Ring-opening polymerization of ɛ-caprolactone catalyzed by a novel lipase Candida sp. 99-125

The ring-opening polymerization of ?-caprolactone catalyzed by a novel lipase Candida sp. 99-125 was achieved in organic solvents. ?-Caprolactone was found to be polymerized in quantitative yield in toluene at 80?°C for 5 days. The poly (?-caprolactone) obtained had a number-average molecular weight of 14,120 (polydispersity index?=?1.161). The effect of enzyme concentration, temperature, reaction time, reaction medium, and water content on monomer conversion and product molecular weight were investigated. Kinetic analysis showed that the enzyme displayed Michaelis–Menten kinetics and showed stronger affinity for ?-caprolactone (with a K_m value of 6.1?mmol/L) than other reported lipases.     

Ring-opening polymerization of ɛ-caprolactone catalyzed by a novel thermophilic esterase from the archaeon Archaeoglobus fulgidus

The ring-opening polymerization of ?-caprolactone catalyzed by a novel thermophilic esterase from the archaeon Archaeoglobus fulgidus was successfully conducted in organic solvents. The effects of enzyme concentration, temperature, reaction time, reaction medium, and water activity on monomer conversion and product molecular weight were investigated. Poly(?-caprolactone) was obtained in almost 100% of the monomer conversion, with a number-average molecular weight of 1400 in toluene at 80 °C for 72 h. Furthermore, the Michaelis–Menten kinetic analysis showed that the enzyme had the highest affinity for ?-caprolactone, with a K_m value of 0.093 mol/l compared with other reported lipases. The possible structural and energetic effects of the enzyme on the K_m value were investigated, using molecular docking studies.     

Heat shock protein DnaK — Substrate of actin-specific bacterial protease ECP32

It has been found that actin-specific bacterial protease ECP32 cleaves prokaryotic heat shock protein DnaK, which belongs to the family of heat shock proteins with molecular weight 70 kDa. We propose a new one-step method for DnaK purification using heat treatment. The technique yields ∼1 mg of partially purified DnaK from 25 g of wet bacterial biomass. Polyclonal antibodies against DnaK were obtained. The degree of ECP32 catalyzed proteolysis of partially purified DnaK and that of DnaK in initial cell extracts was compared.     

Cleavage of bacillus subtilis endo-β-1,4-glucanase by B. megaterium protease

Summary A protease secreted by B. megaterium ATCC 14945 was purified by ammonium sulfate fractionation, Q-Sepharose, Sephadex G-75, and hydroxyapatite chromatography and its molecular weight was estimated to be 38 kD. The purified protease caused the cleavage of 52 kD B. subtilis endo--l,4-glucanase expressed in B. megaterium. The enzyme was most active at pH 7.5 and 55 °C. Calcium ion was required for the enzyme activity and the activity was almost completely inhibited by EDTA. The protease was not inhibited by phenylmethylsulfonyl fluoride.     

Artificial mixed-linked β-glucans produced by glycosynthase-catalyzed polymerization: tuning morphology and degree of polymerization

The glycosynthase derived from Bacillus licheniformis 1,3-1,4-β-glucanase was able to polymerize glycosyl fluoride donors (G4)(m)G3GαF (m = 0-2, G = Glcβ) leading to artificial mixed-linked β-glucans with regular sequences and variable β1,3 to β1,4 linkage ratios. With the E134A glycosynthase mutant, polymers had average molecular masses (M(w)) of 10-15 kDa. Whereas polymer 2 ([4G4G3G](n)) was an amorphous precipitate, the water-insoluble polymers 1 ([4G3G](n)) and 3 ([4G4G4G3G](n)) formed spherulites of 10-20 μm diameter. With the more active E134S glycosynthase mutant, polymerization led to high molecular mass polysaccharides, where M(w) was linearly dependent on enzyme concentration. Remarkably, a homo-polysaccharide [4G4G4G3G](n) with M(w) as high as 30.5 kDa (n ≈ 47) was obtained, which contained a small fraction of products up to 70 kDa, a value that is in the range of the molecular masses of low viscosity cereal 1,3-1,4-β-glucans, and among the largest products produced by a glycosynthase. Access to a range of novel tailor-made β-glucans through the glycosynthase technology will allow to evaluate the implications of polysaccharide fine structures in their physicochemical properties and their applications as biomaterials, as well as to provide valuable tools for biochemical characterization of β-glucan degrading enzymes and binding modules.     

