH 6 was considered for hydrolysis studies at 4 to 10% (w/w) solid loading due to reasonable exoglucanase, endoglucanase, and maximum ß-glucosidase activity levels of 9.3 U/ml, 3.87 U/ml, and 2.65 U/ml respectively. Multi-reaction systematic kinetic modeling was implemented to evaluate enzyme performance during hydrolysis, and the values of inhibitory kinetic parameters (K2r = 7.1 < K1r = 18.5 < K3r = 276.6) suggested that sequential conversion of cellulose to glucose by existing enzyme components was more dominant over direct conversion.

Genomics and cellulolytic, hemicellulolytic, and amylolytic potential of Iocasia fonsfrigidae strain SP3-1 for polysaccharide degradation.

Background: Cellulolytic, hemicellulolytic, and amylolytic (CHA) enzyme-producing halophiles are understudied. The recently defined taxon Iocasia fonsfrigidae consists of one well-described anaerobic bacterial strain: NS-1T. Prior to characterization of strain NS-1T, an isolate designated Halocella sp. SP3-1 was isolated and its genome was published. Based on physiological and genetic comparisons, it was suggested that Halocella sp. SP3-1 may be another isolate of I. fronsfrigidae. Despite being geographic variants of the same species, data indicate that strain SP3-1 exhibits genetic, genomic, and physiological characteristics that distinguish it from strain NS-1T. In this study, we examine the halophilic and alkaliphilic nature of strain SP3-1 and the genetic substrates underlying phenotypic differences between strains SP3-1 and NS-1T with focus on sugar metabolism and CHA enzyme expression. Methods: Standard methods in anaerobic cell culture were used to grow strains SP3-1 as well as other comparator species. Morphological characterization was done via electron microscopy and Schaeffer-Fulton staining. Data for sequence comparisons (e.g., 16S rRNA) were retrieved via BLAST and EzBioCloud. Alignments and phylogenetic trees were generated via CLUTAL_X and neighbor joining functions in MEGA (version 11). Genomes were assembled/annotated via the Prokka annotation pipeline. Clusters of Orthologous Groups (COGs) were defined by eegNOG 4.5. DNA-DNA hybridization calculations were performed by the ANI Calculator web service. Results: Cells of strain SP3-1 are rods. SP3-1 cells grow at NaCl concentrations of 5-30% (w/v). Optimal growth occurs at 37 °C, pH 8.0, and 20% NaCl (w/v). Although phylogenetic analysis based on 16S rRNA gene indicates that strain SP3-1 belongs to the genus Iocasia with 99.58% average nucleotide sequence identity to Iocasia fonsfrigida NS-1T, strain SP3-1 is uniquely an extreme haloalkaliphile. Moreover, strain SP3-1 ferments D-glucose to acetate, butyrate, carbon dioxide, hydrogen, ethanol, and butanol and will grow on L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, D-raffinose, D-xylose, cellobiose, lactose, maltose, sucrose, starch, xylan and phosphoric acid swollen cellulose (PASC). D-rhamnose, alginate, and lignin do not serve as suitable culture substrates for strain SP3-1. Thus, the carbon utilization profile of strain SP3-1 differs from that of I. fronsfrigidae strain NS-1T. Differences between these two strains are also noted in their lipid composition. Genomic data reveal key differences between the genetic profiles of strain SP3-1 and NS-1T that likely account for differences in morphology, sugar metabolism, and CHA-enzyme potential. Important to this study, I. fonsfrigidae SP3-1 produces and extracellularly secretes CHA enzymes at different levels and composition than type strain NS-1T. The high salt tolerance and pH range of SP3-1 makes it an ideal candidate for salt and pH tolerant enzyme discovery.

Cellulase as an "active" excipient in prolonged-release HPMC matrices: A novel strategy towards zero-order release kinetics.

