Fred W. Wagner

Aspergillus niger mutants with increased glucose oxidase production

SummaryAspergillus niger NRRL-3, an organism used for the industrial scale production of d-gluconic acid and glucose oxidase (EC 1.1.3.4), was subjected to mutagenesis and selection for acid production on diagnostic media containing methyl red. The plates contained 0.1 M d-glucose, a concentration that does not produce a color change in the medium surrounding mycelia of the parental strain under the conditions employed. Mutagenized spores yielded occasional colonies which were able to grow rapidly and were surrounded by a reddish zone. A number of such presumptive mutants were selected and isolated. Twenty-six such strains were grown in shaken cultures with liquid media containing 0.01, 0.1 or 0.5 M d-glucose, harvested, disrupted and the specific activity of d-glucose oxidase determined. Seven of the mutant strains had glucose oxidase specific activities markedly higher than the parental strain.

Detection of mercuric ions in water by ELISA with a mercury-specific antibody

An immunoassay that detects mercuric ions in water at concentrations of 0.5 ppb and above is described. The assay utilizes a monoclonal antibody that binds specifically to mercuric ions immobilized in wells of microtiter plates. Within the range of 0.5-10 ppb mercury, the absorbance in the enzyme-linked immunosorbent assay (ELISA) is linear to the log of the mercuric ion concentration. The quantitation of mercury by ELISA correlates closely with results from cold-vapor atomic absorption. Other divalent metal cations do not interfere with the assay, although there is interference in the presence of 1 mM chloride ions. The optimum pH for mercury detection is 7.0, although 2 ppb mercury can be detected over a wide pH range. The assay is as sensitive as cold-vapor atomic absorption for mercury detection and can be performed with only 100 microliters of sample.

Cloning and nucleotide sequence of the Vibrio proteolyticus aminopeptidase gene

The gene encoding the Vibrio proteolyticus aminopeptidase was cloned and sequenced and its amino acid sequence was deduced. The gene encodes a 54 kDa protein, larger than the previously reported size of 30 kDa for the purified aminopeptidase. Sequence alignments revealed a 43-45% homology with two other Vibrio sp. extracellular proteinases.

Bacillus subtilis aminopeptidase: purification, characterization and some enzymatic properties.

Abstract The extracellular aminopeptidase from Bacillus subtilis was purified 300-fold by a simple procedure which gave a high recovery of enzyme. The native enzyme was shown to be a monomer of molecular weight 46,500 and to contain 1 g-atom of Zn 2+ per mole of protein. Amino acid analyses demonstrated the protein to be rich in acidic residues and Lys, to possess about 3 residues of Met, and to be devoid of Cys. When activated with 5 m m Co(NO 3 ) 2 for 90 min the activity of the native enzyme was increased; the amount of activation depended on the identity of the substrate. Cobalt activation involved the reversible binding of 1 g-atom of Co 2+ per mole of protein, without displacing the native Zn 2+ ; K Co was 1.25 m m . Zinc ions competed with Co 2+ during activation, a process characterized by a K Zn of 28 μ m . Ions other than Co 2+ did not appreciably activate the enzyme.

Purification and characterization of a soybean cotyledon aminopeptidase

Abstract An aminopeptidase has been purified from expanded cotyledons of Glycine max [Merr.] cv. Hobbit. The time course for development of aminopeptidase activity correlates with protein loss during seedling growth. Aminopeptidase activity was very low in dry seeds and increased to a maximum 6–8 days after inhibition. Histochemically, a zone of activity with migration identical to the purified enzyme was detected in cotyledon extracts only 2 days after sowing. The enzyme was purified from extracts of 8-day-old cotyledons by ion-exchange, affinity and gel permeation chromatography. The enzyme is a monomer of approximately 85 000 Da and apparently requires free sulfhydryl groups for activity. Fluorometric measurements indicated that the enzyme has high affinity for hydrophobic aminoacylnaphthylamide substrates. Hydrolysis of amino-terminal blocked peptides and large proteins was not detected. The purified aminopeptidase is similar to aminopeptidase characterized from other plant seeds; however, differences exist in substrate specificities and in the time course of the appearance of enzyme activity.