Proofreading of ribonucleotides inserted into DNA by yeast DNA polymerase ɛ

We have investigated the ability of the 3′ exonuclease activity of Saccharomyces cerevisiae DNA polymerase ? (Pol ?) to proofread newly inserted ribonucleotides (rNMPs). During DNA synthesis in vitro, Pol ? proofreads ribonucleotides with apparent efficiencies that vary from none at some locations to more than 90% at others, with rA and rU being more efficiently proofread than rC and rG. Previous studies show that failure to repair ribonucleotides in the genome of rnh201Δ strains that lack RNase H2 activity elevates the rate of short deletions in tandem repeat sequences. Here we show that this rate is increased by 2–4-fold in pol2-4 rnh201Δ strains that are also defective in Pol ? proofreading. In comparison, defective proofreading in these same strains increases the rate of base substitutions by more than 100-fold. Collectively, the results indicate that although proofreading of an ‘incorrect’ sugar is less efficient than is proofreading of an incorrect base, Pol ? does proofread newly inserted rNMPs to enhance genome stability.     

Regulation of lysine ɛ-aminotransferase by carbon source and lack of control by phosphate in Streptomyces clavuligerus

Cephalosporin production by Streptomyces clavuligerus is known to be negatively regulated by carbon sources, e.g., glycerol and starch, and by phosphate at high concentrations. Formation of lysine ɛ-aminotransferase (LAT) activity, the first enzyme of the biosynthetic pathway, was affected by a high concentration of carbon source. Whereas 3% starch more than doubled LAT activity production as compared to 1% starch, 3% glycerol repressed LAT activity formation by 20%–30%. LAT activity production was not affected by 100 mM K₂HPO₄. Our results thus show that the negative effects of 2% glycerol and 3% starch and 100 mM phosphate on cephalosporin production are not due to an effect on production of LAT activity. However, repression of LAT activity by 3% glycerol would be expected to play a negative role in antibiotic production. Received: 13 June 1997 / Received revision: 20 August 1997 / Accepted: 25 August 1997     

Modification of microstructure and selected physicochemical properties of bacterial cellulose produced by bacterial isolate using hydrocolloid-fortified Hestrin–Schramm medium

Bacterial cellulose (BC) is a biopolymer with applications in numerous industries such as food and pharmaceutical sectors. In this study, various hydrocolloids including modified starches (oxidized starch—1404 and hydroxypropyl starch—1440), locust bean gum, xanthan gum (XG), guar gum, and carboxymethyl cellulose were added to the Hestrin-Schramm medium to improve the production performance and microstructure of BC by Gluconacetobacter entanii isolated from coconut water. After 14-day fermentation, medium supplemented with 0.1% carboxymethyl cellulose and 0.1% XG resulted in the highest BC yield with dry BC content of 9.82 and 6.06 g/L, respectively. In addition, scanning electron microscopy showed that all modified films have the characteristic three-dimensional network of cellulose nanofibers with dense structure and low porosity as well as larger fiber size compared to control. X-ray diffraction indicated that BC fortified with carboxymethyl cellulose exhibited lower crystallinity while Fourier infrared spectroscopy showed characteristic peaks of both control and modified BC films.     

Stepwise unfolding of a β barrel protein by the AAA+ ClpXP protease

In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded β barrel of superfolder GFP-ssrA involves extraction of the C-terminal β strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.     

Thermostable alkaline protease produced by Bacillus thermoruber — a new species of Bacillus

Summary The proteolytic activity produced by a new species of Bacillus isolated in our laboratory was investigated. This enzyme was purified to homogeneity from cell-free culture liquids of B. thermoruber. The purification procedure included ion-exchange chromatography on DEAE-Sephadex A-50 and -casein agarose affinity chromatography. The protease consists of one polypeptide chain with a molecular weight of 39000±800. the isoelectric point was 5.3; the optimum pH and temperature for proteolytic activity (on casein) was found to be pH 9 and 45°C respectively. Enzyme activity was inhibited by PMSF and EDTA. The stability was considerably increased by addition of Ca²⁺, and the protease exhibited a relatively high thermal stability. The alkaline protease shows a preference for leucine in the carboxylic side of the peptide bond of the substrate. The K _m value for benzyloxycarbonyl-Ala-Ala-Leu-p-nitroanilide was 2.5 mM.     

Determinants of laminin polymerization revealed by the structure of the α5 chain amino-terminal region

The polymerization of laminin into a cell-associated network--a key step in basement membrane assembly--is mediated by the laminin amino-terminal (LN) domains at the tips of the three short arms of the laminin αβγ-heterotrimer. The crystal structure of a laminin α5LN-LE1-2 fragment shows that the LN domain is a β-jelly roll with several elaborate insertions that is attached like a flower head to the stalk-like laminin-type epidermal growth factor-like tandem. A surface loop that is strictly conserved in the LN domains of all α-short arms is required for stable ternary association with the β- and γ-short arms in the laminin network.     

