Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli.

A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45°C. Specific activity of StmPr2 determined with suc-L-Ala-L-Ala-L-Pro-l-Phe-p-nitroanilide as the substrate was 17±2U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.

Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases.

In the wide field of laundry and cleaning applications, there is an unbroken need for novel detergent proteases excelling in high stability and activity and a suitable substrate range. We demonstrated the large amount of highly diverse subtilase sequences present in metagenomic DNA by recovering 57 non-redundant subtilase sequence tags with degenerate primers. Furthermore, an activity- as well as a sequence homology-based screening of metagenomic DNA libraries was carried out, using alkaline soil and habitat enrichments as a source of DNA. In this way, 18 diverse full-length protease genes were recovered, sharing only 37-85% of their amino acid residues with already known protease genes. Active clones were biochemically characterized and subjected to a laundry application assay, leading to the identification of three promising detergent proteases. According to sequence similarity, two proteases (HP53 and HP70) can be classified as subtilases, while the third enzyme (HP23) belongs to chymotrypsin-like S1 serine proteases, a class of enzymes that has not yet been described for the use in laundry and cleaning applications.

C-terminal truncation of a metagenome-derived detergent protease for effective expression in E. coli.

Recently, a new alkaline protease named HP70 showing highest homology to extracellular serine proteases of Stenotrophomonas maltophilia and Xanthomonas campestris was found in the course of a metagenome screening for detergent proteases (Niehaus et al., submitted for publication). Attempts to efficiently express the enzyme in common expression hosts had failed. This study reports on the realization of overexpression in Escherichia coli after structural modification of HP70. Modelling of HP70 resulted in a two-domain structure, comprising the catalytic domain and a C-terminal domain which includes about 100 amino acids. On the basis of the modelled structure the enzyme was truncated by deletion of most of the C-terminal domain yielding HP70-C477. This structural modification allowed effective expression of active enzyme using E. coli BL21-Gold as the host. Specific activity of HP70-C477 determined with suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide as the substrate was 30 ± 5 U/mg compared to 8 ± 1 U/mg of the native enzyme. HP70-C477 was most active at 40°C and pH 7-11; these conditions are prerequisite for a potential application as detergent enzyme. Determination of kinetic parameters at 40°C and pH=9.5 resulted in K(M)=0.23 ± 0.01 mM and k(cat)=167.5 ± 3.6s(-1). MS-analysis of peptide fragments obtained from incubation of HP70 and HP70-C477 with insulin B indicated that the C-terminal domain influences the cleavage preferences of the enzyme. Washing experiments confirmed the high potential of HP70-C477 as detergent protease.

Heterologous expression and characterization of choline oxidase from the soil bacterium Arthrobacter nicotianae.

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15-70 degrees C). Specific activities were determined toward choline chloride (4.70 +/- 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 +/- 0.45 x 10(-2) U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 +/- 0.12 x 10(-2) U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K (M) = 1.51 +/- 0.09 mM and V (max) = 42.73 +/- 0.42 mU/min for choline chloride and K (M) = 4.77 +/- 0.76 mM and V (max) = 48.40 +/- 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.

Thermostable variants of subtilisin selected by temperature-gradient gel electrophoresis.

Region-specific random mutagenesis in the weak calcium binding site of subtilisin Carlsberg and subsequent screening for variants with enhanced heat stability revealed two variants, which showed significantly enhanced residual activity at 68 degrees C, 0.1 mM CaCl2, pH 8.0. Preselected variants have been studied by temperature-gradient gel electrophoresis (TGGE) and were found to be stabilized due to different effects. Whereas the point mutation (Ser188Pro) mainly enhanced autoproteolytic stability of subtilisin, the double mutation (Ser188Pro; Ala194Glu) additionally increased the apparent Tm-value of the molecule for 2-3 degrees C under a variety of conditions. It was possible to differentiate between the effects of autoproteolysis and structural unfolding to a certain degree by TGGE and to show the complex influence of changed calcium affinity on thermal stability for the double variant.

Substrate specificity of natural variants and genetically engineered intermediates of Bacillus lentus alkaline proteases.

