Selective enhanced cytotoxicity of amino acid deprivation for cancer therapy using thermozyme functionalized nanocatalyst.

BACKGROUND: Enzyme therapy based on differential metabolism of cancer cells has demonstrated promising potential as a treatment strategy. Nevertheless, the therapeutic benefit of reported enzyme drugs is compromised by their uncontrollable activity and weak stability. Additionally, thermozymes with high thermal-stability suffer from low catalytic activity at body temperature, preventing them from functioning independently. RESULTS: Herein, we have developed a novel thermo-enzymatic regulation strategy for near-infrared (NIR)-triggered precise-catalyzed photothermal treatment of breast cancer. Our strategy enables efficient loading and delivery of thermozymes (newly screened therapeutic enzymes from thermophilic bacteria) via hyaluronic acid (HA)-coupled gold nanorods (GNRs). These nanocatalysts exhibit enhanced cellular endocytosis and rapid enzyme activity enhancement, while also providing biosafety with minimized toxic effects on untargeted sites due to temperature-isolated thermozyme activity. Locally-focused NIR lasers ensure effective activation of thermozymes to promote on-demand amino acid deprivation and photothermal therapy (PTT) of superficial tumors, triggering apoptosis, G1 phase cell cycle arrest, inhibiting migration and invasion, and potentiating photothermal sensitivity of malignancies. CONCLUSIONS: This work establishes a precise, remotely controlled, non-invasive, efficient, and biosafe nanoplatform for accurate enzyme therapy, providing a rationale for promising personalized therapeutic strategies and offering new prospects for high-precision development of enzyme drugs.

Increased Expression Levels of Thermophilic Serine Protease TTHA0724 through Signal Peptide Screening in Bacillus subtilis and Applications of the Enzyme.

The thermostable protease TTHA0724 derived from Thermus thermophilus HB8 is an ideal industrial washing enzyme due to its thermophilic characteristics; although it can be expressed in Escherichia coli via pET-22b, high yields are difficult to achieve, leading to frequent autolysis of the host. This paper details the development of a signal peptide library in the expression system of B. subtilis and the optimization of signal peptides for enhanced extracellular expression of TTHA0724. When B. subtilis was used as the host and the optimized signal peptide was used, the expression level of TTHA0724 was 16.7 times higher compared with E. coli. B. subtilis as an expression host does not change the characteristics of TTHA0724. The potential application fields of TTHA0724 are studied. TTHA0724 can be used as a detergent additive at 60 °C, which can sterilize and eliminate mites while thoroughly cleaning protein stains. Soybean meal enzymatic hydrolysis with TTHA0724 at a high temperature produced a higher content of antioxidant peptides. These results indicate that TTHA0724 has great potential for industrial applications.

Constitutive Damage Model of Foamed Lightweight Concrete Using Statistical Damage Theory.

Foamed lightweight concrete has been applied in different fields of civil engineering because of its superior properties, but the related research considering internal pore damage is limited. Based on statistical damage theory and considering the uneven distribution of fracture damage and strength between the pores of light concrete, a damage constitutive model of foamed lightweight concrete was established based on the Weibull function. The parameters of the damage model were determined through a triaxial compression test, and the rationality was verified by combining the existing test data. Comparative tests show that the theoretical calculation results of the proposed statistical damage model of foamed light soil are consistent with the general trend of the experimental results, reflecting the value of the peak stress and strain and describing the overall development law of the stress and strain. The best fit was obtained when the confining pressure was 0.3 MPa and the density was 700 kg·m-3. The suggested damage constitutive method is highly applicable, which is of great significance to the microscopic mechanical analysis of foamed light concrete and the structural design in civil engineering.

Molecular dynamics simulation guided distal mutation of Thermotoga naphthophila ß-glucosidase for significantly enhanced synthesis of galactooligosaccharides and expanded product scope.

