X = S, Se) based nanostructured electrode materials for electrochemical SCs. Different non-metallic oxide materials are highlighted in various categories, such as transition metal sulfides and selenides materials. Finally, the designing strategy and future improvements on metal chalcogenide materials for the application of electrochemical SCs are also discussed.Supercapacitors (SCs) have received a great deal of attention and play an important role for future self-powered devices, mainly owing to their higher power density. Among all types of electrical energy storage devices, electrochemical supercapacitors are considered to be the most promising because of their superior performance characteristics, including short charging time, high power density, safety, easy fabrication procedures, and long operational life. An SC consists of two foremost components, namely electrode materials, and electrolyte. The selection of appropriate electrode materials with rational nanostructured designs has resulted in improved electrochemical properties for high performance and has reduced the cost of SCs. In this review, we mainly spotlight the non-metallic oxide, especially metal chalcogenides (MX;  A substantial global upsurge in the depletion of fossil fuels from the rapid growth of global economy has generated two vital concerns: the first is the exhaustion of existing fossil fuel reserves, and the second is associated with an increase in greenhouse gas emissions, in particular, and environmental pollution, in general. Hence, it is necessary to develop and commercialize sustainable environment friendly energy sources and their related technologies are being developed globally as a matter of urgency ,3,4,5,6.SCs are used in applications which require many charge and discharge cycles, rather than long-term compact energy storage within hybrid vehicles and electronic systems. Depending on the mode of energy storage in SCs, they are classified into three types, namely electrical double layer capacitors (EDLCs), pseudocapacitors, and hybrid capacitors. EDLCs are based on the working principle of the charge being stored electrostatically within the electric double layer formed at the interface of two electrodes. Generally, EDLCs use carbon-based materials, such as activated nanoporous carbon, carbon aerogel, carbon nanosheets, carbon nanotubes (CNTs), and graphene, to store energy .2, RuO2, NiO, MnO2, MoO, V2O5, Fe3O4, etc.), metal chalcogenides , metal nitrides , and conducting polymers , etc.) are the electrode materials which have been employed in pseudocapacitors [Pseudocapacitors are another type of SC in which electrical energy storage is based on the working principle of faradaic charge transfer between the electrode and the electrolyte by reduction and oxidation reactions. Metal oxides –(4) ,19,20.−1) is calculated via a current-voltage (CV) analysis:m is mass of the used electrode material (g), I is the voltammetric current, ∆V is the potential window (V), and v is the scan rate (mV·s−1), respectively. The electrode materials’ specific capacitances are evaluated from a (CD) analysis:I is the discharging current (A), t is the time (s), and V is the potential difference (V), respectively. Furthermore, the energy and power densities are calculated byI, ∆V, ∆t, and m are the current potential difference, discharging time, and mass of an electroactive material, respectively.The electrode materials’ specific capacitance (in F·g(i)Multiple oxidation states(ii)Superior electrical conductivity (iii)High surface area & chemical stability (iv)Electrochemical activity (electrolyte ions can freely interact into the electrode surface)Criteria for electrode materials selection are(i)Doping of the metals to increase the conductivity and redox activity(ii)A wide potential window (iii)High surface area for the redox reaction(iv)High charge/discharge rateTo improve the capacitance of supercapacitors, four key factors are required:In the present review, the recent advances in the fabrication of metal sulfides and metal selenide-based nanostructured electrode materials for electrochemical SCs are discussed. Finally, the benefits of both metal sulfide- and selenide-based nanostructured electrode materials in the designing strategy for electrochemical SC applications are also systematically presented.2, where M is a transition element in groups IV B , V B , VI B , or VII B  and X is a chalcogen atom in the VI A group . The structure and properties of most of the transition MCs almost resemble semimetal pristine graphene, except for the band gap [The industrially vital and scientifically significant metal chalcogenides (MCs)  have received a great deal of attention in the past two decades due to their anisotropic property. In general, transition elements of groups IV to VII B combine with VI A group elements, such as S, Se, and Te to form binary stable layered crystalline structures . These lband gap , which iband gap . In this part of the review article, we particularly describe the application of nanostructured transition MCs in electrochemical SCs. They have gained considerable attention due to their high specific power, and long stability and life cycle, and they offer better safety tolerance relative to batteries in a wide range of applications in consumer electronics, electric tools, buffer powers, hybrid electronic vehicles, and so forth . On the 3S2, NiS2, Ni3S4, Ni7S10, and Ni9S8, and its different morphologies [In recent years, nanometer-sized metal sulfides have played a significant role in the field of electronics, especially optical and optoelectronic devices, due to their distinct excellent physical and chemical properties. Certainly, nickel sulfide is of particular interest because of its different phases, such as NiS, Nihologies . Howeverhologies . Hence, 3S2(a) Ni3S2 has exhibited a better performance as an electrode material for energy storage devices, due to its different types of morphology and advantages, including its low capital cost, high specific capacitance, and simple synthesis route. These are anticipated to help it meet the increasing necessities of energy storage systems, especially for SCs [3S2, unfortunately, has low conductivity, which restricts the fast electron transport required for high rate capability, and can even act as an insulator. This sort of issue has been overcome by way of incorporating highly conductive electrode materials in the pseudocapacitive Ni3S2 material.In the midst of the different types of nickel sulfides, Ni for SCs . In addi3S2 nanostructure on Ni-foam by a simple potentiodynamic deposition method and employed it for SCs. This material showed a maximum specific capacitance of 717 F·g−1 at 2 A·g−1 rate in 1 M KOH solution with remarkable capacitance retention of 91%. On the other hand, Karthikeyan et al. [3S2 to increase the electrochemical properties and specific capacitance of Ni3S2 further. They grew hierarchical Ni3S2 nanostructures in a Ni foam cell and evaluated its capacitance behavior. The cell offered a maximum specific capacitance of 1293 F·g−1 at a current density of 5 mA·cm−2. Moreover, a different kind of preparation method has been extensively studied and reported for other similar type of electrode materials [2 nanosheets coated onto single-crystal Ni3S2 nanorods grown on the surface of three-dimensional (3-D) graphene nanosheets (Ni3S2@Ni(OH)2/3-D-GN), which were able to achieve a relatively high capacitance of 1277 F·g−1 at 2 mV·s−1 and 1037.5 F·g−1 at 5.1 A·g−1. They also investigated the structural evaluation of Ni3S2@Ni(OH)2/3-D-GN with respect to hydrothermal reaction time, and concluded that as the reaction time increases from 6 h to 12 h, the evolution of the structure from Ni3S2 nanorods to Ni3S2@Ni(OH)2 occurred, followed by conversion to pure Ni(OH)2 nanosheets. After a hydrothermal reaction time of 6 h, Ni3S2 nanorods were obtained, as exhibited in Chou et al.  first syn et al.  used a oaterials ,29. Zhouaterials  further 3S2 nanosheets on a CNT backbone with a specific capacitance of 514 F·g−1 at a current density of 4 A·g−1 and excellent cycling stability. Likewise, Pan et al. [3S2 and Ni3S2/graphene on Ni-foam. Obviously, compared to pristine Ni3S2, the Ni3S2/graphene nanocomposites showed better electrochemical behavior and achieved a specific capacitance value of around 278.3 F·g−1 for the first 20 cycles. Afterwards, the capacitance started to decrease to 230.6 F·g−1 over 35 cycles, and finally reached 223 F·g−1 until 50 cycles, which might have been due to the detachment of electrode material from the Ni-foam.Later on, Zhu et al.  reportedn et al.  designed3S2/graphene composites, a simple process controlled by adjusting the extent of sulfidation was proposed by Ou et al. [−1. The same group also studied the one-step hydrogen reduction synthesis of Ni3S2/graphene composites reported elsewhere [3S2 nanospheres/reduced graphene oxide (Ni3S2/rGO) nanocomposites was investigated using l-cysteine as the reducing agent, and their application to SCs characterized [−1 and 761 F·g−1 at 5 A·g−1 and 50 A·g−1 current rates, respectively, with good cycling stability, while bare Ni3S2/rGO on Ni-foam offered a specific capacitance of 2188.8 F·g−1 at 2.9 A·g−1 [To improve the specific capacitance of Niu et al.  who achilsewhere . Moreovecterized . They di.9 A·g−1 .3S2 nanowires, such as Ni3S2-Ni, Ni3S2-NiS, and Ni3S2-NiS-Ni, have been grown on nickel nanowire templates, and their capacitance behavior compared elaborately [3S2-NiS nanowires presented superior redox reactivity with a high specific capacitance of 1077.3 F·g−1 at 5 A·g−1, due to their excellent aspect ratio and electrical conductivity. On the contrary, the other two nanowire electrodes (Ni3S2-Ni and Ni3S2-NiS-Ni) possessed 100% capacitance retention compared to the Ni3S2-NiS electrode (76.3%). A rationally designed two-step method to fabricate self-supported Ni3S2 nanosheet arrays on a metal-organic framework has been investigated by Chen et al. [−1 at a current density of 10 A·g−1.In recent times, a series of Niborately . Among tn et al.  who achi(b) NiS2 phases with a pyrite structure [−1 at a current rate of 0.5 A·g−1. Similarly, Wang et al. [−1 at a current density of 1 A·g−1. This high specific capacitance of the electrode material might have been due to the designed 1-D electron-transport pathway and large specific surface area of NiS. Likewise, successful SC performances of α-NiS and β-NiS were reported by Wei et al. [As discussed earlier, uniform morphology with a pure phase of nickel sulfide is still a challenge, and currently, plenty of research is focused on resolving this problem . Neverthtructure ,42. In atructure ,44,45,46tructure , in whicg et al.  preparedi et al. .