The synthesized nanocatalyst (Pd@CAI@cell@Fe3O4) was employed for Heck-type arylation of different substituted maleimides with iodoarenes in good to excellent yields. This green catalyst was easily recovered and reused several times with no substantial loss of activity, providing a clean and efficient synthetic procedure with excellent yield and reduced time.Pd supported on 5-carboxyoxindole functionalized cell@Fe 3O4 nanoparticles (Pd@CAI@cell@Fe3O4), a new magnetic nanocatalyst, was prepared and characterized.Pd supported on 5-carboxyoxindole functionalized cell@Fe The principal focus of this field is the evolution of catalysts that include both metal nanoparticles and a nanomaterials as the main support. In fact, these nanocatalysts have high specific surface area and surface energy, which result in their high catalytic activity. Also, they have other properties, such as improving the selectivity of the reactions while reducing the reaction temperature, minimizing side reactions, and with higher recycling rates. Hence, these catalysis are of great interest for researchers in the synthesis of many organic compounds.3Nanocatalysis has emerged as a field at the interface between homogeneous and heterogeneous catalysis and suggests exceptional solutions to the various industries for catalyst enhancement.4,5Recently, the majority of scientists increasingly employ biopolymer supports from renewable, durable, and abundant resources as attractive materials for catalysis reactions.6Nanocellulose is the most noteworthy and abundant biopolymer, and it is obtained from plants, bacteria and algae; its known properties include hydrophobicity, biodegradability, economy, biocompatibility, and wide chemical-functionalization capacity.6 In fact, supported nanocellulose is emerging as an attractive protocol to stabilize some transition metal complexes.6–12 In recent times, nanocellulose supporting palladium, platinum, zirconium, copper and nickel nanoparticles have been studied and reported.13–16These abilities of cellulose make it an interesting support, and its application as an efficient support for the catalytic processes in the synthesis of many organic compounds has been studied.17Also, nanocellulose is one of the most perfect coating supports for magnetic nanoparticles (MNPs) due to its ability to not only stabilize nanoparticles in solution but also promote functionalization to produce biopolymer-based catalysts.18,19 The design of new, magnetically separable systems has generated a lot of attention in recent years as an attractive candidate to improve the efficient separation of heterogeneous nanocatalysts from products by their response to an external magnetic field.20,21 In other words, in order to control the fast oxidation and the tendency of MNPs to agglomerate, their surface is generally protected with organic, inorganic or bio-polymeric materials to form core shell structures, which have thus been named bio-based magnetic nanocatalysts.22In fact, MNPs have attracted great attention due to their unique properties, including eco-friendliness, high flexibility, low toxicity, low Curie temperature, easy preparation and functionalization, large surface-to-volume ratio, facile separation using an external magnet, and a high degree of chemical stability.23–25 involve the coupling of an unsaturated halide with an alkene in the presence of palladium catalyst  to form a substituted alkene; they are also referred to as Mizoroki–Heck reactions.26 The first work of Heck-type coupling was reported by Hacksell and Daves in 1985,27 with a new update in 1990.28 In fact, Heck reactions are very significant in industry, since substitution reactions can be accomplished on planar centers.29 The first time the Heck reaction was discovered it was by the American chemist Richard F. Heck,29 and also Mizoroki, who considerably developed this reaction in organic chemistry.30Heck cross-coupling reactions31,32The C–H functionalization of alkenes is presently under intensive investigation as a direct carbon–carbon bond-forming procedure for the preparation of higher substituted alkenes. The prevalently used Heck reaction enables direct C–H to C–C functionalization of alkenes with aryl and vinyl halides, but is rarely performed with alkyl halide coupling partners due to the propensity of the alkyl group to undergo β-hydride elimination.33–35 according to the above-mentioned explanations, in this work, the connected palladium supported on 5-carboxyoxindole functionalized cell@Fe3O4 (Pd@CAI@cell@Fe3O4), an efficient and interesting bio-based magnetic nanocatalyst, was prepared. Then, this nanocatalyst was employed for the Heck-type arylation of maleimides with iodoarenes  in a Bragg's angle range of 10–80° at 25 °C. Fourier-transform infrared (FT-IR) spectroscopy was carried out using a FT-IR spectrometer  in the range of 400–4000 cm−1 at room temperature. Scanning electron microscopy (SEM) analysis was recorded using a VEGA//TESCAN KYKY-EM 3200 microscope (acceleration voltage 26 kV). Transmission electron microscopy (TEM) experiments were done on a Philips EM 208 electron microscope. The elemental analysis spectrum of the catalyst was assessed by energy dispersive X-ray (EDX) spectroscopy (VEGA3 XUM/TESCAN). Thermogravimetric analysis (TGA) was performed on a Stanton Red craft STA-780 . Nuclear magnetic resonance (NMR) spectra were measured using a Bruker DRX-400 AVANCE instrument  in DMSO-d6 as a solvent. Magnetic measurements were carried out using a vibration sample magnetometer . The metal loading was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Melting points were evaluated on electrothermal 9100 apparatus.Powder X-ray diffraction (PXRD) of the prepared catalyst was performed on a Philips PW 1830 X-ray diffractometer using a Cu Kα source , first, acidic hydrolysis of Whatman filter paper was employed as reported in the literature with a slight modification. Hydrolysis of the cellulose was obtained after 3 h at 100 °C using 100 mL of 2.5 M HBr and alternating ultrasonication. After dilution with twice-distilled water, the mixture was subjected to five washing/centrifugation cycles to remove excess acid and water-soluble residues. After neutralization to around pH 5, the fine cellulose nanoparticles started to disperse into the aqueous supernatant and were collected by centrifugation at 12 000 rpm for 60 min to remove the ultrafine particles.2.2.2.3·6H2O , FeCl2·4H2O  and 100 cm3 of CH3OH/deionized water (50/50), then stirred for 3 h. Next, 10 mL of 25% NH4OH (10 mL) was added immediately into the reaction mixture in one portion under a N2 atmosphere at 80 °C, followed by vigorous stirring for around 30 min using a magnetic stirrer. The product was washed with deionized water four times  was first added to a solution containing FeClur times .2.2.3.3O4@cell was mixed with 400 mg of 5-carboxyoxindole (CAI) in 20 mL of DMSO and stirred for 24 h at 100 °C. After the reaction was completed, the mixture was cooled to room temperature, and the obtained precipitate was filtered using an external magnet and rinsed with ethanol several times, then placed in an oven for 24 h to dry, thus the CAI@ Fe3O4@cell nanoparticles were obtained (400 mg of Feobtained .2.2.4.3O4@cell in DMF was added to a solution of Pd(Cl)2  in 10 mL DMF under N2 atmosphere, and the obtained mixture was stirred for 24 h at 60 °C. After the reaction was completed, the mixture was cooled to room temperature, and the resulting product was collected using an external magnet. The solid black product was washed carefully with deionized water (3 × 25 mL), absolute ether (2 × 25 mL) and absolute ethyl alcohol (2 × 25 mL), then dried in a vacuum oven at room temperature . Acetonitrile (10 mL) was then added, and the reaction mixture was vigorously stirred at 80 °C for different lengths of time, according to each substrate. Upon completion of the reaction (as monitored by thin layer chromatography (TLC)), the reaction mixture was cooled to room temperature. After that, the reaction mixture was dissolved in dichloromethane (10 mL) and, subsequently, the Pd@CAI@cell@Fe3O4 nanoparticle catalyst was separated by an external magnet at 5 min. Removal of the solvent under reduced pressure yielded a crude mixture, which was purified by column chromatography (hexanes/EtOAc gradient) to provide the desired product , iodoarene , triethylamine (2.0 eq.) and Pd@CAI@cell@Fe product .Spectroscopic data for the unknown products are as follows:H-pyrrole-2,5-dione : δ = 3.30 , 7.29–7.33 , 7.47–7.52  ppm. 13C NMR : 24.1, 128.2, 128.3, 128.9, 136.6, 170.2 ppm.1-Methyl-3,4-diphenyl-1,5-dione , entry 3H-pyrrole-2,5-dione : δ = 3.31 , 3.81 , 6.89–6.95 , 7.27–7.31  ppm. 13C NMR : 24.2, 54.7, 112.2, 119.9, 131.2, 133.4, 159.2, 170.2 ppm.3,4-Bis(4-methoxyphenyl)-1-methyl-1,5-dione , entry 53.3.1.3.1.1.3O4, CAI@cell@Fe3O4 and Pd@CAI@cell@Fe3O4 are displayed in −1 exhibits vibration of the C–O–C in the pyranone ring. The peaks for C–H and C–O vibrations in the polysaccharide rings of cellulose are around 1200–1050 cm−1. The absorption bands at 3420 cm−1 and 2998–2850 cm−1 are attributed to the O–H and C–H stretching vibrations, respectively. Also, the adsorption bands at 586 and 520 cm−1 are ascribed to Fe–O stretching band, and those at 3442 cm−1 correspond to broad OH groups on the magnetic surface of the MNPs.The FT-IR spectra of cell, cell@Fe3O4, two absorption peaks were observed at 2990 and 2930 cm−1, which can be assigned to symmetric stretching of the C–H group. The bands situated at 1460 and 1629 cm−1 display symmetric and asymmetric stretching adsorption peaks, and one at 1700 cm−1 can be assigned to the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O group related to 5-carboxyoxindole connected to the cell@Fe3O4 nanoparticles.After synthesis of CAI@cell@Fe−1, which is assigned to the bending vibration of C–H in the pyridine heterocyclic ring, reduces after the formation of CAI@cell@Fe3O4 complex with Pd. Comparison of FTIR spectra results