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A peptidase S9 prolyl oligopeptidase domain from Thermotoga petrophila RKU-1 T (TpS9) was over-expressed as an active, soluble and hyperstable lipolytic enzyme in the mesophilic host system. The sequence analysis demonstrated, TpS9 is an esterase/lipase-like protein belongs to alpha/beta (α/β)-hydrolase superfamily with a well-conserved penta-peptide (GLSAG) motif and α/β-hydrolase fold. Various approaches (induction and cultivation) were employed to enrich TpS9 production, 6.04- and 7.26-fold increment was observed with IPTG (0.4 mM) and lactose (200 mM) in the modified 4ZB medium (pH 7.0), but with IPTG-independent auto-induction strategy 9.02-fold augmentation was achieved after 16 h incubation at 24 °C (150 rev min -1 ). Purified TpS9 showed optimal activity in McIlvaine buffer (pH 6.5) at 80-85 °C, and revealed great thermal (30-85 °C) and pH (6.0-9.0) for 8 h. No obvious constraint was perceived with various metal ions, surfactants, commercial laundry detergents, and chemical modulators. Whereas, TpS9 activity was improved with Ca 2+ , Mn 2+ , and Mg 2+ by 210 %, 142.5 %, and 134.3 %, respectively. With 2.5 M NaCl (215 %), 50 % (v/v) methanol (140 %), 50 % (v/v) ethanol (126.6 %), 50 % (v/v) n-butanol (122.3 %), 50 % (v/v) isopropanol (120.4 %), 50 % (v/v) acetone (118.6 %) and 50 % (v/v) glycerol (113.2 %) TpS9 activity was also enriched. TpS9 demonstrated great affinity toward natural oils and p-nitrophenyl ester substrates, but showed peak activity with p-nitrophenyl palmitate (3160 U mg -1 ). K m , V max , k cat , V max K m -1 and k cat K m -1 of TpS9 with pNPP were 0.421 mM, 4015 µmol mg -1 min -1 , 906.4 s -1 , 9536.8 min -1 , and 2152.96 mM -1 s -1 , respectively. Moreover, TPS9 has notable ability to clean stains (5 min) and degrade the animals' fat (3 h). Hence, TpS9 is a favorable candidate as cleaning bio-additive in detergent formulation, fat degradation and various other 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 Inc. All rights reserved.)

It is urgent the transition from a fossil fuel-based economy to a sustainable bioeconomy based on bioconversion technologies using renewable plant biomass feedstocks to produce high chemicals, bioplastics, and biofuels. β-Glucosidases are key enzymes responsible for degrading the plant cell wall polymers, as they cleave glucan-based oligo- and polysaccharides to generate glucose. Monosaccharide-tolerant or -stimulated β-glucosidases have been reported in the past decade. Here, we describe a novel mechanism of β-glucosidase stimulation by glucose and xylose. The glycoside hydrolase 1 family β-glucosidase from Thermotoga petrophila (TpBgl1) displays a typical glucose stimulation mechanism based on an increased V max and decreased K m in response to glucose. Through molecular docking and dynamics analyses, we mapped putative monosaccharide binding regions (BRs) on the surface of TpBgl1. Our results indicate that after interaction with glucose or xylose at BR1 site, an adjacent loop region assumes an extended conformation, which increases the entrance to the TpBgl1 active site, improving product formation. Biochemical assays with TpBgl1 BR1 mutants, TpBgl1 D49A/Y410A and TpBgl1 D49K/Y410H , resulted in decreasing and abolishing monosaccharide stimulation, respectively. These mutations also impaired the BR1 looping extension responsible for monosaccharide stimulation. This study provides a molecular basis for the rational design of β-glucosidases for biotechnological applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A thermostable β-xylosidase gene Tpexyl3 from Thermotoga petrophila DSM 13,995 was cloned and overexpressed by Escherichia coli. Recombinant Tpexyl3 was purified, and its molecular weight was approximately 86.7 kDa. Its optimal activity was exhibited at pH 6.0 and 90 °C. It had broad specificity to xylopyranosyl, arabinopyranosyl, arabinofuranosyl and glucopyranosyl moieties. The β-xylosidase activity of the recombinant Tpexyl3 was 6.81 U/mL in the LB medium and 151.4 U/mL in a 7.5 L bio-reactor. It was applied to transform ginsenoside extract into the pharmacologically active minor ginsenoside 20(S)-Rg3, which was combined with thermostable β-glucosidase Tpebgl3. After transforming under