−1 at 1 A·g−1. Similarly, NiS nanoparticles on Ni-foam, [However, the pure phases of these electrodes suffer from poor cycling stability owing to the agglomeration and pulverization of NiS during consecutive cycling of the CD process. The cycling stability of NiS electrodes has been improved by changing the experimental conditions, and including conducting nanomaterials along with a NiS matrix, as reported earlier, some of which are listed later. The phase-controlled synthesis of α-NiS embedded in carbon nanorods was synthesized by Sun et al. ; the eleNi-foam, ,52,53 acNi-foam, , and rGONi-foam, , have be3S4(c) Ni3S4, exists in nature as polydymite. Still, the scientific community is facing the challenge to obtain the purest phase of Ni3S4 by conventional solid-state reactions for SC applications. Hence, to date, the electrochemical properties of Ni3S4 remain hidden. Only a few studies in the literature discussed earlier are on Ni3S4 for SC applications. In recent times, Zhang et al. [3S4 nanosheet frames by controlled solvothermal synthesis, and evaluated their electrochemical performances for SC applications. Interestingly, the 3-D rigid Ni3S4 nanosheet frames possessed better capacitance performances than that of flat Ni3S4. The 3-D rigid Ni3S4 nanosheet frames achieved a maximum capacitance value of 1213 F·g−1, which was due to high free volume and high compressive length. The proposed mechanism for both flat Ni3S4 and 3-D Ni3S4 nanosheet frames is schematically represented in 3S4, MoS2, and conductive carbon fibers were analyzed by Huang et al. [3S4@amorphous MoS2 nanosphere electrodes have exhibited a high specific capacitance of 1440.9 F·g−1 at 2 A·g−1 [One of the rarely reported nickel sulfide phases, Nig et al.  preparedg et al.  who repo 2 A·g−1 .3S4-MoS2 hetero-junction electrode materials assisted by an ionic liquid 1-butyl-3-methylimidazolium thiocyanate have been prepared for the first time with the electrode attaining high specific capacitance of 985.21 F·g−1 at a current density of 1 A·g−1 [3S4-MoS2 with different precursor reactions. Other phases of nickel sulfides, such as Ni9S8 [2 [Easily self-assembled Ni 1 A·g−1 . The rolas Ni9S8  and NiS2Ni9S8 [2 , were al2S), villamaninite (CuS2), djurleite (Cu1.95S), anilite (Cu1.75S), and covellite (CuS) in nature [The inexpensive, naturally abundant functional semiconductor copper sulfide is available as different phases, such as chalcocite . The CNT-incorporated porous 3-dimensional CuS microsphere composite electrodes had peony-like microspheres with a diameter of 1 μm, and each microsphere was composed of a few tens of bundled nanosheets of 15–30 nm thickness [−1 at 10 mA·cm−2. The electrochemical SC performances of different important metal sulfides are tabulated in Ultrafine CuS nanoneedle arrays grown on a CNT backbone have also been investigated as electrodes for SC applications in the past. Interestingly, these reported 1-D hierarchical electrodes offered better capacitance values with excellent cyclability, owing to the abundant surface area between the electrode and electrolyte. A schematic illustration of the formation of CuS nanoneedles on a CNT backbone is depicted in hickness . They sh−1 at 1 A·g−1. Currently, CuS nanowires on a copper mesh have also served as working electrode in SCs. These CuS-nanowire-based electrodes were free from the binder and conductive material, and had well-arrayed structures with nanosized grains and a high aspect ratio and density. In addition, the other electronically conducting substances like rGO, acetylene black, polyaniline (PANI), and CNTs have also been combined with CuS with the resultant electrodes showing very good capacitance performance and great retention [A high-performance SC based on CuS@PPy composite has been developed by in situ oxidation polymerization recently . The cometention ,75,76,77etention . 1−xS, CoS, CoS2, Co9S8, and Co3S4), it can easily transform from one phase to another phase; (ii) During preparation, it is very difficult to remove impurities such as cobalt oxide and cobalt hydroxide, because cobalt ions have a very strong affinity to oxygen; (iii) Controlling the reaction temperature is challenging for the reason that cobalt sulfides possess a complicated phase diagram. In order to deal with these factors as well to prepare high purity cobalt sulfide nanostructures, various types of synthetic routes have been employed in the past. There are several reports on the synthesis and electrochemical evaluation of nanostructured cobalt sulfides pertinent to SCs and will be discussed in this section.In the past decade, cobalt sulfide has received a great deal of interest, due to its applications in versatile fields such as SCs, lithium ion batteries, alkaline rechargeable batteries, magnetic materials, and catalysts ,89,90,913S4(a) Co3S4 nanosheet arrays on Ni-foam as electrodes, which were prepared by an anion exchange reaction of the Co3O4 nanosheet arrays. Furthermore, they compared the electrochemical performances of Co3S4 nanosheet arrays with its corresponding metal oxide analog Co3O4 nanosheet arrays. Interestingly, the specific capacitance and cycling stability of Co3S4 nanosheet arrays electrodes were 4.1 times higher than that of Co3O4 nanosheet arrays, as shown in −2 at a current density of 24 mA·cm−2. Recently, rGO nanosheets wrapped around Co3S4 nanoflake electrodes were developed, and their electrochemical performance thoroughly investigated by Patil et al. [−1 at 2 mV·s−1.Chen et al.  fabricatl et al. ; the ele(b) CoS−1 at 1 A·g−1 after 1000 cycles [−1 at 3 A·g−1 [Due to the synergic properties of the metallic and layered characteristics of CoS, it has been widely investigated for use in SC electrodes. Different morphologies of CoS nanostructures have been synthesized by various synthetic routes and have exhibited distinct electrochemical performances. 3-D flower-like hierarchical CoS nanostructure electrodes have been prepared using 6 M KOH solution and employed in SCs, which yielded 586 F·g0 cycles . Neverth 3 A·g−1 . Later,  3 A·g−1 , while J 3 A·g−1 , and rec 3 A·g−1 . Accordi 3 A·g−1 ,99,100.1.097(c) CoS1.097 and employed it as an SC electrode, which exhibited high specific capacitances of 555 F·g−1 and 464 F·g−1 at 5 mA·cm−2 and 100 mA·cm−2, respectively, while 1-D hierarchical CoS1.097 on CNT nanostructured electrodes delivered a remarkable specific capacitance of 640 F·g−1 at 8 A·g−1 after 3000 consecutive CD cycles [1.097 nanotube network provided high specific capacitance, good capacitance retention, and excellent coulombic efficiency, due to its hollow structure and large surface area [As with CoS, Wang et al.  developeD cycles . Anotherace area .2(d) CoS2) is intrinsically a conductive metal that has been considered as one of the promising materials for wide potential application in SCs [−1) of CoS2 is much higher than that of other metal sulfides, indicating that it has superior capacitive behavior compared to activated carbon positive electrodes for hybrid SCs [2 ellipsoids, nanoflake thin films, nanowires, octahedrons, and hollow spheres [2 electrodes offered a high specific capacitance of 718.7 F·g−1 at 1 A·g−1 [2 nanoframe electrodes fabricated by anion replacement had a maximum capacitance of 568 F·g−1 at 0.5 A·g−1 [2 was intrinsically unstable, which caused several problems, such as relatively low capacitance, poor cycling stability, and rate capability. These could be overcome by an effective synthetic strategy for direct growth of a CoS2 active material on a conductive support, which dramatically enhanced the capacitance performance [2-rGO composites which possessed better electrochemical properties than pure individual components have been prepared and investigated recently [2/MoS2 on carbon fiber cloth hierarchical electrode exhibited excellent long life cycle stability and achieved a maximum capacitance value of 406 F·g−1 [Pyrite-phase cobalt disulfide  Co9S8 including nanosheets, nanoneedles, nanospheres, a yolk-shell structure, as well as various heterostructures with CNT and rGO were reported as potential anodes for lithium-ion batteries and dye-sensitized solar cells [9S8 nanostructured electrodes leading to SC applications are very scarce. For instance, high purity Co9S8 thin films on Ni foam have been developed by atomic layer deposition and employed as high-performance SC electrodes which possessed a specific capacitance of 1645 F·g−1 at 3 A·g−1 [9S8 nanoflake/graphene composite electrodes that offered a maximum specific capacitance of 808 F·g−1 at 5 mV·s−1 in 6 M KOH electrolyte solution. Mashikwa et al. [9S8 nanoparticle clusters embedded in an activated graphene foam structure using a microwave-assisted hydrothermal method; the electrode was capable of delivering a specific capacitance of 1150 F·g−1 at 5 mV·s−1. Furthermore, 3-D petal-like two-mixed metal sulfide-graphene composite electrodes (Co9S8/rGO/Ni3S2/Ni foam) fabricated for high-performance SCs exhibited superior capacitive performance with the high capability (2611.9 F·g−1 at 3.9 A·g−1), excellent rate capability, and enhanced electrochemical stability with remarkable capacitance retention [Various nanostructures of Coar cells ,119,120. 3 A·g−1 . Later R 3 A·g−1  suggestea et al.  developeetention .Although many transition metal sulfides have been investigated as electrodes for SCs, binary metal sulfides are quite interesting, due to their higher active redox sites, as well as mechanical and thermal stability compared to that of their corresponding single component counterparts. Most binary metal sulfide nanostructures have been synthesized by applying the Kirkendall effect , and rec2S4(a) NiCo2S4 nanotubes have been synthesized and employed as pseudocapacitor electrodes with excellent electrochemical performance in the past [2S4 nanoplates, which exhibited a high specific capacitance of 437 F·g−1 at 1 A·g−1 using 3 M KOH electrolyte solution. CoNi2S4 electrode materials were successfully fabricated by Du et al. [2S4 hollow spheres have shown excellent electrochemical properties, such as an intrinsic electronic conductivity hundreds of times higher than that of its corresponding binary metal oxides [−1 at 2 A·g−1 with remarkable rate capability. In the meantime, NiCo2S4 nanostructures prepared by, for instance, hydrothermal, solvothermal, and polyol methods also exhibited high specific capacitance with fabulous capacitance retention, and were reported as potential pseudocapacitor electrodes for SC applications [2S4 on carbon fiber cloth and carbon fiber paper have been investigated, and their electrochemical performances compared to SC applications. NiCo2S4 carbon fiber paper demonstrated favorable charge-transfer kinetics and fast electron transport compared to NiCo2S4 carbon fiber cloth, and thus showed superior electrochemical performance compared to its counterpart [The urchin-like porous NiCothe past ,108. Latthe past  successfu et al. . Self-tel oxides ; an elecications ,128,129.nterpart .