confirm the well grafted 5-carboxyoxindole (CAI) as ligand connected to the Pd metal on CAI@cell@Fe3O4.Also, the intensity of the adsorption peak at 586 cm3.1.2.3O4 is shown in 3O4. The wide peak at 2θ = 22.5° present in both the Pd@CAI@cell@Fe3O4 and cell@Fe3O4 spectra is concluded to be from nanocellulose. Also, the PXRD pattern revealed that standard Fe3O4 crystal has six diffraction peaks, namely, (220), (311), (400), (422), (511) and (440) at 2θ = 30, 35.5, 43.5, 54, 57, 63 , (200) and (220) present in cubic palladium. The average Pd@CAI@cell@Fe3O4 NP diameter was estimated to be approximately 15 nm from the PXRD results by the Scherrer equation, D = kλ/β cos θ, where k is a constant , λ is the wavelength of Cu Kα (1.54 Å), β is the corrected diffraction line full-width at half-maximum (FWHM), and θ is Bragg's angle.The PXRD spectrum of Pd@CAI@cell@Fe, 57, 63 . Also, t3.1.3.3O4. As shown in 3O4 is stable before 200 °C. The mass weight loss of nearly 50% between 200–500 °C is attributed to thermal decomposition of the organic group.TGA was used to determine the thermostability of Pd@CAI@cell@Fe3.1.4.3O4 is presented in The SEM image of Pd@CAI@cell@Fe3.1.5.3O4 nanoparticles was studied by TEM, which is displayed in 3O4 nanoparticles, which is in good agreement with the crystallite size estimated from PXRD, at 15 nm. As shown in 3O4 nanoparticles, and light shell for the organic group) was concluded. This is a presentation of the almost single crystalline character of the Pd@CAI@cell@Fe3O4 nanoparticles.The morphology of the prepared catalysts consisting of Pd@CAI@cell@Fe3.1.6.3O4 is shown in 3O4 is composed of Fe, C, O and Pd, indicating that Pd has been inserted in the desired catalyst. In other words, there is a Pd peak, which is consistent with the ICP-AES results.The EDX analysis of Pd@CAI@cell@Fe3.1.7.3O4 NPs.The ICP-AES analysis shows the weight percentage of Pd to be 10% in the Pd@CAI@cell@Fe3.1.8.3O4, CAI@cell@Fe3O4 and Pd@CAI@cell@Fe3O4 at 300 K  at different temperatures (50 and 80 °C) with Et3N as a base in various solvents. According to the obtained results, which are reported in 3O4 as a catalyst and Et3N (2.0 eq.) as a base at 80 °C in CH3CN  and 4-iodotoluene (2 mmol) in the presence of the Pd@CAI@cell@FeAfterwards, different substituted maleimides and iodoarene derivatives were applied in the Heck-type arylation to prepare the corresponding products, which led to high to excellent yields . As can 3O4 (10 mol%) at 80 °C (reaction time 12 h and yield 84%) is the better catalyst relative to PdCl2 (10% mol) at 100 °C (reaction time 24 h and yield 28%)36 due to the reusability of the catalyst several times with no substantial loss of activity, its short time reaction and good yields of the product.In order to show the accessibility of the present work in comparison with the only reported result in the literature, we summarized some of the results for the Heck-type arylation of maleimide with 4-iodotoluene. The results show that the Pd@CAI@cell@Fe3O4 nanoparticles were studied using a model reaction . According to the observed results, the recovered catalyst was reused for five runs with no loss of activity  were conducted, as shown in 3O4 NPs. As can be seen, the structure of the recycled nanocatalyst has not changed and is quite similar to that of the newly prepared catalyst.Moreover, the FT-IR, PXRD and ICP analysis of recycled Pd@CAI@cell@Fe4.3O4 nanoparticles (Pd@CAI@cell@Fe3O4) as an efficient, novel and reusable heterogeneous nanomagnetic catalyst. The prepared nanomagnetic catalyst was characterized by XRD, ICP-AES, SEM, TEM, FT-IR, TGA, VSM and EDX techniques and was used successfully for the Heck-type arylation between different substituted maleimides with iodoarenes. The Pd@CAI@cell@Fe3O4 nanomagnetic catalyst demonstrated an average particle size of about 15 nm. The nanocatalyst was recovered by simple separation using an external magnet and reused for subsequent cycles. The prepared nanocatalyst exhibited several advantages, including high specific surface area, more active sites, prominent chemical and thermal stability, decrease in the leaching (the release) of the nanocatalyst into the bioenvironment (ecosystem), the presence of organic groups for easier modification, and lower accumulation with respect to other nanocatalysts.In conclusion, we report the preparation of Pd supported on 5-carboxyoxindole functionalized cell@FeThere are no conflicts to declare.


Lys3. During HIV-1 infection, LysRS is phosphorylated at S207, released from a multi-aminoacyl–tRNA synthetase complex and packaged into progeny virions. LysRS is critical for proper targeting of tRNALys3 to the primer-binding site (PBS) by specifically binding a PBS-adjacent tRNA-like element (TLE), which promotes release of the tRNA proximal to the PBS. However, whether LysRS phosphorylation plays a role in this process remains unknown. Here, we used a combination of binding assays, RNA chemical probing, and small-angle X-ray scattering to show that both wild-type (WT) and a phosphomimetic S207D LysRS mutant bind similarly to the HIV-1 genomic RNA (gRNA) 5′UTR via direct interactions with the TLE and stem loop 1 (SL1) and have a modest preference for binding dimeric gRNA. Unlike WT, S207D LysRS bound in an open conformation and increased the dynamics of both the PBS region and SL1. A new working model is proposed wherein a dimeric phosphorylated LysRS/tRNA complex binds to a gRNA dimer to facilitate tRNA primer release and placement onto the PBS. Future anti-viral strategies that prevent this host factor-gRNA interaction are envisioned.Interactions between lysyl–tRNA synthetase (LysRS) and HIV-1 Gag facilitate selective packaging of the HIV-1 reverse transcription primer, tRNA Lys3 serves as the primer and is selectively packaged into virions along with the other major tRNALys isoacceptors, tRNALys1,2 [Lys isoacceptors, is selectively packaged into HIV-1 [Lys are packaged in equimolar proportions, and siRNA knockdown of LysRS resulted in similarly reduced levels (~70%) of tRNALys packaging and viral infectivity [Lys requires interaction with LysRS [Lys packaging; however, the exact mechanism of selecting non-aminoacylated tRNALys3 remains unclear.The fundamental property that distinguishes retroviruses from other RNA viruses is the use of a specific host tRNA as a primer to reverse transcribe their genomic RNA (gRNA) into double-stranded proviral DNA. In HIV-1, human tRNANALys1,2 . Lysyl–tto HIV-1 . LysRS aectivity . Moreoveth LysRS ,7, but nth LysRS , which wN-terminal region of a scaffolding protein known as aminoacyl-tRNA synthetase-interacting protein 2 (AIMP2) [Lys3 binding, S207D LysRS lacked aminoacylation activity [Lys packaging may be an adaptation by HIV-1 to ensure that the primer has a free 3′ hydroxyl to initiate proviral DNA synthesis.Reports on the primary source of LysRS packaged into HIV-1 have been diverse, leaving this an unsettled question. As a result of alternative splicing, two isoforms of LysRS that differ by only ~20 residues at the N-terminus, cytoplasmic and mitochondrial, are expressed in human cells , and bot (AIMP2) ,12. Anot (AIMP2) . MSC rel (AIMP2) ,12,14. W (AIMP2) . Treatme (AIMP2) . Further (AIMP2) . In vitractivity ,15. TherLys3 is released from LysRS and specifically targeted to the primer-binding site (PBS) in the 5′UTR. Just upstream of the PBS lies a tRNA-like element (TLE), which mimics the U-rich anticodon loop sequence of tRNALys3; this TLE binds preferentially to LysRS [Lys3 directly adjacent to the PBS. Nuclear magnetic resonance (NMR) spectroscopy experiments highlighted the strikingly similar interaction of the anticodon binding domain of LysRS with either the TLE or the anticodon stem-loop of tRNALys3 [105) domain mimics that of the canonical L-shape of tRNALys3 [The large size (~9.1 kb) and structural complexity  of the Hto LysRS , promotitRNALys3 . SAXS antRNALys3 . The afftRNALys3 , suggestEarly mechanistic models of LysRS packaging were based on unmodified cytoplasmic LysRS ,17,19,20E. coli strain BL21(DE3) CP-RIL, and proteins were prepared as follows. Starter cultures were grown overnight in 50 mL of LB media containing 100 µg/mL ampicillin at 37 °C. A 10 mL aliquot of this culture was added to 1 L of LB media containing 100 µg/mL ampicillin and grown for 90 min at 37 °C. After cooling cells to room temperature for 15 min, protein expression was induced using 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and incubated in a room temperature shaker overnight (~16 h). Cells were harvested by centrifugation at 8000× g for 15 min at 4 °C followed by resuspension of the cell pellet in 20 mL of lysis buffer  per liter). The cells were lysed by sonication for 12 cycles of 15 s of sonication followed by 45 s of rest. Polyethylenimine  was added, and the solution was stirred gently for 30 min at 4 °C and cleared of cellular debris by centrifugation at 27,000× g for 15 min at 4 °C. Unwanted protein was precipitated by the addition of 375 mg/mL ammonium sulfate, incubated at room temperature for 15 min, and pelleted by centrifugation at 27,000× g for 15 min at 4 °C. The pellet was washed with 50 mL of lysis buffer supplemented with 375 mg/mL ammonium sulfate and re-centrifuged at 27,000× g for 15 min at 4 °C. The pellet was resuspended in 20 mL of lysis buffer and further centrifuged at 27,000× g for 15 min at 4 °C to remove insoluble debris. The supernatant was loaded onto a Ni-NTA (Sigma) column and washed with lysis buffer containing 20 mM imidazole. LysRS constructs were eluted using increasing amounts of imidazole and fractions containing hLysRSΔN65 were combined and concentrated using 30 K molecular weight cut-off centrifugal filter units (Amicon) by centrifuging at 3200× g at 4 °C. The concentrated protein was then dialyzed overnight in a 10 K molecular weight cut-off Slide-A-Lyzer dialysis cassette (Thermo Scientific) into 2× storage buffer  per liter). After dialysis, the protein solution was further concentrated