Plate Assay for Simultaneous Detection of Alginate Lyases and Determination of Substrate Specificity

Gacesa, Peter; Wusteman, Frederick S.

doi:10.1128/aem.56.7.2265-2267.1990

Cited by 68 publications

(15 citation statements)

References 20 publications

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“…Alginate lyase plate assays were performed as described by Gacesa and Wusteman (31) and Schiller et al (53) with slight modifications. Seaweed alginate and uronic acid polymers isolated from selected bacterial strains were added to LB medium containing ampicillin to a final concentration of 0.3% (wt/vol).…”

Section: Methodsmentioning

confidence: 99%

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae

Kidambi

Sundin

Palmer

et al. 1995

Appl Environ Microbiol

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Plant-associated pseudomonads are commonly exposed to copper bactericides, which are applied to reduce the disease incidence caused by these bacteria. Consequently, many of these bacteria have acquired resistance or tolerance to copper salts. We recently conducted a survey of 37 copper-resistant (Cu r) Pseudomonas spp., including P. cepacia, P. fluorescens, P. syringae, and P. viridiflava, and found that a subset of the P. syringae strains showed a dramatic increase in exopolysaccharide (EPS) production on mannitol-glutamate medium containing CuSO 4 at 250 g/ml. A modified carbazole assay indicated that the EPS produced on copperamended media contained high levels of uronic acids, suggesting that the EPS was primarily alginic acid. Uronic acids extracted from selected strains were further confirmed to be alginate by demonstrating their sensitivity to alginate lyase and by descending paper chromatography following acid hydrolysis. Subinhibitory levels of arsenate, cobalt, lithium, rubidium, molybdenum, and mercury did not induce EPS production, indicating that alginate biosynthesis is not induced in P. syringae cells exposed to these heavy metals. A 200-kb plasmid designated pPSR12 conferred a stably mucoid phenotype to several P. syringae recipients and also increased their resistance to cobalt and arsenate. A cosmid clone constructed from pPSR12 which conferred a stably mucoid phenotype to several P. syringae strains but not to Pseudomonas aeruginosa was obtained. Results obtained in this study indicate that some of the signals and regulatory genes for alginate production in P. syringae differ from those described for alginate production in P. aeruginosa.

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Section: Methodsmentioning

confidence: 99%

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae

Kidambi

Sundin

Palmer

et al. 1995

Appl Environ Microbiol

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“…Among them, only 62 isolates could utilize alginate as the sole carbon source in the liquid alginate medium. All isolates grew as the alginate liquid medium turned cloudy and presented an alginate lyase activity as clear zones were observed when all isolates were infused with a solution of 10% w/v cetylpyridinium chloride liquid (Sigma‐Aldrich) on the plates [16].…”

Section: Resultsmentioning

confidence: 99%

Maximizing the utilization of Laminaria japonica as biomass via improvement of alginate lyase activity in a two‐phase fermentation system

Kim

et al. 2015

Biotechnology Journal

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Brown seaweed contains up to 67% of carbohydrates by dry weight and presents high potential as a polysaccharide feedstock for biofuel production. To effectively use brown seaweed as a biomass, degradation of alginate is the major challenge due to its complicated structure and low solubility in water. This study focuses on the isolation of alginate degrading bacteria, determining of the optimum fermentation conditions, as well as comparing the conventional single fermentation system with the two-phase fermentation system which is separately using alginate and mannitol extracted from Laminaria japonica. Maximum yield of organic acids production and volatile solids reduction obtained were 0.516 g/g and 79.7%, respectively, using the two-phase fermentation system in which alginate fermentation was carried out at pH 7 and mannitol fermentation at pH 8. The two-phase fermentation system increased the yield of organic acids production by 1.14 times and led to a 1.45-times reduction of VS when compared to the conventional single fermentation system at pH 8. The results show that the two-phase fermentation system improved the utilization of alginate by separating alginate from mannitol leading to enhanced alginate lyase activity.

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“…Sodium alginate (Macrocystis pyrifera origin) was from Sigma-Aldrich (St. Louis, MO, USA). Poly(M)-rich, poly(G)-rich, and random(MG) substrates were prepared from the alginate by the method of Gacesa and Wusteman [39].…”

Section: Methodsmentioning

confidence: 99%

cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme

et al. 2011

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Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5'- and 3'-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1.

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Plate Assay for Simultaneous Detection of Alginate Lyases and Determination of Substrate Specificity

Cited by 68 publications

References 20 publications

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae

Copper as a signal for alginate synthesis in Pseudomonas syringae pv. syringae

Maximizing the utilization of Laminaria japonica as biomass via improvement of alginate lyase activity in a two‐phase fermentation system

cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme

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