Molecular characterization of SCO0765 as a cellotriose releasing endo-<Emphasis Type="Italic">β</Emphasis>-1,4-cellulase from <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A(3)

The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward β-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50–60°C toward β-glucan. Thin layer chromatography of the hydrolyzed products of CMC and β-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-β-1,4-cellulase, hydrolyzing the β-1,4 glycosidic bond of cellulose into cellotriose.     

Chromosome Synapsis Alleviates Mek1-Dependent Suppression of Meiotic DNA Repair

Faithful meiotic chromosome segregation and fertility require meiotic recombination between homologous chromosomes rather than the equally available sister chromatid, a bias that in Saccharomyces cerevisiae depends on the meiotic kinase, Mek1. Mek1 is thought to mediate repair template bias by specifically suppressing sister-directed repair. Instead, we found that when Mek1 persists on closely paired (synapsed) homologues, DNA repair is severely delayed, suggesting that Mek1 suppresses any proximal repair template. Accordingly, Mek1 is excluded from synapsed homologues in wild-type cells. Exclusion requires the AAA⁺-ATPase Pch2 and is directly coupled to synaptonemal complex assembly. Stage-specific depletion experiments further demonstrate that DNA repair in the context of synapsed homologues requires Rad54, a repair factor inhibited by Mek1. These data indicate that the sister template is distinguished from the homologue primarily by its closer proximity to inhibitory Mek1 activity. We propose that once pairing or synapsis juxtaposes homologues, exclusion of Mek1 is necessary to avoid suppression of all templates and accelerate repair progression.     

Stimulation of acetoin production in metabolically engineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by increasing ATP demand

Having a sufficient supply of energy, usually in the form of ATP, is essential for all living organisms. In this study, however, we demonstrate that it can be beneficial to reduce ATP availability when the objective is microbial production. By introducing the ATP hydrolyzing F₁-ATPase into a Lactococcus lactis strain engineered into producing acetoin, we show that production titer and yield both can be increased. At high F₁-ATPase expression level, the acetoin production yield could be increased by 10 %; however, because of the negative effect that the F₁-ATPase had on biomass yield and growth, this increase was at the cost of volumetric productivity. By lowering the expression level of the F₁-ATPase, both the volumetric productivity and the final yield could be increased by 5 % compared to the reference strain not overexpressing the F₁-ATPase, and in batch fermentation, it was possible to convert 176 mM (32 g/L) of glucose into 146.5 mM (12.9 g/L) acetoin with a yield of 83 % of the theoretical maximum. To further demonstrate the potential of the cell factory developed, we complemented it with the lactose plasmid pLP712, which allowed for growth and acetoin production from a dairy waste stream, deproteinized whey. Using this cheap and renewable feedstock, efficient acetoin production with a titer of 157 mM (14 g/L) acetoin was accomplished.     

5''-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5''-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography.

Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups.     

A study of the metabolism of l-αγ-diaminobutyric acid in a Xanthomonas species

1. l-αγ-Diaminobutyric acid is metabolized in Xanthomonas sp. to aspartic β-semialdehyde, aspartic acid and oxaloacetic acid. 2. Aspartic β-semialdehyde is formed from diaminobutyric acid by a pyruvate-dependent γ-transamination. 3. The transaminase has a pH optimum of 9 and exhibits a high degree of substrate specificity, as analogues of diaminobutyric acid and pyruvate are inert in the system. The transaminase is inhibited by carbonyl-binding agents such as hydroxylamine. 4. Aspartic acid is formed from aspartic β-semialdehyde by an NAD⁺-dependent dehydrogenation. 5. The dehydrogenase has a pH optimum of 8·5 and is a thiol enzyme. It is specific for aspartic β-semialdehyde but analogues of NAD⁺ such as 3-acetylpyridine–adenine dinucleotide and deamino-NAD are partly active in the system. 6. The significance of these reactions is discussed in relation to diaminobutyric acid metabolism in plants and mammalian systems.     

Study of human cord blood lymphocytes by immobilized metal ion affinity partitioning

The potential of immobilized metal ion affinity partitioning (IMAP) using dextran-PEG+PEG-IDA-M(II) systems to separate mononuclear cells from cord blood has been evaluated. The distribution of B cells, T cells, monocytes and hematopoietic stem cells between PEG and dextran phases was determined by flow cytometry with fluorochrome-labelled specific antibodies. Comparing these values with the post-Ficoll repartition resulted in the determination of enrichment factors, for each subpopulation, in the different phases. We were able to distinguish the partition pattern of B cells, T cells, monocytes and stem cells in different IMAP systems. Their partition was affected by the nature and the concentration of the metal used, but no specificity in distribution for the subpopulations was found.     

Lipoprotein profile during perchlorate toxicity

Perchlorate administration to rats for 45 days alters the lipoprotein profile in plasma. The levels of cholesterol, phospholipids and triglycerides in HDL, LDL and VLDL fractions are significantly increased in perchlorate-treated rats. Post-heparin lipolytic activity of plasma of sodium perchlorate-treated rats is decreased. The risk factor, i.e. the total cholesterol/HDL cholesterol, increases in the experimental animals, indicating that the treatment of rats with perchlorate may develop the susceptibility of the animals to cardiac heart disease.     

Structural study of porcine pancreatic elastase complexed with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin as a nonreactivatable doubly covalent enzyme-inhibitor complex

The complex of porcine pancreatic elastase (PPE) with 7-amino-3-(2-bromoethoxy)-4-chloroisocoumarin, a potent mechanism-based inhibitor, was crystallized and the crystal structure determined at 1.9-A resolution with a final R factor of 17.1%. The unbiased difference Fourier electron density map showed continuous density from O gamma of Ser 195 to the benzoyl carbonyl carbon atom and from N epsilon 2 of His 57 to the carbon atom at the 4-position of the isocoumarin ring in the inhibitor. This suggested unambiguously that the inhibitor was doubly covalently bound to the enzyme. It represents the first structural evidence for irreversible binding of an isocoumarin inhibitor to PPE through both Ser 195 and His 57 in the active site. The PPE-inhibitor complex is only partially activated in solution by hydroxylamine and confirms the existence of the doubly covalently bound complex along with the acyl enzyme. The benzoyl carbonyl oxygen atom of the inhibitor is not situated in the oxyanion hole formed by the amide (greater than NH) groups of Gly 193 and Ser 195. The complex is stabilized by the hydrogen-bonding interactions in the active site (from the N epsilon 2 of Gln 192 to the bromine atom in the inhibitor and the amino group at the 7-position of the isocoumarin ring to the carbonyl oxygen of Thr 41) and by van der Waals interactions. The inhibition rates of several 7-substituted 4-chloro-3-(bromoalkoxy)isocoumarins toward PPE were measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein adsorption on histidyl-aminohexyl-Sepharose 4B: II. Application to the negative one-step affinity purification of human β2-microglobulin and Immunoglobulin G

The adsorption of two human proteins, β2-microglobulin and Immunoglobulin G, from uremic patient’s blood ultrafiltrate and plasma, respectively, was investigated on the histidyl-aminohexyl-Sepharose 4B adsorbent. Both target proteins could be adsorbed on the gel through a low affinity for immobilized histidine ligand. However, a fine adjustment of the operating conditions (ionic strength, buffer, pH) prevented their adsorption and thus allowed their “negative affinity” purification (purity estimated by silver nitrate SDS–PAGE) by the removal of the contaminating proteins. This simple and efficient method provides purification under gentle chromatographic conditions and a further characterization of both molecules.