• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.288-296
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• 1995

To investigate high-level expression of Serratia marcescens metalloprotease (SMP) in Escherichia coli and S. marcescens, we constructed various recombinant plasmids: pSP2, containing SMP gene and lac promoter; pKSP2, containing SMP gene and tac promoter; pTSP2, containing SMP gene, trc99a promoter, and lacI$^{q}$. The recombinant E. coli (pKSP2) strain expressed SMP to a high-level, about 36% of total cellular proteins but accumulated inactive SMP precursors intracellularly, which indicated that E. coli does not have activation and secretion system for SMP. To overproduce active SMP, we transformed S. marcescens with the recombinant plasmids by a modified CaCl$_{2}$ method. The recombinant S. marcescens ATCC27117 (pSP2) containing lac promoter for SMP transcription produced 530 U/ml of active SMP on LB broth, which is about 5.1 times of the SMP yield, 105 U/ml of a control strain, S. marcescens ATCC27117 (pUC19). However, S. marcescens ATCC27117 (pKSP2) containing tac promoter for SMP transcription did not grow healthy and hardly produced SMP. To overcome a harmful effect of the strong tac promoter, we constructed a regulatory plasmid pTSP2 containing a strong trc99a promoter and its repressor gene lacI$^{q}$. When S. marcescens ATCC27117 (pTSP2) was induced with 1.0 mM IPTG after 9 hr cultivation, 2,200 U/ml of SMP was obtained in LB broth, which is about 21 times of that of a control strain.

• Journal of Life Science
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• v.10 no.4
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• pp.397-402
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• 2000

Polyphosphate kinase catalyzes the formation of polyphosphate from ATP. To understand the mechanism of phosphate accumulation, the Serratia marcescens gene encoding ppk was cloned from the genomic library by the method of Southern hybridization. The hybridization positive DNA fragment region from pDH3 was subcloned into the expression vector. The ppk gene product, a polypeptide of 75 kDa, was confirmed by SDS-PAGE. Expression of the Serratia marcescens ppk is regulated by the catabolite repression system. The enzyme activity polyphosphate kinase was increased in the E. coli strain harboring plasmid pMH4 with ppk gene.

• YAKHAK HOEJI
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• v.37 no.2
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• pp.129-135
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• 1993

Serratia sp. Y-4 was isolated from soil. Many characteristics of the strain and optimum cultivation condition for protease production were investigated.,The protease from Serratia sp. Y-4 was purified and studied for the properties of the enzyme. The isolated strain was identified to the genus Serratia. The strain was cultivated in 1%-casein, 0.5%-Na$_{3}$PO$_{4}$.7H$_{2}$O, 0.1%-NaCl, 0.05%-KCI, 0.02%-MgSO$_{4}$.7H$_{2}$O, 0.02%-CaCl$_{2}$.2H$_{2}$O, 0.02%-ZnSO$_{4}$.7H$_{2}$O, 0.02%-MnCl$_{2}$.4H$_{2}$O and 0.5%-soy bean oil at pH 7.0 for 35 hrs. The enzyme was purified about 5.89 fold from the culture broth with 31.1% recovery and 19,613 u/mg through ultrafiltration, ammonium sulfate, DEAE-sephacel and Superose-12 chromatography. The purified enzyme was identified as one band by isoelectric focusing, SDS- and native-PAGE. It has maxium activity at $37^{\circ}C$ and pH 9.0. Molecular weight of it is approx. 50 kD and pl is about 6.70. Its Km value for casein was 20 mg/ml. 5 mM-EDTA, 5mM-SDS, Ag$^{+1}$, Cu$^{+2}$, Hg$^{+2}$ and Pb$^{+2}$ inhibited the enzyme.