For efficient enzymatic production of health-beneficial galactooligosaccharides (GOSs), a glycone (-1)/aglycone (+2) subsite mutation strategy to engineer a thermophilic GH1 ß-glucosidase (Tn0602) from Thermotoga naphthophila RKU-10 was introduced. Six single mutation variants (F226G, N246G, N246E, N222F, N222Y, G224T) and two double mutants (F226GF414S, F226GF414Y) were designed. The +2-subsite variant F226G produced 136 mM galactooligosaccharide 1.2-fold more than the wild type (115 mM). More significantly, a superimposed mutation of the -1/+2 subsites F226G/F414S gave a total GOS production of 314 mM (82.16% lactose conversion), 2.7-fold higher than the total GOS production of the wild type. Furthermore, the variant F226GF414S was profiled 241 mM of trisaccharide (galß (1 â†’ 3)/(1 â†’ 4) lactose) and 73 mM tetrasaccharide (galß (1 â†’ 3)/(1 â†’ 4) galß (1 â†’ 3)/(1 â†’ 4) lactose). According to a 300-ns molecular dynamic simulation, the superimposed mutation increased GOS productivity and expanded the scope of products by changing the structural flexibility and reducing the steric hindrance of the substrate tunnel. Overall, our study successfully demonstrated that a - 1/+2 subsite mutagenesis method could be used in ß-glucosidases Tn0602 to improve enzyme productivity and expand product scope, which could be a potential route to evolve retaining glycosidases towards the desired direction.

Heterologous expression and characterization of a novel subtilisin-like protease from a thermophilic Thermus thermophilus HB8.

Subtilisins are a family of serine proteases used widely throughout the detergent, leather and food industries, with the identification and development of new enzymes holding much potential value. Thermus thermophilus HB8 was examined for serine proteases and found TTHA0724 gene. Sequence analysis of this putative serine protease placed it within the subtilisin family. To obtain active T. thermophilus HB8 subtilisins, three genes encoding prepro-subtilisin, pro-subtilisin and mature-subtilisin were cloned and expressed in Escherichia coli Transetta (DE3). Although direct expression of the mature-subtilisin gene was found to produce inactive inclusion bodies, expression of the pro-subtilisin gene resulted in active mature-subtilisin, indicating that the pro-sequence of translated pro-subtilisin underwent autoproteolysis. The resulting mature-subtilisin exhibited maximal activity between 65 and 85 °C at pH 7.5. The mature-subtilisin showed good stability, maintaining 50% activity after 48 h at 75 °C and >78% activity across the pH range 5.0-9.5. Furthermore, the mature-subtilisin demonstrated broad substrate specificity, with no requirement for the presence of metal ions which are essential for other subtilisin enzymes. Despite this Cu2+ was able to increase enzyme activity, while Ca2+ partially inhibited the activity. These properties suggest that T. thermophilus HB8 mature-subtilisin has potential value in its application in many industries.

ß-Glucosidase from Thermotoga naphthophila RKU-10 for exclusive synthesis of galactotrisaccharides: Kinetics and thermodynamics insight into reaction mechanism.

This work reports a novel thermophilic ß-glucosidase (TN0602) from Thermotoga naphthophila RKU-10, demonstrating exceptionally high catalytic selectivity (100%) for the exclusive synthesis of prebiotic galactotrisaccharides (GOS3) in a high volumetric production yield of 23.28gL-1h-1 (higher than the highest value ever reported) at pH 6.5 and 75°C, with milk processing waste lactose as both the galactosyl donor and acceptor. A comparative study with commercial ß-galactosidase from Aspergillus oryzae (AO) with respect to reaction kinetics, enzyme-substrate thermodynamic binding (substrate induced fluorescence quenching) and molecular docking simulation studies showed that ß-glucosidase TN0602 has a deep catalytic "pocket" with a narrow entrance that prevents simultaneous access of lactose and GOS3 to the catalytic site, explaining its distinct catalytic specificity and reaction kinetics. The findings revealed in this work offer an improved understanding of how enzyme protein structure determines catalytic specificity, which serves as new knowledge to engineer ß-glucosidase for the biosynthesis of designer GOS.

Identification of an archaeal maltooligosyltrehalose trehalohydrolase encoded by an interrupted gene.

Complete genome analysis of the thermoacidophilic Archaeon Sulfolobus tokodaii strain 7 revealed two open reading frames (ORF), namely, ST0926 and ST0927. These ORFs are interrupted by two putative insertions and encode for the N- and C-terminal fragments, respectively, of a putative Sulfolobus sp. maltooligosyltrehalose trehalohydrolase (StMTHase). Two specific deletion mutations, designed on the basis of sequence alignments of the adjacent ORFs and the published Sulfolobus sp. MTHases, allowed soluble expression in Escherichia coli of an active acidic and thermophilic enzyme. The purified enzyme exhibited a maximum amylolytic activity at 70 °C and pH 5.0, which resembled the optimal conditions of the Sulfolobus homologs. Furthermore, we report that these ORFs are actively co-transcribed in vivo, and we confirm the presence of insertions between them at the cDNA level. However, immunization and western blot experiments demonstrated no expression of ST0926 or the putative full-length StMTHase in vivo, indicating that they might exist as nonfunctional pseudogenes.