(b) Manganese Cobalt Sulfides (MCS)2S4, reports on MCS are very few. Previously, Chen et al. [−1 at 5 A·g−1 with long-term cycling stability and capacitance retention have been reported for nano honeycomb-like MCS/3 D-graphene on Ni-foam electrodes [Great attention has been paid to MCS-based electrodes in the past three years, due to its eco-friendly nature and high redox properties. As with NiCon et al.  synthesin et al. . Very reectrodes .2S4 structure confined with nanosheets for high-performance SC applications [Apart from the above binary metal sulfides, there has only been one report on a 3-D yolk-shell NiGaications .2 has received a great deal of attention, due to its unique physical and chemical properties and find applications in various fields including electrochromic devices hydrogen storage, catalysis, capacitors, lubricants, and batteries [2 is a graphene-like 2-D material in which the middle layer of molybdenum is sandwiched between two sulfur layers. All three layers are stacked over each other and held together by weak van der Waals forces [2 to develop high-performance SCs, due to its higher theoretical capacitance (1000 F·g−1) than graphite and fast intrinsic ionic conductivity [In the past decade, MoSatteries ,136,137.s forces ,139. In uctivity ,141. 2 film-based micro-SCs by a low-cost spray painting process and subsequent laser printing. The prepared SCs exhibited a better electrochemical performance than graphene-based micro-SCs and delivered a high voltammetric capacitance of 178 F·cm−3 with better cycling performance. Later on, several groups have also reported the same range of capacitance values for hydrothermally synthesized MoS2 at current density rate of 1 A·g−1 [2 nanosheets as potential electrodes for SCs, and reported that the SC performance of the electrodes was comparable to that of CNT array electrodes. Recently, chemically deposited MoS2 thin films have been synthesized by Pujari et al. [2SO4 as an electrolyte solution. They showed a specific capacitance value of 180 F·g−1 with 82% capacitance retention. Karthikeyan et al. [2-based electrodes prepared using a ball-milling process, and employed them in wire type solid-state SCs. However, in practice, the electronic conductivity of MoS2 (10−5 Ω−1·cm−1) is still lower compared to graphene and the specific capacitance of MoS2 is very limited when used on its own in SCs [Ajayan and co-workers  prepared 1 A·g−1 ,145,146. 1 A·g−1  investigi et al.  using 1 n et al.  reportedn in SCs ,151, a d2 composites in which the short rod PANI was anchored onto the surface of MoS2. The resultant electrode offered a specific capacitance of 575 F·g−1 at 20 mV·s−1. The same group extended their research on MoS2-graphene nanocomposites and concluded that the capacitance behavior of MoS2-graphene composite (243 F·g−1) was quite higher than that of bare MoS2 (120 F·g−1) and bare graphene (35 F·g−1) at 1 A·g−1, and was comparable with other reported results on MoS2-graphene electrodes [2 decorated laser-induced graphene on polyimide foil-based flexible electrodes [−1 for MoS2/rGO composites at 1 A·g−1 current density rate, which implies the superiority of MoS2 nanocomposites for SCs as high-performance electrodes. Meanwhile, multi-walled CNT/MoS2 composites have shown a better specific capacitance and achieved a maximum of (452.7 F·g−1) compared to bare MoS2 (149.6 F·g−1) and bare MWCNT (69.2 F·g−1) at a current density rate of 1 A·g−1 [Huang et al.  fabricatectrodes ,156,157.ectrodes  have beeectrodes  reported 1 A·g−1 .2 composites using porous aluminum oxide as a template were prepared for the first time by Hu et al. [−1 at 1 A·g−1 with a very good cycling rate. In another typical case, hydrothermally synthesized C/MoS2 having flower-like morphology exhibited a capacitance value of 201.4 F·g−1 at 1 A·g−1 [2@C nanofiber electrodes using an electrospinning process, and achieved high capacitance with 100% life cycle, due to their prominent electrochemical properties with improved stability. In addition, conducting templates like Mo foil, PANI, and PPy have been used along with MoS2 as binder-free electrodes for SCs in recent years [2-based SCs are not satisfactory and are still a challenge.Moreover, the utilization of a conducting template along with molybdenum sulfide also improved the surface area and electrochemical performance, and a few classical references are discussed herein. Porous tubular C/MoSu et al. , and the 1 A·g−1 . Kumuthi 1 A·g−1  preparednt years ,165,166,2S3(a) Bi2S3 is a direct band gap (1.4 eV) layered semiconductor material, and exists mostly in orthorhombic form. In recent years, more attention has been paid to it due to its specific electrical and optical properties, and it has found potentially applicable in the fields of SCs, photocatalysis, sensors, and batteries [2S3 leading to SC applications are discussed herein. Rod-like Bi2S3 micro flowers have been synthesized and characterized for their application in SCs; they provided a maximum specific capacitance of 185.7 F·g−1 at 1 A·g−1 [2S3 nanorod/MoS2 nanosheet electrodes showed a specific capacitance of 1258 F·g−1 at 10 A·g−1 with 92.6% of capacitance retention [2S3 thin films on stainless steel using a successive ionic layer adsorption and reaction (SILAR) method, which improved capacitance performance with long-term cyclability [Biatteries ,168. The 1 A·g−1 . Similarlability .2S3(b) La2S3, and La3S4, which possess excellent pseudocapacitive behavior and high electronic conductivity similar to other metal sulfides [2S3 leading to asymmetric SCs have been synthesized using the SILAR method. For instance, Patil et al. [2S3 thin films on a stainless steel substrate using the SILAR method and studied its electrochemical performance. The resultant electrode delivered a specific capacitance of 256 F·g−1 using LiClO4/PC electrolyte, while the same La2S3 electrodes in aqueous electrolytes, such as KOH and Na2SO4, offered a maximum capacitance of 358 F·g−1 at 5 mV·s−1 [2S3 electrodes prepared using chemical bath method, which improved the specific capacitance of the electrodes drastically [2S3 electrodes achieved a maximum of 294 F·g−1 at 0.5 mV·s−1, which was much higher than bare La2S3 electrodes. Due to its stable transition state, the rare earth element lanthanum-based chalcogenides are considered as promising for use in SC electrodes in the current era. Depending on the experimental conditions, lanthanum sulfide exists in different forms, including LaS, Lasulfides . Howeversulfides . Most ofl et al.  prepared5 mV·s−1 . Later ostically . Their a2(c) WS2-based electrode materials are receiving increased attention for applications in SCs, owing to their high specific surface area and adaptable electronic structures. In brief, naturally occurring WS2 possesses a hexagonal crystal structure with space group P63/mmc. Each WS2 monolayer consists of an individual layer of W atoms with six-fold coordination symmetry, which is then hexagonally packed between two trigonal atomic layers of S atoms [2 in SCs. Quite a few reported results are displayed herein. Ratha and coworkers [2/rGO electrodes using a one-step hydrothermal method; these electrodes were capable of delivering 350 F·g−1 at 2 mV·S−1. Furthermore, the mechanism of WS2/rGO nanosheet electrodes was explained by Tu et al. [6+ and W4+ on WS2, as well as by the O-containing surface functionality on the surface of rGO. It showed excellent specific capacitance with remarkable capacitance retention. Later on, a series of 2-D transition metal carbides (TMCs) , including WS2, were investigated, and their strong influence on capacitance studied by Martinez et al. [2 electrodes for SCs that offered a maximum specific capacitance of 3.5 F·g−1 at 10 mV·S−1.WS S atoms . Though oworkers  reportedu et al. . In the z et al. . Bissettz et al.  analyzedThe charge storage mechanism and electrochemical properties of transition metal sulfides for SC applications were discussed in the previous section. This section is purely devoted to a discussion on selenium-based metal chalcogenides for SC applications. Selenium, the nearest neighbor of sulfur in the VI A group, possesses the same valence electrons and oxidation number as sulfur . Hence, −3 Ohm·cm−1, due to their paramagnetic nature, which makes them suitable candidates for energy storage devices, especially for SCs [2-based SCs are very limited, due to their highly complicated synthetic routes. The synthesis of NiSe2 involves multiple steps, which has led to more expensive capital cost in bulk scale preparations. Among the transition metal chalcogenides studied, nickel selenides are of particular interest due to their tunable electronic configuration and multiple oxidation states. In addition, they possess resistivity below 10 for SCs . To date2 single crystals using a simple hydrothermal approach, and deeply studied its electrochemical performance. These electrodes offered a maximum specific capacitance of 1044 F·g−1 at 3 A·g−1 with an excellent rate capability. Similarly, hexapod-like two-dimensional NiSe2 single crystals have been investigated; their electrode delivered a maximum capacitance value of 75 F·g−1 at a current density of 1 mA·cm−2 with a capacitance retention rate of 94% [Recently, Wang et al.  synthesie of 94% .2/Cu electrodes have been synthesized using a simple hydrothermal method, and the reported electrodes delivered a high specific capacitance of 1037.5 F·g−1 at 0.25 mA·cm−2 [−1 [2Se nanodentrites as electrodes for high-performance SCs. However, the electrochemical properties of CuSe have not yet been fully identified, and future research is likely to be in the direction of developing high-performance SCs using CuSe electrodes. Inexpensive, semiconducting CuSe has been applied in the fields of optoelectronics, thermoelectrics, and solar cells ,183. Due mA·cm−2 . Moreovecm−2 [−1 . Shinde cm−2 [−1  reported2, the molybdenum atom is squashed between two selenium atoms by means of strong covalent bonds that characterize the Se-Mo-Se interaction. It has high theoretical capacitance and comprises low-cost and abundant elements. The stacked layers are held together by weak van der Waals forces responsible for ion migration during the CD process. To the best of our knowledge, only very few studies have become available on MoSe2 for SC applications. In MoSe2 in a two-electrode configuration using H2SO4 electrolyte, in which the electrodes possessed very good specific capacitance of 199 F·g−1 at 2 mV·s−1. The electrode cell’s specific capacitance was increased further by combining MoSe2 with rGO to attain a maximum of 211 F·g−1 with excellent cyclability. Later on, Haung et al. [2/graphene composites for SC applications, and the same group recently grew MoSe2-based electrodes on Mo-foil and reported their capacitance behavior [2/MWCNT electrodes have been prepared recently using dip and dry, followed by a chemical bath deposition method [Balasingam et al.  reportedg et al.  studied behavior . Furthern method . The rem2, CoSe, Co0.85Se, Co3Se4, and Co2Se3 [In recent years, cobalt selenide-based materials have been a new research hot spot in the field of electrochemical SCs, due to their cost-effectiveness and highly reversible nature. There are a variety of compounds including CoSed Co2Se3 , which h0.85Se(a) Co0.85Se nanotubes having a hollow nanostructure were successfully prepared and investigated by Wang et al. [0.85Se nanotubes with Co0.85Se nanoparticles [0.85Se nanotubes were superior to those of Co0.85Se nanoparticles. Additionally, Co0.85Se hollow nanowires have been previously efficaciously synthesized and used as efficient pseudocapacitive electrodes for SCs [0.85Se nanosheets as the positive electrode, and nitrogen-doped porous carbon network as the negative electrode, to fabricate asymmetric SCs, which yielded an energy density of 21.1 W·h·kg−1 at a power density of 400 W·kg−1 with excellent capacitance retention of 93.8%. Later on, Zhao et al. [−1 at a power density of 3.57 kW·kg−1 for Co0.85Se/AC asymmetric SCs. Meanwhile, Gong et al. [0.85Se nanosheet positive electrodes with Co0.85Se nanosheet/Ni-foam, which provided a significant increase in energy density with outstanding cycling stability (39.7 W·h·kg−1 at a power density of 789.6 W·kg−1). Polycrystalline Cog et al. . They alarticles , the obt for SCs . Interes for SCs  employedo et al. ,196 usedg et al.  replaced2(b) CoSe2 as an electrode material for SCs, and up until now, very few studies have reported using it. However, systematic investigations on the electrochemical performances of metal chalcogenides, such as CoSe2 and CoTe2, have successfully analyzed and studied their applicability as high performance SCs [2 electrodes delivered a maximum specific capacitance of 951 F·g−1 at 5 mV·s−1, which is three times higher than that of CoTe2. Later on Zhang et al. [2/C as pseudocapacitive electrode whose electrochemical properties have not yet been fully studied. Hence, much effort will be focused on developing these electrodes in the near future.There has hardly been any investigation into using CoSeance SCs ,199. Theg et al.  assemble2, SnSe2, and La2Se3, have been studied for flexible solid-state SC electrodes, but rarely reported [Other metal selenides such as Ni-Co-Se, WSereported ,203,204.0.85 Se was able to deliver a highest areal capacitance of 2.33 F·cm−2 at 4 mA·cm−2 current rate. This super-hydrophilic electrode had metal-like electronic conductivity and offered a maximum conductivity of 1.67 × 106 S·cm−1. Similarly, Ni-Co-Se nanowires have shown a high specific capacitance of 86 F·g−1 at a current density of 1 A·g−1 and excellent cycling stability, with virtually no decrease in capacitance after 2000 continuous CD cycles [2 and Ni0.85Se@MoSe2, using a hydrothermal method, and studied their use in asymmetric SC applications.The binary metal selenides are currently highly fascinating, and only a scarce number of reports on their use in SC applications are available, and their electrochemical performance has not yet been fully studied. Nevertheless, some classical examples are presented here. Xia et al.  showed tD cycles . More reD cycles ,206 prepCurrently, the development of SCs as electrochemical energy storage devices is of major importance, with the spotlight on their high power density. A typical SC is composed of electrode material and electrolyte. In an assessment of electrochemical SC devices other than those with a long lifecycle, both their energy and power densities are the two most essential properties. In view of this, current major research focused on SCs is on increasing these characteristics and their life cycle to decrease the cost of the electrode materials. The choice of suitable electrode materials with rational nanostructured designs has resulted in improved electrochemical properties for high performance and cost reduction of SCs. In this review, we conferred recent progress in the advancement of non-metallic oxides, transition metal sulfides and selenides were especially highlighted for SC applications.(i)Energy density: For practical application, high energy density electrochemical system is required. In view of this, the energy density of electrochemical supercapacitors is less than less than of batteries.(ii)2 are more expensive. Also, the cost of organic electrolytes is far from negligible. Cost efficiency: The commonly employed electrode materials such as high porous surface area carbon materials and RuO(iii)Self-discharge rate: Electrochemical supercapacitors have high in self discharge rate 10–40%/day.The major advantages of electrochemical supercapacitors are high power density (1–10 Kw/kg), lifetime (estimated to be up to 30 years), cycle efficiency (98–100%), operating temperatures (−40 to 70 °C), environmental friendliness, and safety. However, the challenges to be focused on supercapacitor areNanostructured transition metal chalcogenides have gained huge consideration due to their distinctive chemical stability, electronic properties, and remarkable structure. Among these, transition metal sulfides have been proven to exhibit superior electrochemical performance compared to their bulk counterparts, because of their novel properties associated with decreased size, unique shape, and defective nature. Nanoscale structures can effectively improve electrochemical reaction efficiency and utilization of active materials with improved energy and power densities. Extraordinary investigation ought to be done to construct novel electrode materials for SCs, and new ideas and/or design strategies are required in this field. While designing and constructing electrode materials, the researcher ought to take into consideration that they should be abundant, cheap, and eco-friendly for clean technology and potentially be of use in a broad selection of applications.


The use of C3d, the final degradation product of complement protein C3, as a “natural” adjuvant has been widely examined since the initial documentation of its immunogenicity-enhancing properties as a consequence of binding to complement receptor 2. Subsequently it was demonstrated that these effects are most evident when oligomeric, rather than when monomeric forms of C3d, are linked to various test protein antigens. In this study, we examined the feasibility of enhancing the adjuvant properties of human C3d further by utilizing C4b-binding protein (C4BP) to provide an oligomeric arrayed scaffold fused to the model antigen, tetanus toxin C fragment (TTCF).in vitro and in vivo analysis that, in the presence of mouse C3, human C3d does not interact with either mouse or even human complement receptor 2.High molecular weight, C3d-containing oligomeric vaccines were successfully expressed, purified from mammalian cells and used to immunize groups of mice. Surprisingly, anti-TTCF antibody responses measured in these mice were poor. Subsequently we established by These data confirm the requirement to develop murine versions of C3d based adjuvant compounds to test in mice or that mice would need to be developed that express both human C3 and human CR2 to allow the testing of human C3d based adjuvants in mouse in any capacity. On B cesplicing . C3d,g rsplicing , also foesponses .It has been clear for some time that many components of the innate immune system act to activate and enhance the adaptive immune response, and can therefore be considered to be “natural” adjuvants . C3d is We therefore decided to investigate whether multiple copies of C3d arrayed in an oligomeric format , rather than linked as a linear chain, would function as an improved adjuvant. Herein, we provide new evidence that hC3d, even when arrayed with C4BP, does not enhance antigen-specific immune responses as surprisingly hC3d does not bind to mCR2 or even to hCR2 in the presence of mC3(/d). This finding clearly demonstrates the difficulty in testing hC3d-based adjuvants in the mouse. Alternative approaches to combat this include the development of wholly murine versions of such constructs or the development of mice with a fully humanised complement system (hC3 and hCR2), or molecular adjuvants designed to activate complement appropriately in multiple species.22.1+ve, CR2−ve, mouse B cell line CH27, and transfectants expressing mCR2 (CH27.mCR2), a tetanus toxin C fragment (TTCF)-specific BCR (CH27.TTCF-BCR) or mCR2 and a TTCF-specific BCR (CH27.mCR2/TTCF-BCR), previously described (The Chinese hamster ovary (CHO) cell line and human B cell line Raji (expressing high levels of CR2 ), were oescribed , were ma2.2hC4BP. Next, hC3d  was PCR cloned and incorporated in-frame between the CD33 and histag regions, generating phC3d-hC4BP. hCR2 (SCR1-4)-Fc, native hC3d (containing 9th cysteine)-Fc, hC3dg (aa 955-1303) −Fc as well as Cys to Ser variants of these, mCR2 (SCR1-4)-Fc and mC3d-Fc constructs were also generated using PCR cloning utilizing a pDR2EF1α plasmid containing human IgG1 Fc . Recombinant TTCF encoding 444 amino acids of the mature TTCF from the 7th amino acid  instructions. Inclusion of hygromycin B (600 μg/ml) and limiting dilution allowed the selection of clonal transfectants. Recombinant proteins were purified from tissue culture supernatant using immobilized metal ion affinity chromatography, essentially as described for TTCF  genetic background with or without the transgenes and/or genetic deletions described below.tg.hCR2tg.Cr2−/− mice  (2a) , fluorescein isothiocyanate (FITC) or PerCP conjugated RA3-6B2 , FITC conjugated species anti-hCD19 were obtained from Pharmingen . Goat, anti-mouse IgG-HRPO; goat, anti-rabbit IgG − HRPO; donkey, anti-sheep IgG-HRPO conjugated anti-IgM or IgG, streptavidin-FITC and −PE were obtained from Jackson Immunoresearch Laboratories . Rabbit, anti-hC3d and sheep, anti-hC4BP were purchased from Dako, UK and Abcam, UK respectively.Purified, biotin and allophycocyanin (APC) conjugated mAb 171  ; biotin pe) (2a) , biotin ti-TTCF, ) and mou2.62SO4. OD450 nm was measured using SpectraMAX 190 microplate reader .To detect the presence of recombinant proteins in CHO culture supernatant (s/n) or for measurement of anti-TTCF Ig responses, flat-bottom 96 well ELISA plates (Thermo Vinyl) were coated with undiluted s/n or 0.1 μg TTCF (in carbonate coating buffer (pH 9.6)), respectively, and incubated at 4 °C overnight. All plates were washed 3 x with PBS-0.05% Tween20 (wash buffer) and with 1% BSA/0.05% Tween20 (blocking buffer) in PBS for 2 h at room temperature (RT). For the detection of bound protein, primary antibodies were diluted 1/10,000 for the polyclonals or 1/1000 for 10G5. For the detection of anti-TCCF Ig, mouse serum was diluted in blocking buffer (1/50)) and compared to blocking buffer containing mouse anti-TTCF monoclonal antibody 10G5. Plates were then incubated for 1 h at RT and then washed 3x and blocked for additional 2 h as above. Appropriate Ig-HRPO secondary