optimal condition, the 20 g/L of ginsenoside extract was transformed into 6.28 g/L of Rg3 within 90 min, with a corresponding molar conversion of 95.0% and Rg3 productivity of 1793.49 mg/L/h, respectively. This study is the highest report of a GH3 family glycosidase with arabinopyranosidase activity and also the first report on the high substrate concentration bioconversion of ginsenoside extract to ginsenoside 20(S)-Rg3 by using two thermostable glycosidases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A multimodular hyperthermophilic β‑glucuronidase (TpGUS) from Thermotoga petrophila RKU-1 T , belongs to glycoside hydrolase family 2 (GH2), was cloned and overexpressed in Escherichia coli BL21 CodonPlus (DE3)-RIPL. Expression and production of extracellular TpGUS was enhanced through various specific cultivation and induction strategies. Extracellular TpGUS activity was improved by 3.44 and 7 fold in 4 × ZB medium induced with 0.5 mM IPTG and 100 mM lactose, respectively. The enzyme was purified to homogeneity with a single band of 65.6 kDa on SDS-PAGE, using two subsequent steps of anion exchange and hydrophobic interaction chromatography after heat precipitation (70 °C, 1 h). Optimal activity of TpGUS was observed at 95 °C and pH 6.0; and it displayed prodigious thermal stability over a temperature range of 50-85 °C for 12 h at pH 6.0-7.5. K m , V max , V max K m -1 , k cat , and k cat K m -1 were calculated to be 0.7 mM, 227 mmol mg -1  min -1 , 324.3 min -1 , 164,492.7 s - 1 and 234,989.6 mM -1  s -1 , respectively using pNPGU as a substrate. Recombinant TpGUS exhibited favorable properties which make this a promising candidate for various biotechnological and pharmacological applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Cellulose, being the most abundant biopolymer found in nature, can be utilized for bioethanol production to cater the future energy needs. Due to increased usage of fossil fuel it has been predicted that fossil fuel reserves may be depleted by year 2050. These concerns need serious attention and focus should be diverted to renewable fuels that are based on natural resources. Cellulases including exoglucanase (cellobiohydrolases) are the key enzymes that are produced by cellulolytic micro-organisms for the biodegradation of natural resource (cellulose) into fermentable reducing sugars. Many members of genus Clostridium possess supramolecular structures known as cellulosomes which contain various cellulases. Cellulase are composed of catalytic subunits that include endoglucanase, β-glucosidase and cellobiohydrolases which concurrently can catalyse and subsequently convert cellulose into glucose and other sugars. After the action of cellulases, the sugars can be conveniently converted into bioethanol., Objective: In the present study, characterization of a thermostable cellobiohydrolase enzyme from Thermotoga petrophila was carried out. The main purpose of this study is the utilization of thermostable cellobiohydrolase along with other cellulases in the process of saccharification of the cellulosic biomass to produce fermentable sugars that could in turn be converted into bioethanol which is the fuel of the future., Method: In this article, we propose a framework for achieving our a forementioned object. We started with the cloning of thermophilic cellobiohydrolase gene in mesophilic hosts to ease enzyme production. After cloning of cellobiohydrolase gene, submerged fermentation was performed for intracellular enzyme production. Microbial pellet obtained after centrifugation was sonicated and subjected to ammonium sulphate precipitation. The fraction obtained was purified to isoelectric homogeneity through ion exchange chromatography. Finally SDS analysis of purified cellobiohydrolase was carried out alongwith its characterization, kinetics and thermodynamics studies., Results: Purification fold of 4.05 was obtained along with enzyme activity and specific activity of 11.5 U ml-1 min-1 and 66.5 U mg-1, respectively. The molecular mass of purified recombinant enzyme was 37 kDa as calculated by means of SDS-PAGE analysis. The enzyme showed 50% residual activity at 90°C and also at a wide pH range of 4-10. The enzyme retained its activity in the presence of most of the metal ions except