Three natural variants of subtilisin lentus could be differentiated by their amino acid sequence and their specific activity with low molecular weight peptide substrates of the type sAAPFpNA. The variants had amino acid exchanges in five, respective six positions of their amino acid sequence, four of which are located in the substrate loop of the enzyme (positions 92 - 102). Variants of one type of highly alkaline subtilisin (subtilisin 309) were made by site directed mutagenesis, each containing one of the corresponding amino acid exchanges. These intermediate forms were tested for activity, pH-dependence and substrate specificity. The changes in substrate affinity were relatively small for substrates with different amino acids as P1 residue. The differences in activity on peptide-substrates could be related primarily to a single amino acid substitution in the S4 substrate binding pocket in position 102. With substrate variations in the P3 amino acid residue, changes in k(cat) and K(m) revealed the importance of the charged amino acid exchanged between subtilisin 309 and BLAP. By these experiments an interaction of amino acid position 101 and the P3 residue of the substrate could be demonstrated. The substitution of two differently charged amino acids in the substrate binding region resulted in an unchanged pH-profile of the natural enzyme. With the single exchange intermediates differences in the pH-profile could be found, depending on the substrate tested: a characteristic change was observed with casein as substrate, no such change occurred with hemoglobin.

Zymogram of proteases made with developed film from nondenaturing polyacrylamide gels after electrophoresis.

A simple, extremely versatile method for preparing zymograms from proteases after nondenaturing Phast-System gel electrophoresis is described. After completion of the run and before staining, an electropherogram and a piece of developed, single-side coated X-ray film are brought into contact for 5 min at room temperature. The film overlay is then separated and the gel is stained for protein. The zymogram on the X-ray film is generated by simply pouring about 10 ml of a suitable buffer solution at 30 to 50 degrees C over the film strip. Clearing zones appeared within a few seconds to a few minutes depending on protease amount. For the alkaline protease subtilisin BL the lower limit of detectability was 10 ng applied to the gel prior to electrophoresis. For preservation and archiving the zymogram film is simply rinsed with distilled water and air-dried. The film can be cut to size and mounted for a slide projector, or the film can serve as a negative for photographic enlargements. The clearing zone area is proportional to the amount of protease from 10 to 100 ng of protein.

Analysis of the conformational transitions of proteins by temperature-gradient gel electrophoresis.

Temperature-gradient gel electrophoresis (TGGE) is a technique for studying the structural transitions of nucleic acids and proteins. A temperature gradient is formed in a horizontal slab gel perpendicular to the direction of the electric field. Whereas the principle of the TGGE method has previously been applied to proteins, we describe in this report the systematic optimization of TGGE as a routine technique for the quantitative analysis of conformational transitions in proteins. Using alpha-amylase as an example we show the kinds of results which may be obtained from such measurements. Buffers suitable for use in gel electrophoresis were analyzed with respect to the dependence of their pH value upon temperature. The correct pH range for TGGE of a given protein is determined by electrophoretic titration curves. The protein bands are detected by silver and/or activity staining. The thermal denaturation of alpha-amylase from Aspergillus oryzae showed a discontinuous transition into the denatured conformation, which exhibited much slower electrophoretic mobility. The discontinuity is due to an irreversible denaturation process under the gel conditions. The transition temperature was measured as a function of several parameters, e.g., the concentration of Ca(+)+, dithiotreithol, urea and the pH value. The structural transition of alpha-amylase is accompanied by a loss of enzymatic activity as determined by activity staining or by an activity assay carried out in solution. The structural transitions of two other alpha-amylases from Bacillus subtilis and Bacillus licheniformis were also studied. The results show that the TGGE method is simple to perform and allows the analysis of conformational transitions of proteins in a wide variety of conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of Streptomyces erythraeus.

Streptomyces erythraeus strains were transformed efficiently with six different plasmid DNA vectors by a protocol that uses 0.2 mM Ca2+, 5 mM Mg2+ and 10% DMSO to enhance the stability of protoplasts to storage at -80 degrees C and their transformability at 30 degrees C. The primary thiostrepton-resistant (Thior) transformants for most vectors were unstable even when grown selectively. This instability did not appear to be due to incompatibility with indigenous S. erythraeus plasmids. Conversely, stable transformation was not the result of plasmid or host mutations. Transformation instability or plasmid copy number thus prevented successful shotgun-cloning of DNA that complemented the eryD mutation, which blocks the biosynthesis of erythromycin A, because only plasmid DNA without an insert could be isolated from a Thior Ery+ clone.