Cloning, purification, and characterization of inorganic pyrophosphatase from the hyperthermophilic archaea Pyrococcus horikoshii.

The gene encoding inorganic pyrophosphatase (PPiase) from the hyperthermophilic archaea Pyrococcus horikoshii (Pho PPiase) was cloned in the Escherichia coli strain BL21/pET15b, and the recombinant PPiase was purified by Ni-chelating chromatography in only an one-step procedure. The PPiase showed optimal activity at 88°C and pH of 10.3. Kinetic analysis revealed Km, kcat, Vm of 14.27µM, 3436s(-1), and 34.35µmol/min/mg protein, respectively. Pho PPiase was stable against denaturant chemicals as well as heat. It retained 19.61% of the original activity after incubation at 100°C for 12h and 25.96% of the original activity in the presence of 8M urea after incubation at 50°C for 120h. Pho PPiase showed high specificity for inorganic pyrophosphate but low reactivity to sodium tripolyphosphate and sodium tetrapolyphosphate. ADP and ATP could not serve as substrates.

Heterologous expression and characterization of human cellular glutathione peroxidase mutants.

Cellular glutathione peroxidase (GPx1; EC1.11.1.9) is a major intracellular antioxidant selenoenzyme in mammals. However, the complicated expression mechanism of selenocysteine (Sec)-containing protein increases the difficulty of expressing human GPx1 (hGPx1) in Escherichia coli (E. coli). In this study, hGPx1 gene was cloned from a cDNA library of the human hepatoma cell line HepG2. The codon UGA encoding Sec49 of hGPx1 was first mutated to UGC encoding cysteine (Cys) and then biosynthetically converted to Sec during expression in an E. coli BL21(DE3)cys auxotrophic system. Seleno-GPx1Sec displayed a low GPx activity of 522 U/µmol. To improve the activity, the other five Cys residues (C2, C78, C115, C156, C202) were mutated to serine (Ser) in one hGPx1 molecule. The mutant seleno-hGPx1Ser showed a high activity of 5278 U/µmol, which was more than 10-fold enhanced as compared with seleno-GPx1Sec . The activity was the highest among all of those seleno-proteins obtained by this method so far. Kinetic analysis of seleno-hGPx1Ser showed a typical ping-pong mechanism, which was similar to those of natural GPxs. This research will be of value in overcoming the problem of limited sources of natural GPx and substantially promotes the research of the characterization of GPx. © 2014 IUBMB Life, 66(3):212-219, 2014.

Crystal structure of an acylpeptide hydrolase/esterase from Aeropyrum pernix K1.

Acylpeptide hydrolases (APH; also known as acylamino acid releasing enzyme) catalyze the removal of an N-acylated amino acid from blocked peptides. The crystal structure of an APH from the thermophilic archaeon Aeropyrum pernix K1 to 2.1 A resolution confirms it to be a member of the prolyl oligopeptidase family of serine proteases. The structure of apAPH is a symmetric homodimer with each subunit comprised of two domains. The N-terminal domain is a regular seven-bladed beta-propeller, while the C-terminal domain has a canonical alpha/beta hydrolase fold and includes the active site and a conserved Ser445-Asp524-His556 catalytic triad. The complex structure of apAPH with an organophosphorus substrate, p-nitrophenyl phosphate, has also been determined. The complex structure unambiguously maps out the substrate binding pocket and provides a basis for substrate recognition by apAPH. A conserved mechanism for protein degradation from archaea to mammals is suggested by the structural features of apAPH.

Crystallization and preliminary X-ray analysis of inorganic pyrophosphatase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Inorganic pyrophosphatase (PPase; EC 3.6.1.1) from the hyperthermophile Pyrococcus horikoshii was crystallized by the hanging-drop vapour-diffusion method at pH 5.0 using polyethyleneglycol 4000 as the precipitant. The crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 71.7, b = 86.5, c = 92.5 A, alpha = beta = gamma = 90 degrees. There are two molecules in the asymmetric unit. The crystals were stable during exposure to X-rays and a full set of X-ray diffraction data was collected to 2.7 A resolution in-house.