Cellulase production from Pseudoalteromonas sp. NO3 isolated from the sea squirt Halocynthia rorentzi.
ABSTRACT Pseudoalteromonas sp. NO3 was isolated from the hemolymph of diseased sea squirts (Halocynthia rorentzi) with symptoms of soft tunic syndrome. The strain was found to produce an extracellular cellulase (CelY) that consisted of a 1,476 bp open reading frame encoding 491 amino acid residues with an approximate molecular mass of 52 kDa. Homologies of the deduced amino acid sequence of celY with the products of the celA, celX, celG and cel5Z genes were 92.6, 93.3, 92.6, and 59.1%, respectively. Additionally, CelY had 50-80% remnant catalytic activity at temperatures of 10-20 degrees C. Highest carboxymethyl cellulose (CMC) hydrolysis was observed at pH 8.0 and 40 degrees C. CMC activity was determined by zymogram active staining and different degraded product profiles for CelY were obtained when cellotetraose, cellopentaose, and CMC were used as substrates. This study identified a transglycosylation activity in CelY that allows the enzyme to digest G4 to G2 and G3 without the production of G1.
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ABSTRACT: We previously characterized two endoglucanases, CelG and EGD, from the mesophilic ruminal anaerobe Fibrobacter succinogenes S85. Further comparative experiments have shown that CelG is a cold-active enzyme whose catalytic properties are superior to those of several other intensively studied cold-active enzymes. It has a lower temperature optimum, of 25 degrees C, and retains about 70% of its maximum activity at 0 degrees C, while EGD has a temperature optimum of 35 degrees C and retains only about 18% of its maximal activity at 0 degrees C. When assayed at 4 degrees C, CelG exhibits a 33-fold-higher kcat value and a 73-fold-higher physiological efficiency (kcat/Km) than EGD. CelG has a low thermal stability, as indicated by the effect of temperature on its activity and secondary structure. The presence of small amino acids around the putative catalytic residues may add to the flexibility of the enzyme, thereby increasing its activity at cold temperatures. Its activity is modulated by sodium chloride, with an increase of over 1.8-fold at an ionic strength of 0.03. Possible explanations for the presence of a cold-active enzyme in a mesophile are that cold-active enzymes are more broadly distributed than previously expected, that lateral transfer of the gene from a psychrophile occurred, or that F. succinogenes originated from the marine environment.Applied and Environmental Microbiology 04/1999; 65(3):995-8. · 3.68 Impact Factor
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ABSTRACT: Four strains of aerobic, Gram-negative rods, motile by means of a single polar flagellum, that produced phenolic anti-methicillin-resistant Staphylococcus aureus (MRSA) substances and brown-pigmented colonies, were isolated from sea water. The G + C content of the DNA ranged from 39.9 to 40.6 mol%. The isolates grew at 18-37 degrees C and pH 6.5-9.5 (optimal pH 7.5-9) and in medium containing 1-5% (w/v) NaCl (optimal NaCl concentration 2-3.5%). The isolates grew optimally in medium dissolved in 40-100% artificial sea water. Based on 16S rDNA similarities, the novel strains were closely related to Pseudoalteromonas luteoviolacea and Pseudoalteromonas piscicida, with 96.3 and 95.7% sequence similarity, respectively. However, the strains could be differentiated from P. lutioviolacea by seven traits and from P. piscicida by 10 traits. Analysis of DNA-DNA relatedness to these related species revealed low levels of DNA hybridization (19.6% to P. luteoviolacea and 22.4% to P. piscicida). However, the type strain, O-BC30T, and the other three bacterial isolates showed high DNA relatedness to each other, ranging from 84.8 to 93.7%. Based on the results of phenotypic characterization, phylogenetic analysis based on 16S rDNA sequences and DNA-DNA hybridization, it is concluded that these isolates represent a novel species in the genus Pseudoalteromonas. Because the type strain, O-BC30T (=IAM 14989T =KCTC 12086T), produces phenolic anti-MRSA substances, the name proposed for this novel species is Pseudoalteromonas phenolica sp. nov.International journal of systematic and evolutionary microbiology 04/2003; 53(Pt 2):583-8. · 2.11 Impact Factor
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ABSTRACT: Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.Biotechnology Advances 03/2006; 24(5):452-81. · 9.60 Impact Factor