A Single Mutation at the Ferredoxin Binding Site of P450 Vdh Enables Efficient Biocatalytic Production of 25‐Hydroxyvitamin D3

Vitamin D₃ hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two‐step hydroxylation of vitamin D₃ (VD₃) to produce 25‐hydroxyvitamin D₃ (25(OH)VD₃) and 1α,25‐dihydroxyvitamin D₃ (1α,25(OH)₂VD₃). These hydroxylated forms of VD₃ are useful as pharmaceuticals for the treatment of conditions associated with VD₃ deficiency and VD₃ metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin‐binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD₃, a promising intermediate for the synthesis of various hydroxylated VD₃ derivatives, by using nisin‐treated Rhodococcus erythropolis cells containing Vdh_T107A. The gene‐expression cassette encoding Bacillus megaterium glucose dehydrogenase‐IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 μg mL^?1 25(OH)VD₃ was successfully produced by a 2 h bioconversion.     

Fracture Energy of an Aligned Porous Silicon Nitride

The fracture energy of a porous silicon nitride with aligned fibrous grains was investigated, using a chevron-notched-beam technique. A crack was constrained to propagate normal to the grain alignment. The obtained fracture energy was ∼500 J/m², which was ∼7 times larger than that of a dense silicon nitride with randomly oriented fibrous grains. The large fracture energy was attributable primarily to the sliding resistance associated with interlocking grains.     

Plastic Buckling Transition Modes in Moderately Thick Cylindrical Shells

Moderately thick perfect cylindrical shells under axial compression first exhibit an axisymmetric buckling mode, where a localization of buckling patterns, referred to as an elephant foot bulge, is caused by the first plastic bifurcation. However, the transition from the axisymmetric buckling mode to a nonaxisymmetric buckling mode, referred to as a diamond buckling mode, may occur due to the next bifurcation if we continue the loading under displacement control. Herein, this phenomenon is examined, based on a rigorous plastic bifurcation analysis. As a result, it is observed that the circumferential wave number of the diamond buckling mode increases with the decrease of the wall thickness. The boundary conditions also considerably influence the occurrence of diamond buckling. It is found that the strain concentration is intensified for the diamond buckling modes, compared with the axisymmetric modes.     

An anaerobic bacterium isolated from Japanese spiny lobster Panulirus japonicus

Eight strains of bacteria which could not grow under aerobic conditions, were isolated from Japanese spiny lobster. All the strains were Gram-positive, non-sporeforming cocci, and grew well under anaerobic conditions. The strains required a decreased oxygen tension rather than an increased CO₂ tension. Catalase, oxidase, nitrate reductase and urease were not produced. The strains fermented glucose, cellobiose, trehalose and levulose. Indole and H₂S were not produced. Macromolecule substances including starch, esculin, casein, gelatin and chitin were not hydrolyzed. Lactic acid was the sole product from peptone-yeast extract-Fildes solution-glucose broth. Fermentable carbohydrates were not required. The strains could grow at 20 to 37°C, and required NaCl for growth. From the above results, the strains were identified as anaerobic (aerotolerant) Streptococcus sp.     

Solution Property and Irradiation Effect of Random Copolypeptides Composed of Ala and Pro Residues

Summary Poly(proline) and random copolypeptide composed of Pro and Ala residues were synthesized, and their solution properties and molecular conformation were investigated. Aqueous solutions of the polypeptide were irradiated with γ-rays above the transition temperature. It was shown that the transition temperature of the aqueous solution of the copolypeptide is influenced by Ala-residue content and γ-ray irradiation.     

Determination of Mark-Houwink-Sakurada equation for phenolic resins containing alkylenediamine in the main chain and estimation of their molecular conformation in tetrahydrofuran solution

Summary Phenolic resins containing alkylenediamine in the main chain could be prepared by the co-condensation reaction of phenol, formaldehyde and alkylenediamines such as ethylenediamine and hexamethylenediamine. The molecular structures were determined by¹H-NMR spectra, IR spectra and nitrogen content. The molecular conformation of these resins in THF solution were studied from the viscosity measurements, and it became clear that the molecules were considerably compact compared with linear vinyl polymers. To clarify the reason of this phenomenon, the resins whose degrees of branching were different from each other and the resins whose phenolic hydroxyl groups were acetylated were prepared. From viscosity data, it was pointed out that the intramolecular hydrogen bonding contributes to the compaction of the phenolic resins containing alkylenediamine in the main chain more strongly than the branching does.     

Identification and Characterization of D‐Succinylase,and a Proposed Enzymatic Method for D‐Amino Acid Synthesis

Chiral amino acids are important intermediates for the pharmaceutical industry. We have developed a novel one‐pot enzymatic method for D ‐amino acid synthesis by the dynamic kinetic resolution of N‐succinyl‐dl ‐amino acids using D ‐succinylase (DSA) and N‐succinylamino acid racemase (NSAR, EC 4.2.1.113). The DSA from Cupriavidus sp. P4‐10‐C, which hydrolyzes N‐succinyl‐D ‐amino acids enantioselectively to their corresponding D ‐amino acids, was identified for the first time by screening soil microorganisms. Subsequently, the DSA gene was cloned and overexpressed in Escherichia coli. DSA was shown to comprise two subunits with molecular masses of 26 kDa and 60 kDa. Additionally, the NSAR gene from Geobacillus stearothermphilus NCA1503, which racemizes N‐succinylamino acids, was also cloned and overexpressed in E. coli. The highly purified DSA and NSAR prepared from each recombinant E. coli were characterized and used for D ‐amino acid synthesis. A one‐pot enzymatic method converted 100 mM N‐succinyl‐dl ‐phenylalanine to D ‐phenylalanine in 91.1% conversion with 86.7% ee. This novel enzymatic method may be useful for the industrial production of many D ‐amino acids.

     

A neck‐in model in extrusion lamination process

In this study, the experiment of the extrusion lamination process using high‐pressure process low‐density polyethylene (LDPE) was performed. The nonisothermal viscoelastic simulation of the extrusion lamination experiment was also carried out. The simulation results were in good agreement with the experimental data within wide range of take up velocity and air gap length. We developed the theoretical model based on force balance and deformation type of a film to predict the neck‐in behavior in the extrusion lamination or cast film process. It was suggested from the neck‐in model that the neck‐in correlates with the ratio of planar to uniaxial elongational viscosity. It was confirmed that the neck‐in model could predict the film edge shape and neck‐in properly for conventional LDPE. POLYM. ENG. SCI., 2010. © 2009 Society of Plastics Engineers