using 30 K molecular weight cut-off centrifugal filter units (Amicon) by centrifuging at 3200× g at 4 °C. The concentrated protein solution was diluted with 1 volume of 80% glycerol  and the concentration was determined using the Bradford assay (Bio-Rad) and stored at −20 °C. The concentration determined by the Bradford assay was multiplied by a correction factor of 1.35  to yield the true concentration. Since hLysRS∆N65 tends to aggregate after extended periods of storage (>6 months), freshly prepared protein was used for all experiments.Plasmid pCDNA3ΔN65.LysRS  was muta18, antiPBS18+3, antiPBS18+6, and antiPBS18+11) were purchased from IDT  and used without further purification. All RNAs—tRNALys3, PBS/TLE, UTR240, UTR240(ΔDIS), and UTR240—were prepared by in vitro transcription using T7 RNA polymerase and purified using denaturing (8 M urea) polyacrylamide gel electrophoresis (PAGE). Human tRNALys3 was prepared from a plasmid purchased from IDT (pIDTSmart-htRNALys3) containing the tRNALys3 sequence flanked by a T7 promoter on the 5′ side and a FokI restriction site on the 3′ side. PBS/TLE, corresponding to nucleotides (nt) 126 to 224 of the HIV-1 gRNA with an additional three 5′ G and three 3′ C nucleotides to enable efficient transcription, was transcribed from a FokI-digested plasmid as described [240 and UTR240(ΔDIS), corresponding to nt 106–345 of HIV-1 gRNA, were PCR amplified from plasmids containing the WT sequence or a dimerization-defective mutant, where the dimerization initiation site (DIS) palindrome (5′ AAGCGCGCA 3′) was replaced by a GAGA tetraloop (ΔDIS). The plasmids contained two additional mutations corresponding to U106G and U108C, and the reverse primer contained two mutations corresponding to U342C and G345C. The mutations allowed for efficient transcription by encoding two 5′ guanosines and helped to stabilize the UTR240 stem. The PCR products were used as templates for in vitro transcription. To determine an optimal RNA:DNA primer ratio for preparing DNA primer-annealed PBS/TLE, the RNA/DNA complex was heated to 80 °C for 2 min, 60 °C for 4 min, followed by the addition of MgCl2 to 1 mM, incubating at 37 °C for 6 min, and cooling on ice for at least 30 min. Complexes were analyzed by native PAGE . The labeling efficiencies were calculated as described [RNAs used for FA assays were 3′-end labeled with fluorescein-5-semithiocarbazide (FTSC) (Invitrogen) as described ,25. Brie240 RNAs were folded in 50 mM HEPES, pH 7.5 by heating at 80 °C for 2 min, 60 °C for 2 min followed by the addition of 10 mM MgCl2, and 37 °C for 15 min, then incubating them on ice for at least 30 min. PBS/TLE and DNA-primer-annealed PBS/TLE complexes were prepared with FTSC-labeled PBS/TLE alone or in the presence of a 1.5 molar excess of DNA primer in 50 mM HEPES, pH 7.5 and refolding them using the heating strategy described above with the 37 °C incubation step shorted to 6 min. FA binding assays were performed as previously described [2. Anisotropy was measured by excitation at 485 nm with the emission read at 525 nm. The data points were fit assuming 1:1 binding [FTSC-labeled UTRescribed  with min binding . All FA 240 RNAs in complex with WT and S207D LysRS(ΔN65) were probed by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) using N-methylisotoic anhydride (NMIA) (Sigma-Aldrich) as described, with minor variations [2, as described above, and then diluted to 0.5 µM in 20 mM HEPES, pH 7.5, 60 mM NaCl, 10 mM KCl and 1 mM MgCl2, as well as different concentrations of the corresponding LysRS variant . Each reaction (10 µL) was incubated at room temperature for 30 min to allow the binding to reach equilibrium. Reactions were initiated with 1 μL NMIA (80 mM stock in DMSO) or neat DMSO for the (+) and (−) reactions, respectively, and incubated at 37 °C for 30 min followed by phenol–chloroform extraction and ethanol precipitation. For RNA-only controls, protein storage buffer was added instead of protein. The following six reactions were included in each experiment: a control reaction of RNA with DMSO, RNA with NMIA, and RNA with NMIA in the presence of the four noted concentrations of LysRS.Purified in vitro transcribed WT UTRriations ,27. Brie2. Non-crosslinked control samples were incubated on ice for the same amount of time. The following six reactions were included in each experiment: a no protein/no UV background, RNA only with crosslinking, and RNA incubated with the four different concentrations of LysRS noted above. Following the crosslinking reactions, protein was denatured with 1 µL of 5% SDS  followed by the addition of 1 µL of Proteinase K  and incubation at 55 °C for 60 min. RNA was recovered by phenol–chloroform extraction and ethanol precipitation.For crosslinking analyses, RNA:LysRS complexes were prepared as described for SHAPE. Samples (10 µL) were crosslinked on ice in a Stratalinker 2400 UV crosslinker (Stratagene) using a total energy of 200 mJ/cmTM labeled primer  and incubation at 85 °C for 1 min, 60 °C for 5 min, 35 °C for 5 min and 50 °C for 10 min. Reverse transcription mix (8 µL) containing 1 µL of SuperScript III (200 U/µL), 4 µL 5× first-strand buffer, 2 µL 0.1 M DTT and 1 µL MiliQ H2O was then added. Samples were incubated at 55 °C for 1 h and inactivated at 70 °C for 15 min following the manufacturer’s protocol (Invitrogen). RNA was hydrolyzed with 1 µL of 4 M NaOH and incubated at 95 °C for 3 min. Samples were cooled on ice and neutralized with 2 µL of 2 M HCl. A description of the primers used for these experiments can be found in RNA samples from both SHAPE and crosslinking experiments were resuspended in 9 µL RNase-free water followed by the addition of dNTP mix (1 µL of 10 mM each) and 2 µL of 5 µM 5′-NED240 constructs, using the Thermo Sequenase Cycle Sequencing Kit (Affymetrix) and the same NEDTM-labeled primers as those used for primer extension . Sequencing reactions were performed independently with each NEDTM-labeled primer. Primer extension products from the SHAPE experiments, crosslinking experiments and sequencing reactions were analyzed by capillary electrophoresis on an Applied Biosystems 3730 DNA Analyzer  after resuspension of the cDNA pellets in formamide and mixing with GeneScan™ 600 LIZ® Size Standard  for inter-capillary alignment as described [Dideoxy sequencing reactions were conducted on the same DNA template used for the in vitro transcription of the UTRxtension . Sequencescribed .t-test. Statistical significance was attributed to peaks characterized by an absolute difference in SHAPE reactivity of ≥ 0.3 arbitrary units and a p-value < 0.05 between the RNA-only control and the highest protein concentration sample [Raw electropherograms were converted to normalized SHAPE reactivity using the ribonucleic acid capillary–electrophoresis analysis tool (RiboCAT) . Peak arn sample . In addi240(ΔDIS) and UTR240 constructs (400 µg) were refolded in a volume of 200 µL by heating in 50 mM HEPES, pH 7.4, to 80 °C for 2 min, 60 °C for 2 min, followed by addition of 10 mM MgCl2. The samples were then incubated at 37 °C for 15 min, and on ice for at least 30 min. The folded RNAs were purified by size exclusion chromatography (SEC) on a 24-mL Superdex 200 10/300 GL Increase column  using a GE ÄKTA purifier in a buffer containing 150 mM NaCl, 50 mM HEPES, pH 7.4, 1 mM MgCl2, and 3% glycerol (wt/vol) at a flow rate of 0.5 mL/min. The absorbance of the eluate was monitored at 260 nm. Fractions corresponding to the peak of interest were concentrated to ~40 µL and serially diluted to yield three different concentrations for the analysis of each sample. Prior to shipment to the SIBYLS beamline at Lawrence Berkeley National Labs for data collection [The UTRllection . There wg of the RNA, and Kratky analysis was performed to determine the extent of folding [max and P(r)-based Rg was determined using the AUTOGNOM feature. Ab initio envelopes were generated largely as previously described using the ATSAS software suite [2 fits and reproducibility (NSD) values were determined. These 50 envelopes were averaged into one envelope, which was then packed with additional “dummy atoms” with an atomic radius of 2.0 Å. This was then used as the starting point for an additional 50 ab initio envelope calculations, generated in expert mode with no symmetry restraints imposed. These envelopes were averaged and filtered to generate the final envelope, and their χ2 fits and NSD values were calculated.SAXS data analysis steps were carried out as previously described ,31 using folding , and there suite ,31,32. B18, antiPBS18+3, antiPBS18+6, and antiPBS18+11 DNA primers) were prepared using 1 mg total nucleic acid with a 1.5 molar excess of DNA primer and refolded in a volume of 200 µL by heating the sample in 50 mM HEPES, pH 7.4, to 80 °C for 2 min, 60 °C for 4 min, followed by the addition of 10 mM MgCl2. The sample was then incubated at 37 °C for 6 min and on ice for at least 30 min. For LysRS-bound PBS/TLE complexes, PBS/TLE was first refolded as described for the primer-annealed PBS/TLE complexes and purified by SEC on a 24-mL Superdex 200 10/300 GL Increase column  using a GE ÄKTA purifier in a buffer containing 150 mM NaCl, 50 mM HEPES, pH 7.4, 1 mM MgCl2, and 3% glycerol (wt/vol) at a flow rate of 0.5 mL/min. The absorbance of the eluate was monitored at 260 nm. Fractions corresponding to the peak of interest were concentrated to ~40 µL. Purified PBS/TLE was then incubated with a 1:1 molar ratio of either WT or S207D LysRS for 30 min prior to SEC-SAXS analysis.Size-exclusion chromatography coupled with SAXS (SEC–SAXS) was used to analyze complexes to alleviate problems with sample heterogeneity. Each of the DNA primer-annealed PBS/TLE RNA complexes  at a flow rate of 0.5 mL/min. The absorbance of the eluate was monitored at 260 nm. The ÄKTA Pure M is coupled to the beamline such that the sample goes directly from the absorbance detector into a flow-cell in the beamline. The X-ray source was a 1.5 m CHESS Compact Undulator, and X-rays