Contribution of volatiles to the antifungal effect of Lactobacillus paracasei in defined medium and yogurt.

Lactic acid bacteria with antifungal properties can be used to control spoilage of food and feed. Previously, most of the identified metabolites have been isolated from cell-free fermentate of lactic acid bacteria with methods suboptimal for detecting possible contribution from volatiles to the antifungal activity. The role of volatile compounds in the antifungal activity of Lactobacillus paracasei DGCC 2132 in a chemically defined interaction medium (CDIM) and yogurt was therefore investigated with a sampling technique minimizing volatile loss. Diacetyl was identified as the major volatile produced by L. paracasei DGCC 2132 in CDIM. When the strain was added to a yogurt medium diacetyl as well as other volatiles also increased but the metabolome was more complex. Removal of L. paracasei DGCC 2132 cells from CDIM fermentate resulted in loss of both volatiles, including diacetyl, and the antifungal activity towards two strains of Penicillium spp. When adding diacetyl to CDIM or yogurt without L. paracasei DGCC 2132, marked inhibition was observed. Besides diacetyl, the antifungal properties of acetoin were examined, but no antifungal activity was observed. Overall, the results demonstrate the contribution of diacetyl in the antifungal effect of L. paracasei DGCC 2132 and indicate that the importance of volatiles may have been previously underestimated.

A group of alpha-1,4-glucan lyase genes from the fungi Morchella costata, M. vulgaris and Peziza ostracoderma. Cloning, complete sequencing and heterologous expression.

We here report genes encoding a newly discovered class of starch- and glycogen-degrading enzyme, alpha-1,4-glucan lyase (EC 4.2.2.13), which degrades starch and glycogen to 1,5-anhydro-D-fructose. Two lyases were purified and partially sequenced from the macrofungi Morchella costata and M. vulgaris. The obtained lyase amino acid sequences were used to generate PCR primers, which were further used to probe the fungal genomic libraries. Two lyase genes (Agll1;Mo.cos and Agll1;Mo.vul) from the two fungi were fully sequenced and found to contain a coding region of 3201 bp and 3213 bp, respectively. A total of 13 small introns were found in each of the two genes with identical positions. The two lyase genes share 86% identity at the amino acid level. They encode mature lyases with 1066 and 1070 amino acids, respectively. The deduced molecular masses of 121,530 and 121,971 Da agree with the values found for the two purified lyases. A structure analysis of the promoter regions of the lyase genes revealed a number of putative regulatory DNA elements, such as the AREA and CREA sites, which are related to nitrogen and carbon metabolism, respectively, and the CCAAT/CAAT boxes, which are related to basal expression of genes. A third lyase gene (Agll1;Pe.ost) from the fungus Peziza ostracoderma was partially sequenced to 557 bp. The amino acid sequence deduced from this nucleotide fragment shares 76% identity with the M. costata lyase. Heterologous expression of the M. costata lyase gene was achieved intracellularly in Pichia pastoris and Aspergillus niger.

alpha-1,4-glucan lyases producing 1,5-anhydro-D-fructose from starch and glycogen have sequence similarity to alpha-glucosidases.

In the past few years a novel enzyme alpha-1,4-glucan lyase (EC 4.2. 2.13), which releases 1,5-anhydrofructose from starch and glycogen, has been cloned and characterized from red algae and fungi. Accumulated evidence indicates that the lytic degradation of starch and glycogen also occurs in other organisms. The present review focuses on the biochemical and molecular aspects of eight known alpha-1,4-glucan lyases and their genes from red algae and fungi. While the amino acid sequence identity is 75-80% among the alpha-1, 4-glucan lyases from each of the taxonomic groups, the identity between the algal and fungal alpha-1,4-glucan lyases is only 25-28%. Notably database searches disclosed that the alpha-1,4-glucan lyases have a clear identity of 23-28% with alpha-glucosidases of glycoside hydrolase family 31, thus for the first time linking enzymes from the class of hydrolases with that of lyases. The alignment of lyases and alpha-glucosidases revealed seven well-conserved regions, three of which have been reported to be involved in catalysis and substrate binding in alpha-glucosidases. The shared substrate and inhibitor specificity and sequence similarity of alpha-1,4-glucan lyases with alpha-glucosidases suggest that related structural elements are involved in the two different catalytic mechanisms.

A group of alpha-1,4-glucan lyases and their genes from the red alga Gracilariopsis lemaneiformis: purification, cloning, and heterologous expression.

