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The microflora isolated from food-packaging board is dominated by paenibacilli; a number of these micro-organisms have been characterized using a polyphasic approach. The highest 16S rRNA gene similarity was found between these isolates and Paenibacillus azotofixans ATCC 35681(T) (97.7 %). The main fatty acid of the paperboard isolates was C(16 : 0) (34-45 %); straight-chain fatty acids made up 41-60 % of the total cellular fatty acids, thus distinguishing these strains from other Paenibacillus species. The paperboard isolates produced cyclodextrins from starch. The spore surface had a characteristic ribbed ornamentation. Spores and vegetative cells frequently had pilus-like appendages. Based on phylogenetic data and phenotypic and chemotaxonomic characteristics, it is proposed that the isolates represent a novel species, Paenibacillus stellifer sp. nov., with IS 1(T) (=DSM 14472(T)=CCUG 45566(T)) as the type strain.

The cellular fatty acid content of 22 cyanobacterial strains belonging to the genera Anabaena, Aphanizomenon, Calothrix, Cylindrospermum, Nostoc, Microcystis and Planktothrix were analysed. The identities of the major peaks were confirmed by MS. Correspondence analysis of the data revealed three distinct groups formed by the Microcystis strains, the Nostoc/Planktothrix strains and the Anabaena/Aphanizomenon/Cylindrospermum strains. The Calothrix strain did not cluster with the other heterocystous cyanobacteria, supporting its morphological classification separate from the Nostocaceae family. The presence of large amounts of the fatty acids 18:30omega6,9,12c and 18:0 iso distinguished the Microcystis strains from the other cyanobacteria studied. The high content of 16:1omega7c grouped the Nostoc strains with the Planktothrix strains. A free-living strain of Nostoc contained 16:1omegao5c and 16: 1omega7c (about 1: 1), separating it from the symbiotic Nostoc strain and the Planktothrix strains. the strains of Anabaena, Aphanizomenon and Cylindrospermum grouped tightly and were characterized by the presence of 16:1omega9c and 16:0 anteiso fatty acids. Correspondence analysis of Anabaena, Aphanizomenon and Cylindrospermum showed that all hepatotoxic Anabaena strains grouped together, whereas the non-toxic and neurotoxic Anabaena strains grouped with the non-toxic Aphanizomenon strains.

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 degrees C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1-10 microg ml(-1) (EC50) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.

The ligninolytic enzyme system of Phanerochaete chrysosporium is able to decolorize several recalcitrant dyes. Three lignin peroxidase isoenzymes, LiP 3.85, LiP 4.15, and LiP 4.65, were purified by preparative isoelectric focusing from the carbon-limited culture medium of P. chrysosporium. Based on amino terminal sequences, the purified isoenzymes correspond to the isoenzymes H8, H6, and H2, respectively, from the N-limited culture. The purified isoenzymes were used for decolorization of an azo dye, Crocein Orange G (COG). According to the kinetic data obtained, the oxidation of COG by lignin peroxidase appeared to follow Michaelis-Menten kinetics. Kinetic parameters for each isoenzyme were determined. The inactivating effect of ascending H2O2 concentrations on COG oxidation is shown to be exponential within the used concentration range. The best degree of decolorization of 100 microM COG was obtained when the H2O2 concentration was 150 microM. This was also the lowest H2O2 concentration for maximal decolorization of 100 microM COG, regardless of the amount of lignin peroxidase used in the reaction.

The soluble form of human placental alkaline phosphatase (PLAP) was expressed in the methylotrophic yeast Pichia pastoris and the expression product was purified and characterized. Yeast-derived PLAP (yPLAP) was secreted into the medium to the level of 2 mg/liter. yPLAP displayed kinetic properties similar to those reported earlier for the membrane-bound PLAP. Purified yPLAP had specific activity of 774 U/mg and appeared in two subunit sizes, ca. 62 and 65 kDa. This difference was due to heterogenous N-glycosylation. Purified yPLAP appeared as multiple forms in isoelectric focusing in pI range of 4.2 to 5.2. The expression system is discussed in comparison to previously reported expression systems., (Copyright 1998 Academic Press.)

In this paper we report the expression in Pichia pastoris, purification, and characterization of the Aspergillus awamori glucoamylase catalytical domain (GAc). Pichia pastoris produced GAc to the level of 0.4 g per liter medium. This production level is about the same level as that gained for recombinant GA from Aspergillus and about 100-fold more than previously achieved by Saccharomyces cerevisiae. The GAc expressed in Pichia pastoris was purified by two independent chromatographic methods employing ion exchange or affinity chromatography to apparent homogeneity. The purified protein has a molecular weight of about 75,000 and specific activity of 78 units per milligram protein. The propeptide present in the glucoamylase N terminus was found to be removed correctly by P. pastoris. Glucoamylase produced by P. pastoris is N- and O-glycosylated, with 23% carbohydrate content. The N-linked oligosaccharides appear to be larger than in invertase, another glycoprotein heterologously expressed in P. pastoris. O-glycosides (studied to our knowledge for the first time in P. pastoris in this report) contribute about half of the total carbohydrate content in GAc. Purified GAc appears as multiple hands on isoelectric focusing with p1 values around 3.5, a value that is little higher than that for GAc produced in S. cerevisiae. GAc could be used as a versatile tool in studying protein expression in P. pastoris: as an affinity handle for other secreted proteins produced in P. pastoris, as a reporter gene when studying gene expression, and as a model protein in studying protein secretion and processing in P. pastoris.