antibody was incubated in blocking buffer for 1 h at RT. Plates were washed twice with wash buffer and finally two times with PBS. Freshly made 3,3,5,5′-Tetramethylbenzidine (TMB) substrate was added to each well and the reaction stopped using 10% H2.7Mouse splenocytes were isolated as described in . Human pSplenocytes (following red blood cell lysis) or cell lines were washed in PBS containing 1% fetal calm serum (flow buffer) and then re-suspended in 10 μg/ml Fc Block for 15 min on ice. Cells were subsequently washed and re-suspended in flow buffer containing primary antibody (Ab) (0.1–3 μg/ml) for 30 min on ice. Cells were then washed 3 times in flow buffer and incubated with the appropriate secondary reagent for 30 min. Cells were washed and resuspended in flow buffer containing 1% formaldehyde. Samples were analysed using a FACS Canto ™ II  and FlowJo  or Cyflogic .2.8Chips were purchased from GE Healthcare and all buffers were 0.2 μm filtered prior to use. Immobilisation: standard amine coupling reactions were carried out at 30 μl/min. Ligands were prepared in 10 mM sodium acetate at a final concentration of 2 μg/ml. Immobilisation was optimised using the pH scouting wizard program using sodium acetate at pH 5.4, 5.0, 4.6, 4.2 and 3.7. The immobilisation wizard controlled the coupling reactions. Optimal reaction conditions were set up using equal volumes of N-hydroxysuccinimide  and 1-ethyl-3-(dimethylaminopropyl)carbodiimide  the ligand in the appropriate buffer, 1 M ethanolamine-HCl pH 8.5 and 50 mM NaOH. The coupling was automatically terminated when the response unit (RU) target was reached. Typically, flow cell one (FC1) was left blank to act as control flow cell and only received NHS/EDC and 1 M ethanolamine-HCl pH 8.5. The dextran matrix in FC2 was activated with NHS/EDC, followed by ligand. Remaining free binding sites in the matrix were blocked with 1 M ethanolamine-HCl pH 8.5. Binding analysis was carried out immediately after coupling. Prior to loading of the samples, flow was allowed to stabilise for one minute. Analyte concentrations were blank buffer control , 0.195 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.125 nM, 6.25 nM 12.5 nM, 25 nM, 50 nM. Analytes were injected in triplicate for 360 s. The injection was changed from sample to buffer and dissociation was monitored for 240 s. Typically the flow path was FC1 followed by FC2, which allowed subtraction of any secondary interactions with the dextran matrix. Chip surfaces were regenerated with two brief pulses of 0.5 M NaCl for 30 s.2 values (<5) as calculated by the evaluation software.Results were analysed using the BIAevaluation Software version 4.1. Single flow cells were analysed following blank flow cell background subtraction (e.g. FC2-FC1). Curves were aligned by y-axis transformation and values obtained for buffer controls were subtracted from the curve to compensate for buffer-surface interactions. Sensorgram data was analysed with a steady state affinity model. Good fits were indicated by low residuals and χ33.1Taking into account previous oligomerisation approaches , we used3.2+ve Raji B cells were incubated with the various fusion proteins in the presence or absence of an hCR2 blocking antibody . Both hmAb 171, a. Using mAb 171, b.Fig. 2h3.3Having established both antibody and receptor binding for the various domains in the hC3d-TTCF-hC4BP fusion protein, and following several previous reports that document hC3d interaction with mouse (m) CR1/2 , we next3.41 BCR . As seen in 3.5ex-vivo B cells isolated from either B6 non-transgenic, or hCR1/2 transgenic mice. Similar to that seen with mouse B cell lines (−/−)C3. Similar to that seen with both human B cell lines and PBMC (−/−)C3.As a result of our demonstration that hC3d-TTCF-C4BP  does not bind mCR2, we re-examined the ability of the hC3d-TTCF-C4BP candidate vaccine to enhance anti-TTCF antibody production following the immunization of transgenic mice expressing hCR2. Using an essentially identical immunization and boost protocol to that shown in ll lines , hC3d-TTll lines b. In addll lines , Fig. 6band PBMC , Fig. 6b4The key role played by complement in enhancing the adaptive immune response is now well accepted. Over 40 years ago, the seminal studies by Pepys et al. using C3 activating/depleting agents including cobra venom factor and zymosan clearly demonstrated intact C3 function was important for the T-dependent response to various antigens including sheep red blood cells and ovalbumin . A refinFollowing further investigations we, somewhat surprisingly, established that the hC3d component of this construct, along with purified hC3d, does not bind mCR1/2. This finding apparently contradicts studies using polymerised human complement C3dg (pC3dg) and its specific binding to COS and K562 cells expressing recombinant mCR2 . These din vivo setting, and that unknown factors may have influenced the studies using recombinant mouse CR2 expressed on K562  hCR2.The oligomerisation domain on the complement regulator C4BP  has prev−/− background would be possible given time. However, these experiments will be hampered by the loss of endogenous C3 to drive and support the immune response and therefore give a true idea of how that will translate to the clinical setting that these pre-clinical studies are supposed to emulate. We concede that only construction of mC3d fused to mC4BP constructs will allow the adjuvant capabilities of similar oligomeric C3d compounds to be fully tested in mouse models. This remains an important line of investigation as there are still many unknowns regarding the mode of action of C3d as an adjuvant. The linear trimer construct of C3d has been shown to be an effective stimulator of B cell responses but monomeric C3d-Ag is considered immunosuppressive (The full testing of these oligomeric hC3d antigen compounds in the recently generated hCR2 BAC mice  crossed pressive . Thus, wpressive .In summary, these experiments clearly demonstrate that hC3d does not bind to mCR2 as previously reported, while mC3d does bind hCR2 with an increased affinity to that measured for hC3d. Importantly we also demonstrate for the first time that even following the replacement of mCR2 with the hCR2 homologue in transgenic animals no hC3/hCR2 binding is seen in the present of mC3. These 2 new findings severely hamper the testing of novel strategies to improve the efficiencies of hC3d-containing recombinant adjuvants in mice. Importantly these studies thus highlight the importance of extensive testing of candidate C3-based vaccines and that, even when attempts are made to maximize species conservation in animal models, considerable care needs to be taken when analyzing cross species receptor/ligand interactions.There are no conflicts of interest.


Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongated state of the factor. Binding of Initiation Factor IF1 triggers closure of IF3 bound to the 30S complex, while both aminoglycosides revert the IF1-dependent conformation. Our results uncover dynamic perturbations across the 30S subunit, from the A-site to the platform, and suggest that both aminoglycosides could interfere with prokaryotic translation initiation by modulating the interaction between IF3 domains with the 30S platform.Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR)  Mycobacterium tuberculosis, is a devastating disease with higher incidence in underdeveloped countries than in their developed counterparts  of the minor ribosomal subunit (30S) and inhibit protein synthesis mainly by causing misreading of the mRNA  or transE. coli cells [Due to streptomycin’s effects, it was proposed that this drug could trigger an “error catastrophe” during protein elongation . Howeverli cells , and thali cells .Streptomycin, kanamycin and initiation factor IF1 bind nearby within the decoding center of the 30S subunit and promote diverse local and long range conformational perturbations ,15,16,17DL) to develop an intramolecular Förster resonance energy transfer (FRET) system capable of sensing rapid conformational changes of the factor and/or its binding sites at the 30S platform. In combination with pre-steady state kinetics, the FRET signal of IF3DL responds to the interaction of streptomycin, kanamycin, and IF1 with the A-site of the 30S subunit. Our data, in combination with recent structural studies, suggest a novel molecular mechanism for the aminoglycosides as capable of perturbing IF3 binding sites on the 30S platform.The 30S platform greatly contributes to the association of the small subunit with the major ribosomal subunit (50S) through the formation of several inter-subunit bridges . The intIF3 binds across the 30S platform, interacts with several intersubunit bridges, and responds to conformational states of the small subunit  17,19,2,28,29. T0 between the dyes is Å 64, providing a wide range of distances to be monitored by changes of FRET efficiencies  contained a non-emitting acceptor (quencher) at the CTD and a fluorescent dye at the NTD  recruitment [IF3 and IF1 cooperatively increase their affinity for the 30S subunit ,33. Alonruitment . This muruitment . The cooruitment ,36.DL to the 30S subunit and the influence of IF1 in the resulting 30S–IF3DL complex  resulted in proportional increased emission fluorescence  and measured fluorescence emission after passing a 515 nm long-pass optical filter.Indeed, fluorescence equilibrium measurements of IF3rescence b, indicaurements . BindingDL with 30S subunits, the fluorescence increased with time with a biphasic behavior ). Analysis is consistent with an initial bimolecular encounter between IF3DL and the 30S subunit, kapp1 = 16 ± 1 s−1, followed by a conformational rearrangement of the factor, kapp2 = 2 ± 0.05 s−1. These measurements are in agreement with previous rapid kinetic studies, although different fluorescent reporters, 30S subunits, and IFs purification methods were used [Upon mixing of IF3behavior c. In conbehavior . Non-linere used ,38.DL pre-bound to the 30S subunit could monitor IF1 interactions with the ribosome. Equilibrium titrations of IF3DL–30S with increasing concentrations of IF1 resulted in a decrease of emitted fluorescence, indicating that IF3 domains reach closer distances, consistent with previous single-molecule studies [DK of about 100 nM, consistent with measurements by pre-steady state methods [Then we investigated whether IF3 studies  ) (kapp = 0.26 ± 0.01 s−1. Previous studies reported an association constant for the bimolecular encounter of IF1 with the 30S–IF3 complex of 20 µM−1·s−1 [−1 (IF1 = 1 µM). Then, IF3DL reports an IF1-dependent FRET change that is ≥75-fold slower than the initial binding, suggesting that IF3DL is monitoring a successive step, i.e., a conformational rearrangement of IF3 on the platform resulting in the accommodation of one of IF3 domains.Equilibrium measurements of the interaction of