Chemosynthesis of poly(ε-lysine)-analogous polymers by microwave-assisted click polymerization

Poly(ε-lysine) (ε-PL)-analogous click polypeptides with not only similar α-amino side groups but also similar main chain to ε-PL were chemically synthesized for the first time through click polymerization from aspartic (or glutamic)-acid-based dialkyne and diazide monomers. With microwave-assisting, the reaction time of click polymerization was compressed into 30 min. The polymers were fully characterized by NMR, ATR-FTIR, and SEC-MALLS analysis. The deprotected click polypeptides had similar pK(a) value (7.5) and relatively low cytotoxicity as ε-PL and could be used as substitutes of ε-PL in biomedical applications, especially in endotoxin selective removal. Poly(ethylene glycol) (PEG)-containing alternating copolymers with α-amino groups were also synthesized and characterized. After deprotection, the polymers could be used as functional gene vector with PEG shadowing system and NCA initiator to get amphiphilic graft polymers.     

Extraction of protease from Calotropis procera latex by polyethylene glycol–salts biphasic system

The protease from the latex of Calotropis procera was isolated by an aqueous two-phase system (ATPS). The systems consist of polyethylene glycol (PEG 4000, 6000 and 8000) at concentrations of 9, 12 and 15% (w/w) with salts (Na-citrate, MgSO₄, K₂HPO₄, and (NH₄)₂SO₄) at concentrations of 11, 14 and 17% (w/w) were investigated. The highest protease recovery was found in the PEG-rich phase of the system, comprising of 12% PEG 4000–17% MgSO₄. For optimization of the system to obtain the higher yield of protease, the system pH (4, 7 and 10) or NaCl addition (2, 4 and 6%, w/w) was studied. At acidic (pH 4.0) and alkaline (9.0) conditions of the systems the reduction of K_E and protease recovery was clearly observed compared to that of the neutral pH (7.0). The addition of NaCl up to a final concentration of 6% (w/w) significantly increased the yield to 107% of the control. Molecular weight distribution and activity staining showed that the isolated protease had the molecular weight of ∼38 kDa. However, the isolated protease had no activity under reducing condition (βME). Under cathodic electrophoresis, protease from C. procera showed the same protein pattern to purified papain.     

Potent inhibition of norovirus 3CL protease by peptidyl α-ketoamides and α-ketoheterocycles

A series of structurally-diverse α-ketoamides and α-ketoheterocycles was synthesized and subsequently investigated for inhibitory activity against norovirus 3CL protease in vitro, as well as anti-norovirus activity in a cell-based replicon system. The synthesized compounds were found to inhibit norovirus 3CL protease in vitro and to also exhibit potent anti-norovirus activity in a cell-based replicon system.     

Determination of residual water in freeze-dried bacterial cultures by Fischer’s method

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Enzyme-catalyzed ring-opening polymerization of β-propiolactone

Summary -Propiolactone (BPL) was polymerized in bulk using lipase as a catalyst to form the polyester having a molecular weight of greater than 50000. The molecular weight of the polyesters produced by the lipase-catalyzed polymerization of BPL was inversely dependent on the concentration of lipase. Both molecular weight and polymerization speed were increased with increasing reaction temperature from 40 to 60 °C.     

On the inhibition of HIV-1 protease by hydrazino-ureas displaying the N→CO interaction

To create novel HIV-1 protease (HIV PR) inhibitors, we have extended our investigations of the N→CO interaction as a moiety that reproduces electrostatic properties of the transition state of peptidolysis. Consequently, we prepared a series of compounds with an unusual hydrazino-urea core. In polar protic media, these adopt solely a cyclic constitution displaying the interaction on one side of the molecule while offering a urea moiety on the opposite side meant to hydrogen-bond with the enzyme flaps. Each inhibitor candidate was obtained via a key series of three synthetic steps employing carbonyl-di-imidazole (CDI). It was thus possible to efficiently fuse two independent building blocks, a hydrazine and a protected aminoaldehyde in a convergent manner. NMR and UV analysis proved that all compounds, when dissolved in polar protic media, existed exclusively in the cyclic constitution exhibiting the N→CO interaction. In total, five inhibitor candidates were tested with HIV PR for their potency. The one carrying the least bulk in peripheral substituents showed the highest activity. Its very low molecular weight (365 g/mol) holds great promise for future improvements in affinity without violating Lipinski’s rule of remaining within the limit of 500 g/mol.