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Purification and characterization of thermoactive serratiopeptidase from Serratia marcescens AD-W2.

ABSTRACT: Serratiopeptidase is a proteolytic enzyme extensively used as an anti-inflammatory and analgesic drug. Present work reports a thermoactive serratiopeptidase from Serratia marcescens AD-W2, a soil isolate from the North-Western Himalayan region of India. The extracellular metalloprotease has been purified by a simple two-step procedure resulting in a specific activity of 20,492 Units/mg protein with 5.28-fold purification. The molecular mass of the metalloprotease, as determined by SDS-PAGE was ~ 51 kDa. The purified serratiopeptidase presented optimum activity at pH 9.0, temperature 50 °C and stability in wide pH and temperature range. Critical temperature of 50 °C confirmed the thermoactivity of the purified serratiopeptidase. The kinetic studies of the purified serratiopeptidase revealed V_max and K_m of 57,256 Units/mL and 1.57 mg/mL, respectively, for casein. The purified serratiopeptidase from S. marcescens AD-W2 was found to be 100% identical to serralysin from Serratia marcescens ATCC 21074/E-15. The catalytic domain comprising of Zn coordinated with three histidine residues (His192, His196, His202), along with glutamate (Glu193) and tyrosine (Tyr232) residues, further confirmed that the purified protein is identical to serralysin.

SUBMITTER: Chander D

PROVIDER: S-EPMC8035408 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Chitoporin from Serratia marcescens: recombinant expression, purification and crystallization.

Project description:Serratia marcescens is an opportunistic pathogen that commonly causes hospital-acquired infections and can utilize chitin-enriched nutrients as an alternative energy source. This study reports the identification of a chitoporin (ChiP), termed SmChiP, from the outer membrane of S. marcescens. Sequence alignment with genetically characterized ChiPs suggests that SmChiP is more closely related to the monomeric EcChiP from Escherichia coli than to the trimeric VhChiP from Vibrio campbellii. A single crystal of SmChiP grown under the condition 22%(w/v) PEG 8000, 0.1 M calcium acetate, 0.1 M MES pH 6.0 diffracted X-ray synchrotron radiation to 1.85 Å resolution. SmChiP co-crystallized with chitohexaose under the condition 19%(w/v) PEG 1500, 2 M ammonium phosphate monobasic, 0.1 M HEPES pH 7.0 diffracted X-rays to 2.70 Å resolution. Preliminary crystallographic analysis shows that both SmChiP crystal forms contain one molecule per asymmetric unit and that they belong to the tetragonal space groups P42212 and P41212, respectively. The SmChiP crystal has unit-cell parameters a = 82.97, b = 82.97, c = 189.53 Å, α = β = γ = 90°, while the crystal of SmChiP in complex with chitohexaose has unit-cell parameters a = 73.24, b = 73.24, c = 213.46 Å, α = β = γ = 90°. Initial assessment of the complex structure clearly revealed electron density for the sugar ligand. Structure determination of SmChiP in the absence and presence of chitohexaose should reveal the molecular basis of chitin utilization by S. marcescens.

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Serratia marcescens BJL200 secretome

Project description:Study the secretome of Serratia marcescens BJL200 upon growth on chitin.

2018-05-30 | PXD005225 | Pride

Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens.

Project description:As suggested by Y. Suh and M.J. Benedik (J. Bacteriol. 174: 2361-2366, 1992), Serratia marcescens ATCC 27117 produced very small amounts (0.8 U ml-1) of an inhibitor protein (SmaPI) that shows an inhibitory activity against extracellular 50-kDa metalloprotease (SMP) of S. marcescens and that is localized in the periplasm of cells at the optimal growth temperature of 25 degrees C. A recombinant S. marcescens harboring plasmid pSP2 encoding SMP and SmaPI genes produced 20 U of SmaPI ml-1 that is also localized in the periplasm of cells at 25 degrees C. However, a large amount of SmaPI (86 Uml-1) was extracellularly produced at the supraoptimal growth temperature 37 degrees C from the recombinant S. marcescens (pSP2). We purified SmaPI from the culture supernatant of S. marcescens (pSP2) grown at 37 degrees C, and some biochemical properties were characterized. SmaPI had a pI value of about 10.0 and was a monomeric protein with a molecular mass of 10,000. SmaPI was produced from a precursor SmaPI by cleavage of a signal peptide (26 amino acid residues). The inhibitor was stable in boiling water for up to 30 min. The thermostability of SmaPI can be attributed to its reversible denaturation. SmaPI inhibited SMP by formation of a noncovalent complex with a molar ratio of 1:1 and showed a high protease specificity, which inhibited only SMP among the various proteases we examined.