Fe+2, Hg+2 and Pb+2. EDTA has an inhibitory effect on the function of the enzyme. The catalytic activity of the enzyme was maintained in the presence of the organic solvents. The enzyme had a Km and Vmax of 4.6 mM and 25.64±1.87 µM min-1 for PNP-β- D-cellobioside under optimal conditions., Conclusion: The present study demonstrated that cellobiohydrolase produced from Thermotoga petrophila can be employed in many industries like paper and pulp and food processing. Most recent application of the cellobiohydrolases is their utilization in the production of bioethanol., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Thermostable α-amylases have many industrial applications and are therefore continuously explored from novel sources. We present the characterization of a novel putative α-amylase gene product (Tp-AmyS) cloned from Thermotoga petrophila. The purified recombinant enzyme is highly thermostable and able to hydrolyze starch into dextrin between 90 and 100°C, with optimum activity at 98°C and pH8.5. The activity increased in the presence of Rb 1+ , K 1+ and Ca 2+ ions, whereas other ions inhibited activity. The crystal structure of Tp-AmyS at 1.7Å resolution showed common features of the GH-13 family, however was apparently found to be a dimer. Several residues from one monomer interacted with a docked acarbose, an inhibitor of Tp-AmyS, in the other monomer, suggesting catalytic cooperativity within the dimer. The most striking feature of the dimer was that it resembled the dimerization of salivary amylase from a previous crystal structure, and thus could be a functional feature of some amylases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Non-productive adsorption of cellulases onto lignins is an important mechanism that negatively affects the enzymatic hydrolysis of lignocellulose biomass. Here, we examined the non-productive adsorption of two bacterial β-glucosidases (GH1 and GH3) on lignins. The results showed that β-glucosidases can adsorb to lignins through different mechanisms. GH1 β-glucosidase adsorption onto lignins was found to be strongly pH-dependent, suggesting that the adsorption is electrostatically modulated. For GH3 β-glucosidase, the results suggested that the fibronectin type III-like domain interacts with lignins through electrostatic and hydrophobic interactions that can partially, or completely, overcome repulsive electrostatic forces between the catalytic domain and lignins. Finally, the increase of temperature did not result in the increase of β-glucosidases adsorption, probably because there is no significant increase in hydrophobic regions in the β-glucosidases structures. The data provided here can be useful for biotechnological applications, especially in the field of plant structural polysaccharides conversion into bioenergy and bioproducts., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A putative α-amylase gene of Thermotoga petrophila was cloned and expressed in Escherichia coli BL21 (DE3) using pET-21a (+), as an expression vector. The growth conditions were optimized for maximal expression of the α-amylase using various parameters, such as pH, temperature, time of induction and addition of an inducer. The optimum temperature and pH for the maximum expression of α-amylase were 22 °C and 7.0 pH units, respectively. Purification of the recombinant enzyme was carried out by heat treatment method, followed by ion exchange chromatography with 34.6-fold purification having specific activity of 126.31 U mg(-1) and a recovery of 56.25%. Molecular weight of the purified α-amylase, 70 kDa, was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 100 °C temperature and at pH of 7.0. The enzyme activity was increased in the presence of metal ions especially Ca(+2) and decreased in the presence of EDTA indicating that the α-amylase was a metalloenzyme. However, addition of 1% Tween 20, Tween 80 and β-mercaptoethanol constrained the enzyme activity to 87, 96 and 89%, respectively. No considerable effect of organic solvents (ethanol, methanol, isopropanol, acetone and n-butanol) was observed on enzyme activity. With soluble starch as a substrate, the enzyme activity under optimized conditions was 73.8 U mg(-1). The α-amylase enzyme was active to hydrolyse starch forming maltose.