Thermostable phycocyanin from the red microalga Cyanidioschyzon merolae, a new natural blue food colorant.

The demand for natural food colorants is growing as consumers question the use of artificial colorants more and more. The phycobiliprotein C-phycocyanin of Arthospira platensis is used as a natural blue colorant in certain food products. The thermoacidophilic red microalga Cyanidioschyzon merolae might provide an alternative source of phycocyanin. Cyanidioschyzon merolae belongs to the order Cyanidiophyceae of the phylum Rhodophyta. Its natural habitat are sulfuric hot springs and geysers found near volcanic areas in, e.g., Yellowstone National Park in the USA and in Java, Indonesia. It grows optimally at a pH between 0.5 and 3.0 and at temperatures up to 56 °C. The low pH at which C. merolae grows minimizes the risk of microbial contamination and could limit production loss. As C. merolae lacks a cell wall, phycocyanin with a high purity number of 9.9 could be extracted by an osmotic shock using a simple ultrapure water extraction followed by centrifugation. The denaturation midpoint at pH 5 was 83 °C, being considerably higher than the A. platensis phycocyanin (65 °C). The C. merolae phycocyanin was relatively stable at pH 4 and 5 up to 80 °C. The high thermostability at slightly acidic pH makes the C. merolae phycocyanin an interesting alternative to A. platensis phycocyanin as a natural blue food colorant.

The unique branching patterns of Deinococcus glycogen branching enzymes are determined by their N-terminal domains.

Glycogen branching enzymes (GBE) or 1,4-alpha-glucan branching enzymes (EC 2.4.1.18) introduce alpha-1,6 branching points in alpha-glucans, e.g., glycogen. To identify structural features in GBEs that determine their branching pattern specificity, the Deinococcus geothermalis and Deinococcus radiodurans GBE (GBE(Dg) and GBE(Dr), respectively) were characterized. Compared to other GBEs described to date, these Deinococcus GBEs display unique branching patterns, both transferring relatively short side chains. In spite of their high amino acid sequence similarity (88%) the D. geothermalis enzyme had highest activity on amylose while the D. radiodurans enzyme preferred amylopectin. The side chain distributions of the products were clearly different: GBE(Dg) transferred a larger number of smaller side chains; specifically, DP5 chains corresponded to 10% of the total amount of transferred chains, versus 6.5% for GBE(Dr). GH13-type GBEs are composed of a central (beta/alpha) barrel catalytic domain and an N-terminal and a C-terminal domain. Characterization of hybrid Deinococcus GBEs revealed that the N2 modules of the N domains largely determined substrate specificity and the product branching pattern. The N2 module has recently been annotated as a carbohydrate binding module (CBM48). It appears likely that the distance between the sugar binding subsites in the active site and the CBM48 subdomain determines the average lengths of side chains transferred.

Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal alpha-amylase enzymes.

Currently known fungal alpha-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal alpha-amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal alpha-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall alpha-glucan. To study the biochemical characteristics of this novel cluster of alpha-amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg(-1)), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall alpha-glucan synthesis is discussed.

Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.

In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing alpha-(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.

Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes.

Bacterial fructosyltransferase enzymes belonging to glycoside hydrolase family 68 (GH68) are not known to require a metal cofactor. Here, we show that Ca2+ ions play an important structural role in the Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes. Analysis of the Bacillus subtilis Lev 3D structure [Meng, G. and Futterer, K. (2003) Nat. Struct. Biol. 10, 935-941] has provided evidence for the presence of a bound metal ion, most likely Ca2+. Characterization of site-directed mutants in the putative Ca2+ ion-binding sites of Lb. reuteri Lev and Inu revealed that the Inu Asp520 and Lev Asp500 residues play an important role in Ca2+ binding. Sequence alignments of family GH68 proteins showed that this Ca2+ ion-binding site is (largely) present only in proteins of Gram-positive origin.

Glucan synthesis in the genus Lactobacillus: isolation and characterization of glucansucrase genes, enzymes and glucan products from six different strains.