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Partial purification and characterization of serine protease produced through fermentation of organic municipal solid wastes by Serratia marcescens A3 and Pseudomonas putida A2.

Project description:Proteolytic bacteria isolated from municipal solid wastes (MSW) were identified as Serratia marcescens A3 and Pseudomonas putida A2 based on 16S rDNA sequencing. Protease produced through fermentation of organic MSW by these bacteria under some optimized physicochemical parameters was partially purified and characterized. The estimated molecular mass of the partially purified protease from S. marcescens and P. putida was approximately 25 and 38 kDa, respectively. Protease from both sources showed low Km 0.3 and 0.5 mg ml-1 and high Vmax 333 and 500 µmole min-1 at 40?°C, and thermodynamics analysis suggested formation of ordered enzyme-substrate (E-S) complexes. The activation energy (Ea) and temperature quotient (Q10) of protease from S. marcescens and P. putida were 16.2 and 19.9 kJ/mol, and 1.4 and 1.3 at temperature range from 20 to 40 °C, respectively. Protease of the both bacterial isolates was serine and cysteine type. The protease retained approximately 97% of activity in the presence of sodium dodecyl sulphate. It was observed that the purified protease of S. marcescens could remove blood stains from white cotton cloth and degrade chicken flesh remarkably. Our study revealed that organic MSW can be used as raw materials for bacterial protease production and the protease produced by S. marcescens A3 might be potential for applications.

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Nuclease overexpression mutants of Serratia marcescens.

Project description:A family of mutants overexpressing the Serratia marcescens extracellular nuclease has been known for decades. A number of these alleles are characterized here at the molecular level, and the mutant genes are identified, yielding a likely model for their phenotype. The known mutations exert their effect indirectly on nucA expression by elevating the basal SOS response of the cell. Mutations have been found in xerC and uvrD, both of which result in partial SOS induction. A classic nucsu allele, that of strain W1050, is also likely to be in xerC.

| S-EPMC107160 | biostudies-literature

Purification, crystallization and preliminary X-ray analysis of the outer membrane complex HasA-HasR from Serratia marcescens.

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In silico Functional Annotation and Characterization of Hypothetical Proteins from Serratia marcescens FGI94.

Project description:Serratia marcescens, rod-shaped Gram-negative bacteria is classified as an opportunistic pathogen in the family Enterobacteriaceae. It causes a wide variety of infections in humans, including urinary, respiratory, ocular lens and ear infections, osteomyelitis, endocarditis, meningitis and septicemia. Unfortunately, over the past decade, antibiotic resistance has become a serious health care issue; the effective means to control and dissemination of S. marcescens resistance is the need of hour. The whole genome sequencing of S. marcescens FGI94 strain contains 4434 functional proteins, among which 690 (15.56%) proteins were classified under hypothetical. In the present study, we applied the power of various bioinformatics tools on the basis of protein family comparison, motifs, functional properties of amino acids and genome context to assign the possible functions for the HPs. The pseudo sequences (protein sequence that contain ?100 amino acid residues) are eliminated from the study. Although we have successfully predicted the function for 483 proteins, we were able to infer the high level of confidence only for 108 proteins. The predicted HPs were classified into various classes such as enzymes, transporters, binding proteins, cell division, cell regulatory and other proteins. The outcome of the study could be helpful to understand the molecular mechanism in bacterial pathogenesis and also provide an insight into the identification of potential targets for drug and vaccine development.

| S-EPMC7394047 | biostudies-literature

Cloning, sequencing, and characterization of the SdeAB multidrug efflux pump of Serratia marcescens.

Project description:Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

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The A component (SmhA) of a tripartite pore-forming toxin from Serratia marcescens: expression, purification and crystallographic analysis.

Project description:Tripartite α-pore-forming toxins are constructed of three proteins (A, B and C) and are found in many bacterial pathogens. While structures of the B and C components from Gram-negative bacteria have been described, the structure of the A component of a Gram-negative α-pore-forming toxin has so far proved elusive. SmhA, the A component from the opportunistic human pathogen Serratia marcescens, has been cloned, overexpressed and purified. Crystals were grown of selenomethionine-derivatized protein and anomalous data were collected. Phases were calculated and an initial electron-density map was produced.

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