Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ_RpiT1) consists of three functional domains to effectively degrade solid PHB materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate. We performed the directed evolution of PhaZ_RpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved p-nitrophenyl butyrate (pNPC4) activity. Mutated PhaZ_RpiT1 genes generated by error-prone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface. Some cells displaying the mutant enzymes showed a two- to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wild-type enzyme. These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five- to eightfold higher pNPC4 hydrolysis activity than the wild type. To further evaluate the mutation effects, unfused and undisplayed enzymes were prepared and applied to the hydrolysis of p-nitrophenyl esters having different chain lengths (pNPCn; n?=?2–6) and PHB degradation. One specific second-generation mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis, although its PHB degradation efficiency at 1 μg/mL of enzyme concentration was approximately 3.5-fold lower than that of the wild type. Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved second-generation mutants, indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residues.     

Synthesis and biological evaluation of new chlorin derivatives as potential photosensitizers for photodynamic therapy

Three new water-soluble chlorin derivatives 3, 5 and 8 for potential use as photosensitizers in photodynamic therapy (PDT) for cancer were synthesized from photoprotoporphyrin IX dimethyl ester (1). The in vivo biodistribution and clearance of chlorin derivatives 3, 5 and 8 were investigated in tumor-bearing mice. Iminodiacetic acid derivative 8 showed the greatest tumor-selective accumulation among the new chlorin derivatives with maximum accumulation in tumor tissue at 3 h after intravenous injection and rapid clearance from normal tissues within 24 h after injection. The in vivo therapeutic efficacy of PDT using 8 was evaluated by measuring tumor growth rates in tumor-bearing mice with 660 nm light-emitting diode irradiation at 3 h after injection of 8. Tumor growth was significantly inhibited by PDT using 8. These results indicate that iminodiacetic acid derivative 8 is useful as a new photosensitizer to overcome the disadvantages of photosensitizers that are currently in clinical use.     

Structure-based design,synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.     

Mycelial β-N-Acetylglucosaminidase of Streptomyces sp. Having β-N-Acetylgalactosaminidase Activity

Formation of transfer products from soybean arabinogalactan and glycerol by endo-1,4-β-d-galactanase from Penicillium citrinum was described. The amount of transfer products depended on the glycerol concentration. About 50% of the galactose residues which could be liberated from the polysaccharide by the enzyme were transferred to glycerol at an acceptor concentration of 2.5% (w/v). Transfer products with various polymerization degrees were accumulated at the beginning of the reaction and then those with higher polymerization degrees were degraded gradually. At a final stage of the reaction, two transfer products in addition to two hydrolysis products (galactose and galactobiose) were mainly accumulated. The two transfer products were isolated and their structures were examined. They were 2-O-β-d-galactosyl glycerol and O-β-d-galactosyl-(1 → 4)-O-β-d-galactosyl-(1 → 2)glycerol.     

Release,Regeneration and Mutant Induction of Pleurotus cornucopiae (‘Tamogitake’) Protoplasts

Both enantiomers of aliphatic and aromatic cyanohydrins were hydrolyzed with the aid of Rhodococcus butanica ATCC 21197 to afford optically active α-hydroxy acids. The usefulness of this reaction is demonstrated by the synthesis of optically pure (R)-4-dodecanolide, a defensive secretion of rove beetles, starting from (R)-2-hydroxydecanenitrile.     

Metabolism of Glycine and Threonine in Growing Rats at Various Dietary Protein Levels

The metabolic fates of the carbon skeletons of [U-¹⁴C]glycine and l-[U-¹⁴C]threonine were investigated in growing rats fed with diets containing different percentages of protein calories (0, 5, 10, 15, and 30PC%) at 4100 kcal of metabolizable energy per kg of diet.

The incorporation of ¹⁴C into the body protein at 12 hr after the injection of ¹⁴C-glycine was about 58% of the dose in rats fed with the 10 or 15 PC% diet, and the values were reduced in both the lower and higher PC% groups. A considerable amount of ¹⁴C was recovered in the soluble fraction, and it was attributed to labeled glycine and serine in the free amino acid pools of the tissues.

The incorporation of ¹⁴C into the body protein from ¹⁴C-threonine was extremely high in the dietary groups of 0 to 10 PC%, and it decreased in the 30PC% group. Conversely, the expired ¹⁴C0₂ production was much less until the dietary protein level reached at 10PC%, and it increased with higher PC% in the diets. The change in the activity of hepatic threonine dehydratase in rats fed diets with increasing protein levels was similar to that of the expired ¹⁴C0₂ production from ¹⁴C- threonine.

These results indicate that, though the metabolic patterns for glycine and threonine differ from each other, their responses to dietary protein levels change at 10 to 15 PC%, where the growth rate reached its approximate maximum.     

S-Carboxymethylcysteine Synthase from Escherichia coli

An enzyme that catalyzes the synthesis of S-carboxymethyl- l-cysteine from 3-chloro- l-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S- carboxymethyl-l-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzyme has a molecular weight of 84,000 and gave one band corresponding to a molecular weight of 37,000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the β-replacement reactions between 3-CI-AIa and various thiol compounds. The apparent Km values for 3-Cl-Ala and thioglycolic acid were 40 mM and 15.4 mM. The enzyme showed very low activity as to the α,β-elimination reaction with 3-Cl-Ala and l-serine. It was not inactivated on the incubation with 3-Cl-Ala. The absorption spectrum of the enzyme shows a maximum at 412 nm, indicating that it contains pyridoxal phosphate as a cofactor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.