IF1 with the 30S–IF3ion (4)) e. FittinµM−1·s−1 , thus weDL allows monitoring equilibrium and real-time kinetics of factor binding, dissociation, as well as conformational changes induced by IF1. The intramolecular FRET sensibility and versatility of IF3DL provide solid bases to test 30S subunit binders, with a special emphasis for those targeting the A-site. Among them, streptomycin and kanamycin stood out because of their medical importance as second-line drugs to treat MDR TB.IF3DL complexes to monitor real-time conformational perturbations of IF3 on the platform upon the binding of streptomycin and kanamycin to the A-site  and an associated fluorescent amplitude (F) (Equation (4)). Analysis by non-linear regression fitting returned apparent rates for streptomycin and kanamycin kappStr = 4.6 ± 0.1 s−1 and kappKan = 1.4 ± 0.1 s−1  perturbation if compared to the aminoglycosides. Consequently, we probed whether the interaction of streptomycin and kanamycin with the 30S subunit could revert IF1-dependent closing up of IF3DL  showed an increased amplitude of fluorescence change and slower apparent rates , suggesting they are rate-limited by a similar reaction.Rapid mixing of 30S–IF3rescence c. Comparnt rates d. Nonlinectively d. On theectively . In addiThe intrinsic flexibility and dynamics of IF3 seem to sample different conformational states of the 30S subunit, at the 30S platform where the factor binds. With opposing directions, IF1 together with streptomycin and kanamycin alter the relative disposition of IF3 domains, revealing molecular mechanisms of antibiotic action at an unexpected site but with potential functional implications. Perturbing IF3 binding sites by streptomycin, even in the presence of IF1, provides a rationale to previous reports where the absence of IF1 or the addition of streptomycin increased the rates of non-canonical translation initiation .in vivo formation of unproductive 70S complexes. This postulation is supported by experiments from the late 1960s which indeed showed streptomycin to cause an accumulation of 70S monomers concomitantly to a reduction of polysomes, consistent with streptomycin preferentially inhibiting early steps of translation [Both, streptomycin and kanamycin, disturb the positioning of IF3 at the 30S platform, possibly affecting translation initiation  in additnslation ,44.DL (increased FRET), interpreted as a closing up of IF3 domains  with either expression vector pET24c InfA, pET24c InfC wt, or pET24c InfC E166C, coding for IF1, IF3 wt, or IF3E166C, respectively. pET24c vectors containing wt and mutant genes were commercially acquired . Typically, 2 L of Luria–Bertoni (LB) medium were used to grow BL21DE3 pET24c InfA or pET24c InfC to an OD600nm of 0.5. Protein expression was induced by adding 1 mM Isopropyl β-d-1-thiogalactopyranoside . Cells were allowed to express IF1 or IF3 for 3 h prior to harvesting by centrifugation at 5000× g at 4 °C. Cells were lysed in Lysis Buffer  supplemented with 0.1 mg/mL of Lysozyme . After five cycles of freezing and thawing, 1 U/mL DNAse I was added to reduce the viscosity in 20 min of incubation at 4 °C. Membranes and supernatant were separated by centrifugation at 15,000× g for 30 min.Competent 4Cl gradient (0.05–1 M) in a Jasco HPLC system . The gradient was prepared in BufferA . IF3 and IF1 were eluted at 700 mM and 400 mM of NH4Cl, respectively, in  followed by concentration on a HiTrap SP HP (Amersham), single step eluted with 1 M NH4CL BufferA . Supernatants were manually loaded to the column (1 mL column volume) and subsequently subjected to a linear NHvely, in . The besvely, in . While Ively, in . Full el BufferA .30S subunits purification methods are described in detail in .E166C was subjected to extensive dialysis in labeling buffer  in a D-Tube™ Dialyzer Maxi (Merck) to remove traces of 2-mercaptoethanol as the reducing agent strongly inhibits the coupling of maleimide-linked dyes to cysteines. First, the C-terminal was labeled at the recombinant cysteine (166) as it is exposed and efficiently reacts with maleimide derivatives [E166C was incubated in labeling buffer for 20 min. The reaction was stopped by the addition of 6 mM 2-mercaptoethanol. The modified IF3CTD540Q was purified from unreacted dyes on a HiTrap SP HP column. After 10 CV washes with BufferA containing 100 mM NH4Cl, a single step elution was applied using 3 mL of 1 M NH4Cl in BufferA. Typically, full protein recovery is achieved in 0.5 mL and elution of the labeled protein is readily visible. IF3 CTD540Q was subsequently dialyzed as mentioned above in a labeling buffer containing 2 M UREA.IF3ivatives . A 10-foCTD540Q–NTD488 (IF3DL) was purified from the unreacted dye as described above using HiTrap SP HP column (Merck). Eluted proteins were dialyzed against storage buffer  and small aliquots were stored at −80 °C. Purity and efficiency of labeling was assayed by 15% SDS-PAGE, where fluorescence was observed under a UV trans-illuminator and total protein by blue Coomassie staining . 30S titrations of IF3DL (0.5 µM) were incubated with varying concentrations of 30S subunits . Reactions were incubated for 10 min at 37 °C. Fluorescence was measured in a NanoDrop 3000 fluorimeter (Thermo) using blue LED excitation and emission at maximum for Atto-488 (518 nm) at room temperature. Typically, five independent measurements were performed for each reaction to calculate mean and standard deviation values. Binding of IF1 to 30S–IF3DL complexes was performed as above after pre-incubating IF3DL with 30S subunits for 10 min at 37 °C. IF1 influence was measured at varying concentration of the factor . 30S subunits were MgCl2 (20 mM) activated for 30 min at 42 °C prior to being used.All reactions were performed in HAKM10 buffer (50 mM Tris (pH: 7.5), 70 mM NH4Cl, 30 mM KCl, 10 mM MgCl2, 6 mM 2-Mercaptoethanol) at 25 °C; 30S and IFs concentrations are given in the figure legends.Fluorescence stopped-flow measurements were performed using a SF-300X stopped-flow apparatus (KintekCorp) by rapidly mixing equal volumes (30 µL each) of reacting solutions a. ExcitaDL; C = 30S–IF3DL and DK is the dissociation constant.DL; B = IF1; C = 30S–IF3DL–IF1.Non-linear regressions by Prism 6.0  were performed using the following equations:Thermus thermophiles , streptDB 1HR0; ), kanamyDB 1HR0; ) and theDB 1HR0; ) (Table DB 1HR0; ,57. MoleDB 1HR0; .


G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay.Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex.When G-quadruplex-forming sequences from the Dele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.The online version of this article (doi:10.1186/s12867-017-0094-z) contains supplementary material, which is available to authorized users. Although genomic DNA usually adopts the canonical double helix structure –3, formac-MYC  or pGL4.10 [luc2]  and then transformed into E. coli DH5α . The plasmids were purified by PureYield Plasmid Miniprep System . E. coli HST04 dam−/dcm−  was transformed by the plasmids to prepare DNA methylation-free plasmids. All plasmids were sequenced using a 3730xl DNA analyzer . The Dele and Cdc6 CGIs were amplified from C57BL/6 mouse genomic DNA by PCR. The PCR primers are shown in Additional file SfiI . The products were cloned in the SfiI site of pGL4.10 [luc2] and the plasmids were prepared as described above. To construct the mutant-types vectors, the site-directed mutagenesis was performed using KOD -plus- mutagenesis kit  according to manufacturer’s protocol  at 37 °C in 5% CO2.NIH3T3 cells  were cultured in dulbecco modified eagle medium (DMEM) medium  containing 10% fetal bovine serum , 1× penicillin–streptomycin-The NIH3T3 cells were transfected with 100 ng of the firefly luciferase reporter vector and 100 ng of the Renilla luciferase control vector by Lipofectamine 3000  according to the manufacturer’s protocol. After 48 h, cells were harvested, and luciferase activities were measured using the Dual-Luciferase Reporter Assay System  and SPARK 10 M microplate reader . The ratio of firefly luciferase activity to Renilla luciferase activity was utilized to calculate the firefly luciferase expression level in each cell and then these results were normalized with that of the pGL4.10 or pGL4.23 luciferase reporter vector. All reporter assays were performed in triplicate.In the experiment to analyze the G4 ligand effect, two methods were used. In the first method, the 100 ng of the firefly luciferase reporter vector, and 100 ng of the Renilla control vector were transfected into NIH3T3 cells by Lipofectamine 3000. After 24 h, the medium was changed to one with 10 µM G4 ligand L1H1-7OTD. After 24 h, the luciferase activity was measured as described above. In the second method, the culture medium was changed to one with 10 µM G4 ligand L1H1-7OTD prior to transfection. The NIH3T3 cells were transfected with 100 ng of the firefly luciferase reporter vector, 100 ng of the Renilla control vector, and 10 µM of the G4 ligand L1H1-7OTD by Lipofectamine 3000, after which the luciferase activity was measured as described above. The luciferase expression level in the presence or absence of L1H1-7OTD was normalized with that of the pGL4.23 luciferase reporter vector in the presence or absence of L1H1-7OTD, respectively. All reporter assays were performed in triplicate.Dele and Cdc6 G4 oligonucleotides were purchased from Macrogen, South Korea (Additional file Dele and Cdc6 G4 DNAs, at 264 nm and 262 nm, respectively. To determine Tm values, the molar ellipticity at 25 °C was set as 100%, and the molar ellipticity at 95 °C was set as 0%. Using GhraphPad Prism7 software, curve fitting was performed to yield normalized molar ellipticities. Tm values were recorded as temperatures equivalent to 50% of the normalized molar ellipticity.Wild-types and mutant-types of the Additional file 1. G4 DNA sequences used in the reporter assay.Additional file 2. Luciferase reporter assay results of G4 DNA sequences cloned in pGL4.23 vector.Additional file 3. Luciferase reporter assay results of G4 DNA sequences cloned in pGL4.10 vector.Additional file 4. Luciferase reporter assay results of CGI DNA sequences cloned in pGL4.10 vector.Additional file 5. Luciferase reporter assay results with or without L1H1-7OTD for G4 DNA sequences cloned in pGL4.23 vector.Additional file 6. Luciferase reporter assay results with or without L1H1-7OTD for G4 DNA sequences cloned in pGL4.23 vector.Additional file 7. G4 DNA sequences used in the CD Spectroscopy.Additional file 8. CD spectra results of Dele G4 DNA wild or mutant-type sequences with or without L1H1-7OTD.Additional file 9. CD spectra results of Cdc6 G4 DNA wild-type sequence with or without L1H1-7OTD.Additional file 10. Detailed molar ellipticity of wild-types of Dele and Cdc6 G4 DNAs in the absence and the presence of L1H1-7OTD.Additional file 11. Primer sequences used for CGI vectors construction and mutagenesis.