were detected using a Finger Lakes charged–coupled device (CCD). The beam diameter was 250 × 250 µm with a flux of 8.4 × 1011 photons s−1 and an energy of 9.962 keV. X-ray exposures were taken continuously every 4 s while the SEC eluent passed through the SAXS flow cell. Initial SAXS data processing, including radial averaging and buffer subtraction, was performed using the BioXTAS RAW software program [SEC–SAXS data were collected at station G1 at the Cornell High Energy Synchrotron Source (CHESS) using the experimental set-up described . Briefly program . SAXS ex program . Average program .N-terminal 65 residues deleted is more sensitive to specific RNA binding interactions [Lys3, the 105-nt PBS/TLE105 domain alone or constructs (UTR240 and UTR240(ΔDIS)) containing both the PBS/TLE and Psi domains. The first nine nucleotides of the gag coding region were included because they contribute to the structure and stability of the packaging signal [240 RNA showed complete dimerization under the conditions used for FA assays (Lys3 and PBS/TLE105 with similar affinity but bound the Psi-containing WT UTR240 with approximately two-fold higher affinity (Lys3 and PBS/TLE105 for both LysRS(ΔN65) variants. Therefore, the higher binding affinity is either due to specific interactions with the DIS sequence or gRNA dimerization/conformational differences.A LysRSΔN65) variant with the ractions , and a t variant ractions ,18,19, bractions ,37. Therg signal , but theg signal . Native A assays . The ΔDIA assays B. To proA assays A. Both Waffinity . This claffinity B reducedMAX). Signs of aggregation, radiation damage or heterogeneity were absent from the obtained SAXS data (MAX = 123 Å), the binding of WT and S207D LysRS(ΔN65) resulted in more extended envelopes with DMAX of 139 and 162 Å, respectively (Results of the binding assays suggest that WT and S207D LysRS(ΔN65) have similar RNA binding affinities, displaying somewhat higher affinity binding to the dimeric gRNA. SAXS was next used to investigate the structures formed upon WT and S207D LysRS(ΔN65) binding to the PBS/TLE RNA. SAXS yields ab initio molecular envelopes that reflect the global fold of the molecule or complex with a calculated maximum dimension  binding sites and RNA conformational changes upon protein binding, XL-SHAPE studies were carried out using the WT UTRcriptase ,40. SHAPcriptase ,41. SHAPcriptase . To iden240  binding when compared to WT LysRS(ΔN65) . Indeed, 22 of the 25 differences upon titration of S207D LysRS(ΔN65) are increases in SHAPE reactivity, located predominantly in the PBS loop and SL1 stem  gRNA construct. The SAXS data showed no signs of aggregation, radiation damage or heterogeneity  RNA with a GAGA tetraloop inserted in place of the PBS/TLE domain. PBS/TLE truncation resulted in a structural model wherein the L-shaped region is missing from the ab initio envelope (240(ΔDIS) with the PBS/TLE domain replaced with a tetraloop, termed the “core encapsidation signal”, was previously determined by NMR [The binding assays and XL data suggest that both LysRS(ΔN65) constructs directly bind the TLE and SL1 stems of the UTRogeneity . The resogeneity A. Furthed by NMR . To assi240(ΔDIS). This orientation is too distant for a single WT or S207D LysRS(ΔN65) homodimer (240(ΔDIS) relative to the WT UTR240 is not due to misfolding of the RNA. Rather, tighter binding of both LysRS(ΔN65) variants to the UTR240 requires gRNA dimerization.Based on our SAXS data, we conclude that the TLE and SL1 are oriented in opposite directions in the monomeric UTRomodimer D to bindLys3 in close proximity to the PBS due to competition by the TLE for LysRS binding [Previous work demonstrated that the TLE hairpin is the primary determinant for LysRS(ΔN65) binding to gRNA via anticodon loop sequence mimicry and structure mimicry of the canonical tRNA L-shaped fold ,19,37. I binding ,19. Ther18 and antiPBS18+3 should not significantly alter the TLE structure, but annealing of antiPBS18+6 and antiPBS18+11, with complementary regions that extend into the TLE stem, would be expected to cause gradual unfolding of the TLE hairpin. Reverse transcription pauses were observed at +3 and +5 sites upon primer extension, due to the stability of the TLE stem [To test this hypothesis and characterize the conformational changes that occur during reverse transcription initiation, binding assays were performed between WT and S207D LysRS(ΔN65) variants and PBS/TLE constructs annealed to short DNA oligonucleotides resembling different stages of initial reverse transcription products A. The anTLE stem . As expe18 annealed  RNA construct adopts a cruciform-like structure with the PBS/TLE and SL1 on opposite ends of the structure (The fact that LysRS binds to both the PBS/TLE and SL1 suggests that these regions of the 5′UTR may be in close proximity in the three-dimensional structure. However, SAXS data revealed that the UTRtructure . Based oThe first secondary structure element that reverse transcriptase encounters during minus-strand strong-stop cDNA synthesis is the TLE stem , which rBased on these results, a new working model is proposed . In this


The simultaneous increasein the electrical conductivity (σ) and Seebeck coefficient (α)with WC (tungsten carbide) volume fraction (z) resultsin an enhanced power factor (α2σ) in the composite.The rise in σ is attributed to the creation of favorable currentpaths through the WC phase located between grains of Ge0.87Mn0.05Sb0.08Te, which leads to increased carriermobility in the composite. Detailed analysis of the obtained electricalproperties was performed via Kelvin probe force microscopy (work functionmeasurement) and atomic force microscopy techniques  characteristics), which are further supported by density functionaltheory (DFT) calculations. Furthermore, the difference in elasticproperties  between Ge0.87Mn0.05Sb0.08Te and WC results in a high AIM, and hence,a large interface thermal resistance (Rint) between the phases is achieved. The correlation between Rint and the Kapitza radius depicts a reducedphonon thermal conductivity (κph) of the composite,which is explained using the Bruggeman asymmetrical model. Moreover,the decrease in κph is further validated by phonondispersion calculations that indicate the decrease in phonon groupvelocity in the composite. The simultaneous effect of enhanced α2σ and reduced κph results in a maximumfigure of merit (zT) of 1.93 at 773 K for (1-z)Ge0.87Mn0.05Sb0.08Te-(z)WC composite for z = 0.010. It resultsin an average thermoelectric figure of merit (zTav) of 1.02 for a temperature difference (ΔT) of 473 K. This study shows promise to achieve higher zTav across a wide range of composite materials.The preparation ofcomposite materials is a promising methodologyfor concurrent optimization of electrical and thermal transport propertiesfor improved thermoelectric (TE) performance. This study demonstrateshow the acoustic impedance mismatch (AIM) and the work function ofcomponents decouple the TE parameters to achieve enhanced TE performanceof the (1- Therefore, technologythat can utilize it is essential for the efficient use and productionof energy in general. Thermoelectric (TE) energy conversion technologythat can harvest waste heat energy using temperature gradient withoutemitting pollution is a propitious solution for waste heat recoveryand niche power resources.21 The simultaneous filtering of the charge carrier and enhanced phononscattering at the interface between the Bi0.4Sb1.6Te3-Cu2Se nanocomposite results in an enhanced zT (∼1.6 at 488 K).16 Kimet al. reported an improved zT (∼1.85) inthe PbTe-PbSe composite because of the synergistic effect of reducedκph and enhanced α2σ.22 A notable reduction in κph wasalso observed in several composites with nanostructured secondaryphase and is mainly attributed to the quantum size effects.27 In composite materials, the interface thermal resistance (Rint), which originates from the acoustic impedancemismatch (AIM) between the phases is rarely considered for the optimizationof κph.28 However, thermalresistance at the phase boundary is often used in the descriptionof the heat transport in ceramic and polymer composites, includingZnS/diamond,28 SiC/Al29, andglass/epoxy.30 These reports along withour previous studies on composite materials demonstrate that interfacethermal resistance between the phases of the composite (and Rint as a parameter) is crucial in designingTE composite materials with the desired κph.12Moreover, the preparation of compositematerials is promising forsimultaneous optimization of electrical and thermal transport propertiesto obtain an improved TE performance. This approach allows the utilizationof a few transport phenomena that can result in improvement of theσ/κ ratio.21 cm–3), high thermal conductivity(∼8 W·m–1·K–1), and low Seebeck coefficient (∼30 μV·K–1) and hence poor zT.31 Several innovative approaches have been demonstrated in recent timesto achieve enhanced thermoelectric performance in GeTe including manipulationof Ge vacancies,32 band convergence,35 crystal structure modification,37 resonance-leveldoping39 high-entropy concept,40 and so forth and are based on the atomic doping strategies.Herein, we demonstrate a novel composite approach that considers theoptimized GeTe (Ge0.87Mn0.05Sb0.08Te via band-structure and lattice dynamics engineering41) as matrix and tungsten carbide  as the second phase. In composite,interface between phases plays a vital role in determining the electricaland thermal conductivity. However, the role of interface has beenneglected or underestimated in optimizing the performance of a thermoelectricmaterial. The present study focuses on the effect of WC addition onthermoelectric properties of the Ge0.87Mn0.05Sb0.08Te-WC composite considering the role of workfunctionand interface thermal resistance on electrical and thermal conductivityin the Ge0.87Mn0.05Sb0.08Te-WC composite.The effect of WC on electrical transport has been analyzed using theKelvin probe and atomic