Proteases of psychrotrophic bacteria isolated from glaciers

Margesin, Rosa; Palma, Norbert; Knauseder, Franz; Schinner, Franz

doi:10.1002/jobm.3620310515

Cited by 16 publications

(7 citation statements)

References 24 publications

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“…The SDS-PAGE profile showed predominant crystal fractions with ∼45 and ∼35 kDa, which are close to the findings of Wei et al [34] and Hasan [35], The larger Cry proteins would generally form crystalline inclusions in the mother cell compartment by their self-assembly into the characteristic crystal due to the cysteine-rich C-terminal half of the Cry protoxins contributes to crystal structure through the formation of disulfide bonds [36]. However, smaller proteins need the help of accessory proteins to form crystals [37]. The crystal toxin of Btk is composed primarily of the glycoprotein protoxin [38], and when whole crystals are solubilized by either reducing or denaturing agents or by mild alkali, several smaller proteins would be formed.…”

Section: Discussionsupporting

confidence: 83%

Biphasic Fermentation Is an Efficient Strategy for the Overproduction of δ-Endotoxin from Bacillus thuringiensis

Jisha

Smitha

Priji

et al. 2014

Appl Biochem Biotechnol

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This study illustrates a biphasic solid-state fermentation (SSF) strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk) and also purification of δ-endotoxin from the solid-fermented medium. The fermentation strategy had two phases (biphasic); i.e., the first short phase was semisolid state (12 h), and the remaining long phase was strict SSF. To achieve the biphasic SSF, after 12 h (150 rpm, 37 °C) fermentation of the medium [Luria-Bertani (LB) supplemented with 30 % (w/v) raw soybean flour (phase I)], the supernatant in it was completely centrifuged out (1,000 × g, 10 min) aseptically for harvesting the extracellular enzymes as by-product. The resultant wet solid matter without free-flowing liquid but with embedded Btk was incubated 60 h more (phase II) for enhancing δ-endotoxin production at static condition (37 °C). Coupled with this, δ-endotoxin was purified by the modified phase separation method, and its purity was physically confirmed by both staining and microscopic techniques. The maximum δ-endotoxin yield from solid medium (48 h) was 15.8 mg/mL (recovery was 55-59 %) LB-equivalent, while that of LB control (recovery was 95 %) was only 0.43 mg/mL (72 h), i.e., thus, in comparison, 36.74-fold more yield in solid medium obtained by 24 h less gestation period. The purified crystal proteins showed apparent molecular weights (MWs) of 45, 35, and 6 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, this unique study physically demonstrates how Btk δ-endotoxin is purified (95-99 % purity) from solid-fermented matter for the first time, coupled with its overproduction at the expense of only 21.5 % higher production cost.

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Section: Discussionsupporting

confidence: 83%

Biphasic Fermentation Is an Efficient Strategy for the Overproduction of δ-Endotoxin from Bacillus thuringiensis

Jisha

Smitha

Priji

et al. 2014

Appl Biochem Biotechnol

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“…3), for cell‐free proteases derived from either psychrophilic or psychrotolerant bacteria. The apparent activation energy for the enzyme preparation (50.5 kJ mol −1 , calculated from the linear portion, 5–15°C, of an Arhennius plot of the data) falls within the range of activation energies reported for other cold‐active cell‐free proteolytic extracts ( Margesin et al ., 1991 ).…”

Section: Resultsmentioning

confidence: 99%

Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice

Huston

Krieger-Brockett

Deming

2000

Environmental Microbiology

162

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Extracellular degradative enzymes released by psychrophilic marine bacteria (growing optimally at or below 15 degrees C and maximally at 20 degrees C) typically express activity optima at temperatures well above the upper growth limit of the producing strain. In the present study, we investigated whether or not near-zero Arctic environments contain extracellular enzymes with activity optimized to temperatures lower than previously reported. By applying fluorescently tagged substrate analogues to measure leucine-aminopeptidase and chitobiase activity, the occurrence of extracellular enzymatic activity (EEA) with remarkably low temperature optima (15 degrees C) was documented in sea-ice samples. An extremely psychrophilic bacterial isolate, strain 34H, yielded an extract of cell-free protease with activity optimized at 20 degrees C, the lowest optimum yet reported for cell-free EEA from a pure culture. The use of zymogram gels revealed the presence of three proteolytic bands (between 37 and 45 kDa) in the extract and the release of the greatest quantities of the proteases when the strain was grown at -1 degrees C, suggesting a bacterial strategy for counteracting the effects of very cold temperatures on the catalytic efficiency of released enzymes. The detection of unusually cold-adapted EEA in environmental samples has ramifications not only to polar ecosystems and carbon cycling but also to protein evolution, biotechnology and bioremediation.

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“…Bacteria capable of growing at low temperature were isolated first by Forster in 1887 from preserved fish (4). Many kinds of cold-active enzymes from cold-adapted microorganisms have been reported (5)(6)(7)(8). We have also reported cold-active protease, lipase, and amylase from cold environments (9).…”

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confidence: 86%

Purification and characterization of a cold‐active protease from psychrotrophic Serratia marcescens AP3801

Morita

Kondoh

Hasan³

et al. 1997

J Americ Oil Chem Soc

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Protease activity was detected in the culture medium of Serratia marcescens AP3801 grown at 10˚C, which was isolated from soil collected from the top of a mountain. The enzyme, designated as CP-58 protease, was purified to homogeneity from the culture broth by ion exchange and gel filtration chromatographies. The molecular mass of the protease was 58 kDa, and its isoelectric point was close to 6.0. Maximal activity toward azocasein was observed at 40˚C and from pH 6.5 to 8.0. The activity was strongly inhibited by 1,10-phenanthroline, suggesting that the enzyme is a metalloprotease. The Nterminal amino acid sequence was Ser-Leu-Asn-Gly-Lys-ThrAsn-Gly-Trp-Asp-Ser-Val-Asn-Asp-Leu-Leu-Asn-Tyr-His-Asn-ArgGly-Asn (or Asp)-Gly-Thr-Ile-Asn-Asn-Lys-Pro-Ser-Phe-AspIle-Ala. A search through databases for sequence homology aligned CP-58 protease with metalloprotease. The result of the cleavage pattern of oxidized insulin B-chain suggests that CP-58 protease has a broader specificity than other proteases against the peptide substrate.

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Proteases of psychrotrophic bacteria isolated from glaciers

Cited by 16 publications

References 24 publications

Biphasic Fermentation Is an Efficient Strategy for the Overproduction of δ-Endotoxin from Bacillus thuringiensis

Biphasic Fermentation Is an Efficient Strategy for the Overproduction of δ-Endotoxin from Bacillus thuringiensis

Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice

Purification and characterization of a cold‐active protease from psychrotrophic Serratia marcescens AP3801

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