Hilton Jorge Nascimento

Lignin peroxidase isoforms from Streptomyces viridosporus T7A: are they a monomer based structure?

Five fractions with lignin peroxidase activity were isolated by FPLC-Mono Q from a Streptomyces viridosporus culture. F4 and F5 showed the highest specific activity and degree of protein homogeneity by chromatofocusing, IEF- and gradient-PAGE. The individual analysis of F4 and F5 by FPLC-Superdex 75, showed MW that were multiples to each other (68,000; 23,000; 12,000), although by SDS PAGE a sole MW of 13,500 was obtained, indicating a monomer based structure. The amino-acid composition of F5 showed absence of sulfur amino acids.

Glucoamylase isoenzymes tailoring through medium composition

Two major glucoamylase isoenzymes (GAI and GAII) have been identified in culture supernatants of Aspergillus awamori. It has been suggested that a stepwise degradation of a native enzyme during the fermentation by proteases and/or glucosidases results in the formation of isoenzymes that have different characteristics concerning substrate specificity and stability to pH and temperature. In this study, the glucoamylase isoenzymes produced by Aspergillus awamori using liquid media with C/N 10 (2.0% starch, 0.45% (NH4)2 SO4) and C/N 26 (5.2% starch, 0.45% (NH4)2 SO4) were analyzed. In both cases, GAI and GAII were characterized concerning its hydrolitic activities, mol wt, and isoeletric point. Using HPLC gel filtration and FPLC chromatofocusing, it was obtained for GAI a mol wt of 110,000 Da, pi 3.45 and for GAII a mol wt of 86,000 Da, pI 3.65. A different isoenzymes proportion was observed by the use of the two C/N ratios. In the lower carbohydrate content, fermentation of the GAI form predominated, whereas in the C/N 26 medium, GAII was prevalent. Gel eletrophoresis, amino acid analysis, and structural data confirmed that both preparations were glucoamylases with a high degree of homogeneity.

Complete amino acid sequence and location of Omp-28, an important immunogenic protein from Salmonella enterica serovar typhi

Omp-28 isolated from Salmonella enterica serovar typhi presented a subunit molecular mass of 9,632 Da by MALDI-TOF MS. It was denatured, S-alkylated, and 1) directly submitted to Edman sequencing, 2) cleaved with CNBr, and 3) hydrolyzed either with endoproteinase Glu-C or Asp-N. The major CNBr peptide containing the C-terminal portion of Omp-28 was isolated by tricine-SDS-PAGE and electroblotted whereas Omp-28 enzymatic peptides were isolated by C18-RP-HPLC. All peptides were sequenced. This approach allowed the elucidation of the complete primary structure of Omp-28. Its amino acid sequence is identical to that deduced from part of the DNA of the “putative periplasmic transport protein” of either S. enterica serovar typhimurium and a multiple drug resistant S. enterica serovar typhi. Omp-28 homologous protein sequences were also deduced from Escherichia coli and Yersinia pestis genomic DNA. All proteins had their secondary structures predicted. Immunogold cytochemistry indicated that Omp-28 is found on the bacterium outer membrane.

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp‐28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp‐28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp‐28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N‐terminal amino acid sequence of Omp‐28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c‐DNA. ELISA and Western blotting identified Omp‐28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp‐28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp‐28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp‐28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.

Extracellular proteolytic processing of Aspergillus awamori GAI into GAII is supported by physico-chemical evidence.

The proportion of glucoamylases, GAI and GAII, in the culture supernatant ofAspergillus awamori fermentations depends on the medium C/N ratio in such a way that the transformation of GAI into GAII is favored by the existence of a surplus of the carbon source in the growth medium. This condition also favors the appearance of the proteolytic activity. The authors report the observation that the shift in the isoenzyme proportion was concomitant to the peak of proteolytic activity. A peptide that may have resulted from the continuous degradation of the GAI C-terminal peptide, Gp-1, was isolated by gel filtration and purified by reversephase chromatography. This peptide matched with the region G14-A34 of the substrate-binding domain of GAI, thus reinforcing the hypothesis of the extracellular proteolytic processing of GAI.