Members of the genera Streptococcus and Leuconostoc synthesize various alpha-glucans (dextran, alternan and mutan). In Lactobacillus, until now, the only glucosyltransferase (GTF) enzyme that has been characterized is gtfA of Lactobacillus reuteri 121, the first GTF enzyme synthesizing a glucan (reuteran) that contains mainly alpha-(1-->4) linkages together with alpha-(1-->6) and alpha-(1-->4,6) linkages. Recently, partial sequences of glucansucrase genes were detected in other members of the genus Lactobacillus. This paper reports, for the first time, isolation and characterization of dextransucrase and mutansucrase genes and enzymes from various Lactobacillus species and the characterization of the glucan products synthesized, which mainly have alpha-(1-->6)- and alpha-(1-->3)-glucosidic linkages. The four GTF enzymes characterized from three different Lb. reuteri strains are highly similar at the amino acid level, and consequently their protein structures are very alike. Interestingly, these four Lb. reuteri GTFs have relatively large N-terminal variable regions, containing RDV repeats, and relatively short putative glucan-binding domains with conserved and less-conserved YG-repeating units. The three other GTF enzymes, isolated from Lactobacillus sakei, Lactobacillus fermentum and Lactobacillus parabuchneri, contain smaller variable regions and larger putative glucan-binding domains compared to the Lb. reuteri GTF enzymes.

Biochemical and molecular characterization of Lactobacillus reuteri 121 reuteransucrase.

Lactobacillus reuteri strain 121 uses sucrose for synthesis of a unique, soluble glucan ('reuteran') with mainly alpha-(1-->4) glucosidic linkages. The gene (gtfA) encoding this glucansucrase enzyme had previously been characterized. Here, a detailed biochemical and molecular analysis of the GTFA enzyme is presented. This is believed to be the first report describing reuteransucrase enzyme kinetics and the oligosaccharides synthesized with various acceptors. Alignments of the GTFA sequence with glucansucrases from Streptococcus and Leuconostoc identified conserved amino-acid residues in the catalytic core critical for enzyme activity. Mutants Asp1024Asn, Glu1061Gln and Asp1133Asn displayed 300- to 1000-fold-reduced specific activities. To investigate the role of the relatively large N-terminal variable domain (702 amino acids) and the relatively short C-terminal putative glucan-binding domain (267 amino acids, with 11 YG repeats), various truncated derivatives of GTFA (1781 amino acids) were constructed and characterized. Deletion of the complete N-terminal variable domain of GTFA (GTFA-Delta N) had little effect on reuteran characteristics (size, distribution of glycosidic linkages), but the initial transferase activity of the mutant enzyme increased drastically. Sequential C-terminal deletions (up to six YG repeats) in GTFA-Delta N also had little effect on reuteran characteristics. However, enzyme kinetics drastically changed. Deletion of 7, 8 or 11 YG repeats resulted in dramatic loss of total enzyme activity (43-, 63- and 1000-fold-reduced specific activities, respectively). Characterization of sequential C-terminal deletion mutants of GTFA-Delta N revealed that the C-terminal domain of reuteransucrase has an important role in glucan binding.

Exploring and exploiting starch-modifying amylomaltases from thermophiles.

Starch is a staple food present in water-insoluble granules in many economically important crops. It is composed of two glucose polymers: the linear alpha-1,4-linked amylose and amylopectin with a backbone of alpha-1,4-glycosidic bonds and alpha-1,6-linked side chains. To dissolve starch completely in water it needs to be heated; when it cools down too much the starch solution forms a thermo-irreversible gel. Amylomaltases (EC 2.4.1.25) are enzymes that transfer a segment of an alpha-1,4-D-glucan to a new 4-position in an acceptor, which may be glucose or another alpha-1,4-D-glucan. Acting upon starch, amylomaltases can produce cycloamylose or a thermoreversible starch gel, both of which are of commercial interest.

Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri.