We present here the first report of a group of alpha-1,4-glucan lyases (EC 4.2.2.13) and their genes. The lyases produce 1, 5-anhydro-D-fructose from starch and related oligomers and polymers. The enzymes were isolated from the red alga Gracilariopsis lemaneiformis from the Pacific coasts of China and USA, and the Atlantic Coast of Venezuela. Three lyase isozymes (GLq1, GLq2 and GLq3) from the Chinese subspecies, two lyase isozymes (GLs1 and GLs2) from the USA subspecies and one lyase (GLa1) from the Venezuelan subspecies were identified and investigated. GLq1, GLq3, GLs1 and GLa1 were purified and partially sequenced. Based on the amino acid sequences obtained, three lyase genes or their cDNAs (GLq1, GLq2 and GLs1) were cloned and completely sequenced and two other genes (GLq3 and GLs2) were partially sequenced. The coding sequences of the lyase genes GLq1, GLq2 and GLs1 are 3267, 3276 and 3279 bp, encoding lyases of 1088, 1091 and 1092 amino acids, respectively. The deduced molecular masses of the mature lyases from the coding sequences are 117030, 117667 and 117790 Da, respectively, close to those determined by mass spectrometry using purified lyases. The amino acid sequence identity is more than 70% among the six algal lyase isozymes. The algal GLq1 gene was expressed in Pichia pastoris and Aspergillus niger, and the expression product was identical to the wild-type enzyme.

Efficient purification, characterization and partial amino acid sequencing of two alpha-1,4-glucan lyases from fungi.

alpha-1,4-Glucan lyases from the fungi Morchella costata and M. vulgaris were purified by affinity chromatography on beta-cyclodextrin-sepharose, followed by ion exchange and gel filtration. The purified enzymes produced 1,5-anhydro-D-fructose from glucose oligomers and polymers with alpha-1,4-glucosidic linkages, such as maltose, maltosaccharides, amylopectin, and glycogen. The lyases were basically inactive towards glucans linked through alpha-1,1, alpha-1,3 or alpha-1,6 linkages. For both enzymes the molecular mass was around 121,000 Da as determined by matrix-assisted laser desorption mass spectrometry. The pI for the lyases from M. costata and M. vulgaris was 4.5 and 4.4, respectively. The lyases exhibited an optimal pH range of pH 5.5 to pH 7.5 with maximal activity at pH 6.5. Optimal temperature was between 37 degrees C and 48 degrees C for the two lyases, depending on the substrates. The lyases were examined with 12 inhibitors to starch hydrolases and it was found that they were inhibited by the -SH group blocking agent PCMB and the following sugars and their analogues: glucose, maltitol, maltose, 1-deoxynojirimycin and acarbose. Partial amino acid sequences accounting for about 35% of the lyase polypeptides were determined. In the overlapping region of the sequences, the two lyases showed 91% identity. The two lyases also cross-reacted immunologically.

Analysis of 3',5'-cAMP and adenylyl cyclase activity in higher plants using polyclonal chicken egg yolk antibodies.

Polyclonal antibodies were raised in chicken against an adenosine 3',5'-monophosphate-diphtheria toxoid antigen construct. The antibodies obtained show selectivity and high affinity toward 3',5'-cyclic nucleotides while exhibiting negligible affinity for 2',3'-cyclic nucleotides and other related adenine compounds. This paper reports on the development of an immunoaffinity purification procedure allowing both adenosine 3':5'-monophosphate (3',5'-cAMP) and adenylyl cyclase activity measurement in plant tissue samples. Basically, the technique consists of sequential purification of samples on solid-phase columns, the newly developed immunoaffinity columns, and quantitative analysis in ion-suppression HPLC coupled to photo diode array detection. The described method results in a drastic reduction of processing time compared to existing procedures and combines high yields (70-80%) and thorough purification, hence significantly increasing the sensitivity of quantification of 3',5'-cAMP content in higher plant material. Used in adenylyl cyclase activity measurement it also allows for a routine positive identification of the newly formed compound, 3',5'-cAMP, a feature generally lacking in existing adenylyl cyclase assays.

Chemical synthesis of 6'-alpha-maltosyl-maltotriose, a branched oligosaccharide representing the branch point of starch.

Chemical synthesis of the branched pentasaccharide 6'-alpha-maltosyl-maltotriose (15) is reported, based on the use of one synthon as a glycosyl acceptor and another synthon as a glycosyl donor. The synthon used as glycosyl acceptor was phenyl 2,3,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside (7) and was synthesized from D-glucose with phenyl 2,3-di-O-acetyl-4,6-O-benzylidene-1-thio-beta-D-glucopyranoside and phenyl 2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-beta-D-glucopyranoside as key intermediates. The synthon used as glycosyl donor was O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O -benzyl - alpha-D-glucopyranosyl)-(1-->6)-O-[(2,3,4,6-tetra-O-benzyl-alpha-D- glucopyranosyl)-(1-->4)]-2,3-di-O-benzyl-alpha,beta-D-glucopyranosyl trichloroacetimidate (12) and was synthesized from phenyl O-2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O- benzyl- alpha-D-glucopyranosyl)-(1-->6)-O-[(2,3,4,6-tetra-O-acetyl-alpha-D- glucopyranosyl)-(1-->4)]-2,3-di-O-acetyl-1-thio-beta-D-glucopyranoside with O-(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)-O-(2,3,6-tri-O - benzyl-alpha-D-glucopyranosyl)-(1-->4)]-2,3-di-O-benzyl-D-glucopyranose as an intermediate. Condensation of compounds 7 and 12 followed by removal of the phenylthio group and debenzylation provided the branched pentasaccharide 15. Alternatively, the branched pentasaccharide was produced from amylopectin by consecutive alpha- and beta-amylase treatments and purified by chromatography. The identity of the products obtained by chemical synthesis and enzymatic hydrolysis is documented by 1H and 13C NMR spectra.