• Journal of Life Science
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• v.8 no.3
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• pp.263-271
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• 1998

One of the better-characterized transcription factor of E. coli is the cAMP receptor protein(CRP) and the CRP binds cAMP and DNA. The cAMP-CRP complex is involved in regulation of many genes at bacteria. The cAMP-CRP regulatory element represents, in some respects, a global regulatory network. The aim of this work was to study the structure and the mechanisms controlling the expression of CRP in Serratia marcescens. We have been get 5 different clones from Serratia which stimulated the cells to use maltose as a sole carbon source in E. coli TP2139. The crp gene clone, pCKB12, was confirmed by Southern hybridization with E. coli crp gene. The location of the crp gene was determined by construction subclones carrying various portions of pCKB12. To investigate the potential role of CRP in E. coli, lacZ fused plasmids were constructed and investigated the ${\beta}$-galactosidase activity of the fused plasmid. The Serratiamarcescens cAMP receptor protein can substitute the E. coli CRP in transcriptional activation at the lacZ gene. These results suggest that Serratia marcescens cAMP receptor protein complex functions to regulate several promoters in E. coli.

• Microbiology and Biotechnology Letters
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• v.5 no.4
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• pp.177-184
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• 1977

Arylsulfatase catalyzes the release of SO$\sub$4//sup2/- from sulfate esters of simple phenols. Arylsolfatase occurs widely in animal tissues and in microorganisms including soil bacteria. Its widespread distribution suggests that it has a rather fundamental function and environmental meaning. It has been shown previously that arylsulfatase of Klebsiella was purified and characterized. A condition of arylsulfatase synthesis was tested with several strains of Serratia. Serratia marcescens could not utilize some sugars, such as xylose, rhamnose, glucosamine and arabinose hut glucose and mannitol as a sole carbon source. However, arylsulfatase synthesis was repressed by glucose but not by mannitol. The enzyme synthesis was repressed ob inorganic sulfate and methionine, and this repression was relieved by addition of tyramine. Arylsulfatase of S. marcescen was purified by fractionation with ammonium sulfate and followed by chromatographies on DEAE-Cellulose CM-Cellulose, and DEAE-Sephadex A-25. The molecular weight of arylsulfatase was determined to be 46,000 by SDS-Gel electrophoresis and 49,000 by Sephadex G-100 column chromatography. The enzyme showed some different properties with that of K. aerogenes. The activity was maximum at pH 6.8. The Km and Vmax values for p-nitrophenyl sulfate were 2.5${\times}$10$\^$－4/ M and 20 nmoles/min/mg protein, respectively. The enzyme showed high activities toward phenyl sulfate, ο-and p-nitro phenyl sulfates, and p-nitrocatechol sulfate. The inhibition of enzyme was strongly affected by hydroxylamine, inorganic fluoride, sulfide and phosphate, but by inorganic sulfate. Like Klebsiella arylsulfatase, tyramine, octopamine, and dopamine gave signifcant inhibitory effect.

• BMB Reports
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• v.32 no.2
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• pp.210-213
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• 1999

In Serratia marcescens, acetolactate produced by the catabolic acetolactate synthase (ALS) is converted into acetoin, its physiological role of which is to maintain intracellular pH homeostasis. In this study, the expression mode of catabolic ALS by aeration and branched-chain amino acids was examined by the ELISA method. The amount of catabolic ALS decreased approximately 93% under aerobic conditions. We also showed that the expression of catabolic ALS decreased approximately 34 % and 65 % in the presence of 2.5 mM and 10 mM leucine, respectively. The repression of catabolic ALS by leucine has not been reported previously. In contrast to leucine, catabolic ALS levels increased approximately 13% and 38% by treatment with 2.5 mM and 10 mM isoleucine, respectively, while valine alone did not have any significant effect on the synthesis of catabolic ALS. The amount of catabolic ALS was also reduced to approximately 32% and 45% in the presence of 10 mM Leu+Ile and Leu+Ile+Val, respectively. The regulatory mode of the Serratia catabolic ALS suggests that catabolic ALS may also have a role in supplying acetolactate as an intermediate of valine and leucine biosynthesis in addition to the maintenance of internal pH.

