Schematic of the synthetic route for fluorescent CDs-BC and their applications in detection of<italic>p</italic>-NP bioimaging.

In immature, 3-week-old female rats, 5 mg equol given by subcutaneous injection increased uterine wet weight 24 h later to the same degree as did μg oestradiol-17β. At this dose there was more receptor complex binding nucleus in equol-injected rats than injected with oestradiol-17β even after 6 h. However, equol–receptor that bound extractable 0·3 m -KC1. duration of growth shorter and less replenishment synthesis protein DNA 30 either injection. It concluded is a weakly oestrogenic compound...

Genetically engineered bacteria represent a promising drug delivery tool for disease treatment. The development of new strategies specific and independent protein regulation is necessary, especially combination therapy. Using the well-studied Escherichia coli phage λ as model system, we applied noncanonical amino acids (ncAAs) novel inducers in bacteria-based system. Screening permissive sites Cro revealed that incorporation AlocK at K8 site with MbPylRS-349F/tRNAPyl system produced...

Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little known about effects of metal ions on properties pyrolysin. Here, we report that supplementation Na(+), Ca(2+), or Mg(2+) salts concentrations similar to those seawater destabilizes recombinant pyrolysin leads increase activity. The destabilizing effect appears be related disturbance surface electrostatic interactions...

By directed evolution and subsequent site-directed mutagenesis, cold-adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four-amino acid substitution variant RTN29 displayed sixfold increase in caseinolytic activity the temperature range 15-25 degrees C, down-shift optimum by approximately 15 as well decrease thermostability, indicating it follows general principle trade-off between stability. Nevertheless, to some extent remained its nature, no...

Natrinema sp. J7-2 is an extreme haloarchaeon capable of growing on synthetic media without amino acid supplements. Here we report the complete genome sequence which composed a 3,697,626-bp chromosome and 95,989-bp plasmid pJ7-I. This first member genus Natrinema. We demonstrate that can use gluconate, glycerol, or acetate as sole carbon source its encodes metabolic pathways for assimilating these substrates. The biosynthetic all 20 acids have been reconstructed, discuss possible...

The incorporation of the structural elements thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, process engineering with both high thermostability and activity remains an important challenge. Here, we report that a thermophilic subtilase were simultaneously improved by incorporating psychrophilic subtilase. There 64 variable regions/residues (VRs) in alignment WF146 protease, mesophilic sphericase, S41. protease was...

A gene encoding a spore-associated subtilase, designated protease CDF, was cloned from Thermoactinomyces sp. CDF and expressed in Escherichia coli. The enzyme is translated as proform consisting of 94 aa propeptide 283 mature domain. Phylogenetic analysis revealed that this belonged to the subtilisin family, but could not be grouped into any its six known subfamilies. has an unusually high content charged residues, which are mainly distributed on surface. recombinant formed inclusion bodies,...

Summary Halolysins are T at‐dependent extracellular subtilases of haloarchaea. Whether halolysins can be activated before transport across the cytoplasmic membrane in a folded state and how haloarchaea minimize risk intracellular activation proteolysis cellular proteins unknown. Here, we report that both precursor proform halolysin S pt A from N atrinema sp. J 7‐2 mature autocatalytically, maturation proceeds less efficiently presence KCl than C l. When produced H aloferax volcanii , most...

Here, the gene encoding a subtilisin-like protease (protease Als) was cloned from Thermoactinomyces vulgaris strain CDF and expressed in Escherichia coli . The recombinant enzyme released into culture medium of E. as mature form (mAls). Purified mAls displayed optimal activity at 60–70°C pH 10.0 using azo-casein substrate, showed half-life 13.8 h 70°C. Moreover, thermostable comparable to or higher than those mesophilic subtilisin Carlsberg proteinase K low temperatures (10–30°C). Protease...

Halolysin SptA from haloarchaeon Natrinema sp. J7 consists of a subtilisin-like catalytic domain and C-terminal extension (CTE) containing two cysteine residues. In this report, we have investigated the function CTE using recombinant enzymes expressed in Haloferax volcanii WFD11. Deletion greatly reduced but did not abolish protease activity, which suggests that is essential for enzyme folding. Mutational analysis residues Cys303 Cys338 within form disulfide bond make resistant to...

ABSTRACT The haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains halolysin gene, sptC . encodes precursor composed of signal peptide, an N-terminal propeptide consisting core domain (N*) linker subtilisin-like catalytic domain, polycystic kidney disease (PkdD), chitin-binding (ChBD). Here we report autocatalytic maturation SptC is initiated by cis -processing N* yield autoprocessed complex...

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 S39) mesophilic SSII sphericase) homologs. Deletion of the insertion 3 (positions 193–197) or 4 210–221) resulted in significant decrease enzyme stability. In addition, substitution residues Pro211 Ala212 residue Glu221 which localized vicinity Ca 2+ binding site by corresponding subtilisin remarkably reduced half‐life at 70 °C, suggesting that three...

The proform of the WF146 protease, an extracellular subtilase produced by thermophilic Bacillus sp. WF146, matures efficiently at high temperatures. Here we report that proform, which contains N-terminal propeptide composed a core domain (N*) and linker peptide, is intrinsically able to mature via multiple pathways. One autocatalytic pathway initiated cis-processing N* generate autoprocessed complex N*-IWT, this step followed truncation peptide degradation N*. Another trans-processing Unlike...

Although in silico predictions have revealed that haloarchaea can be distinguished from other organisms the Tat pathway is used more extensively than Sec for haloarchaeal protein secretion, only a few haloarchaeal-secreted proteins been experimentally confirmed. Here, culture supernatant and membrane fraction of haloarchaeon Natrinema sp. J7-2 grown at 23% salt concentration were subjected to RPLC-ESI-MS/MS analysis. In total, 46 predicted substrates, 14 3 class III signal peptide-bearing...

ABSTRACT Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis . Its precursor composed of signal peptide, an N-terminal propeptide, catalytic domain, and long C-terminal extension (CTE). Several active forms Bpr have been previously reported, but little known about the maturation this enzyme. Here, gene encoding (BprL) was cloned from B. LZW expressed in WB700, three mature with apparent molecular masses 45, 75, 85 kDa were identified culture supernatant....

Proteolytic degradation of collagen at high temperatures has the advantages increasing efficiency and minimizing risk microbial contamination. Reports on thermostable collagenolytic proteases are limited, their maturation catalytic mechanisms remain to be elucidated.

Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from thermophile Bacillus sp. WF146, revealed that was translated as a 416-amino acid precursor consisting putative 18-amino signal peptide, 10-kDa N-terminal propeptide 32-kDa mature region. The shares high degree amino sequence identity with two psychrophilic subtilisins, S41 (68.2%) S39 (65.4%), mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to...

Pyrolysin-like proteases from hyperthermophiles are characterized by large insertions and long C-terminal extensions (CTEs). However, little is known about the roles of these extra structural elements or maturation enzymes. Here, recombinant proform Pyrococcus furiosus pyrolysin (Pls) several N- deletion mutants were successfully expressed in Escherichia coli. Pls was converted to mature enzyme (mPls) at high temperatures via autoprocessing both N-terminal propeptide portion CTE, indicating...