Purification and Characterization of 50 kDa ExtracellularMetalloprotease from Serratia sp. ZF03

Salarizadeh, Navvabeh; Hasannia, Sadegh; Noghabi, Kambiz Akbari; Sajedi, Reza H.

doi:10.15171/ijb.1009

Cited by 12 publications

(10 citation statements)

References 36 publications

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“…MGS13. PMSF at 1, 5, and 10 mM had little effect on serratiopeptidase activity confirming that the enzyme is not a serine protease, because serine proteases are strongly inactivated by PMSF at a concentration ranging from 0.1 to 1 mM [29]. However, the activity of purified protease was impaired by EDTA indicating that metal ion plays an indispensable role in the catalytic action of an enzyme which is conclusively indicating that this enzyme comes under metalloproteases class.…”

Section: Discussionmentioning

confidence: 95%

Purification, characterization, and structural elucidation of serralysin-like alkaline metalloprotease from a novel source

Nageswara

Guntuku

Yakkali

2019

Journal of Genetic Engineering and Biotechnology

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BackgroundSerratiopeptidase is an alkaline metalloendopeptidase, which acquired wide significance because of its therapeutic applications. The present study was undertaken for purification, characterization, and structural elucidation of serratiopeptidase produced from Streptomyces hydrogenans var. MGS13.ResultThe crude enzyme was purified by precipitating with ammonium sulfate, dialysis, and Sephadex gel filtration, resulting in 34% recovery with a 12% purification fold. The purified enzyme S.AMP13 was spotted as a single clear hydrolytic band on casein zymogram and whose molecular weight was found to be 32 kDa by SDS-PAGE. The inhibitor and stability studies revealed that this enzyme is metalloprotease, thermostable, and alkaline in nature. The maximum serratiopeptidase activity was observed at 37 °C and pH 9.0. The partial amino acid sequence of the purified enzyme S.AMP13 by LC-MS/MS analysis shows the closest sequence similarities with previously reported alkaline metalloendopeptidases. The amino acid sequence alignment of S.AMP13 shared a conserved C-terminus region with peptidase-M10 serralysin superfamily at amino acid positions 128–147, i.e., ANLSTRATDTVYGFNSTAGR revealed that this enzyme is a serralysin-like protease. The kinetic studies of the purified enzyme revealed a Km of 1 mg/mL for its substrate casein and Vmax of 319 U/mL/min. The 3D structure of the purified enzyme was modeled by using SWISS-MODEL, and the quality of the structure was authenticated by assessing the Ramachandran plot using PROCHECK server, which suggested that the enzyme was stable with good quality.ConclusionInhibitor, stability, electrophoretic, and bioinformatic studies suggested that the purified enzyme obtained from S. hydrogenans var. MGS13 is a serralysin-like protease.Electronic supplementary materialThe online version of this article (10.1186/s43141-019-0002-7) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 95%

Purification, characterization, and structural elucidation of serralysin-like alkaline metalloprotease from a novel source

Nageswara

Guntuku

Yakkali

2019

Journal of Genetic Engineering and Biotechnology

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“…The resultant pellet was dissolved in 5 ml of distilled water. The procedure was repeated for the left supernatant again with ammonium sulfate to achieve 40, 60, and 70% (w/v) saturation 21 . Both enzyme activity and protein content were determined for the precipitated fraction.…”

Section: Serrapeptase Purification Ammonium Sulfate Fractionationmentioning

confidence: 99%

Enhancement of Serrapeptase Hyper Producing Mutant by Combined Chemical and UV Mutagenesis and its Potential for Fibrinolytic Activity

Gopinath¹,

Venkataprasad²,

Rajnish³

et al. 2020

J Pure Appl Microbiol

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Serrapeptase is a proteolytic enzyme with many favourable biological properties like anti-inflammatory, analgesic, anti-bacterial, fibrinolytic properties and hence, is widely used in clinical practice for the treatment of many diseases. The activity of microbial enzymes is usually low and hence enhancing the enzyme activity is an integral and crucial area of research. In this study, a mutant with higher serrapeptase activity was developed by multiple exposures of Serratia marcescens to Ultra Violet radiation (UV) and Ethyl methyl sulfonate (EMS) individually and also a combination of both the methods was used. The mutant exposed only to UV radiation for 40 seconds indicated an increase in enzyme activity of 3234.9 EU/mL in comparison to wild-type strain (2770 EU/mL). The mutant exposed to only EMS indicated a very small increase in enzyme activity of 2797 EU/mL in comparison to wildtype strain (2770 EU/mL) but a lower enzyme activity than the UV mutant. Combinational exposure of wild-type to UV and EMS gave a better mutant with an enzyme activity of 3437.6 EU/mL which was higher than all the above methods. Optimization of temperature, incubation period and pH was studied in the wild type and mutant strains and found slight increase in activity in the mutant strain than the wild strain. The mutant serrapeptase has a molecular weight of approximately 50kDa and also exhibited fibrinolysis activity with a maximum blood clot lysis of 35% with 100 U/mL of Serrapeptase.

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“…Therefore, the protease production was inhibited when S. marcescens IBBPo15 cells were exposed to aromatics at 37°C. Characterizations of extracellular proteases from various strains of S. marcescens indicate that most strains produce a very similar metalloprotease (N. SALARIZADEH & al [17]).…”

Section: Cell Growthmentioning

confidence: 99%

“…Modifications in the extracellular proteins profile (including 50, 40, 20, 18 and 16 kDa protein) were observed in S. marcescens IBBPo15 cells grown at 37°C and exposed to 5% organic solvents, as compared with proteins profile of control cells grown at 37°C and 30°C. The S. marcescens metalloprotease (50 kDa protein) is a member of the serralysin family of proteolytic enzymes which is important for different biomedical applications (N. SALARIZADEH & al [17]).…”

Section: Extracellular Proteinsmentioning

confidence: 99%

Effect of high growth temperature on Serratia marcescens

Stancu¹

2020

Rom Biotechnol Lett.

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The purpose of this study was to investigate some parameters of S. marcescens IBBPo15 cells adaptation to toxic organic solvents. S. marcescens IBBPo15 cells incubated at 37°C exhibited a slower growth when they were exposed to 5% organic solvents (alkanes, aromatics), as compared with the control cells. S. marcescens IBBPo15 cells grown at 37°C produced protease and the enzyme production decreased in cells exposed to aromatic compounds, as compared with the control cells. Surfactant serrawettin and red pigment prodigiosin were produced by S. marcescens IBBPo15 cells grown at 37°C. Modifications in the pigment and extracellular proteins profile were observed in S. marcescens IBBPo15 cells grown at 37°C during exposure to organic solvents (alkanes, aromatics). Alkane hydroxylase (i.e., alkB1) and toluene dioxygenase (i.e., todM) catabolic genes, 4'-phosphopantetheinyl transferase (i.e., pswP) and serralysin metalloproteinase (i.e., ser) genes were detected by polymerase chain reaction in DNA extracted from S. marcescens IBBPo15 control cells grown at 37°C, as well as in cells exposed to organic solvents (excepting todM in n-decane exposed cells).

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Purification and Characterization of 50 kDa ExtracellularMetalloprotease from Serratia sp. ZF03

Cited by 12 publications

References 36 publications

Purification, characterization, and structural elucidation of serralysin-like alkaline metalloprotease from a novel source

Purification, characterization, and structural elucidation of serralysin-like alkaline metalloprotease from a novel source

Enhancement of Serrapeptase Hyper Producing Mutant by Combined Chemical and UV Mutagenesis and its Potential for Fibrinolytic Activity

Effect of high growth temperature on Serratia marcescens

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