Enzymatic generation of hydrogen peroxide shows promising antifouling effect.

The antifouling (AF) potential of hydrogen peroxide (H(2)O(2)) produced enzymatically in a coating containing starch, glucoamylase, and hexose oxidase was evaluated in a series of laboratory tests and in-sea field trials. Dissolved H(2)O(2) inhibited bacterial biofilm formation by eight of nine marine Proteobacteria, tested in microtiter plates. However, enzymatically produced H(2)O(2) released from a coating did not impede biofilm formation by bacteria in natural seawater tested in a biofilm reactor. A field trial revealed a noticeable effect of the enzyme system: after immersion in the North Sea for 97 days, the reference coating without enzymes had 35-40 barnacles, 10% area coverage by diatoms and 15% area coverage by tunicates. The enzyme containing coating had only 6-12 barnacles, 10% area coverage by diatoms and no tunicates. The enzyme system had a performance similar to a copper-based commercial coating and thus appears to have potential as a non-persistent AF agent.

Purification and characterisation of a malto-oligosaccharide-forming amylase active at high pH from Bacillus clausii BT-21.

Bacillus clausii BT-21 produced an extracellular malto-oligosaccharide-forming amylase active at high pH when grown on starch substrates. The enzyme was purified to homogeneity by affinity and anion-exchange chromatography. The molecular weight of the enzyme estimated by sodium dodecyl sulfate polyacrylamide electrophoresis was 101 kDa. The enzyme showed an optimum of activity at pH 9.5 and 55 degrees C. Maltohexaose was detected as the main initially formed starch hydrolysis product. Maltotetraose and maltose were the main products obtained after hydrolysis of starch by the enzyme for an extended period of time and were not further degraded. The enzyme readily hydrolysed soluble starch, amylopectin and amylose, while cyclodextrins, pullulan or dextran were not degraded. The mode of action during hydrolysis of starch indicated an exo-acting type of amylolytic enzyme mainly producing maltohexaose and maltotetraose. Amino acid sequencing of the enzyme revealed high homology with the maltohexaose-forming amylase from Bacillus sp. H-167.

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.

Characterization of chitinases able to rescue somatic embryos of the temperature-sensitive carrot variant ts 11.

To characterize the acidic endochitinase EP3, able to rescue somatic embryos of the carrot cell line ts11, the enzyme was purified from the medium of wild-type suspension cultures. Peptide sequences, deduced amino acid sequences of corresponding PCR-generated cDNA clones, serological relation and biochemical properties showed that there were at least five closely related chitinases, four of which could be identified as class IV EP3 chitinases with an apparent size of 30 kDa. Two other proteins were identified as a serologically related class I acidic chitinase (DcChitI) of 34 kDa, and a serologically unrelated 29 kDa class II acidic chitinase (DcChitII), respectively. Additional cDNA sequences, Western and Southern analysis showed the presence of a least two, but possibly more, highly homologous class IV EP3 genes in the carrot genome. Two class IV EP3 chitinases were tested and found to be able to increase the number of ts11 globular embryos formed under non-permissive conditions. One of the class IV EP3 chitinases as well as the class I chitinase DcChitI promoted the transition from globular to heart-stage ts11 embryos. The class II endochitinase and a heterologous class IV chitinase from sugar-beet were not active on ts11. This suggests that there are differences in the specificity of chitinases in terms of their effect on plant somatic embryos.

Characterization and localization of new antifungal cysteine-rich proteins from Beta vulgaris.

Two novel antifungal proteins, AX1 and AX2, were isolated from leaves of sugar beet infected with Cercospora beticola. AX1 (MW = 5078 +/- 3D) and AX2 (MW = 5193 +/- 3D) were N-terminally sequenced and identified as monomeric, basic proteins consisting of 46 amino acid residues, of which eight are cysteines. Both AX proteins strongly inhibit growth of C. beticola and other filamentous fungi, but have little or no effect against bacteria. Based on primary sequence homology (24 to 46% identity), they are related to the superfamily of gamma-thionins, which have been isolated recently from seeds of monocotyledons and Brassicaceae. Specific antibodies were raised against the AX proteins after conjugation to diphtheria toxoid. Using immunoblotting and immunohistology, we detected high concentrations of AX proteins extracellularly in cell walls and in globular bodies around necrotic lesions in sugar beet leaves infected with C. beticola, suggesting that AX proteins are involved in antifungal defense. Furthermore, AX proteins or serologically related proteins were detected in xylem, stomata, and stomatal cells as well as in sugar beet styles.

