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Multifunctionality, processivity, and thermostability are critical for the cost-effective enzymatic saccharification of non-food plant biomass polymers such as β-glucans, celluloses, and xylans to generate biofuels and other valuable products. We present molecular insights into a processive multifunctional endo-1,3-1,4-β-d-glucanase (Tt_End5A) from the hyperthermophilic bacterium Thermogutta terrifontis. Tt_End5A demonstrated activities against a broad spectrum of β-polysaccharides, including barley glucan, lichenan, carboxymethyl cellulose, regenerated amorphous cellulose (RAC), Avicel, xylan, laminarin, mannan, curdlan, xanthan, and various chromogenic substrates at pH 7 and temperatures ranging from 70 to 80°C. The enzyme exhibited a high level of processivity on RAC and retained over 90% activity at 80°C for an extended period, indicating exceptional thermal stability. The 1.20 Å crystal structure of the Tt_End5A catalytic domain revealed an archetypal glycoside hydrolase family 5 (GH5) catalytic TIM-(β/α) 8 -barrel, supplemented with additional β-strands, elongated α-helices, and a rare cis-non-Pro (His481-cis-Ala482) peptide. A large central cleft was observed in the 3D structure, which is likely related to the enzyme's multifunctionality and processivity. The catalytic domain is preceded by a novel N-terminal multivalent carbohydrate-binding module (CBM) that enhances the enzymatic degradation of insoluble polysaccharides. Mutagenesis studies, ligand interaction analyses, and the structurally conserved positions of E329 and E448 in Tt_End5A suggest that these residues function as the proton donor and nucleophile in the catalytic mechanism. Owing to its multifunctionality and processivity, Tt_End5A can reduce the need for multiple saccharification enzymes to generate fermentable sugars from plant biomass for bioethanol production. Additionally, it holds promise for applications in the pharmaceutical, feed, and food industries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The genomic screening of hyper-thermophilic Pyrococcus abyssi showed uncharacterized novel α-amylase sequences. Homology modelling analysis revealed that the α-amylase from P. abyssi consists of an N-terminal GH57 catalytic domain, α-amylase central, and C-terminal domain. Current studies emphasize in-silico structural and functional analysis, recombinant expression, characterization, structural studies through CD spectroscopy, and ligand binding studies of the novel α-amylase from P. abyssi. The soluble expression of PaAFG was observed in the E. coli Rosetta™ (DE3) pLysS strain upon incubation overnight at 18 °C in an orbital shaker. The optimum temperature and pH of the PaAFG were observed at 90 °C in 50 mM phosphate buffer pH 6. The K m value for PaAFG against wheat starch was determined as 0.20 ± 0.053 mg while the corresponding V max value was 25.00 ± 0.67 μmol min -1 mg -1 in the presence of 2 mM CaCl 2 and 12.5 % glycerol. The temperature ramping experiments through CD spectroscopy reveal no significant change in the secondary structures and positive and negative ellipticities of the CD spectra showing the proper folding and optimal temperature of PaAFG protein. The RMSD and RMSF of the PaAFG enzyme determined through molecular dynamic simulation show the significant protein's stability and mobility. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Alpha amylase belonging to starch hydrolyzing enzymes has significant contributions to different industrial processes. The enzyme production through recombinant DNA technology faces certain challenges related to their expression, solubility and purification, which can be overcome through fusion tags. This study explored the influence of SUMO, a protein tag reported to enhance the solubility and stability of target proteins when fused to the N-terminal of the catalytic domain of amylase from Pyrococcus abyssi (PaAD). The insoluble expression of PaAD in E. coli was overcome when the enzyme was expressed in a fusion state (S-PaAD) and culture was cultivated at 18 °C. Moreover, the activity of S-PaAD increased by 1.5-fold as compared to that of PaAD. The ligand binding and enzyme activity assays against different substrates demonstrated that it was more active against 1 % glycogen and amylopectin. The analysis of the hydrolysates through HPLC demonstrated that the enzyme activity is mainly amylolytic, producing longer oligosaccharides as the major end product. The secondary structure analyses by temperature ramping in CD spectroscopy and MD simulation demonstrated the enzymes in the free, as well as fusion state, were stable at 90 °C. The soluble production, thermostability and broad substrate specificity make this enzyme a promising choice for various foods, feed, textiles, detergents, pharmaceuticals, and many industrial applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Alpha amylases catalyse the hydrolysis of α-1, 4-glycosidic bonds in starch, yielding glucose, maltose, dextrin, and short oligosaccharides, vital to various industrial processes. Structural and functional insights on α-amylase from Methanocaldococcus jannaschii were computationally explored to evaluate a catalytic domain and its fusion with a small ubiquitin-like modifier (SUMO). The recombinant proteins' production, characterization, ligand binding studies, and structural analysis of the cloned amylase native full gene (MjAFG), catalytic domain (MjAD) and fusion enzymes (S-MjAD) were thoroughly analysed in this comparative study. The MjAD and S-MjAD showed 2-fold and 2.5-fold higher specific activities (μmol min -1  mg -1 ) than MjAFG at 95 °C at pH 6.0. Molecular modelling and MD simulation results showed that the removal of the extra loop (178 residues) at the C-terminal of the catalytic domain exposed the binding and catalytic residues near its active site, which was buried in the MjAFG enzyme. The temperature ramping and secondary structure analysis of MjAFG, MjAD and S-MjAD through CD spectrometry showed no notable alterations in the secondary structures but verified the correct folding of MjA variants. The chimeric fusion of amylases with thermostable α-glucosidases makes it a potential candidate for the starch degrading processes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

TpXyl10B is a glycoside hydrolase family 10 xylanase of hyperthermophile Thermotoga petrophila RKU-1. This enzyme is of considerable importance due to its thermostability. However, in its native state, this enzyme does not possess any carbohydrate-binding module (CBM) for efficient binding to plant biomass. In this study CBM6 from Clostridium thermocellum was attached to the N- and C-termini of TpXyl10B, thereby producing the variants TpXyl10B-B6C and TpXyl10B-CB6, respectively. TpXyl10B-B6C showed 5-7 folds increased activity on Beechwood xylan and the different types of plant biomass as compared to that from the catalytic domain only. However, the activity of TpXyl10B-CB6 decreased 0.6-0.8 folds on Beechwood xylan and plant biomass compared to the catalytic domain. We explained these results through molecular modeling, which showed that binding residues of CBM6's cleft B, which were previously reported to show no contribution towards binding due to steric hindrance from a loop region, were exposed in a favorable position in TpXyl10B-B6C such that they efficiently bound the substrate. In contrast, these binding residues of CBM6 in TpXyl10B-CB6 were exposed opposite to the catalytic residues; thus, binding to the substrate resulted in decreased exposure of catalytic residues to the substrate. CD spectroscopy and thermostability assays showed that TpXyl10B-B6C was highly thermostable, having a melting point > 90 °C, which is relatively higher than that of the other variant, TpXyl10B-CB6. In addition, this xylanase variant showed synergism with cellulases for the hydrolysis of plant biomass. Therefore, TpXyl10B-B6C, an engineered xylanase in this study, can be a valuable candidate for industrial applications., Competing Interests: Competing interests The authors declare no competing financial interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Xylanase XynB of the hyperthermophile Thermotoga maritima, which belongs to glycoside hydrolase family 10 (GH10), does not have an associated carbohydrate binding module (CBM) in the native state. CBM6 and CBM22 from a thermophile Clostridium thermocellum were fused to the catalytic domain of XynB (XynB-C) to determine the effects on activity and other properties. XynB-B22C and XynB-CB22, produced by fusing CBM22 to the N- and C-terminal of XynB-C, showed 1.7- and 3.24-fold increase in activity against the insoluble birchwood xylan, respectively. Similarly, CBM6 when attached to the C-terminal of XynB-C resulted in 2.0-fold increase in activity, whereas its attachment to the N-terminal did not show any increase of activity. XynB-B22C and XynB-CB22 retained all the activity, whereas XynB-B6C and XynB-CB6 lost 17 and 11% of activity, respectively, at 60°C for 4h. Thermostability data and the secondary structure contents obtained by molecular modelling are in agreement with the data from circular dichroism analysis. Molecular modelling analysis showed that the active site residues of the catalytic domain and the binding residues of CBM6 and CBM22 were located on the surface of molecule, except XynB-B6C, where the binding residues were found somewhat buried. In the case of XynB-CB22, the catalytic and the binding residues seem to be located favorably adjacent to each other, thus showing higher increase in activity. This study shows that the active site residues of the catalytic domain and the binding residues of