Overexpression of Chitinase A Gene from Serratia marcescens in Bacillus subtilis and Characterization of Enhanced Chitinolytic Activity

Abstract Chitinase enzymes possess various usages in agriculture, biotechnology and medicine due to their chitin degrading property. Thus, efficient production of chitinase enzymes with desired properties has importance for its use. In this study, chitinase A (chiA) gene from Serratia marcescens Bn10 was cloned and heterologously overexpressed using pHT43 vector in Bacillus subtilis 168. The recombinant chitinase was characterized in terms of temperature, pH, and various effectors. The extracellular chitinase activity in recombinant B. subtilis was found 2.15-fold higher than the parental strain after 2 h of IPTG induction. Optimum temperature and pH for the extracellular chitinase activity in the recombinant B. subtilis were determined as 60 oC and pH 9.0, respectively. NaCl, Ca2+, Mn2+, Cu2+, Zn2+, sodium dodecyl sulfate (SDS), Tween-20, and ethanol increased the chitinase activity whereas Mg2+ caused an inhibition. The most notable increment on the chitinase activity was provided by Zn2+ (3.2 folds) and then by SDS (2.9 folds). The chitinase, overproduced by the recombinant B. subtilis 168 heterologously expressing chiA, was determined to have optimum activity at high temperature and alkaline conditions as well as various effectors increase its activity. The extracellular chitinase of recombinant B. subtilis might be a promising source for agricultural, biotechnological and medical applications.

Enhanced alkaline catalase production by Serratia marcescens FZSF01: enzyme purification, characterization, and recombinant expression

Background: Catalase (CAT) is an important enzyme that degrades H2O2 into H2O and O2. To obtain an efficient catalase, in this study, a new strain of high catalase-producing Serratia marcescens, named FZSF01, was screened and its catalase was purified and characterized. Results: After optimization of fermentation conditions, the yield of catalase produced by this strain was as high as 51,468 U/ml. This catalase was further purified using two steps: DEAE-fast flow and Sephedex-G150. The purified catalase showed a specific activity of 197,575 U/mg with a molecular mass of 58 kDa. This catalase exhibited high activity at 20­70°C and pH 5.0­11.0. Km of the catalase was approximately 68 mM, and Vmax was 1886.8 mol/min mg. This catalase was further identified by LC­MS/MS, and the encoding gene was cloned and expressed in Escherichia coli BL21 (DE3) with a production of 17,267 ± 2037 U/ml. Conclusions: To our knowledge, these results represent one of the highest fermentation levels reported among current catalase-producing strains. This FZSF01 catalase may be suitable for several industrial applications that comprise exposure to alkaline conditions and under a wide range of temperatures.

Draft genome sequence of a GES-5-producing Serratia marcescens isolated in southern Brazil

Abstract Serratia marcescens is a Gram-negative rod intrinsically resistant to polymyxins and usually associated with wound, respiratory and urinary tract infections. The whole genome of the first GES-5-producing S. marcescens isolated from a Brazilian patient was sequenced using Ion Torrent PGM System. Besides blaGES-5, we were able to identify genes encoding for other β-lactamases, for aminoglycoside modifying enzymes and for an efflux pump to tetracyclines.

Transferecia de de ß-lactamasas de espectro extendido desde cepas hospitalarias de Klebsiella pneumoniae a otras especies de enterobacterias / Transference of extended-spectrum ß-lactamases from nosocomial strains of Klebsiella pneumoniae to other species of Enterobacteriaceae

Background: Klebsiella pneumoniae is an important pathogenic bacterium, frequently isolated from nosocomial samples, that exhibits wide antimicrobial resistance profiles, including third generation cephalosporins (3GC), aminoglycosides and quinolones. The resistance to 3GC is mainly due to the synthesis of extended spectrum beta lactamases (ESBL), encoded by conjugative plasmids. Aim: To investigate the potential transference of resistance to 3GC from nosocomial strains of K. pneumoniae to other clinical strains of various species of Enterobacteriaceae. Material and methods: The mating experiments were carried out in liquid media and three nosocomial strains of K. pneumoniae were used as donors. These strains were ESBL-producers and resistant to, at least, one of the 3GC assayed. One strain of Citrobacter freundii, Salmonella typhimurium, Serratia marcescens and Escherichia coli, isolated from clinical specimens, were used as recipients. The presence of bla genes was investigated by PCR. Results: The three nosocomial strains of K. pneumoniae were able to transfer the resistance to 3GC and the genes encoding the ESBL to the susceptible recipient strains of enterobacteria. The frequency of transference was as high as 3.2 x 10-2 transconjugants/recipient cell when the strain of Citrobacter freundii was used as recipient. All transconjugants exhibited high level of resistance to the 3GC assayed. Conclusions: Strains of K. pneumoniae isolated from Chilean hospitals are able to disseminate the ESBL genes to clinical strains of others species of Enterobacteriaaceae.

Purificación de la enzima de restricción Sma I / Purification of Sma I restriction enzyme

La enzima de retricción Sma I, producida por el microorganismo Serratia marcescens, ha sido purificada en nuestro laboratorio utilizando precipitación con PEG 6000 y cromatografía de intercambio iónico en fosfocelulosa (P-11 Whatman), obteniéndose una preparación final con una actividad específica de 2971 U/mg proteína y 35 % de recobrado total del proceso de purificación apta para ser utilizada en los experimentos de clonación y otras técnicas del ADN recombinante

Proteasas de Pseudomonas aeruginosa: caracterización parcial y relaciones inmunológicas con proteasas de diferentes taxas / Proteases of pseudomonas aeruginosa: partial characterization and immunological relations with proteases of different rates

El presente trabajo tuvo el propósito de aislar, caracterizar y estudiar inmunológicamente, dos enzimas extracelulares proteolíticas de una cepa de Pseudomonas aeruginosa (Pa) tomada como prototipo, para luego compararlas con las producidas por cepas de la misma o diferentes especies bacterianas. La cepa prototipo mostró por cromatografía de intercambio iónico las dos enzimas proteolíticas identificadas como A y B, sobre la base de su mayor o menor electronegatividad. La enzima A fue activa sobre caseína y no sobre elastina y necesitó fuerza iónica adicional para eluir del intercambiador. la enzima B fue activa sobre caseína y elastina y no necesitó fuerza iónica adicional para su elución. Fue la responsable del 80% o más de la actividad total. Con las dos enzimas obtenidas por poliacrilamida, se inmunizaron conejos, obteniéndose antisueros (As A y As B), con los que se efectuaron reacciones inmunológicas. Los As A y As B inmunoprecipaitaron las enzimas respectivas, tanto de la solución enzimática concentrada como de las obtenidas por cromatografía. Estos dos componentes enzimáticos no mostraron identidad inmunológica. Los antisueros homólogos fueron capaces de inhibir la actividad enzimática in vitro. Seis cepas adicionales de Pa mostraron un comportamiento inmunoenzimático similar a la prototipo. Dos de Serratia marcescens, aisladas de pacientes, mostraron por zimograma una especie proteolítica coincidente con la A de Pa, activa sólo sobre caseína y que reaccionó solamente con el As A. Una cepa de Pseudomonas fluorescens conservada en laboratorio no mostró actividad en el zimograma y tampoco reactividad con los As A y As B