Presently, enzymes still constitute a major part of the cost of biofuel production from lignocellulosic biomass. Processive endoglucanases, which possess both endoglucanase and exoglucanase activity, have the potential to reduce the costs of biomass saccharification when used together with commercial cellulases. Therefore, the exploration of new processive endoglucanases has attracted much attention with a view to accelerating the industrialization of biofuels and biochemicals.Bacillus subtilis BS-5 were expressed and characterized. EG5C was a typical endoglucanase, comprised of a family 5 catalytic domain and family 3 carbohydrate-binding domain, and which had high activity toward soluble cellulosic substrates, but low activity toward insoluble cellulosic substrates. Importantly, the truncated form EG5C-1 was a processive endoglucanase that hydrolyzed not only carboxymethyl cellulose (CMC), but also insoluble cellulosic substrates. The hydrolytic activities of EG5C-1 towards CMC, phosphoric acid-swollen cellulose (PASC), p-nitrophenyl-β-d-cellobioside, filter paper and Avicel are 4170, 700, 2550, 405 and 320 U/μmol, respectively. These data demonstrated that EG5C-1 had higher activity ratio of exoglucanase to endoglucanase than other known processive endoglucanases. When PASC was degraded by EG5C-1, the ratio of soluble to insoluble reducing sugars was about 3.7 after 3 h of incubation with cellobiose and cellotriose as the main products. Importantly, EG5C-1 alone was able to hydrolyze filter paper and PASC. At 5% substrate concentration and 10 FPU/g PASC enzyme loading, the saccharification yield was 76.5% after 60 h of incubation. Replacement of a phenylalanine residue (F238) by an alanine at the entrance/exit of the substrate binding cleft significantly reduces the ability of EG5C-1 to degrade filter paper and Avicel, but this mutation has little impact on CMCase activity. The processivity of this mutant was also greatly reduced while its cellulose binding ability was markedly enhanced.The endoglucanase EG5C and its truncated form EG5C-1 from B. subtilis BS-5 exhibits excellent properties that render it a suitable candidate for use in biofuel and biochemical production from lignocellulosic biomass. In addition, our studies also provide useful information for research on enzyme processivity at the molecular level.The processive endoglucanase EG5C-1 from The online version of this article (10.1186/s13068-018-1022-2) contains supplementary material, which is available to authorized users. Clostridium thermocellum, which employs a large multiprotein complex (cellulosome) to hydrolyze cellulose [Cytophaga hutchinsonii and Saccharophagus degradans were found to be able to rapidly digest cellulose [Cellulose, the most abundant renewable feedstock for biofuels and chemicals production, consists of linear chains of β-1,4-linked glucose units that form a higher order crystalline structure . An alteellulose . In bothellulose , 6. Howeellulose , 6. Furtellulose –7. Here,ellulose . In the ellulose . TherefoVolvariella volvacea consists of the N-terminal CBM1 and the C-terminal GH5 catalytic domain. Deletion of CBM1 domain significantly reduces the ratio of soluble to insoluble products from filter paper, supporting a critical role of CBM1 for EG1 processivity [S. degradans 2–40 is not affected by the absence/presence of the CBM6 module. It is thus speculated that in this enzyme the catalytic domain alone is essential for the processivity mechanism [C. hutchinsonii, has identified the aromatic amino acid W197 as essential for processivity [Presently, most of the known processive endoglucanases belong to GH9 family, which displays a modular structure with a catalytic domain from family 9 and various carbohydrate-binding modules (CBMs) . These Cessivity . In contechanism , 15. A sB. subtilis BS-5 with a complete cellulolytic system was isolated and characterized; this system was shown to rapidly and efficiently degrade cellulose [Bacillus species commonly lack significant exoglucanase activity. Indeed, an analysis of the genome sequence of B. subtilis 168 indicated that the lignocellulolytic system of this strain contained an endoglucanase, two xylanases and five β-glucosidases, but no genes encoding proteins homologous to any known family of exoglucanases has been found [B. subtilis BS-5 to degrade cellulose thus remains unknown. We hypothesized that B. subtilis BS-5 might produce a processive endoglucanase, which might substitute for the apparent deficiency in exoglucanase activity. However, to the best of our knowledge, no processive endoglucanase from B. subtilis has yet been reported. To substantiate our hypothesis, we purified and characterized three secreted proteins from B. subtilis BS-5 using a cellulose-binding strategy. A multidomain enzyme, EG5C, containing a family 5 catalytic domain and a family 3 CBM, and its two truncated forms, EG5C-1  and EG5C-2 (translocating CBM3 domain from C-terminus of EG5C to its N-terminus), were expressed in E. coli, and characterized to analyze the contribution of the CBM to the catalytic activity and mode of action of these enzymes. In addition, mutants of EG5C-1 were generated to probe its processivity mechanism and cellulose binding ability. Finally, the potential of EG5C-1 for applications in saccharification of cellulosic substrates was evaluated.Until recently, most commercial lignocellulolytic enzymes were produced by filamentous fungi. However, the use of bacteria as highly potent sources for cellulase production has gained increasing attention due to the more complex glycoside hydrolases in these organisms, great natural diversity and higher growth rate . In our ellulose . Howeveren found . The mecBacillus subtilis BS-5 was isolated by combined carboxymethyl cellulose agar plate, xylan agar plate and filter paper hydrolysis assays, and cultivation was performed as previously described [E. coli DH5α was used for plasmid constructions and propagation. pET 28a (Novagen) and E. coli BL21 were used as the host and vector for the expression of recombinant enzymes. Bacterial strains were grown at 37 °C in Luria-Bertani (LB) medium, supplemented with 100 μg/mL ampicillin.escribed . E. coliB. subtilis BS-5 was mixed with Avicel at a final concentration of 10% (w/v) at 4 °C under gentle shaking for 4 h. Subsequently, the mixture was centrifuged to separate the cellulosic substrate. The pelleted fraction was washed several times with distilled water to limit nonspecific protein binding. Bound proteins were eluted with SDS-PAGE buffer, and subjected to SDS-PAGE analysis on a 12% gel. The Coomassie-stained bands of the bound proteins were excised from the gel for LC/MS–MS analysis, and the sequences of the fragments were submitted to the Mascot program for possible identity matching.Fermentative supernatant of B. subtilis 168 was used to design the primer pairs to amplify the genes encoding EG5C and EG5C-1 [Nco I and Xho I, and then ligated into a similarly treated pET 28a vector to generate the expression vectors pET-EG5C and pET-EG5C-1. The variant EG5C-2 was formed by the translocation of CBM3 domain from C-terminus of EG5C to its N-terminus. Therefore, the pET-EG5C-2 system was constructed by inserting a fragment consisting of CBM3 and a 22-amino acid linker (TKDSTKDIPETPSKDSHTQENG) upstream of the catalytic domain of EG5C-1. For this purpose, the fragment encoding CBM3, the linker and the catalytic domain was amplified by PCR using the primer pair EG5C-2-F and EG5C-2-CBM-R, the primer pair EG5C-2-LIN-F and EG5C-2-LIN-R, the primer pair EG5C-2-GH-F and EG5C-2-R, respectively  of d EG5C-1 . For EG5E. coli BL21 to produce recombinant mutant proteins. These point mutations were purified and characterized following the procedures described above.Site-directed mutagenesis of EG5C-1 was carried out by overlap extension PCR and the required primers were listed in Additional file E. coli BL21 cells harboring the plasmids described above were cultured in LB medium and the target genes were expressed as described above [Recombinant ed above , 15. Afted above .2HPO4/KH2PO4 buffer (pH 6.5) and 0.5 mL of suitably diluted enzyme at 45 °C [pNPC (10 mmol/L) was assayed by monitoring the concentration of released p-nitrophenol at 410 nm after addition of NaOH at a final concentration of 0.1 mmol/L as described by Ghatge [The hydrolytic activity of EG5C and its derivatives were measured in 2 mL of a reaction mixture containing 2.5% of the desired substrates  in 50 mmol/L Naat 45 °C . The polat 45 °C . After iat 45 °C . One uniy Ghatge .For viscosity measuring, the reaction was performed at 45 °C in 15 mL total volume containing purified enzyme (15 μg) and 2.5% CMC solution at pH 6.5. Then, samples were taken periodically and boiled for 5 min, and viscosity values were determined using an Ubbelohde viscometer tube .The effects of pH and temperature on the activity of EG5C and its derivatives were measured using CMC as substrate by changing the reaction pH value from 3 to 10 at 45 °C and temperature from 30 to 80 °C at a pH of 6.5, respectively. Thermal stability was estimated by measuring the residual activities after 2 h incubation at a specific temperature ranging from 30 to 80 °C with temperature increments of 10 °C. Enzyme activity prior to incubation was expressed as 100%. pH stability was evaluated by measuring the residual activities after 2 h of incubation in different buffers with pH values ranging from 3 to 9 at 45 °C.Km, Vmax) were determined using CMC (at concentrations from 1 to 20 mg/mL), Avicel and PASC (ranging from 1 to 200 mg/mL) as substrates under optimum reaction conditions, respectively. The data were analyzed using nonlinear regression with GraphPad Prism 5.0 software (http://www.graphpad.com/prism/). Turnover rate (kcat) and catalytic efficiency ratio (kcat/Km) were calculated from the