the CBM are arranged in a unique fashion, not reported before., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Early diagnosis of colorectal cancer (CRC) can be of value for increasing the survival rate of patients. Recently, proteomic strategies to identify markers for the diagnosis of cancer at an early stage have been employed with noteworthy results. To extend these studies, we utilized two dimensional gel electrophoresis and mass spectrometry for expression profiling of proteins extracted from the freshly frozen human colorectal cancer tissue specimens and the comparable regions of adjacent normal mucosa (serving as controls). Four gel spots were determined to be differentially stained between the tumor and the control samples on a consistent basis. Following mass spectrometric analysis of these spots, six proteins were identified; five of these had previously been reported to be associated with colorectal cancer. One protein actin beta-like 2 (ACTBL2), not linked with colorectal cancer in the earlier reports, was however found to be at higher abundance in colorectal tumor samples both by proteomics and immunohistochemistry analysis. Thus ACTBL2 association and differential upregulation in colorectal cancer is novel, and as such may contribute to our understanding of the colorectal carcinogenesis and potentially serve a function in developing markers for colorectal cancer., Biological Significance: Colorectal cancer (CRC) is a major cause of death world-wide and good markers for early detection are lacking. In this study we conducted a proteomic analysis of tumor vs. normal tissue. We corroborated the finding of a number of previously identified proteins associated with CRC and more importantly identified a novel protein, ACTBL2, which we demonstrated to be upregulated in CRC. As additional proteins associated with CRC are identified the potential for developing panels of markers may be realized with better outcomes in early cancer detection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Removal and recovery of zirconium from dilute aqueous solutions by Candida tropicalis used as biosorbent, was studied by performing biosorption-desorption tests. This biosorbent was selected after screening a range of microbial species. The process was found to be highly dependent on initial pH and concentration of metal solution. At optimized experimental parameters, the maximum zirconium biosorption capacity of C. tropicalis was 179 mg Zr g(-1) dry weight of biosorbent. The adsorption distribution coefficient value of 3968 ml g(-1) was obtained for zirconium biosorption by C. tropicalis. Different theoretical thermodynamic models governing the adsorption behavior of zirconium were also tested. Zirconium biosorption was found to closely follow the Langmuir model. At low biomass concentrations it was found to follow pseudo-first-order kinetics. However when higher biomass concentrations were used kinetics was changed to pseudo-second-order. The zirconium bound to the biomass was stripped out (60.2% at S/L of 1.0 g of zirconium loaded biomass/l of eluent) using sodium bicarbonate and the biomass could be used for multiple sorption-desorption cycles.

The study reports removal of heavy metals when present singly or in binary and ternary systems by the milling agrowaste of Cicer arientinum (chickpea var. black gram) as the biosorbent. The biosorbent removed heavy metal ions efficiently from aqueous solutions with the selectivity order of Pb>Cd>Zn>Cu>Ni. The biosorption of metal ions by black gram husk (BGH) increased as the initial metal concentration increased. Biosorption equilibrium was established within 30 min, which was well described by the Langmuir and Freundlich adsorption isotherms. The maximum amount of heavy metals (qmax) adsorbed at equilibrium was 49.97, 39.99, 33.81, 25.73 and 19.56 mg/g BGH biomass for Pb, Cd, Zn, Cu and Ni, respectively. The biosorption capacities were found to be pH dependent and the maximum adsorption occurred at the solution pH 5. Efficiency of the biosorbent to remove Pb from binary and ternary solutions with Cd, Cu, Ni and Zn was the same level as it was when present singly. The presence of Pb in the binary and ternary solutions also did not significantly affect the sorption of other metals. Breakthrough curves for continuous removal of Pb from single, binary and ternary metal solutions are reported for inlet-effluent equilibrium. Complete desorption of Pb and other metals in single and multimetal solutions was achieved with 0.1 M HCl in both shake flask and fixed bed column studies. This is the first report of removal of the highly toxic Pb, Cd, and other heavy metals in binary and ternary systems based on the biosorption by an agrowaste. The potential of application for the treatment of solutions containing these heavy metals in multimetal solutions is indicated.