Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing

Souzu, Hiroshi

doi:10.1016/0005-2736(80)90387-9

Cited by 39 publications

(15 citation statements)

References 36 publications

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“…Three successive cycles of freezing and thawing were conducted for the specific extraction of periplasmatic proteins [39]. After precipitation of the remaining cell content, the cytoplasm was extracted in the same buffer by FastPrep (Bio101) treatment including a 1:1 volume ratio of 102 nm glass beads (Sigma) at speed 5.5 for three times 20 sec.…”

Section: Methodsmentioning

confidence: 99%

Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239

2010

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BackgroundChitosanases (EC 3.2.1.132) hydrolyze the polysaccharide chitosan, which is composed of partially acetylated β-(1,4)-linked glucosamine residues. In nature, chitosanases are produced by a number of Gram-positive and Gram-negative bacteria, as well as by fungi, probably with the primary role of degrading chitosan from fungal and yeast cell walls for carbon metabolism. Chitosanases may also be utilized in eukaryotic cell manipulation for intracellular delivery of molecules formulated with chitosan as well as for transformation of filamentous fungi by temporal modification of the cell wall structures.However, the chitosanases used so far in transformation and transfection experiments show optimal activity at high temperature, which is incompatible with most transfection and transformation protocols. Thus, there is a need for chitosanases, which display activity at lower temperatures.ResultsThis paper describes the isolation of a chitosanase-producing, cold-active bacterium affiliated to the genus Janthinobacterium. The 876 bp chitosanase gene from the Janthinobacterium strain was isolated and characterized. The chitosanase was related to the Glycosyl Hydrolase family 46 chitosanases with Streptomyces chitosanase as the closest related (64% amino acid sequence identity). The chitosanase was expressed recombinantly as a periplasmic enzyme in Escherichia coli in amounts about 500 fold greater than in the native Janthinobacterium strain. Determination of temperature and pH optimum showed that the native and the recombinant chitosanase have maximal activity at pH 5-7 and at 45°C, but with 30-70% of the maximum activity at 10°C and 30°C, respectively.ConclusionsA novel chitosanase enzyme and its corresponding gene was isolated from Janthinobacterium and produced recombinantly in E. coli as a periplasmic enzyme. The Janthinobacterium chitosanase displayed reasonable activity at 10°C to 30°C, temperatures that are preferred in transfection and transformation experiments.

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Section: Methodsmentioning

confidence: 99%

Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239

2010

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“…Approximately 70% of the product was released during the freeze thaw process; the rest was released upon homogenisation (see Table 1 and 2). This is an interesting observation as it shows extensive cell damage during the freeze thaw cycle which may have been made worse due to a slow freeze thaw cycle caused by the large mass of cells being freezethawed [24]. The question of the contaminant profile created by homogenisation is related to the size of debris created and the formation or release of fouling materials such as lipids as this will have a significant impact on subsequent stages such as centrifugation (smaller debris will require more effort to gain the same level of clarification), filtration (potential for foulants to blind filters and/or increase the filter area requirement) and chromatography (foulants generated at homogenisation could bind and reduce column capacity and/or physically foul the column causing an increase in pressure drop across the column).…”

Section: Resuspension and Homogenisationmentioning

confidence: 97%

Addressing a whole bioprocess in real-time using an optical biosensor-formation, recovery and purification of antibody fragments from a recombinant E. coli host

Bracewell

Brown

Hoare

2004

Bioprocess Biosyst Eng

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The use of biosensor technology is described to address in real-time the production and subsequent purification of a bioactive recombinant protein product. The product, D1.3 Fv antibody fragment, was expressed in Escherichia coli and purified via two process routes, one for extracellular and one for intracellular product material. The cells were harvested by centrifugation in a solid bowl CARR Powerfuge and stored at -70 degrees C. Clarification of the supernatant was performed by depth filtration, followed by affinity chromatography for final purification of the extracellular product. To purify the intracellular product the harvested cells were resuspended and homogenised. Removal of debris in the CARR Powerfuge was followed by depth filtration and affinity chromatography. In this work we have shown the rapid determination of bioactive product levels, and the impact this has on improved accountability and confidence is demonstrated in process mass balances on the product using the data acquired during process operation.

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“…Therefore, storage at 8oC reduces S. aureus viability either using a random suspension (data not shown) or 4 th degree of McFarland's scale suspension, indicating that the CFU counting method with 24 h storage will result in reduced S. aureus load. Indeed, temperature and time of storage also affects other bacteria such as Vibrio spp (SOUZU, 1980) and Clostridium sporogenes (MAH, KANG, TANG, 2009). This fact can be attributed to damage of the cytoplasmic membrane as well as to outer membrane of bacteria (MATCHES, LISTON, DANEAULT, 1971).…”

Section: Discussionmentioning

confidence: 99%

Influência do armazenamento de suspensão bacteriana na resposta inflamatória em camundongos

Staurengo‐Ferrari

Saito

Pelayo

et al. 2013

Semin. Cienc. Biol. Saude

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The preparation of bacterial suspension is an important procedure used in laboratories for inflammatory evaluation protocols and can be obtained by different methods that need or not need step storage. The aim of this work was investigate the influence of storage of Staphylococcus aureus suspension in bacterial viability and its influence in bacteria-induced inflammation in vivo. The bacterial suspension of S. aureus ATCC 6538 was prepared accordingly to 4 th degree of McFarland's Scale by visual comparison. This suspension was used to determine by CFU counting the bacterial viability and for administration to the animals to induce septic arthritis and peritonitis. Twenty four hours of storage reduced the S. aureus CFU. As a consequence of reduced bacterial viability, was detected reduced mechanical hyperalgesia, edema and leukocyte recruitment in septic arthritis and leukocyte recruitment and cytokine production bacterial peritonitis. These results demonstrate that storage of bacterial suspension affected bacterial viability, which resulted in diminished inflammatory response in vivo, raising the importance of standard procedures for bacterial suspension preparation. A conceivable approach would be to determine the number of CFU at a specific McFarland's scale degree, which will allow the preparation and use a bacterial suspension in the same day for in vivo testing. Keywords

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Studies on the damage to Escherichia coli cell membrane caused by different rates of freeze-thawing

Cited by 39 publications

References 36 publications

Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239

Isolation, characterization and heterologous expression of a novel chitosanase from Janthinobacterium sp. strain 4239

Addressing a whole bioprocess in real-time using an optical biosensor-formation, recovery and purification of antibody fragments from a recombinant E. coli host

Influência do armazenamento de suspensão bacteriana na resposta inflamatória em camundongos

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