• Journal of Microbiology and Biotechnology
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• v.20 no.4
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• pp.732-736
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• 2010

Five strains of tannic acid degrading bacteria were isolated and identified by phenotypic characterization. All the five isolates showed cell-associated activity, whereas only three showed extracellular activity. Serratia ficaria DTC, showing the highest cell-associated activity (0.29 U/l), was selected for further shake-flask studies. Tannase synthesis was growth associated and reached the peak in the late stationary phase of growth. Organic nitrogen sources enhanced the tannase production. Peak tannase production of 0.56 U/l was recorded in the medium having the initial pH of 6. The pH and temperature optima of the enzyme were found to be 8.9 and $35^{\circ}C$, respectively. This is the first report of cell-associated activity in the case of bacterial tannase. Cell-associated tannase of Serratia ficaria DTC could be industrially important from the perspective of its activity at broad temperature and pH ranges, and its unusually high activity at pH 8.9.

• Microbiology and Biotechnology Letters
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• v.20 no.1
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• pp.25-33
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• 1992

One acetolactate synthase isozyme which has Rf value of 0.83 on polyacrylamide gel electrophoresis was purified from Sewatia marcescens ATCC 25419 by ammonium sulfate fractionation, DEAE-Sephacel chromatography, Phenyl-Sepharose chromatography, Sephacryt S-400 gel filtration followed by native gel elution. The native molecular weight of the enzyme was determined to be 531,400 by gel filtration method, and SDS-polyacrylamide gel electrophoresis separated the native enzyme into two polypeptides having molecular sizes of 55,000 and 38,900 respectively. In kinetic parameters, $K_m$ value for pyruvate was 2.54 mM, and $V_{max}$ was 21.75 nmoie/min/mg. The enzyme showed maximal activity around pH 8.0 and optimal temperature of the acetolactate formation was $37^{\circ}C$. Feedback inhibition studies indicate that the purified enzyme is rather resistant to branched chain amino acids when compared with acetolactate synthase isozymes of plants or other enterobacteria.

• Journal of Applied Biological Chemistry
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• v.42 no.1
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• pp.7-11
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• 1999

An extracellular chitinase of the selected strong antifungal microorganism, Serratia sp. 3095, was purified by salting out, affinity adsorption, Sepadex G-100 gel fitration, Sepadex G-75 gel fitration and DEAE Sepadex A-50 chromatography. The molecular weight of the purified chitinase was estimated to be 62,000 dalton by SDS-PAGE. Optimal pH and temperature of the chitinase were pH 7.5 and 45, respectively. The enzyme retained more than 80% of the activity between pH 5.5 and pH 10.5, and below $50^{\circ}C$ but was unstable above $60^{\circ}C$, below pH 5.0. The activity of the chitinase was inhibited about 60% by $Sn^{2+}$, 40% by $Hg^{2+}$ and $Ag^+$, 70% by AHA, 40% by iodoacetate, 35% by thiourea and p-CMB, but stabilized by SDS. $K_m$ value of the purified chitinase was 3.68 mg/ml for colloidal chitin. The chitinase from Serratia sp. 3095 showed antifungal activity to Fusariurm solani.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.695-701
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• 1995

An alkaline lipase producing bacteria was isolated from soil and identified as Serratia liquefaciens AL-11. from the results of analysis of its morphological, biochemical and physiological properties. This strain showed the highest productivity of alkaline lipase when grown at pH 9.0 and 30$\circ$C for 42 hours in the medium of 1% peptone, 0.5% tryptone, 0.9% yeast extract, 1% starch, 1% tween 80, 0.05% CaCl$_{2}$ and 0.05% NaCl. The enzyme was purified by ammonium sulfate treatment, Sephadex G-100 gel filtration and DEAE-Sephadex A-50 column chromatography. The specific activity of the purified enzyme was 27 unit/mg protein and the yield of enzyme activity was 61.3%. The homogeneity of the purified enzyme was verified by polyacrylamide gel disc electrophoresis. Molecular weight of the purified enzyme was estimated about 53,000 by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. This enzyme is composed of 17 amino acids of which glycine, proline and glutamic acid were three miajor acids.