Regulation of enzymes involved in the biosynthesis of the sesquiterpene antibiotic pentalenolactone in Streptomyces arenae.

The production of the sesquiterpenoid antibiotic pentalenolactone in the producer strain Streptomyces arenae TU 469 is controlled by the activity of the enzyme farnesylpyrophosphate cyclase. In contrast to the activity of this enzyme, the specific activities of all other enzymes of the mevalonoid pathway tested so far, proved to be not rate-limiting. Several metabolites of the pentalenolactone pathway were tested for inhibitory effects on the activity of the HMG-CoA reductase and farnesylpyrophosphate cyclase. The activity of the cyclase was inhibited by low concentrations of pentalenolactone and its derivatives, thus suggesting an end product inhibition of the starting enzyme of the pentalenolactone pathway. The activity of HMG-CoA reductase was not inhibited by pentalenene or any pentalenolactone-derivatives. According to these results, an end-product inhibition of the first enzyme which is specific for pentalenolactone synthesis seems to be a mechanism involved in the regulation of pentalenolactone biosynthesis.

Characterization of two glyceraldehyde-3-phosphate dehydrogenase isoenzymes from the pentalenolactone producer Streptomyces arenae.

Pentalenolactone (PL) irreversibly inactivates the enzyme glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) and thus is a potent inhibitor of glycolysis in both procaryotic and eucaryotic cells. We showed that PL-producing strain Streptomyces arenae TU469 contains a PL-insensitive glyceraldehyde-3-phosphate dehydrogenase under conditions of PL production. In complex media no PL production was observed, and a PL-sensitive glyceraldehyde-3-phosphate dehydrogenase, rather than the insensitive enzyme, could be detected. The enzymes had the same substrate specificity but different catalytic and molecular properties. The apparent Km values of the PL-insensitive and PL-sensitive enzymes for glyceraldehyde-3-phosphate were 100 and 250 microM, respectively, and the PL-sensitive enzyme was strongly inhibited by PL under conditions in which the PL-insensitive enzyme was not inhibited. The physical properties of the PL-insensitive enzyme suggest that the protein is an octamer, whereas the PL-sensitive enzyme, like other glyceraldehyde-3-phosphate dehydrogenases, appears to be a tetramer.

Hydroxyapatite phagocytosis by human polymorphonuclear leucocytes.

Hydroxyapatite crystals were incubated with human polymorphonuclear leucocytes and samples examined by light and electron microscopy after 3, 8, 30, and 120 minutes' incubation. Phagocytosis of crystals occurred at 3 minutes and increased with incubation. Degranulation of neutrophils, loss of cytoplasmic density, and cell necrosis were greatest in cells mixed with apatite and increased with time. Avid in vitro phagocytosis of hydroxyapatite crystals lends further support to the potential of these crystals as causes of human arthritis.

Further compounds from the pedal gland of the bontebok (Damaliscus dorcas dorcas).

The identification of four further major constituents of the pedal gland exudate of the bontebok, Damaliscus dorcas dorcas, viz. alpha-terpineol, 2-n-heptylpyridine, m-cresol and (A)-6-dodecen-4-olide and the investigation of the stereochemistry of the double bond in (Z)-6-dodecen-4-olide by means of iterative computer analysis are described. An improved synthesis of this compound is outlined.

Identification of drugs using a gas chromatography-mass spectrometry system equipped with electron impact-chemical ionization and electron impact-field ionizationfield desorption combination sources.

In a case of toxicological analysis by gas chromatography (GC)-mass spectrometry-data system, chemical ionization (CI), field ionization (FI) and electron impact data are shown to lead rapidly to assignments for all significant GC peaks. Valuable data include not only full spectra in the three ionization modes, but also exact mass measurements in the FI or CI mode. Such measurements, obtained by a dynamic peak-matching technique, lead to the elemental composition of the compounds of interest. This knowledge makes the assignment of key GC peaks unequivocal and provides an extremely high level of confidence regarding the identity of whole metabolite series. It is also shown that the nature of the FI information is very similar to that obtained from CI data.