force microscope via measuring the work functionof both materials, current–voltage characteristics, and currentdistribution map. Furthermore, the electronic band structure for theMn-Sb co-doped GeTe-WC composite is calculated using the density functionaltheory (DFT). The large difference in elastic properties between Ge0.87Mn0.05Sb0.08Te and WC has been usedto control κph in the composite using the interfacethermal resistance (Rint) between thephases, estimated from the acoustic impedance model (AIM) and theDebye model. The κph in the composite is furtheranalyzed using the Bruggeman asymmetrical model, which considers Rint between the phases. Furthermore, the phonondispersion calculation for the Mn-Sb doped GeTe-WC composite is alsoperformed to establish the decrease in κph.GeTe-based materials are promising forTE application in the mid-temperaturerange (500–800 K). However, pristine GeTe suffers from theintrinsic Ge vacancies that result in a high hole carrier concentration(∼1022.1z)Ge0.87Mn0.05Sb0.08Te-(z)WC compositeis shown in 0.87Mn0.05Sb0.08Te has a rhombohedralstructure (space group: R3m) at 300 K, with the latticeparameters a = b = 4.1709 Å, c = 10.5612 Å in a hexagonal configuration. The reflectionintensity of WC is not prominently observed because of its low volumefraction in the composite. However, the main reflections (001) and(100) of WC are revealed in the log-scale (inset of z) in the composite.X-raydiffraction pattern of the (1-inset of 1 and conz)Ge0.87Mn0.05Sb0.08Te-(z)WC composite. The sintered Ge0.87Mn0.05Sb0.08Te is homogenous andone does not observe pores and impurity phases, as well as grain boundariesin nonchemically etched samples 0.87Mn0.05Sb0.08Te (z). One can also observeirregular pores at points of contact of three or more grains. However,the pores are rarely observed at grain boundaries between two adjacentgrains. Therefore, the samples’ relative density is very highand lies in the range of 98–99%, and the pores should not significantlydetermine transport properties. z = 0.010. The zoom-inimage for the same sample is shown in the inset of 0.87Mn0.05Sb0.08Te-WC sample areconfirmed by the EDS analysis, as shown in 0.87Mn0.05Sb0.08Te grains. The structuraland microstructural analysis shows the existence of both individualphases in the composite samples. It further confirms the satisfyingpurity of the composite constituent and the high density of sinteredmaterials. and GeTe-WCcomposites are shown in z =0) polycrystalline Ge0.87Mn0.05Sb0.08Te phase (having GeTe structure) is homogenous and fine grains arewell sintered, ensuring good electrical and thermal contacts  analysis reveals moredetails concerningthe microstructure and charge transport phenomena near the grain boundaries.The surface topology and corresponding current distribution for GeTeGe0.87Mn0.05Sb0.08Te-(z)WC with z = 0.02 composite was subjected to the additional analysis utilizingthe AFM method to determine the current distribution on the surfaceof the material (I–V) characteristic (0.87Mn0.05Sb0.08Te (marked with a dashed closed path) in comparison to within thegrains. Nevertheless, there is still a noticeable amount of the currentflowing through the volume of the Ge0.87Mn0.05Sb0.08Te. This indicates that the percolation thresholdis not reached.Single-phase Gematerial 3c, d andteristic 4. The cuteristic 3c is disteristic 3d, the cI–V characteristicfor single-phase Ge0.87Mn0.05Sb0.08Te shows a typical semiconductor behavior in combination with the presence of partial percolation paths of WCgrains.0.87Mn0.05Sb0.08Te and between GeTe-WC composite, a semi-logplot is presented in ca. −500to 500 nA), measured current differs between materials by a few ordersof magnitude, which shows how much easier the current flows throughthe polycrystalline Ge0.87Mn0.05Sb0.08Te when metallic WC is located at the grain boundaries. The aboveobservations are consistent with the current distribution map shownin To further emphasize how significant is the differencein the mechanismof the current flow between a single-phase Ge2.3z)Ge0.87Mn0.05Sb0.08Te-(z)WC (0 ≤ z ≤ 0.02) as a function oftemperature is shown in 0.87Mn0.05Sb0.08Te at 300 K is 1150 S·cm–1, andit increases to 1342 S·cm–1 for z = 0.010 and 1501 S·cm–1 for z = 0.020. This increase in σ may be attributed to the highσ of the dispersed phase .42Theelectrical conductivity (σ) for  as a function of WCvolumefraction at 300 K is shown in m and σ represent the electricalconductivity of the matrix and the composite, respectively, z is the volume fraction, and s is a constant,with a well-established and universal value of 0.87.43 This model was fitted to the experimental data using Originsoftware with percolation threshold as a parameter. The solid linein z)Ge0.87Mn0.05Sb0.08Te-(z)WC composite using percolation theory with percolation threshold(zc) of 0.073 (Pearson’s correlationcoefficient R2 = 0.971). This indicates that the volumefraction of WC required for the creation of continuous percolationpaths in the composite is relatively small (∼7%). The volumefraction used in the present study (z = 0.005–0.02)is lower than the threshold value (∼7%), and hence, partialpercolation is observed in the current distribution map, as shownin Here, σn) and carrier mobility (μ) with an increase in WC volume fraction(z) in the composite using the relation σ = neμ, where e is the electronic charge.44 The carrier concentration (n) measured using Hall measurement and corresponding carrier mobility(μ) estimated using the σ and n are shownin the inset of 45 The increase in μ in the composite sample may be attributedto the filtering of high-energy carriers at the interface betweenthese two phases in the composite (discussed later).47Next, we discuss the change in carrier concentration (z)Ge0.87Mn0.05Sb0.08Te-(z)WC (0 ≤ z ≤0.02) is shownin z = 0.010. The α for Ge0.87Mn0.05Sb0.08Te at 300 K is ∼93 μV·K–1 and increases to (∼100 μV·K–1) up to z = 0.010. However, witha further increase in WC volume fraction, α decreases to 78μV·K–1 for z = 0.020.These values of α are consistent with the αav obtained from the scanning thermoelectric microprobe (STM) analysis(Supporting Information (SI), Figure S1). The increase and decrease in α are consistent with the carrierconcentration change in the composite ,as shown in 2σ increases with the increase intemperature up to ∼700 K and then decreases. The power factorincreases from 1.32 mW·m–1·K–2 at 300 K and reaches ∼3.8 mW·m–1·K–2 at 700 K for the composite with z = 0.010. Because of a significant decrease in α for higherWC volume fraction (z = 0.020), α2σ decreases.The temperature-dependent Seebeck coefficient (α) for forboth the individual phases using the Kelvin probe force microscopy(KPFM) technique.48 KPFM is a useful toolto estimate the relative position of the Fermi level in solids.49 The work function for individual phases is calculatedby measuring the contact potential difference (CPD) using the KPFM.The CPD is defined astip and φsample are the AFM probe and sample work functions,respectively. The work function of the AFM probe (φtip = 4.15 eV) was calibrated by measuring the potential map of freshlycleaved and highly oriented pyrolytic graphite HOPG with the knownvalue of work function (φHOPG = 4.6 eV).50 Furthermore, the work functions of the samples(φsample) are obtained using φsample = φtip + CPD. The surfaces of both the samples(Ge0.87Mn0.05Sb0.08Te and WC) andHOPG were scanned alternatively to determine the CPD values.It is noted that the increase in α0.87Mn0.05Sb0.08Te and WC phases areshown in 0.87Mn0.05Sb0.08Te and WC is 4.5 ±0.14 and 4.37 ± 0.06 eV, respectively. It is worth noting thatthe mean values of CPD for HOPG analyzed before and after measurementsare close (449 vs 461 mV). The work function obtained from the KPFMmeasurement is used to design the band diagram for both the phasesand is shown in 0.87Mn0.05Sb0.08Te)–metal (WC) junction, therecan be either Schottky contact  or Ohmic contact .52 The present study shows that the work function for WC is smallerthan that of Ge0.87Mn0.05Sb0.08Te,indicating an Ohmic contact and hence further supports the linearnature of the I–V curve in 0.87Mn0.05Sb0.08Te, supporting the enhanced σ in the system. Also,the energy difference (ΔEf) betweenthese two materials is quite small (0.16 eV), which helps to enhanceσ in the composites. This small difference in ΔEf may scatter the lower energy carrier at theinterface and allows the high energy carriers with increased μto improve α due to energy filtering.53 This indicates that the slight mismatch in ΔEf enhances both σ and α in the composite.The spatial variation of CPD and two-dimensional surface topographyobserved for both phases are shown in 2.3.20.87Mn0.05Sb0.08Te-WC composite,density functional theory (DFT) calculations are performed. The detailsof electronic structure calculations are shown in the SI . First, we have examinedthe stability of the Ge19MnSb2Te24-WC composite by calculating its binding energy (Eb), which is defined asE(Ge19MnSb2Te24-WC), E(Ge19MnSb2Te24) and E(WC) are, respectively,the total energies of the Ge19MnSb2Te24/WC composite, Ge19MnSb2Te24 matrix,and WC particles. Ge19MnSb2Te24 correspondsto Ge0.87Mn0.05Sb0.08Te, consideringthe intrinsic Ge vacancies during calculations, as shown in the earlierstudy.4119MnSb2Te24, WC, and layered Ge19MnSb2Te24-WC system, which represents an interfacebetween these materials in the composite.For further exploration ofthe enhancement of electrical conductivityin the Ge19MnSb2Te24-WC composite is Eb = −1.85 eV. The negative value of Eb implies that the composite is thermodynamicallystable.Subsequently, we have calculated the band structures for Ge19MnSb2Te24, WC, and Ge19MnSb2Te24-WC supercells  behavior.The result agrees well with the linear I–V characteristic recorded for the composite, presented in the insetof The obtained value of the binding energy for theGepercells 7d–f. As 19MnSb2Te24, we have plotted the electrostatic potentialenergy for both materials separately. 