obtained kinetic data.Kinetic constants  was performed using a mixture of n-butanol, acetic acid and water in a volume ratio of 3:2:1. Then, the hydrolysis products were visualized by spraying the plates with an ethanol/sulfuric acid mixture  and heating at 120 °C for 10 min. Hydrolysis of cello-oligosaccharides with the enzymes was carried out for 1 h and analyzed according to the method described above.Hydrolysis of CMC, Avicel and PASC by EG5C and its derivatives was performed for 6 h under the same conditions used for the activity assays and terminated by heating the samples in boiling water bath for 10 min. After centrifugation at 10,000×2HPO4/KH2PO4 buffer (pH 6.5). After incubation at 4 °C for 1 h, the samples were centrifuged  to remove Avicel and the bound protein. The concentration of unbound protein remaining in the supernatant was measured by the method of Bradford [The binding capacity of EG5C and its derivatives to the cellulosic substrate Avicel was assayed in Eppendorf tubes containing recombinant protein and substrate as described by Zhang et al. . The conBradford . Bound pBradford . The iniProcessivity of EG5C and its derivatives was determined by measuring the ratio of soluble to insoluble reducing sugar from PASC , 15. In 2HPO4/KH2PO4 buffer  at a final volume of 20 mL. EG5C and EG5C-1 were added to the reaction mixture at a concentration of 40 μg/mL for each. The mixture was shaken at 200 rpm at 40 °C for 24 h. The control assay without enzyme was conducted under the same conditions.With filter paper, the hydrolysis reactions were carried out at a 2.5% (w/v) solid loading in Na2HPO4/KH2PO4 buffer  with 5% (w/v) of PASC and 10 FPU/g PASC of EG5C-1. The reaction mixture (20 mL) was incubated in a water bath at 40 °C for 60 h. Samples from the reaction were collected every 12 h and quantified by high performance liquid chromatography (HPLC), equipped with a UltiMate 3000 pump  and a refractive index detector . A 100-5 NH2 column  was used for HPLC analysis. The mobile phase comprised acetonitrile and water in a ratio of 75:25 and at a flow rate of 0.7 mL/min. Synergistic hydrolysis by the commercial β-glucosidase  and EG5C-1 under the same conditions was implemented as a control. The loading rates of β-glucosidase and EG5C-1 were 20 U/g PASC and 10 FPU/g PASC, respectively. The hydrolysis yield was defined as the ratio of the total equivalents of glucan hydrolyzed to the total potential glucan available in PASC.The hydrolysis of PASC was performed in a 100-mL Erlenmeyer flask and in Nahttp://www.cbs.dtu.dk/services/SingnalP/). Multiple sequence alignments were carried out using Clustal Omega program suite . Homology modeling of EG5C-1 was performed with Discovery studio (DS) 3.5 MODELER based on the template of the crystal structure of the catalytic domain of BsCel5A from B. subtilis 168 [Nucleotide and amino acid sequences were analyzed using the BLASTn and BLASTp programs. The signal peptide of EG5C was predicted using SignalP 4.0 web program  [B. subtilis 168 (NCBI Accession No. NP 389744.1). Surprisingly, the fragments linked to the 33 kDa protein also displayed high homology to the endoglucanase of B. subtilis 168. However, the matched peptides only appeared in the catalytic domain of this enzyme  . The fraB. subtilis 168, B. subtilis A53, B. amyoliquefaciens DL-3 and B. subtilis UMC7  was composed of a glycoside hydrolase family 5 catalytic domain of 302 amino acids, a linker region of 22 amino acids and a CBM3a module of 146 amino acids  enzyme was ascribed to the low stability of CBM3 in comparison to the catalytic domain [While the three variants had a similar temperature optimum Fig. c, they dlucanase . Chiriacterminus . A serieterminus . Furtherterminus . To dateterminus . Anotherc domain . Due to c domain , 25.p-NPC (330 U/μmol). With respect to insoluble cellulose substrates, EG5C was able to hydrolyze PASC (110 U/μmol), but its activity towards other insoluble substrates was either very low (FP and Avicel) or non-existent (xylan and chitosan). These trends and observations were in agreement with those reported for typical endoglucanases from B. subtilis 168, B. subtilis LH and B. subtilis UMC7 [pNPC (no activity was detected for xylan or chitosan).In order to assess the catalytic potential of EG5C and its derivatives, their activities towards a range of substrates were tested. As shown in Fig. lis UMC7 , 24, 26.yze PASC 10 U/μmolKm, kcat and kcat/Km of EG5C were 5.7 mg/mL, 218/s and 38 mL/mg/s, respectively, while these values for EG5C-1 were 6.5 mg/mL, 193/s and 30 mL/mg/s. From a comparison between EG5C and EG5C-1, it was apparent that the CBM3 domain contributed to a moderate enhancement of substrate affinity  and catalytic efficiency (determined from kcat/Km ratio) in the reaction with CMC. However, only the truncated variant retained significant catalytic activity towards the insoluble polysaccharides PASC and Avicel. Usually, Avicel and p-NPC are used as substrates to measure the activity of exoglucanases as they are not commonly hydrolyzed by endoglucanases [The catalytic parameters recorded for the reactions with CMC, PASC and Avicel were summarized in Table ucanases . The datucanases , 21, 30,vs insoluble reducing ends generated from cellulosic substrates [A. ochraceus MTCC1810, the processivity ratio increased from 1.79 to 3.1 when the incubation time was prolonged from 3 h to 9 h [vs insoluble products was observed for endoglucanase EG1 (8.6), the incubation time was 24 h [The processivity of endoglucanases has been mainly determined from the ratio of soluble bstrates , 21. In bstrates . Furtherbstrates , 12. Andwas 24 h . Generalwas 24 h . For EG5V. volvariella had a CBM1 module and a family 5 catalytic domain, and the deletion of CBM1 significantly decreased its processivity [S. degradans were expressed and characterized. These CBM6 modules only promoted strong binding of the polysaccharide chains but were not necessary for processivity [G. trabeum and CHU_2103 from C. hutchinsonii, lacking a CBM domain have been reported [Since processivity is critical for cellulases to catalyze the degradation of crystalline cellulose, exoglucanases have been playing a key role in the solubilization of cellulose in both the complexed and non-complexed cellulolytic systems , 32. Theessivity . Howeveressivity , 14, 15.reported . The advreported , 35, 36.C. hutchinsonii [The degradation of insoluble cellulosic material by cellulases is thought to involve an initial adsorption of the enzyme onto the cellulose surface, followed by the formation of a protein-cellulose complex. Therefore, the binding ability of EG5C, EG5C-1 and EG5C-2 to Avicel was examined . Similarly, EG5C-1 also hydrolyzed cellotetraose and cellopentaose with cellobiose and cellotriose being the main products formed Fig. a. Howevevolvacea , 13, 21)volvacea . While svolvacea . To the volvacea , 15.Fig.Unlike classical endoglucanases that randomly cleave cellulose polymers to form a variety of degradation products, EG5C-1 released predominantly cellobiose and cellotriose as end products when hydrolyzing the insoluble cellulose substrates PASC and Avicel Fig. b, while The capacity of EG5C and EG5C-1 to solubilize Whatman NO.1 filter paper was demonstrated in Additional file The hydrolysis of PASC was monitored either in presence of only EG5C-1 or in presence of a combination of EG5C-1 and the commercial β-glucosidase Novozyme 188 (an enzyme that degrades predominantly cellobiose) to evaluate saccharification capacity of EG5C-1 on PASC and its potential synergy with β-glucosidase Fig. a. The reB. subtilis 168 [8-barrel fold and a deep substrate-binding cleft formed by the C-terminal ends of β-strands (Fig. In bacterial systems, the majority of processive endoglucanases belong to the GH9 family . Insofarilis 168 . Based opNPC, filter paper and Avicel was observed (Fig. In this study, there were seven aromatic amino acid residues, W69, Y70, Y96, W207, Y231, F238 and W291, distributed in the binding cleft of enzyme EG5C-1 (Fig. ved Fig. . F238 thC. hutchinsonii to alanine. The mutated enzyme still retained nearly 80% specific activity on CMC and PASC compared with wild enzyme, but the processivity was markedly decreased. Moreover, this mutant almost completely lost the cellulose binding activity [To date, experimental and computational investigations indicated that aromatic residues at the entrance and exit of exoglucanase played an important role in binding, processivity and stabilization of carbohydrate ligands , 41, 44.activity . In contB. subtilis BS-5 were expressed in E. coli, purified and characterized for their potential in biomass conversion. EG5C was a typical endoglucanase consisting of a family 5 catalytic domain and a CBM3 domain, and was only an efficient catalyst for the hydrolysis of soluble cellulosic substrates. However, EG5C-1 exhibited high activities for soluble CMC as well as insoluble amorphous and crystalline cellulose substrates, indicating that EG5C-1 was a processive endoglucanase. Moreover, EG5C-1 alone was capable to efficiently hydrolyze filter paper and PASC, releasing cellobiose as the main product. Site-directed mutagenesis demonstrated that the aromatic amino acid F238 at the entrance of the substrate binding cleft played an important role not only for processive activity, but also for cellulose binding ability. Our combined data demonstrate that EG5C-1 has strong industrial potential for the degradation of cellulosic biomass for biofuel production. In addition, due to the distinct functional differences of EG5C, EG5C-1 and the mutant F238, these systems provide an excellent background to investigate the molecular-level details for the mechanism of action of a poorly studied group of enzymes. Efforts towards these goals are in progress.In the present study, the endoglucanase EG5C and its truncated form EG5C-1 from