Chemical synthesis and NMR spectra of a protected branched-tetrasaccharide thioglycoside, a useful intermediate for the synthesis of branched oligosaccharides.

Acid-catalyzed thiophenolysis of per-O-acetylated 1,6-anhydromaltose (3) gave phenyl 2,3-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-alpha-D- glucopyranosyl)-1-thio-beta-D-glucopyranoside (4) in quantitative yield. Phenyl 4-O-alpha-D-glucopyranosyl-1-thio-beta-D-glucopyranoside (5) was obtained by acid-catalyzed thiophenolysis of maltose octaacetate (2), using trimethylsilyl triflate as catalyst, and subsequent deacetylation. Standard benzylation of 5 gave phenyl 2,3-di-O-benzyl-4-O- (2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-1-thio-beta-D-glucopy ran oside (6) which upon treatment with N-bromosuccinimide in aqueous acetone gave 2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-alpha-D- glucopyranosyl)-D-glucopyranose (8). Compound 8 was treated with trichloroacetonitrile in the presence of anhydrous potassium carbonate to give 2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl- alpha-D-glucopyranosyl) -alpha,beta-D-glucopyranosyl trichloroacetimidate (9), which was effectively used as the glycosyl donor in the condensation reaction with compound 4, using trimethylsilyl triflate as catalyst, to obtain the branched tetrasaccharides phenyl O-[2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)- (1-->4)]-O-(2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl)-(1-->6)-O-(2,3,4, 6- tetra-O-acetyl-alpha-D-glucopyranosyl)-(1-->4)-2,3-di-O-acetyl-1-thio-be ta-D- glucopyranoside (10) and phenyl O-[(2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranosyl)-(1-->4)]- O-(2,3,4-tri-O-benzyl-beta-D-glucopyranosyl)-(1-->6)-O-(2,3,4,6-tetra-O- acetyl- alpha-D-glucopyranosyl)-(1-->4)-2,3-di-O-acetyl-1-thio-beta-D-glucopy ran oside (11) in 67 and 21% yield, respectively. A complete NMR interpretation of 10 is presented. Alternative methodologies for the synthesis of the branched tetrasaccharides were investigated. Chemical synthesis of the phenyl thioglycoside 5 was achieved by deacetylation of 4. Reaction of 6 with diethylaminosulfur trifluoride in the presence of N-bromosuccinimide gave 2,3,6-tri-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-alpha-D- glucopyranosyl)-alpha,beta-D-glucopyranosyl fluoride (7) in 78% yield. Subsequent condensation of 7 and 4, using the combination silver perchlorate-stannous chloride as catalyst, gave the corresponding branched tetrasaccharides 10 and 11 in 55 and 10% yield, respectively.

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.) : I. Spatial distribution of endogenous cytokinins in relation to leaf growth.

Immunoaffinity purification of zeatin, dihydrozeatin and isopentenyl-type cytokinins from expanding leaves of sweet pepper was accomplished using a single immobilized monoclonal antibody. Isopentenyl adenosine, zeatin, zeatin riboside and the N9-glucosides of zeatin and isopentenyl adenine were found to be the dominating endogenous cytokinins while the dihydrozeatin cytokinins were either absent or constituted a very minor group of cytokinin metabolites in pepper. Leaves were selected for analysis at an age where a range of developmental stages exist within a single leaf. The spatial distribution of endogenous cytokinins in rapidly expanding leaves at this stage was markedly different from the almost uniform distribution in expanded leaves. The distribution of zeatin and zeatin riboside in rapidly expanding leaves was found to be correlated with the rate of leaf expansion which is high (∼40%/24 h) in the basal leaf tissue and low (∼10%/24 h) near the leaf tip. Applied growtn factors supported a rate of expansion of excised discs comparable to the growth rates observed in situ, but did not affect the ability of the tissue to retain assimilated amino acids. The results are discussed in relation to sink-strength stimultation as a potential mode of cytokinin action in leaf development.

Immunoaffinity purification using monoclonal antibodies for the isolation of indole auxins from elongation zones of epicotyls of red-light-grown Alaska peas.