Hydrophilic matrices are of utmost interest for oral prolonged release of drugs. However, they show decreasing release rate over time, mainly due to lengthening of the diffusional pathway across the gel formed upon glass-rubber transition of the polymer. Therefore, achievement of zero-order release kinetics, which could reflect in constant drug plasma levels, is still an open issue. With the aim of improving the release performance of hydroxypropyl methylcellulose (HPMC) systems, the use of cellulolytic enzymes was proposed to aid erosion of the swollen matrix, thereby counteracting the release rate decrease particularly toward the end of the process. The effectiveness of this strategy was evaluated by studying the mass loss and drug tracer release from tableted matrices consisting of high-viscosity HPMC (Methocel® K4M), Acetaminophen and increasing amounts (0.5-10% on HPMC) of a cellulolytic product (Sternzym® C13030). A faster erosion and progressive shift to linearity of the overall release profiles were observed as a function of the enzyme concentration. Release was markedly linear from matrices containing 5 and 10% Sternzym® C13030. In partially coated matrices with these cellulase concentrations, such results were in agreement with data of erosion and swelling front movement, which exhibited early and long-lasting synchronization.

Complete genome sequence of the cellulolytic planctomycete Telmatocola sphagniphila SP2T and characterization of the first cellulolytic enzyme from planctomycetes.

Planctomycetes of the family Gemmataceae are strictly aerobic chemo-organotrophs that display a number of hydrolytic capabilities. A member of this family, Telmatocola sphagniphila SP2T, is the first described planctomycete with experimentally proven ability for growth on cellulose. In this study, the complete genome sequence of strain SP2T was obtained and the genome-encoded determinants of its cellulolytic potential were analyzed. The T. sphagniphila SP2T genome was 6.59 Mb in size and contained over 5200 potential protein-coding genes. The search for enzymes that could be potentially involved in cellulose degradation identified a putative cellulase that contained a domain from the GH44 family of glycoside hydrolases. Homologous enzymes were also revealed in the genomes of two other Gemmataceae planctomycetes, Zavarzinella formosa A10T and Tuwongella immobilis MBLW1T. The gene encoding this predicted cellulase in strain SP2T was expressed in E. coli and the hydrolytic activity of the recombinant enzyme was confirmed in tests with carboxymethyl cellulose but not with crystalline cellulose, xylan, mannan or laminarin. This is the first experimentally characterized cellulolytic enzyme from planctomycetes.

Preparation and Application of Light-Colored Lignin Nanoparticles for Broad-Spectrum Sunscreens.

Recently, natural sun blockers have been drawing considerable attention because synthetic UV filters could have adverse effects not only on humans but also on the environment. Even though lignin, the second most abundant renewable resource on earth, is a natural UV-absorbing polymer, its unfavorable dark color hampers its applications in sunscreens. In this work, we obtained light-colored lignin (CEL) from rice husks through cellulolytic enzyme treatment and subsequent solvent extraction under mild conditions and compared CEL to technical lignin from rice husks using the International Commission on Illumination L*a*b* (CIELAB) color space. Spherical nanoparticles of CEL (CEL-NP) were also prepared using a solvent shifting method and evaluated for broad-spectrum sunscreens. A moisturizing cream blended with CEL-NP exhibited higher sun protection factor (SPF) and UVA PF (protection factor) values than that with CEL. In addition, CEL-NP had synergistic effects when blended with an organic UV-filter sunscreen: CEL-NP enhanced the SPF and UVA PF values of the sunscreen greatly. However, there was no synergistic effect between CEL-NP and inorganic sunscreens. We expect nanoparticles of light-colored lignin to find high-value-added applications as a natural UV-blocking additive in sunscreens and cosmetics.

Draft genome sequence data of Paenbacillus curdlanolyticus B-6 possessing a unique xylanolytic-cellulolytic multienzyme system.

Paenibacillus curdlanolyticus B-6 is a facultative anaerobic bacterium that efficiently produces a lignocellulolytic multienzyme complex. The whole genome of P. curdlanolyticus B-6 was sequenced on an Ion GeneStudio S5 system, which yielded 74 contigs with a total size of 4,875,097 bp, 4,473 protein-coding sequences, and a G+C content of 49.7%. The genome data have been deposited in DDBJ/ENA/GenBank under accession numbers BLWM01000001-BLWM01000074. Analyses of average nucleotide identities and phylogenetic relationships of 16S rRNA sequences of Paenibacillus species revealed that strain B-6 is most closely related to Paenibacillus xylaniclasticus TW1. P. curdlanolyticus B-6 should thus be reclassified as a strain of P. xylaniclasticus.

