An improved method to measure lipase activity in aqueous media.

An improved method based on the p-nitrophenyl long chain esters method is proposed for measuring lipase hydrolytic activity in aqueous media. Using ethylene glycol as co-solvent for hydrophobic p-nitrophenyl substrates in aqueous buffer, lipase activity is measured by following the release of p-nitrophenol. This fast and easy to handle method improves the solubility of both substrate and product, and also the stability of the substrate. It avoids the use of solvents such as ethanol or propanol, permits the comparison of all the p-nitrophenol acyl ester substrates and allows the influence of ions like Ca+2 to be studied, while avoiding turbidity in the reaction medium.

Improving the production, activity, and stability of CLEAs with diepoxides.

Among enzyme immobilization techniques, the preparation of cross-linked enzyme aggregates has shown promising results in biocatalysis, because they are easy to prepare, versatile, and cheap. The method involves the precipitation of enzymes with ammonium sulfate or an organic solvent and subsequent cross-linking with glutaraldehyde. However, the Schiff base produced with glutaraldehyde is reversible and can be broken with acids or bases, releasing proteins to the reaction medium. To solve this problem, we propose replacing glutaraldehyde with diepoxide compounds to obtain an irreversible secondary amine bond. Such a substitution avoids protein leakage during the biocatalytic process, contamination of the final products, and loss of enzyme. It also improves the synthesis of the biocatalyst, because, while the Schiff base is favored at mildly acidic pH, the epoxide reaction can be made at the optimal enzyme pH, assuring its structural stability and catalytic performance. The proposed method has been successfully used in the production and optimization of aldolase epoxy-cross-linked aggregates, which retain 98% activity. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1425-1429, 2017.

Comparative study of bacterial translocation control with nitric oxide donors and COX2 inhibitor / Estudio comparativo del control de la translocación bacteriana con donadores de óxido nítrico e inhibidor de la COX2

OBJECTIVE AND DESIGN: To evaluate the beneficial effects of exogenous NO and an inhibitor of the COX2, and their action levels in a model of SIRS/bacterial translocation (BT) induced by Zymosan A®. MATERIAL AND METHODS: Ninety Wistar rats were submitted to different treatments, and after 12h and 24h they were anaesthetized in order to collect blood, mesenteric lymph nodes, and kidney for subsequent biochemical analyses and microbiological examinations. Treatments. A nitric oxide donor, Molsidomine®, was compared with a COX2 inhibitor, Celecoxib®. METHODS: Zymosan A® was administered to Wistar rats. The animals were divided into 6 groups: one group for survival study, Group (1) No manipulation (BASAL); Group (2) vehicle of Zymosan A® given intraperitoneally (SHAM); Group I (control), with Zymosan A® (0.6g/kg) intraperitoneally; Group II (Molsidomine), with Molsidomine® (4mg/kg) through the penis dorsal vein, 30min prior to administration of the Zy® (0.6g/kg); Group III (Celecoxib), with Celecoxib® (400mg/kg) orally through a stomach tube, 6h prior to administration of the Zy (0.6g/kg). Determinations. The parameters survival, bacterial translocation, renal function, neutrophil accumulation, oxygen free radicals (OFR), detoxifying enzymes, and cytokines were measured at different times after Zymosan administration. RESULTS: The model established induced a mortality rate of 100% and generated BT and systemic inflammatory response syndrome (SIRS) in all samples. It also significantly increased all variables, with p<.001 for MPO and all pro-inflammatory cytokines, and p<.01 for all OFR. Treatment with Molsidomine reduced mortality to 0%, decreased BT, MPO, pro-inflammatory cytokines and OFR (p<.001) significantly and increased IL-10 and IL-6 production. Moreover, the Celecoxib® showed a lower capacity for SIRS regulation. CONCLUSIONS: The exogenous administration of NO prevented BT and controlled SIRS. Therefore these results suggest that Molsidomine could be used as a therapeutic strategy to protect against BT