The endogenous indole auxins of red-light grown pea (Pisum sativum L.) epicotyls were investigated. Immunoaffinity purification of indole-3-acetic acid (IAA) and its methylester was achieved using two monoclonal antibodies. Antibodies against free IAA were raised against IAA-C5-BSA, a hapten-carrier-conjugate giving rise to highly specific antibodies for indole auxins with a free acetic-acid group at position 3. Immunoaffinity adsorbents prepared with these antibodies were used for single-step purification of extracts of Alaska pea epicotylar tissue prior to quantification by high-performance liquid chromatography (HPLC) with on-line fluorescence detection. Monoclonal antibodies against a hapten-carrier-conjugate with IAA linked to bovine serum albumin through the carboxyl group (IAA-C1'-BSA) were used for the isolation of IAA esters. Indol-3-acetic acid was identified in the elongation zone of the third internode of red-light-grown Alaska pea. 4-Chloro-indole-3-acetic acid, a constituent of immature pea seeds which is considered to be a very active auxin, was absent from the elongation zone. Several compounds were retained by the column based on antibodies against IAA-C1'-BSA. Of these the methylester of IAA was identified by HPLC with on-line fluorescence detection, by co-migration in thin-layer chromatography and by gas chromatography-mass spectrometry. The methyl ester of IAA was very active in promoting elongation of pea third-internode segments. When fed to the epicotylar segments the IAA methylester was rapidly metabolized with IAA being the major metabolite. The methylester of IAA should therefore be classified as a labile auxin conjugate.

A nondestructive method for peptide bond conjugation of antigenic haptens to a diphtheria toxoid carrier, exemplified by two antisera specific to acetolactate synthase.

A method for the preparation of an activated protein carrier is described: Protein carboxyl groups are transformed into N-hydroxysulfosuccinimide esters, a structure that will react with primary amino groups under amide bond formation. Although the activated ester is unstable under aqueous conditions, a significant amount of hapten molecules can be bound covalently to the carrier under very mild conditions. Ligands can be peptides or other molecules possessing a primary amino group. The method avoids the risk of ligand polymerization and no derivatization of the ligand prior to conjugation is needed. Residual unreacted ligand molecules can therefore be recovered in their native form by size exclusion chromatography. The method was used to conjugate two synthetic sugar beet acetolactate synthetase (E.C. 4.1.3.18) peptides to diphtheria toxoid. Antibodies were raised against both of the conjugates. The specificity of these antibodies against sugar beet acetolactate synthetase was verified using immunoblotting, ELISA, and immunoprecipitation.

Monoclonal antibodies toward a stable cyclic AMP-C8 antigen.

A cyclic AMP antigen was prepared utilizing a stable thioether linkage from C8 of the purine moiety. Monoclonal antibodies were raised toward the cyclic AMP-C8-diphtheria toxoid antigen and found to be of high specificity, with only very low cross-reactivity against related compounds, less than 0.03% against cGMP being the most significant cross-reaction. The stability of the thioether linkage enabled the preparation of an enzyme tracer, which was used in an ELISA. The assay had an effective working range from 0.1 to 100 pmol cAMP. Due to the structure of the antigen, the need for sample derivatization was eliminated.

The potential of perturbed angular correlation of gamma rays as a tool for dynamic studies of peptides/proteins.

Four peptides and serum albumin have been derivatized with the bicyclic anydride of diethylenetriamine-pentaacetic acid. Procedures were developed to isolate the labelled species and determine the degree of derivatization. By using perturbed angular correlation of gamma-ray spectroscopy it is possible, through the determination of rotational correlation times, to decide whether labelled peptides interact with other molecules (receptors). In the case of the peptide cholecystokinin it is shown that the interaction between the peptide and its corresponding polyclonal antibody can be determined down to 1 pmol hormone. Experiments on 111In- and 111mCd-labelled Gly-Trp showed that, where the 111Cd PAC spectrum directly reflected the rotational motion of the molecule, the 111In PAC spectrum was affected by the nuclear transitions to 111mCd.

Preparation and Properties of Antibodies against Indoleacetic Acid (IAA)-C5-BSA, a Novel Ring-Coupled IAA Antigen, as Compared to Two Other Types of IAA-Specific Antibodies.

Two types of indoleacetic acid (IAA) antigens have been described in the literature which show marked differences with respect to antibody specificity. In this communication an alternative antigen design is described. In the so-called IAA-C5-BSA conjugate, both the acetic acid group and the pyrrole moiety are presented free, as the covalent linkage between IAA and the carrier molecule is introduced in the benzene moiety. Antibodies were elicited in rabbits against this novel antigen. Using cross-reactivity data and the strategy of successive approximation for the measurements of auxin levels in the internodes of light-grown broad bean (Vicia faba L. cv Superfine), the three types of antibodies are compared.