Crystallization and its effect on the mechanical properties of a medium chain length polyhydroxyalkanoate.

Medium chain length polyhydroxyalkanoates (mcl-PHAs) could play a role in the growing demand for highly elastic and biodegradable materials in the medical field. In this study, a poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) (P(3HO-co-3HH)) was first fully characterized in terms of molecular weight, microstructural chain parameters and chemical structure by means of gel permeation chromatography (GPC), nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR). As determined by NMR, the synthesized polymer contained 94.3% and 5.7% molar content of 3-hydroxyoctanoate and 3-hydroxyhexanoate, respectively. Since mechanical properties are closely related to thermal history, the effect of crystallization on tensile properties was also investigated in the present study. Three crystallization temperatures were selected (0, 23 and 37°C), the conclusion reached is that the maximum crystallization rate for this copolymer was achieved at 0°C. On the other hand, evolution of tensile properties of P(3HO-co-3HH) films stored at room temperature demonstrated that, as crystallization occurred toward the equilibrium state, the polymer underwent a stiffening process. In this sense, secant modulus and tensile strength increased respectively from 8.3 ± 1.0 MPa and 6.4 ± 0.8 MPa after 1 day stored at room temperature to 36.2 ± 3.3 MPa and 16.3 ± 2.1 MPa after 16 weeks.

Cloning and characterization of a beta-galactosidase encoding region in Lactobacillus coryniformis CECT 5711.

A chromosomal DNA fragment of 7.8 kb from Lactobacillus coryniformis CECT 5711 was cloned in Escherichia coli K-12 and was found to express a functional beta-galactosidase. Nucleotide sequence analysis showed that this fragment contained two partially overlapping genes, the lacL (1,881 bp) and the lacM (960 bp), that encode the subunits of a heterodimeric beta-galactosidase, with estimated molecular masses of 72,129 and 35,233 Da, respectively. Other three incomplete open reading frames showing homology to another beta-galactosidase, an alpha-galactosidase, and a galactokinase, respectively, were also found. The L. coryniformis beta-galactosidase was overproduced in E. coli by using an isopropyl-beta-D: -thiogalactopyranoside (IPTG) expression system. Two new proteins with an estimated M (r) s of approximately 72,000 and 35,000 appeared upon induction with IPTG, and extracts of the recombinant E. coli strain showed beta-galactosidase activity.

Performance of a recombinant strain of Streptomyces lividans for bioconversion of penicillin G to deacetoxycephalosporin G.

We examined the performance of Streptomyces lividans strain W25 containing a hybrid expandase (deacetoxycephalosporin C synthase; DAOCS) gene, obtained by in vivo recombination between the expandase genes of S. clavuligerus and Nocardia lactamdurans for resting-cell bioconversion of penicillin G to deacetoxycephalosporin G. Strain W25 carried out a much more effective level of bioconversion than the previously used strain, S. clavuligerus NP1. The two strains also differed in the concentrations of FeSO(4) and alpha-ketoglutarate giving maximal activity. Whereas NP1 preferred 1.8 mM FeSO(4 )and 1.3 mM alpha-ketoglutarate, recombinant W25 performed best at 0.45 mM FeSO(4) and 1.9 mM alpha-ketoglutarate.

Extracellular production of biologically active deacetoxycephalosporin C synthase from Streptomyces clavuligerus in Pichia pastoris.

We have successfully expressed and observed secretion of the Streptomyces clavuligerus deacetoxycephalosporin C synthase (DAOCS) using the Pichia pastoris expression system. Two clones having multiple copies of the expression cassette were selected and used for protein-expression analysis. SDS-PAGE showed efficient expression and secretion of the bacterial recombinant DAOCS. The highest yield (120 microg/mL) was obtained when expression was induced with 2% methanol. Free and immobilized protein were assayed for biological activity and found to expand penicillin N (its natural substrate) and penicillin G to deacetoxycephalosporin C (DAOC) and deacetoxycephalosporin G (DAOG), respectively.

Stimulatory effect of growth in the presence of alcohols on biotransformation of penicillin G into cephalosporin-type antibiotics by resting cells of Streptomyces clavuligerus NP1.

Growth of Streptomyces clavuligerus NP1 in the presence of methanol or ethanol resulted in a marked increase in production of cephalosporin(s) from penicillin G by resting cells. The mycelium produced in alcohol-supplemented medium was fragmented and dispersed as compared with growth in control medium. HPLC analysis showed that at least two products were present in the biotransformation supernatant fluid after 1 h incubation. One of them has been identified as deacetoxycephalosporin G (DAOG).

Further studies on the bioconversion of penicillin G into deacetoxycephalosporin G by resting cells of Streptomyces clavuligerus NP-1.

Resting cells of Streptomyces clavuligerus NP-1, which possess deacetoxycephalosporin C synthase activity, have been shown previously to perform oxidative ring expansion of penicillin G in the presence of iron, ascorbic acid, and alpha-ketoglutaric acid to form deacetoxycephalosporin G. Further studies on this bioconversion indicated that use of MOPS or HEPES buffer at pH 6.5 more than doubled the extent of the reaction observed with the previously used Tris-HCl at pH 7.4. Levels of bioconversion as high as 16.5% were achieved at low penicillin G concentrations. Previously, conversion yields were < 1%.

Elucidation of conditions allowing conversion of penicillin G and other penicillins to deacetoxycephalosporins by resting cells and extracts of Streptomyces clavuligerus NP1.

Using resting cells and extracts of Streptomyces clavuligerus NP1, we have been able to convert penicillin G (benzylpenicillin) to deacetoxycephalosporin G. Conversion was achieved by increasing by 45x the concentration of FeSO4 (1.8 mM) and doubling the concentration of alpha-ketoglutarate (1.28 mM) as compared with standard conditions used for the normal cell-free conversion of penicillin N to deacetoxycephalosporin C. ATP, MgSO4, KCl, and DTT, important in cell-free expansion of penicillin N, did not play a significant role in the ring expansion of penicillin G by resting cells or cell-free extracts. When these conditions were used with 14 other penicillins, ring expansion was achieved in all cases.

Electrophoretic karyotype of the astaxanthin-producing yeast Phaffia rhodozyma.

The electrophoretic karyotype of three different strains of Phaffia rhodozyma was determined by contour-clamped homogeneous electric field (CHEF)-gel electrophoresis. Significant differences in electrophoretic karyotyping patterns were found among the three strains studied. Between nine and 17 bands were observed. The size of these bands, based on their migration relative to the chromosomal DNA of Schizosaccharomyces pombe, Hansenula wingei was estimated to be between 0.48 and 3.1 Mb.