19MnSb2Te24 and WC along thec-direction from 19MnSb2Te24 and WC are 4.51 and 4.36eV, respectively, which are in good agreement with the experimentalresults given using the KPFM method. The smaller work function φof WC in relation to the Ge19MnSb2Te24 matrix indicates that charge can flow from WC inclusions to thematrix, leading to an increase in the effective electrical conductivityof the composite. To better visualize the charge transfer, we haveplotted the 3D charge density difference at the interface betweenthe Ge19MnSb2Te24 and WC, as shownin 19MnSb2Te24-WC composite.To examine the charge transfer between WC and Ge2.4T) for the (1-z)Ge0.87Mn0.05Sb0.08Te-(z)WC composite is shown in z of WC in the composite. A noticeable increasein κ in the whole temperature range is seen for the sample with z = 0.020. This increase in κ seems to be obviousbecause WC possesses extremely high κ (∼170 W·m–1·K–1 at 300 K).42 However, this sample does not follow the trendwith materials with lower WC amounts. Because κ consists oftwo components, electronic thermal conductivity (κe) and phonon thermal conductivity (κph), that is,κ = κe + κph, the additionof WC to the composite improves σ and hence must increase κe. To clarify the nature of the electron and phonon contributionto thermal conductivity, both κe and κph in the composite system are separated from κ by calculatingthe κe using Wiedemann Franz law κe = LσT, where the Lorenz number (L) was calculated using the equation proposed by Snyder and co-workers: L =–8 W·Ω·K–2, where α is the Seebeck coefficient in μV·K–1.54Temperature-dependenttotal thermal conductivity κ(2.4.1ph for the(1-z)Ge0.87Mn0.05Sb0.08Te-(z)WC composite is shown in ph of the composite decreases with the increasein temperature, and interestingly, it is overall lower for a highervolume fraction of WC. The changes in electronic (κe) and phonon thermal conductivity (κph) as a functionof the WC volume fraction at 300 K are shown in the inset of ph of WC is relatively high (∼135 W·m–1·K–1 at 300 K)42 compared to Ge0.87Mn0.05Sb0.08Te despite the fact that κph of the composite decreaseswith the addition of the WC phase. This peculiar decrease in κph is analyzed using Bruggeman’s asymmetrical modelthat considers the interface thermal resistance (Rint) between the phases in the composite. The Rint for the composite is estimated using theacoustic impedance model (AIM) and the Debye model.56 The AIM shows that the phonon propagating from one material to anothercan be reflected if there is a mismatch in the acoustic impedance Z = v·ρ between the two materials.29 The sound velocity (v) andsample density (ρ) measured for both Ge0.87Mn0.05Sb0.08Te and WC and their calculated acousticimpedance are presented in 0.87Mn0.05Sb0.08Te (matrix) and WC (dispersed phase) is η= 4.36%.It is given by the formula η = pq, where vm) and dispersed phase(vd)49 andthe acoustic impedance for the matrix Zm and that of the dispersed phase Zd.Temperature-dependent κRint betweenthe Ge0.87Mn0.05Sb0.08Te-WC phasesfollowing the Debye model Rintcp isthe specific heat capacity of the matrix and cp, ρ, vD, and η, the estimated Rint for Ge0.87Mn0.05Sb0.08Te and WCis 2.535·10–6 m2·K·W–1. This value of Rint ishigher than that for several other TE composites and is beneficialfor improving phonon scattering between the phases in the composite.12 The Rint also givesan important parameter of the critical grain size called Kapitza radius aK calculated from the formula aK = Rint·κph,m. If highly conductive particles are introduced into thelow-conducting matrix, the effective conductivity of the compositecan be decreased if the size of the particles is lower than that of aK.28 Using the phononthermal conductivity of the matrix  and Rint, aK for Ge0.87Mn0.05Sb0.08Te-WC composite is 3.40μm at 300 K. A high Rint providesa larger aK and it suggests that κph of the composite can be reduced if the particle size ofWC is smaller than 3.40 μm. The consideration of AIM and theDebye model is important to estimate the critical size for a compositeto reduce its κph.The probability of phonon transmission obtainedfrom the AIM modelis used to calculate the ph of the Ge0.87Mn0.05Sb0.08Te/WC composite, using aK obtained for the composite, by the formula30ph,d and κph are the thermal conductivity of the dispersedphase (WC) and the composite, respectively, and s = a/aK, where a is the actual particle size of the dispersed phase. κph calculated using Bruggeman’s asymmetrical model forGe0.87Mn0.05Sb0.08Te-WC compositewith z = 0.020 at 300 K as a function of differentparticle sizes of WC is shown in the inset of ph decreases with the decrease in the particle size of WC.It reduces below κph,m (1.35 W·m–1·K–1) when a < aK  for different particle sizes of WC at 300 K. The κph increases when a > aK. It indicates that even a relatively high acoustic mismatchbetween Ge0.87Mn0.05Sb0.08Te andWC will cause an increase of κph when the particlesize of WC is too large. However, κph remains constantfor a ≈ aK, andit decreases when a < aK and indicates that Rint becomes veryprominent due to the very high contact area between the particlesand the matrix and hence reduces the κph. It alsosuggests a strong correlation between Rint and aK.12 In other words, the smaller particle size of the dispersed phaseenhances the surface-to-volume ratio and hence strongly reduces κph. A schematic of the Ge0.87Mn0.05Sb0.08Te-WC composite with different particle sizes of WC isshown in ph even for high Rint. On the otherhand, if the particle size of the dispersed phase is smaller than aK, the Rint becomesprominent with an increased interface surface area to the volume ratioand reduces κph.2.4.219MnSb2Te24-WC were plottedusing the Phonopy code , where vg = dω/dk. As can beseen in 19MnSb2Te24/WC is lower than that of WC and Ge19MnSb2Te24.41 This suggeststhat the Ge19MnSb2Te24/WC has lowerlattice thermal conductivity in comparison to Ge19MnSb2Te24 and WC. These findings are in good agreementwith the experimental results.Next,to examine the thermal stability, phonon dispersion curves for WCand Gecode see 10a, b. A2.5zT) of the composite is calculatedand is shown in zT increases with temperature forall the samples. The zT also increases with WC volumefraction and shows a maximum of 1.93 at 773 K for the sample with z = 0.010. This enhancement in zT is attributedto the simultaneous rise in σ and α for composite alongwith reduced κph owing to the AIM between the phases.However, the WC fraction higher than 0.010 reduces zT because of a significant reduction in α.Usingthe experimentally observed TE parameters α, σ, and κ,the figure of merit (zTav (T = TH – TC) between the hot (TH) andcold (TC) side of the device. The zTav calculated for the (1-z)Ge0.87Mn0.05Sb0.08Te-(z)WC composite with 300 and 773 K as TC and cold TH, respectively, is shownin zTav for the sample with z =0.010 is the highest. It reaches ∼1.02 for a temperature differenceof ΔT = 473 K. The obtained zTav in the present study is higher than that previouslyreported in several literature reports.61 A comparison for the same is shown in av in GeTesuch as Sc-Bi co-doped GeTe,36 Cr-Bi co-doped GeTe (zTav ∼ 1.2 from 300 to 723 K),32 Bi-Zn co-doped GeTe (zTav = 1.35 from 400 to 800 K);33 however,because of differences in their temperature range studies, they arenot included in zTav obtained in the present study for the p-type material and assuminga corresponding similar n-type leg, theoretical energyconversion efficiency (η) is calculated for the temperaturedifference (ΔT) and shown in z)Ge0.87Mn0.05Sb0.08Te-(z)WC for z = 0.010 composite and is higher than thoseof several other promising TE materials reported in the literature.61The energy conversion efficiency of TE devices made up ofTE materialsis defined as 30.87Mn0.05Sb0.08Te-WC composite.X-ray diffraction analysis confirms the individual phases in the composite,which is further supported by electron microscopy images and energy-dispersiveX-ray spectroscopy analysis. Enhancement in the composite’selectrical conductivity (σ) is attributed to the increase incarrier mobility (μ). It is also analyzed using the work functionmeasurement using the Kelvin probe force microscopy technique. Thelower work function of WC compared to Ge0.87Mn0.05Sb0.08Te gives high mobility charge carriers to the systemand hence increases σ. Additional AFM analysis showed highercurrent flow near the boundary of Ge0.87Mn0.05Sb0.08Te grains for composites in comparison to the single-phasematerial. The current–voltage (I–V) characteristics indicate the Ohmic contact between Ge0.87Mn0.05Sb0.08Te and WC grains. Thedensity functional theory (DFT) calculations further support the increasein electrical conductivity and linear I–V characteristics in the composite. The acoustic impedancemismatch (AIM) between the composite phases leads to a high interfacethermal resistance (Rint), beneficialfor improving phonon scattering. A correlation between Rint and the Kapitza radius (aK) decreases the phonon thermal conductivity (κph) of the composite, supported by Bruggeman’s asymmetricalmodel. Furthermore, the phonon dispersion calculations suggest a decreasein phonon group velocity in the composite. The simultaneous effectof the work function and AIM shows an improved power factor and reducedκph. As a result, a maximum zT of1.93 at 773 K with a zTav ∼ 1.02for a temperature difference of 473 K is obtained for (1-z)Ge0.87Mn0.05Sb0.08Te-(z)WC with z = 0.010. A maximum energy conversionefficiency (η) of ∼14% is calculated, considering a similarn-type material. This study shows promise to further develop efficientthermoelectric composite over a wide range of materials having similarelectronic structures and different elastic properties (consideringthe correlation between Rint and aK).This study demonstratesthe influence of work function and AIMon electronic and phonon transport properties, respectively, in theGe44.10.87Mn0.05Sb0.08Te has been carried out by the direct melting of elements  with purity >99.99% (Alfa