Purification and partial characterization of a bacteriocin from Serratia marcescens

Bacteriocin JF246 has been purified from mitomycin C-induced Serratia marcescens cells by salt extraction, ammonium sulfate fractionation, and chromatography on QAE-Sephadex and SP-Sephadex. The purified material is homogeneous on polyacrylamide gel electrophoresis in the presence of 2% sodium dodecyl sulfate or 6 m urea. In the absence of these agents, the bacteriocin associates into aggregates which can be dissociated with 0.4 m NaCl. The bacteriocin is probably composed of a single subunit with a molecular weight of 64,000 daltons. Analytical studies show the bacteriocin to be essentially protein in nature containing less than one residue of glucose or phosphorus per 64,000 daltons.     

Purification and characterization of a chitosanase from Serratia marcescens TKU011

A chitosanase was purified from the culture supernatant of Serratia marcescens TKU011 with shrimp shell wastes as the sole carbon/nitrogen source. Zymogram analysis revealed the presence of chitosanolytic activity corresponding to one protein, which was purified by a combination of ion-exchange and gel-filtration chromatography. The molecular weight of the chitosanase was 21 kDa and 18 kDa estimated by SDS–PAGE and gel-filtration, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase were 5, 50 °C, pH 4–8, and <50 °C, respectively. The chitosanase was inhibited completely by EDTA, Mn²⁺, and Fe²⁺. The results of peptide mass mapping showed that three tryptic peptides of the chitosanase were identical to a chitin-binding protein Cbp21 from S. marcescens (GenBank accession number gi58177632) with 63% sequence coverage.     

Purification and characterization of the extracellular proteinase of Serratia marcescens.

The extracellular proteinase produced by a depressed strain of Serratia marcescens ATCC 25419 was purified and characterized. This produces more than 10-times the amount of extracellular proteinase produced by other strains of Serratia tested. The purified enzyme was prepared from the culture supernatant by (NH4)2SO4 fractionation and DEAE-cellulose chromatography. The purified enzyme has an so20,w of 3.95 and is a monomer of molecular weight 51,900. The proteinase has a broad pH optimum in the alkaline range with a maximum at pH 9.5. The enzyme will utilize a number of proteins as substrate. The products of digestion are primarily in the size range of tripeptides to hexapeptides. Peptides containing a sensitive bond and a minimum size of size amino acids are hydrolyzed. The proteinase is inhibited by chelating agents but unaffected by sulfhydryl or serine reagents and is devoid of esterase activity.     

Formation and Purification of Serratia marcescens Arylsulfatase

The effects of culture conditions on arylsulfatase production by six strains of the genus Serratia were studied. Synthesis of arylsulfatases in all six strains was repressed in media with inorganic sulfate or methionine as the sole source of sulfur and derepressed by the addition of tyramine. Serratia marcescens IFO 3046 grew most rapidly and produced a high level of arylsulfatase when cultured on mannitol with inorganic sulfate and tyramine. The derepressed synthesis of arylsulfatase in S. marcescens was not subject to strong catabolite repression. The molecular weight of purified arylsulfatase was determined to be between 46,000 and 49,000. Arylsulfatase from S. marcescens differed in K_m and V_max values, substrate specificities, fluoride inhibition, and electrophoretic mobility from the enzyme from K. aerogenes, but had the same molecular weight as the latter.     

Purification and characterization of four proteases from a clinical isolate of Serratia marcescens kums 3958.