An acidic class III chitinase in sugar beet: induction by Cercospora beticola, characterization, and expression in transgenic tobacco plants.

An acidic chitinase (SE) was found to accumulate in leaves of sugar beet (Beta vulgaris) during infection with Cercospora beticola. Two isoforms, SE1 and SE2, with MW of 29 kDa and pI of approximately 3.0 were purified to homogeneity. SE2 is an endochitinase that also exhibits exochitinase activity, i.e., it is capable of hydrolyzing chito-oligosaccharides, including chitobiose, into N-acetyl-glucosamine. Partial amino acid sequence data for SE2 were used to obtain a cDNA clone by polymerase chain reaction. The clone was used to isolate a cDNA clone encoding SE2. The deduced amino acid sequence for SE2 is 58-67% identical to the class III chitinases from cucumber, Arabidopsis, and tobacco. A transient induction of SE2 mRNA during the early stages of infection with C. beticola is much stronger in tolerant plants than in susceptible plants. Transgenic tobacco (Nicotiana benthamiana) plants constitutively accumulate SE2 protein in the intercellular space of their leaves. In a preliminary infection experiment, the transgenic plants did not show increase in resistance against C. nicotianae.

Antifungal activity of chitin-binding PR-4 type proteins from barley grain and stressed leaf.

Antifungal activity in vitro has been associated with barley leaf and grain proteins which are homologous with pathogenesis related proteins of type 4 (PR-4) from tobacco and tomato and with C terminal domains of potato win and Hevea hevein precursor proteins. One protein (pI approximately 9.3, M(r) approximately 13.7 kDa) from barley grain and two very similar proteins from leaves infected with Erysiphe graminis were isolated by chitin affinity chromatography, but none of the proteins showed chitinase activity in vitro. The leaf proteins were increased several fold in response to either Erysiphe infection or NiCl2 infiltration and accumulated extracellularly. The three barley proteins were found to inhibit growth of Trichoderma harzianum in microtiter plate assays using approximately 10 micrograms/ml concentrations and in lower concentrations in a synergistic way when mixed either with barley chitinase C (a PR-3 type protein) or with barley protein R (a PR-5 type protein). Structurally similar proteins were detected in wheat, rye and oats grain extracts.

Silencer region of a chalcone synthase promoter contains multiple binding sites for a factor, SBF-1, closely related to GT-1.

Bean nuclear extracts were used in gel retardation assays and DNase I footprinting experiments to identify a protein factor, designated SBF-1, that specifically interacts with regulatory sequences in the promoter of the bean defense gene CHS15, which encodes the flavonoid biosynthetic enzyme chalcone synthase. SBF-1 binds to three short sequences designated boxes 1, 2 and 3 in the region -326 to - 173. This cis-element, which is involved in organ-specific expression in plant development, functions as a transcriptional silencer in electroporated protoplasts derived from undifferentiated suspension-cultured soybean cells. The silencer element activates in trans a co-electroporated CHS15-chloramphenicol acetyl-transferase gene fusion, indicating that the factor acts as a repressor in these cells. SBF-1 binding in vitro is rapid, reversible and sensitive to prior heat or protease treatment. Competitive binding assays show that boxes 1, 2 and 3 interact cooperatively, but that each box can bind the factor independently, with box 3 showing the strongest binding and box 2 the weakest binding. GGTTAA(A/T)(A/T)(A/T), which forms a consensus sequence common to all three boxes, resembles the binding site for the GT-1 factor in light-responsive elements of the pea rbcS-3A gene, which encodes the small subunit of ribulose bisphosphate carboxylase. Binding to the CHS15 -326 to -173 element, and to boxes 1, 2 or 3 individually, is competed by the GT-1 binding sequence of rbcS-3A, but not by a functionally inactive form, and likewise the CHS sequences can compete with authentic GT-1 sites from the rbcS-3A promoter for binding.(ABSTRACT TRUNCATED AT 250 WORDS)