Characteristics of Modified Leghemoglobins Isolated from Soybean (Glycine max Merr.) Root Nodules

Hemoprotein derivatives of an abundant soybean (Glycine max Merr.) root nodule leghemoglobin, Lba, were studied for their modified spectral characteristics and physical properties. Three modified hemoprotein derivatives of Lba (Lbam1, Lbam2, and Lbam3) were purified by preparative isoelectric focusing. The ferric forms of these pigments were green and exhibited anomalous spectra in the visible region as compared to the Lba3+ forms. These modified pigments showed a hypochromic shift of 10 nm for the charge transfer absorption maximum; however, differences were not apparent in the Soret region. Upon binding with nicotinate, the [alpha] and [beta] bands were shifted significantly into the red region as compared to the Lba3+ nicotinate complex. The three Lbam fractions were reduced by dithionite or by NADH in the presence of riboflavin. Lbam2+ also bound nicotinate and displayed absorption spectra indistinguishable from those of Lba2+ nicotinate. In contrast to Lba2+, Lbam2+ displayed aberrant spectra when bound with either O2 or CO. These complexes exhibited a prominent charge transfer band at approximately 620 nm and failed to exhibit spectra characteristic of Lba2+O2 and Lba2+CO. The protein moiety of these modified pigments was intact because their tyrosine/tryptophan ratios and their amino acid compositions were identical with those of Lba, nor were differences observed in the peptide profiles resulting from trypsin digests of purified Lba and Lbams. Automated Edman degradation of selected peaks further confirmed the intactness of the protein backbone including the absence of deamination. Pyridine hemochromogen for heme from Lbams could be formed, and the spectra displayed distinct differences compared to those of Lba. A new peak at 580 nm and a loss of a peak at 480 nm were observed for all three Lbams.

Detection and purification of modified leghemoglobins from soybean root nodules

Abstract A new procedure was developed to purify leghemoglobins (Lbs) from Glycine max L. (Merr). root nodules. This procedure resulted in the purification of a hemoprotein fraction, Lbam as detected by ion-exchange-HPLC was found in nodule extracts prepared in the presence of peptidase inhibitors, and was unstable in acidic solutions. Acidic conditions also emhanced proteolysis of Lba by a nodule enriched in peptidases as observed using a denaturing peptide gel developed for this purpose. In contrast, there was little degradation of Lba by endogenous peptidases at pH 8.0. We incorporated these observations into the purification protocol as follows: (1) nodule extracts were first chromatographed on hydroxylapatite at pH 6.8. Greater than 98% of the peptidases bound to the column and the Lbs were recovered in the wash fraction; (2) subsequent ion-exchange chromatography of the wash fraction at pH 8.0 yielded several fractions related to Lba, including Lbam; and (3) The crude Lbam fraction was resolved into four distinct protein bands by isoelectric focusing using a new buffer system and gel formulation. One of these bands exhibited a p I value identical to that of Lba, whereas the other three proteins displayed more acidic p I values of 4.91, 4.87 and 4.85, respectively. All of these purified Lbam proteins had molecular weights indistinguishable from Lba, similar amino acid compositions and possessed a sequence identical to that of Lba in the first 15 residues from the amino-terminal end.

Light sensitive zinc content of protein fractions from bovine rod outer segments.

Abstract Bovine retinas, isolated rod outer segments and emulphogene extracts of rod outer segments have been shown to contain appreciable amounts of Zn2+, Ca2+ and Mg2+ when isolated in the absence of added metal ions. Chromatography of emulphogene extracted rod outer segments in Sephadex G-25 showed virtually all the Ca2+, Zn2+ and protein to elute with the void volume. Levels of Zn2+ but not Ca2+ were light sensitive. The Zn2+ contents of protein fractions were about 60% higher when samples were bleached. Under optimal conditions protein fractions contained 1.4 – 1.8 g atoms Zn2+/mole rhodopsin for dark adapted samples and 2.1 to 3.2 g atoms Zn2+/mole of rhodopsin for bleached samples.

Ligand exchange amino acid analysis: resolution of some amino sugars and cysteine derivatives.

A new buffer system has been developed for the Hitachi Perkin-Elmer model KLA-3B ligand-exchange amino acid analyzer (protein-hydrolyzates analysis) which allowed for virtually complete resolution of glucosamine and either mannosamine or galactosamine from the basic amino acids tyrosine and phenylalanine. The same buffer resolved S-(β-aminoethyl)cysteine from histidine. Although the resolution of the amino sugars was not affected by small pH changes in the buffer, the retention time of histidine was markedly changed. Elevation of the pH by 0.06 units caused histidine to elute with S-(β-aminoethyl)cysteine, while a similar decrease in pH caused it to elute with ammonia. A modified procedure for sample preparation was also tested that allowed for the complete resolution of S-carboxymethylcysteine from aspartic acid.

Chromatographic separations of sucrose monostearate structural isomers

Abstract High-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and gas—liquid chromatography (GLC) methods are described for separating sucrose monostearate isomers. The HPLC procedure provides baseline separation of purified monoester isomers into three main peaks at room temperature, and completely separates monoesters with different acyl chain lengths (C14, C16, C18). The TLC method separates up to six of the eight possible positional monostearate isomers, which can be further differentiated by specific color development with a visualizing agent. The GLC technique used resolves monoesters with different acyl chain lengths, and partially separates monostearate isomers. Isomers collected from HPLC were subjected to treatment with invertase, then resolved and detected by TLC to determine fatty acid substitution patterns on the sucrose molecule.