Four distinct proteases were purified to homogeneity from culture filtrates of Serratia marcescens kums 3958, a fresh isolate from a patient with a severe corneal ulcer. Purification was achieved by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex gel filtration chromatography. The proteases were differentiated from each other by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate and by immunodiffusion in agarose gels. The molecular weights of these purified proteases were estimated to be 56 X 10(3), 60 X 10(3), and 73 X 10(3) (hereafter designated 56K, 60K, and 73K proteases, respectively). The 73K protease was separated into 73Ka and 73Kb upon isoelectricfocusing. The isoelectric points of the 56K (major) and 60K, 73Ka, and 73Kb proteases (minors) were approximately 5.3, 4.4, 5.8, and 7.3, respectively. Both 56K and 60K enzymes were completely inactivated by EDTA at pH 5.0 and were reactivated by zinc ion; thus, they are metalloenzymes, whereas 73K (73Ka and 73Kb) enzymes appear to be thiol proteases. Carbohydrate, cysteine, and cystine were not detected in the 56K and 60K proteases. Amino acid compositions, partial amino acid sequence, and enzymological and immunological properties revealed that these four enzymes are distinct from each other.     

Purification and Characterization of Metallo-β-Lactamase from Serratia marcescens

Carbapenem-hydrolyzing β-lactamase from Serratia marcescens FHSM4055 was purified 926-fold by means of carboxylmethyl Sephadex C-50, Sephacryl S-200, and Mono S column chromatography. The molecular weight was 30,000 by SDS-PAGE and the isoelectric point was 8.7. The enzyme activity was inhibited by EDTA, and restored by adding zinc (II) or manganese (II). It was inhibited by p-chloromercuribenzoate and iodine as well as the heavy metals, Hg (II), Fe (II), Fe (III), and Cu (II). These results indicate that the enzyme is a metallo-β-lactamase and that the SH-group of only one cysteine residue probably binds to the metal ion, thus contributing to the stability of the enzyme active center. The specific constant (k_cat/K_m) showed that the enzyme hydrolyzed various β-lactam antibiotics such as carbapenems, cephalosporins, moxalactam, cephamycins, and penicillins other than monobactams. Ampicillin and piperacillin with respective amino- and imino-groups, ceftazidime with a carboxypropyloxyimino-group, and cefclidin with a carbamoylquinuclidine-group were poor substrates among the β-lactam antibiotics other than the monobactams tested. The plots of the turnover number (k_cat) against pH for the hydrolysis of cephaloridine gave an asymmetrical curve with the ‘tail’ on the acid side (pK₁, 5.9; pK₂, 9.0; pK₃, 10.8), whereas those of k_cat/K_m gave a bell-shaped curve (pK₁, 5.8; pK₂, 9.8). Both results suggest that two ionic forms of an intermediate yield the same product at different rates and that the enzyme is stable under alkaline conditions. Since the N-terminal amino acid sequence of 27 residues determined was consistent with that of the metalloenzyme (Antimicrob. Agents Chemother., 1994, 38: 71-78), the above enzymatic characteristics seem to coincide.     

Cloning of the Serratia marcescens recA gene and construction of a Serratia marcescens recA mutant

A recombinant plasmid, pSM2513, containing an 8.5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2.7 kb Bg/II-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.     

Purification and characterization of zinc-binding protein from the liver of the partially hepatectomized rat.

Zn-binding protein in liver of the partially hepatectomized rat was purified by column chromatography on Sephadex G-75 and DEAE-cellulose. Homogeneity was judged by polyacrylamide-disc-gel electrophoresis. The molecular weight determined by gel-permeation chromatography in 6 M-guanidine hydrochloride was 6700. This value is in good agreement with the molecular weight calculated from the amino acid composition, which was 6073. Zn-binding protein was composed of 61 amino acid residues, and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and an absolute absence of aromatic amino acids as well as of histidine, leucine and arginine. The amino acid composition was similar to that of the metallothioneins previously isolated from rat liver and mouse liver. These observations suggest that the Zn-binding protein can be classified as a type of metallothionein. Zn-binding protein contained 8.2g-atoms of zinc per mol and traces of copper, but no cadmium. The molar ratio of thiol groups to zinc was calculated to be 2.5:1. Possible roles of this Zn-binding protein in the transport and storage of zinc in the liver are discussed.     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin (4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400 μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties. Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997