Lactobacillus reuteri strain 121 employs a fructosyltransferase (FTF) to synthesize a fructose polymer [a fructan of the levan type, with beta(2-->6) linkages] from sucrose or raffinose. Purification of this FTF (a levansucrase), and identification of peptide amino acid sequences, allowed isolation of the first Lactobacillus levansucrase gene (lev), encoding a protein (Lev) consisting of 804 amino acids. Lev showed highest similarity with an inulosucrase of L. reuteri 121 [Inu; producing an inulin polymer with beta(2-->1)-linked fructosyl units] and with FTFs from streptococci. Expression of lev in Escherichia coli resulted in an active FTF (Lev Delta 773His) that produced the same levan polymer [with only 2-3 % beta(2-->1-->6) branching points] as L. reuteri 121 cells grown on raffinose. The low degree of branching of the L. reuteri levan is very different from bacterial levans known up to now, such as that of Streptococcus salivarius, having up to 30 % branches. Although Lev is unusual in showing a higher hydrolysis than transferase activity, significant amounts of levan polymer are produced both in vivo and in vitro. Lev is strongly dependent on Ca(2+) ions for activity. Unique properties of L. reuteri Lev together with Inu are: (i) the presence of a C-terminal cell-wall-anchoring motif causing similar expression problems in Escherichia coli, (ii) a relatively high optimum temperature for activity for FTF enzymes, and (iii) at 50 degrees C, kinetics that are best described by the Hill equation.

Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121.

Bacterial fructosyltransferases (FTFs) are retaining-type glycosidases that belong to family 68 of glycoside hydrolases. Recently, the high-resolution 3D structure of the Bacillus subtilis levansucrase has been solved [Meng, G. and Futterer, K., Nat. Struct. Biol. 10 (2003) 935-941]. Based on this structure, the catalytic nucleophile, general acid/base catalyst, and transition state stabilizer were identified. However, a detailed characterization of site-directed mutants of the catalytic nucleophile has not been presented for any FTF enzyme. We have constructed site-directed mutants of the three putative catalytic residues of the Lactobacillus reuteri 121 levansucrase and inulosucrase and characterized the mutant proteins. Changing the putative catalytic nucleophiles D272 (inulosucrase) and D249 (levansucrase) into their amido counterparts resulted in a 1.5-4x10(5) times reduction of total sucrase activity.

Kinetic properties of an inulosucrase from Lactobacillus reuteri 121.

Inulosucrases catalyze transfer of a fructose moiety from sucrose to a water molecule (hydrolysis) or to an acceptor molecule (transferase), yielding inulin. Bacterial inulin production is rare and a biochemical analysis of inulosucrase enzymes has not been reported. Here we report biochemical characteristics of a purified recombinant inulosucrase enzyme from Lactobacillus reuteri. It displayed Michaelis-Menten type of kinetics with substrate inhibition for the hydrolysis reaction. Kinetics of the transferase reaction is best described by the Hill equation, not reported before for these enzymes. A C-terminal deletion of 100 amino acids did not appear to affect enzyme activity or product formation. This truncated form of the enzyme was used for biochemical characterization.

Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique, highly branched glucan with alpha-(1-->4) and alpha-(1-->6) glucosidic bonds.

Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the alpha-(1-->4) glucosidic type. The glucan also contains alpha-(1-->6)-linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M(r) of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.

Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight inulin and inulin oligosaccharides.

Fructosyltransferase (FTF) enzymes produce fructose polymers (fructans) from sucrose. Here, we report the isolation and characterization of an FTF-encoding gene from Lactobacillus reuteri strain 121. A C-terminally truncated version of the ftf gene was successfully expressed in Escherichia coli. When incubated with sucrose, the purified recombinant FTF enzyme produced large amounts of fructo-oligosaccharides (FOS) with beta-(2-->1)-linked fructosyl units, plus a high-molecular-weight fructan polymer (>10(7)) with beta-(2-->1) linkages (an inulin). FOS, but not inulin, was found in supernatants of L. reuteri strain 121 cultures grown on medium containing sucrose. Bacterial inulin production has been reported for only Streptococcus mutans strains. FOS production has been reported for a few bacterial strains. This paper reports the first-time isolation and molecular characterization of (i) a Lactobacillus ftf gene, (ii) an inulosucrase associated with a generally regarded as safe bacterium, (iii) an FTF enzyme synthesizing both a high molecular weight inulin and FOS, and (iv) an FTF protein containing a cell wall-anchoring LPXTG motif. The biological relevance and potential health benefits of an inulosucrase associated with an L. reuteri strain remain to be established.

Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced.

Fructosyltransferase (FTF) enzymes have been characterized from various Gram-positive bacteria, but not from Lactobacillus sp. In a screening of 182 lactobacilli for polysaccharide production only one strain, Lactobacillus reuteri strain 121, was found to produce a fructan being a levan. Here we report the first-time identification and biochemical characterization of a Lactobacillus FTF enzyme. When incubated with sucrose the enzyme produced a levan that is identical to that produced by Lb. reuteri strain 121 cells.

Paenibacillus granivorans sp. nov., a new Paenibacillus species which degrades native potato starch granules.

From a native potato starch-degrading enrichment culture, strain A30 had been isolated and had tentatively been identified as a member of the Bacillus firmus/lentus group (Wijbenga et al. Appl. Microbiol. Biotechnol. 35, 180-184, 1991). In this paper the isolate A30 is further characterized using phylogentic analysis of the 16S rDNA and determination of a number of additional phenotypic characteristics. These data are compared to those of Paenibacillus amylolyticus, P. chibensis, and P. thiaminolyticus. It is concluded that strain A30 is a new Paenibacillus species, for which the name Paenibacillus granivorans is suggested.

Biomarker evidence for widespread anaerobic methane oxidation in Mediterranean sediments by a consortium of methanogenic archaea and bacteria. The Medinaut Shipboard Scientific Party.

Although abundant geochemical data indicate that anaerobic methane oxidation occurs in marine sediments, the linkage to specific microorganisms remains unclear. In order to examine processes of methane consumption and oxidation, sediment samples from mud volcanoes at two distinct sites on the Mediterranean Ridge were collected via the submersible Nautile. Geochemical data strongly indicate that methane is oxidized under anaerobic conditions, and compound-specific carbon isotope analyses indicate that this reaction is facilitated by a consortium of archaea and bacteria. Specifically, these methane-rich sediments contain high abundances of methanogen-specific biomarkers that are significantly depleted in (13)C (delta(13)C values are as low as -95 per thousand). Biomarkers inferred to derive from sulfate-reducing bacteria and other heterotrophic bacteria are similarly depleted. Consistent with previous work, such depletion can be explained by consumption of (13)C-depleted methane by methanogens operating in reverse and as part a consortium of organisms in which sulfate serves as the terminal electron acceptor. Moreover, our results indicate that this process is widespread in Mediterranean mud volcanoes and in some localized settings is the predominant microbiological process.

Detection of methanogenic archaea in seawater particles and the digestive tract of a marine fish species.

A methanogen-specific nested PCR approach was used to detect methanogenic archaea in seawater particles of the North Sea and the feces and the digestive tract of flounder (Platichthys flesus), a fish found in the North Sea. A number of 16S rDNA sequences with 97.6-99.5% similarity to Methanococcoides methylutens were found in the seawater particles as well as the digestive tract and fecal samples.

Association of marine archaea with the digestive tracts of two marine fish species.

Recent studies have shown that archaea which were always thought to live under strict anoxic or extreme environmental conditions are also present in cold, oxygenated seawater, soils, the digestive tract of a holothurian deep-sea-deposit feeder, and a marine sponge. In this study, we show, by using PCR-mediated screening in other marine eukaryotes, that marine archaea are also present in the digestive tracts of flounder and grey mullet, two fish species common in the North Sea, in fecal samples of flounder, and in suspended particulate matter of the North Sea water column. No marine archaea could be detected in the digestive tracts of mussels or the fecal pellets of a copepod species. The archaeal 16S ribosomal DNA clone libraries of feces of flounder and the contents of the digestive tracts of grey mullet and flounder were dominated by group II marine archaea. The marine archaeal clones derived from flounder and grey mullet digestive tracts and feces formed a distinct cluster within the group II marine archaea, with 76.7 to 89. 8% similarity to previously described group II clones. Fingerprinting of the archaeal community of flounder digestive tract contents and feces by terminal restriction fragment length polymorphism of archaeal 16S rRNA genes after restriction with HhaI showed a dominant fragment at 249 bp, which is likely to be derived from group II marine archaea. Clones of marine archaea that were closely related to the fish-associated marine archaea clones were obtained from suspended particulate matter of the water column at two stations in the North Sea. Terminal restriction fragment length polymorphism fingerprinting of the archaeal community present in suspended particulate matter showed the same fragment pattern as was found for the archaeal community of the flounder digestive tract contents and feces. These data demonstrate that marine archaea are present in the digestive tracts and feces of very common marine fish. It is possible that the marine archaea associated with the digestive tracts of marine fish are liberated into the water column through the feces and subsequently contribute to the marine archaeal community of suspended particulate matter.