Aesar) in evacuated quartzampoulesfollowing our previous report in GeTe.41 Furthermore, the (1-z)Ge0.87Mn0.05Sb0.08Te-(z)WC composite with z = 0.000, 0.005, 0.010, and 0.020 is prepared by mixingthe different volume percentage of WC . Thecomposite mixture was ground to mix homogeneously in a liquid medium(acetone) using a mortar and a pestle. The mixture was then sinteredusing the pulsed electric current sintering (PECS) technique in theAr (5 N) atmosphere at 873 K for 5 min under a uniaxial pressure of50 MPa with a heating and a cooling rate of 70 and 50 K/min, respectively.The obtained cylindrical pellets of 10 mm diameter and 12 mm lengthwere cut to proper dimensions using a precise wire saw for furthermeasurements. The surface morphology and chemical analysis of thepolished sample surface were done using a scanning electron microscope equipped with an EDXS analyzer.The X-ray diffraction of samples was obtained by the D8 ADVANCE (BRUKER)diffractometer using Ni-filtered Cu-Kα radiation(λ = 1.5406 Å). The bulk density of the sintered pelletswas measured using sample mass and their geometrical volume.The synthesis of Ge4.2z)Ge0.87Mn0.05Sb0.08Te-(z)WC composite was done using a scanning thermoelectricmicroprobe at 300 K with a spatial resolution of 50 μm. Thermaldiffusivity for all the samples was measured using the laser flashanalysis  apparatus in the Ar (5 N) atmosphere (30mL/min). Specific heat was determined simultaneously with the thermaldiffusivity using pyroceram 9606 as a reference material. The sampledensity was measured using sample mass and its geometric volume. Electricalconductivity and Seebeck coefficients were measured under the Ar (5N) atmosphere (50 mL/min) using the SBA 458 (NETZSCH) apparatus. Theuncertainty of the Seebeck coefficient and electrical conductivitymeasurements is 7 and 5%, respectively. The estimated uncertaintyin thermal conductivity is 7%. The carrier concentration was measuredat 300 K using a physical properties measurement system  under the magnetic field of ±3 T. Work function measurementswere performed using an atomic force microscope working in the Peak Force KPFM mode. PFQNE-AL probes  were used for capturing topography andpotential maps under ambient conditions. The images were capturedwith a resolution of 84 × 256 pixels. The work function of theAFM tip (ϕtip = 4.15 eV) was determined by measuringthe CPD of freshly cleaved HOPG with the known value of the work function(4.6 eV). The CPDs of HOPG before and after measurements were virtuallythe same (0.45 and 0.46 V), excluding tip contamination. The workfunction of the analyzed sample was calculated using I–V curves were performed usingthe same AFM probes.The spatialvariation of the Seebeck coefficient for the .65 The self-consistency loop was convergedwith a total energy threshold of 0.01 meV. The structures were fullyrelaxed until the Heymann–Feynman forces on each atom wereless than 10–5 eV/Å. The structural optimizationwas carried out using generalized gradient approximation (GGA) expressedby the Perdew–Burke–Ernzerhof (PBE)66 exchange-correlation functional. The effects of dopingwere considered by substituting Mn and Sb atoms at the specific sitesof Ge atoms. 2 × 2 × 1 and 3 × 3 × 1 supercellswere employed for GeTe and WC so that there exists a minimum latticemismatch of ∼3.25%. A 6 × 6 × 1 k-mesh was used for Brillouin zone sampling for the Ge0.87Mn0.05Sb0.08Te-WC composite. The periodic unitswere separated by a vacuum layer with 20 Å thickness along the Z-direction to prevent spurious interactions between periodicimages. The two-body vdW interaction as devised by Tkatchenko-Schefflerhas been employed.68 The correction parameter is basedon the Hirshfield partitioning of the electron density. The electronwave function was expanded in a plane-wave basis set with an energycutoff of 600 eV. Spin–orbit coupling interactions owing toheavy atoms were included when calculating the electronic structures.Phonon calculations were obtained within the harmonic approximationand using a finite displacement method.69 A 2 × 2 × 2 supercell was set for the calculations. Thestarting parameters for the calculations were the values obtainedfrom the refinement.The DFT


Fervidobaterium pennivorans DSM9078 which had biotransformation activity for protopanaxadiol (PPD)-type ginsenosides. Sequence analysis of BcelFp revealed that it could be classified into glycoside hydrolase family 5 (GH5). The gene encoding a 323-amino acid protein was cloned and expressed in Escherichia coli. The recombinant enzyme was purified, and its molecular weight was approximate 37 kDa. The recombinant BcelFp exhibited an optimal activity at 95 oC and pH 5.5 and showed high thermostability. The endoglucanase had high selectivity for cleaving the outer glucose moiety at the C3 carbon of ginsenoside Rb1, Rb2, Rc and Rd, which produced stronger pharmacologically active gypenoside XVII (GypXVII), Compound O (CO), Compound Mc1 (CMc1) and F2, respectively. The Km values for Rb1, Rb2, Rc and Rd were 3.66 ± 0.04 µM, 4.02 ± 0.12 µM, 5.95 ± 0.03 µM, 0.67 ± 0.006 µM, respectively. The kcat/Km value of BcelFp for ginsenoside Rd was 27.91 mM-1s-1, which was much higher than that of the previously enzymes. This study was the first report of the highly efficient and selective transformation of GypXVII, CO, CMc1 and F2 from Rb1, Rb2, Rc and Rd by a GH5-family thermophilic endoglucanase.A novel endoglucanase (BcelFp) was identified from The online version contains supplementary material available at 10.1186/s13568-022-01473-z. Panax ginseng C. A. Mey., has been widely used as a kind of valuable Chinese-traditional medicine in the Eastern Asian for thousands of years. Ginsenosides are the principal components responsible for the diverse and significant effects of ginseng  strains are commonly applied for the ginsenoside conversion. For instance, the recombinant β-glucosidase from Bifidobacterium breve ATCC 15,700 was identified to produce compound K(CK) from ginsenoside Rd , respectively  family 1, 2, 3, 39, 42, 51, and 78  medium  with 50 mg/L kanamycin. Reference standard of ginsenoside Rb1, Rb2, Rc, Rd, GypXVII and F2 were purchased from Shanghai Yuanye Biological Technology Co. Ltd. .http://web.expasy.org/computepi/). . The glycoside hydrolases in GH5 family were chosen at the CAZy web (http://www.cazy.org/fam/GH5.html), and the sequence information of those proteins were collected by means of the CAZy web page links. Multiple alignments of BcelFp and three characterized glycosidases from GH5 family were performed using the ClustalX program  and isoelectric point (pI) of BcelFp were estimated on the ExPasy server  and used as a template of the gene cloning. The endoglucanase gene, bcelfp, from F. pennivorans DSM9078 was amplifed via polymerase chain reaction (PCR). The primers were designed based on genomic sequence (Genbank No. WP_245530410.1): upstream (5’-CAGCAGGGATCCATGGATCAGTCAGTTGCT-3’) and downstream (5’- CAGCAGCTCGAGTTATTCTTTGCTTTCTCCAA-3’) with BamHI and XhoI restriction sites (underlined), respectively. The PCR amplified DNA fragment with a His-tag at N terminal was purified and inserted into the pET28a vector digested with BamHI and XhoI. The recombinant pET28a-bcelfp was transformed into E. coli BL21-CodonPlus(DE3)-RIL electrocompetent cells. Cells were grown at 37 °C in LB medium supplemented with 50 µg.ml− 1 kanamycin until the OD600 reached 0.6–0.8; protein expression was induced by the addition of 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and the culture was grown for 12 h at 16 °C.Genomic DNA from oC to remove the debris. The supernatants containing the target proteins were loaded onto a Ni-NTA affinity chromatography column  and purified using a 20–100 mM imidazole gradient. The purified enzyme was dialyzed against 50 mM phosphate-citrate buffer (pH 6.0) and concentrated to 1.0 mg/mL. The protein homogeneity was confirmed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).The recombinant strains were collected by centrifugation at 6,000×g for 20 min and resuspended in a lysis buffer . The cells were then sonicated and centrifuged at 14,000×g for 30 min at 4 oC for 5 min, 3,5-Dinitrosalicylic acid (DNS) was added to terminate the action, and the mixture was boiled in 100 oC water for 5 min. The absorption of the reaction mixture was measured at 540 nm. One unit (IU) of enzyme activity was defined as the amount of enzyme that released 1 µmol reducing sugars per min.The hydrolytic activity of the endoglucanase BcelFp was assayed according to the Ghose Ghose  and DNS oC  at the optimum pH. Thermal stability of BcelFp was studied by incubating about 1.5 mg/mL purified enzyme solutions at 85 oC, 90 oC or 95 oC for various lengths of time in 50 mM phosphate-citrate buffer (pH 6.0). The residual activity on CMC was determined at 95 oC in 50 mM phosphate-citrate buffer (pH 6.0). pH stability of BcelFp was tested by incubating about 1.5 mg/mL purified enzyme solutions in pH 4.0, pH 5.0, pH 6.0 and pH 7.0 for various lengths of time at 85 oC. The residual activity on CMC was determined at 95 oC in 50 mM phosphate-citrate buffer (pH 6.0).Using CMC as the substrate, the effects of temperature and pH on enzyme activity were investigated by assaying the endoglucanase activity in a 2 ml reaction mixture according to the method described previously. The pH optima of BcelFp was tested over the pH range of 4.0–8.0 in 50 mM phosphate-citrate buffer. The temperature optima of BcelFp was measured between 30 and 100 4+, Na+, K+, Co2+, Ca2+, Zn2+, Ba2+, Mn2+, and Fe3+), and EDTA on enzyme activity were investigated. The enzyme activity with no addition for the control was set as 100%. After incubation the enzyme with various metal ions (5 mM and 10 mM), and EDTA (5 mM and 10 mM) at room temperature for 30 min, the residual activity was measured according to the method described previously using CMC as substrate at pH 6.0 and 95 oC in a 2 ml reaction mixture.The effects of metal ions  and concentrated to 1.5 mg/mL. The