Oxidative Depolymerization of Cellulolytic Enzyme Lignin over Silicotungvanadium Polyoxometalates.

The aim of this study was to explore the catalytic performance of the oxidative depolymerization of enzymatic hydrolysis lignin from cellulosic ethanol fermentation residue by different vanadium substituted Keggin-type polyoxometalates (K5[SiVW11O40], K6[SiV2W10O40], and K6H[SiV3W9O40]). Depolymerized products were analyzed by gel permeation chromatography (GPC), gas chromatography⁻mass spectrometer (GC/MS), and two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D HSQC NMR) analysis. All catalysts showed an effective catalytic activity. The best result, concerning the lignin conversion and lignin oil production, was obtained by K6[SiV2W10O40], and the highest yield of oxidative depolymerization products of 53 wt % was achieved and the main products were monomer aromatic compounds. The HSQC demonstrated that the catalysts were very effective in breaking the ß-O-4 structure, the dominant linkage in lignin, and the GPC analysis demonstrated that the molecular of lignin was declined significantly. These results demonstrate the vanadium substituted silicotungstic polyoxometalates were of highly active and stable catalysts for lignin conversion, and this strategy has the potential to be applicable for production of value-added chemicals from biorefinery lignin.

Designing cellulolytic enzyme systems for biorefinery: From nature to application.

Cellulolytic enzymes play a key role on conversion of lignocellulosic plant biomass to biofuels and biochemicals in sugar platform biorefineries. In this review, we survey composite carbohydrate-active enzymes (CAZymes) among groups of cellulolytic fungi and bacteria that exist under aerobic and anaerobic conditions. Recent advances in designing effective cellulase mixtures are described, starting from the most complex microbial consortium-based enzyme preparations, to single-origin enzymes derived from intensively studied cellulase producers such as Trichoderma reesei, Talaromyces cellulolyticus, and Penicellium funiculosum, and the simplest minimal enzyme systems comprising selected sets of mono-component enzymes tailor-made for specific lignocellulosic substrates. We provide a comprehensive update on studies in developing high-performance cellulases for biorefineries.

Biochemical Characterization of a Novel Monospecific Endo-ß-1,4-Glucanase Belonging to GH Family 5 From a Rhizosphere Metagenomic Library.

Cellulases have a broad range of different industrial applications, ranging from food and beverages to pulp and paper and the biofuels area. Here a metagenomics based strategy was used to identify the cellulolytic enzyme CelRH5 from the rhizosphere. CelRH5 is a novel monospecific endo-ß-1,4-glucanase belonging to the glycosyl hydrolase family 5 (GH5). Structural based modeling analysis indicated that CelRH5 is related to endo-ß-1,4-glucanases derived from thermophilic microorganisms such as Thermotoga maritima, Fervidobacterium nodosum, and Ruminiclostridium thermocellum sharing 30-40% amino acid sequence identity. The molecular weight of the enzyme was determined as 40.5 kDa. Biochemical analyses revealed that the enzyme displayed good activity with soluble forms of cellulose as a substrate such as ostazin brilliant red hydroxyethyl cellulose (OBR-HEC), carboxymethylcellulose (CMC), hydroxyethyl cellulose (HEC), and insoluble azurine cross-linked hydroxyethylcellulose (AZCL-HEC). The enzyme shows highest enzymatic activity at pH 6.5 with high pH tolerance, remaining stable in the pH range 4.5-8.5. Highest activity was observed at 40°C, but CelRH5 is psychrotolerant being active and stable at temperatures below 30°C. The presence of the final products of cellulose hydrolysis (glucose and cellobiose) or metal ions such as Na+, K+, Li+, and Mg2+, as well as ethylenediaminetetraacetic acid (EDTA), urea, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) or glycerol, did not have a marked effect on CelRH5 activity. However, the enzyme is quite sensitive to the presence of 10 mM ions Zn2+, Ni2+, Co2+, Fe3+ and reagents such as 1 M guanidine HCl, 0.1% sodium dodecyl sulfate (SDS) and 20% ethanol. Given that it is psychrotolerant and retains activity in the presence of final cellulose degradation products, metal ions and various reagents, which are common in many technological processes; CelRH5 may be potential suitability for a variety of different biotechnological applications.