Demethylation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by marine sulfate-reducing bacteria.

The initial step in the anaerobic degradation of the algal osmolyte dimethylsulfoniopropionate (DMSP) in anoxic marine sediments involves either a cleavage to dimethylsulfide and acrylate or a demethylation to 3-S-methylmercaptopropionate. Thus far, only one anaerobic bacterial strain has been shown to carry out the demethylation, namely, Desulfobacterium sp. strain PM4. The aims of the present work were to study how common this property is among certain groups of anaerobic bacteria and to obtain information on the affinities for DMSP of DMSP-demethylating strains. Screening of several pure cultures of sulfate-reducing and acetogenic bacteria showed that Desulfobacterium vacuolatum DSM 3385 and Desulfobacterium niacini DSM 2059 are also able to demethylate DMSP; a very slow demethylation of DMSP was observed with a salt-tolerant strain of Eubacterium limosum. From a 10(5) dilution of intertidal sediment a new marine DMSP-demethylating sulfate-reducing bacterium (strain WN) was isolated. Strain WN was a short, gram-negative, nonmotile rod that grew on betaine, sarcosine, palmitate, H2 plus CO2, and several alcohols, organic acids, and amino acids. Extracts of betaine-grown cells had hydrogenase, formate dehydrogenase, and CO dehydrogenase activities but no alpha-ketoglutarate oxidoreductase activity, indicating the presence of the acetyl coenzyme A-CO dehydrogenase pathway. Analysis of the 16S rRNA gene sequence of strain WN revealed a close relationship with Desulfobacter hydrogenophilus, Desulfobacter latus, and Desulfobacula toluolica. Strain PM4 was shown to group with Desulfobacterium niacini. The K(m) of strain WN for DMSP, as derived from substrate progress curves in cell suspensions, was approximately 10 microM. A similar value was found for D. niacini PM4.

Selection of Pseudomonas sp. strain HBP1 Prp for metabolism of 2-propylphenol and elucidation of the degradative pathway.

A mutant of Pseudomonas sp. strain HBP1, originally isolated on 2-hydroxybiphenyl, was selected for the ability to grow on 2-propylphenol as the sole carbon and energy source. In the mutant strain, which was designated as Pseudomonas sp. strain HBP1 Prp, the cellular induction mechanism involved in the synthesis of the NADH-dependent monooxygenase is changed. 2-Propylphenol, which is known to be a substrate of the monooxygenase, does not induce formation of the monooxygenase in the wild type but does have an induction effect in the mutant strain. Furthermore, in contrast to the wild type, mutant strain HBP1 Prp constitutively produces a small amount of monooxygenase and metapyrocatechase. The enzymes from strain HBP1 Prp catalyzing the first three steps in the degradation of 2-propylphenol--the NADH-dependent monooxygenase, the metapyrocatechase, and the meta fission product hydrolase--were partially purified, and their activities were measured. The product of the monooxygenase activity was identified by mass spectrometry as 3-propylcatechol. The metapyrocatechase used this compound as a substrate and produced a yellow meta fission product that was identified by mass spectrometry as 2-hydroxy-6-oxo-nona-2,4- dienoate. Butyrate could be detected as a product of the meta fission product hydrolase in crude cell extract of 2-propylphenol-grown cells, as well as an intermediate in culture broths during growth on 2-propylphenol. All three enzymes expressed highest activities for the metabolites of the degradation of 2-hydroxybiphenyl.