corresponding buffer was used as a reference. The equilibrated enzyme was scanned at a rate of 2.0 K/min. The scans were analyzed after subtraction of an instrument baseline recorded with buffer in both cells using the software package Origin provided by the manufacturer.Thermal inactivation of BcelFp was analyzed by DSC on a VP-Capillary differential scanning calorimeter  over a temperature range from 35 to 120 oC in 50 mM phosphate-citrate buffer (pH 6.0) in the presence of 1% (w/v) polysaccharide substrates , RAC (regenerated amorphous cellulose)  was used to test the β-glucosidase activity of BcelFp. The hydrolytic reaction was performed at 95 oC in 50 mM phosphate–citrate buffer (pH 6.0) containing 1 mM pNPG and 1 mg/ml enzyme. The activity was determined by measuring the increase in absorbance at 405 nm due to the release of p-nitrophenol. One unit (IU) of activity was defined as the amount of enzyme liberating 1 µmol of p-nitrophenol per min.The 18 chromatographic column . The column oven temperature was maintained at 35 °C, and the mobile phases A and B were water with 0.1% formic acid and acetonitrile, respectively. The gradient elution program was determined as follows: 0-2.5 min, 19% (B); 2.5-5 min, 19–30% (B); 5–11 min, 30–33% (B); 11–20 min, 33–45% (B); 20–25 min, 45–65% (B). The injection volume was 5 µL, and the flow rate was 0.4 mL/min. One unit (IU) of activity was defined as the amount of enzyme catalyzing the conversion of 1 µmol of ginsenoside substrate per min.The substrate specificity of BcelFp towards different PPD- and PPT-type ginsenosides were also studied. In reaction mixture (2 ml), enzyme solution (2 mg/mL) was reacted with equal volume of ginsenoside solution (1 mg/mL) in 50 mM phosphate–citrate buffer (pH 6.0) at 95 °C for 5–30 min. The mixture was then extracted with 2 ml n-butanol for 2 min using a vortex. After centrifugation at 13,000×g for 5 min, the upper organic phase as n-butanol fraction was transferred into clean tube and evaporated to dryness in the centrifugal evaporator. The residue was then reconstituted by methanol for analysis. Ginsenosides were assayed by the High Performance Liquid Chromatography (HPLC) system  coupled with a Syncronis CThe biotransformation ability of recombinant BcelFp towards PPD-type ginsenosides was studied. Ginsenoside Rb1, Rb2, Rc, and Rd were used as substrates. In reaction mixture (2 ml), enzyme solution (2 mg/mL) was reacted with equal volume of ginsenoside solution (1 mg/mL) in 50 mM phosphate–citrate buffer (pH 6.0) at 95 °C. The reaction solution without enzyme served as blank control. Samples were obtained at regular intervals and analyzed via HPLC.m and Vmax were determined using the enzyme kinetics program described by Cleland  at 95 °C. One unit (IU) of activity was defined as the amount of protein required to convert 1 µmol of substrate per min. All assays were performed in triplicate. The parameters K Cleland .18 chromatographic column . The column oven temperature was maintained at 35 °C, and the mobile phases A and B were water with 0.1% formic acid and acetonitrile, respectively. The gradient elution program was determined as follows: 0-2.5 min, 19% (B); 2.5-5 min, 19–30% (B); 5–11 min, 30–33% (B); 11–20 min, 33–45% (B); 20–25 min, 45–65% (B). The injection volume was 5 µL, and the flow rate was 0.4 mL/min.Chromatographic separation was performed on an Agilent 1200 series HPLC system  coupled with a Syncronis Cm/z in negative ionization mode. The parameters of ion source were set as follows: drying gas, N2; flow rate, 4.0 mL/min; drying gas temperature, 350 oC; nebulizer, 30 psig; capillary voltage, 3500 V; fragmentor, 350 V; cone voltages, 65 V. Data analysis was performed by Mass Hunter Qualitative (MHQ) software, version B.03.01 .Mass spectrometric detection was carried out on an Agilent 6520 Q-TOF-MS/MS  equipped with electrospray ionization source operated. The scan range for MS acquisition was from 100 to 2200 18 OBD™ column . Isocratic elution was performed at room temperature using a mixture of H2O/ACN  for product 1, H2O/ACN  for product 2, H2O/ACN  for product 3 and H2O/ACN  for product 4, respectively. The flow rate was 15 mL/min, and the UV detection was at 203 nm. The injection volume was set at 200 µL. The purity of the products were evaluated by HPLC in an Agilent 1200 series HPLC system . The separation conditions were the same as that used in the HPLC analysis. The 13 C NMR spectra of the purified products were recorded on a Bruker AVIII 600 MHz spectrometer at an operating frequency of 151 MHz .The biotransformed products were isolated using a Waters prep-HPLC system  with a Waters Sunfire Prep CBcelFp consisted of 969 bp encoding 323 amino acids with a theoretical molecular mass of 37.89 kDa and a theoretical pI value of 5.43. The amino acid sequence of BcelFp (Genbank No. AFG35892.1) exhibited the highest similarity with GH 5 proteins from Fervidobacterium islandicum  and Fervidobacterium changbaicum . These proteins have not yet been characterized. The nearest characterized glycoside hydrolase  in the CAZy database was cellulase FnCel5A from Fervidobacterium nodosum. The BcelFp and FnCel5A were both from thermophilic bacteriums belonging to genus Fervidobacterium. FnCel5A was the first cellulase of the genus Fervidobacterium that had been cloned and expressed  from Fervidobacterium nodosum Rt17-B1.In order to gain a better understanding of the evolutionary position of BcelFp in glycoside hydrolase family 5, we constructed the phylogenetic tree using the neighbor-joining method in the MEGA4 program with bootstrap values based on 1,000 replications. The resulting consensus tree is presented in Fig. F. pennivorans DSM9078 was cloned and expressed in Escherichia coli under the control of the IPTG-inducible promoter T7. After being induced under 16 oC for 12 h with 1 mM IPTG, the recombinant enzyme was solubly overexpressed in E.coli cells. The recombinant BcelFp was purified by His-trap affinity chromatography with a final purification of 2.3-fold and a specific activity of 297 U/mg for CMC. The expressed enzyme was determined as a single band by SDS-PAGE, with a molecular mass of approximately 38 kDa , which was almost consistent with the molecular weight of 37,892 Da calculated from 323 amino acids.The putative endoglucanase gene from oC, while it also displayed high activity between 60 and 100 oC  of BcelFp, it was 96 oC  and 916 u (peak 2) is 162 u, which corresponds to a glucose moiety. So, BcelFp catalyzed the release of one glucose residue from Rb2 to generate peak 2. As Rb2 has an outer glucose linked to C3 position and an outer arabinopyranose linked to C20 position, the deglycosylation should occur at C3 position of Rb2 to generate CO. Similarly, peak 3 was identified to be CMc1 . Combination with the 13 C NMR analysis, product 1–4 were finally confirmed to be GypXVII, CO, CMc1 and F2 , respectively. Based on the structural analysis, GypXVII, CO, CMc1, and F2 were generated by hydrolysis of the outer glucose linked to the C3 position of ginsenoside Rb1, Rb2, Rc and Rd, respectively. The biotransformation pathways of ginsenoside Rb1, Rb2 Rc and Rd catalyzed by BcelFp were presented in Fig. For the verification of the biotransformation pathways of the four PPD-type ginsenosides  by using BcelFp, the HPLC analyses were performed. As shown in Fig. m), Maximum Velocity (Vmax), and catalytic efficiencies (kcat/Km) for Rb1, Rb2, Rc, and Rd were listed. The order of the kcat/Km values of BcelFp for ginsenosides (Rd > Rb1 > Rb2 > Rc) was the same as that observed for relative activity. However, the Vmax values and the substrate affinity of the enzyme followed the orders Rb1 > Rc > Rb2 > Rd, and Rd > Rb1 > Rb2 > Rc, respectively. BcelFp had higher catalytic efficiency for Rd rather than Rb1, Rb2 and Rc. The kcat/Km value of BcelFp for ginsenoside Rd was 27.91 mM-1s-1, which was higher than that of β-glucosidase from Gordona terrae (2.21 mM-1s-1)    was higher than that of S. alaskensis glucosidase (4.8 mM− 1 min− 1) and CcBgl1A (8.64 mM− 1 S− 1) from C. cellulans sp. 21.Biotranformation of ginsenoside Rb1, Rb2, Rc and Rd by glycosidases have been attempted previously. These enzymes can be divided into three groups according to the hydrolysis activity on the linked positions of sugar moieties in ginsenosides. Group I enzymes catalyzed the simultaneous hydrolysis of C3 and C20 sugars in Rb1, Rb2, Rc or Rd. For instance, β-glucosidase from Paenibacillus mucilaginosus KCTC 3870T can hydrolyze Rd into F2 with the catalytic efficiency of 1.78 mM− 1 S− 1 (Cui et al. Sphingomonas sp. 2F2 showed F2 production abilities from ginsenoside Rd but the low conversion activity limited its application for F2 production (Wang et al. Penicillium aculeatum and Bifidobacterium breve ATCC 15,700 can hydrolyze Rb1 and Rd into F2, but the enzymes can continuously hydrolyze F2 into C-K (Lee et al. Besides, a few ginsenoside hydrolyzing recombinant enzymes have been characterized to convert ginsenoside Rd to F2. A β-glucosidase from Escherichia coli. The recombinant BcelFp exhibited optimal activity at pH 6.0 and 95 oC and showed high thermostability. BcelFp displayed high specificity and catalytic efficiency for biotransformation of ginsenosides Rb1, Rb2, Rc and Rd. Further investigation must be performed on large scale production of GypXVII, CO, CMc1, and F2 by using BcelFp.In a conclusion, in the current study, a novel GH5 endoglucanase BcelFp was successfully cloned and expressed in Below is the link to the electronic supplementary material.Additional File 1Table S1: 13 C NMR dates of the products transformed from Rb1, Rb2, Rc and RdFig. S1: Purification of BcelFp. Lane M, molecular mass marker; Lane 1, BcelFp after Ni-NTA affinity chromatography purificationFig. S2: MS/MS spectrum in negative ion mode of product 2 transformed from Rb2 using HPLC-Q-TOF-MS/MS analysisFig. S3: MS/MS spectrum in negative ion mode of product 3 transformed from Rc using HPLC-Q-TOF-MS/MS analysis