Bacillus stearothermophilus alpha-amylase has a signal peptide typical for proteins exported by Gram-positive bacteria. There is only one signal peptidase processing site when the protein is exported from the original host, but when it is exported by Escherichia coli, two alternative sites are utilized. Site-directed mutagenesis was used to study the processing in E. coli. Processing sites for 13 B. stearothermophilus alpha-amylases carrying mutations in their signal peptide were determined. Processing of the signal peptide was remarkably tolerant to mutations, because switching between the alternative sites was possible. The length and the sequence of the region between the hydrophobic core and the cleavage site was crucial for determining the choice of the processing site. Some mutations more distal to the cleavage site also affected the site preference.

A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases. The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues. The longer the truncation, the lower the specific activity of the enzyme. Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme. The Km values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding. The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile. The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling. The liquefying amylases seem to require approximately 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity.

Genetically engineered versions of beta-galactosidase were constructed through the addition of charged polypeptide fusion tails for the purpose of enhancing polyelectrolyte precipitation. Negatively charged aspartic acid tails and positively charged poly(arginine) tails were added to beta-galactosidase from Escherichia coli. These fusion proteins were all shown to possess specific activity equal to that of the native enzyme. Gel permeation and ion-exchange chromatography provided evidence concerning the integrity of the tails as well as their altered charge characteristics. All enzymes containing charged tails displayed enhanced polyelectrolyte precipitation over the native enzyme. An optimal number of charged residues, beyond which no further enhancement of precipitation was observed, was found to be approximately 10 residues for each type of tail. No interference from nucleic acids was observed in the precipitation of positively tailed beta-galactosidase.

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.

Several fusion tail systems have been developed to promote efficient recovery and purification of recombinant proteins from crude cell extracts or culture media. In these systems, a target protein is genetically engineered to contain a C- or N-terminal polypeptide tail, which provides the biochemical basis for specificity in recovery and purification. Tails with a variety of characteristics have been used: (1) entire enzymes with affinity for immobilized substrates or inhibitors; (2) peptide-binding proteins with affinity to immunoglobulin G or albumin; (3) carbohydrate-binding proteins or domains; (4) a biotin-binding domain for in vivo biotination promoting affinity of the fusion protein to avidin or streptavidin; (5) antigenic epitopes with affinity to immobilized monoclonal antibodies; (6) charged amino acids for use in charge-based recovery methods; (7) poly(His) residues for recovery by immobilized metal affinity chromatography; and (8) other poly(amino acid)s, with binding specificities based on properties of the amino acid side chain. Fusion tails are useful at the lab scale and have potential for enhancing recovery using economical recovery methods that are easily scaled up for industrial downstream processing. Fusion tails can be used to promote secretion of target proteins and can also provide useful assay tags based on enzymatic activity or antibody binding. Many fusion tails do not interfere with the biological activity of the target protein and in some cases have been shown to stabilize it. Nevertheless, for the purification of authentic proteins a site for specific cleavage is often included, allowing removal of the tail after recovery.

Beta-galactosidase served as a model system to explore the feasibility of enhancing the selectivity of a low-cost, easily scaled separation method-precipitation. Enhanced selectivity was sought by fusing the enzyme with polypeptide tails including 5 and 11 aspartates. The unfused protein could not be selectively removed from the Escherichia coli cell extract by precipitation with polyethylenimine (PEI), but the longest fusion could be selectively removed. The presence of nucleic acids limited the purification attainable. Pretreatment with nuclease followed by diafiltration resulted in an extract from which the same fusion could be precipitated with greater than fivefold enrichment, while the untailed enzyme remained unenriched by the same precipitation step. Selectivity is attributed to the binding strength of the polyanionic tails to the polycationic PEI.

The relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus alpha-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and temperature optima. Replacement of Asp-331 by Glu in the active site caused almost total inactivation. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.

Beta-lactamase encoded by a plasmid pBR 322 was produced during the active growth phases of Escherichia coli IA 199. The maximal specific activity was about 15 times higher in shock fluid than in the cells disrupted by sonic disintegration. beta-Lactamase activity found in the membrane preparations increased gradually parallel to the cell growth. The amount of beta-lactamase in the membrane fraction, however, was only 0.2-0.4% of that found in shock fluid. beta-Lactamase was purified to homogeneity from shock fluid by a one-step procedure in a DEAE-Sepharose column. Most of the beta-lactamase activity present in the membrane fraction was released by salt extraction. beta-Lactamase solubilized treatment after salt extractions had the same molecular weight and immunological properties as beta-lactamase purified from the periplasmic space. Membrane associated beta-lactamase did not contain any covalently linked phospholipid.

Overexpression of Bacillus stearothermophilus gene coding for thermostable alpha-amylase in Escherichia coli was shown to cause outer-membrane damage leading to extracellular location of periplasmic proteins. Prolonged high expression of the alpha-amylase gene under lacZpo control eventually also lysed cells. Surprisingly, expression controlled by the pL promoter of phage lambda allowed specific release of periplasmic proteins into the growth medium without total cell lysis. Accumulation of alpha-amylase in the growth medium continued for at least 24 h under lambda pL control, whereas beta-lactamase activity ceased to increase beyond the exponential growth phase. The extent of outer membrane damage caused by alpha-amylase expression was monitored by following growth kinetics in the presence of lysozyme and by electron microscopy of the cells. Supplementing growth medium with Mg2+ restored the normal growth kinetics. It is suggested that periplasmic protein release caused by alpha-amylase overexpression is a stress response of the cell. A role for induced autolytic activity of the cell as a final effector of protein release is also proposed.