OBJETIVO Y DISEÑO. Evaluar los efectos beneficiosos del ON exógeno y de un inhibidor de la COX2 y sus niveles de acción en un modelo de síndrome de respuesta inflamatoria sistémica (SIRS)/traslocación bacteriana (TB) inducida por Zymosan A®. MATERIALES Y MÉTODOS: Noventa ratas Wistar fueron sometidas a diferentes tratamientos, y después de 12 y 24 horas fueron anestesiadas para recoger sangre, nódulos linfáticos mesentéricos y tejido renal, para analizarlos bioquímica y microbiológicamente. Tratamientos. Un donador de óxido nítrico, Molsidomina®, fue comparado con Celecoxib®, inhibidor de la COX2. MÉTODOS: Se administró Zymosan A® a las ratas Wistar. Estas fueron divididas en CINCO grupos: grupo 1 (basal), sin manipulaciones; grupo 2 (sham), vehículo de Zymosan A® administrado intraperitonealmente; grupo I (control), con Zymosan A® (0,6 g/kg) intraperitoneal; grupo II (Molsidomina) con Molsidomina® (4 mg/kg) administrada a través de la vena dorsal del pene, 30 minutos antes de la administración de Zymosan® (0,6 g/kg); y grupo III (Celecoxib) con Celecoxib® (400 mg/kg) administrado oralmente por tubo esomacal, 6 horas antes de la administración de Zymosan A® (0,6 g/kg). Determinaciones. Se midieron los parámetros supervivencia, traslocación bacteriana, función renal, acumulación de neutrófilos, radicales libres de oxígeno, enzimas detoxificantes y citoquinas, a diferentes tiempos después de la administración de Zymosan®. RESULTADOS: El modelo establecido inducía una tasa de mortalidad del 100%, y se generaba traslocación bacteriana y síndrome de respuesta inflamatoria sistémica en todas las muestras. También se incrementaban significativamente todas las variables, con p < 0,001 para mieloperoxidasa y todas las citokinas proinflamatorias, y p < 0,01 para todos los radicales libres de oxígeno. El tratamiento con Molsidomina reducía la mortalidad al 0%, disminuía la traslocación bacteriana, mieloperoxidasas, citokinas proinflamatorias y radicales libres de oxígeno (p < 0,001), e incrementaba la producción de IL-10 e IL-6. Además, Celecoxib® mostró una menor capacidad para la regulación del síndrome de respuesta inflamatoria sistémica. CONCLUSIONES: La administración exógena de óxido nítrico evita la traslocación bacteriana y controla el síndrome de respuesta inflamatoria sistémica. Estos resultados sugieren que Molsidomina podría usarse como estrategia terapéutica frente a la traslocación bacteriana

Cambios en la epidemiología de la infección por Clostridium difficile durante 2005-2014 en Salamanca, España / Changes in the epidemiology of Clostridium difficile infection during 2005-2014 in Salamanca, Spain

Introducción. Conocer los rasgos epidemiológicos más relevantes de la infección por Clostridium difficile (ICD) ocurrida en la provincia de Salamanca (España) entre 2005-2014. Métodos. Estudio descriptivo transversal realizado a partir del archivo informático del Servicio de Microbiología del Complejo Asistencial Universitario de Salamanca. La detección se realizó según la metodología habitual. Resultados. El 2,6% de las muestras de heces analizadas para detección de toxinas de C. difficile (9.103) fueron positivas. La prevalencia media global fue de 6,8 casos por 100.000 habitantes y año. La media de edad fue de 65 ± 21,4 años y la mediana 70 años. El 59% de los casos se produjo en mayores de 64 años, con una prevalencia media anual de 16,5 (4 veces superior a la del grupo de 15-64). La mayoría de casos (86.4%) se produjeron en pacientes hospitalizados, siendo el grupo de mayores de 64 años el de mayor porcentaje de ICD hospitalaria, con un 55%. Conclusiones. Se observa un incremento significativo del número de peticiones y de la prevalencia de ICD a lo largo de la década estudiada y unas tasas de prevalencia bastante inferiores a las de otros estudios. El porcentaje de ICD aumentó de manera significativa tanto en pacientes hospitalizados como en los comunitarios. La edad y la hospitalización fueron factores de riesgo para desarrollar ICD. Tras la introducción de una técnica molecular de detección en 2014, la prevalencia aumentó, siendo 2.5 veces superior a la del 2013 (AU)

Background. To know the most relevant epidemiological features of Clostridium difficile infection (CDI) between 2005-2014 in the province of Salamanca (Spain). Methods. Descriptive cross-sectional study carried out through review of the clinical microbiologic records at Complejo Asistencial Universitario de Salamanca. Detection was performed according to standard methodology. Results. 2.6% of stool samples analyzed for detection of C. difficile toxins (9.103) were positive. The average prevalence was 6.8 cases per 100,000 people per year. The mean age was 65 ± 21.4 years and the median 70 years. 59% of cases occurred in patients over 64 years, with an average prevalence of 16.5 (4 times higher than the 15-64 group). Most cases (86.4%) occurred in hospitalized patients, and the group of over 64 had the highest percentage of hospital CDI, with 55%. Conclusions. A significant increase in the number of requests and in the prevalence of CDI over the decade studied is observed, and prevalence rates were significantly lower than those of other studies. The percentage of CDI increased significantly in both inpatient and community. Age and hospitalization were risk factors for developing CDI. After the introduction of a molecular detection technique in 2014, the prevalence increased, being 2.5 times higher than 2013 (AU)

Cambios en la epidemiología de las gastroenteritis causadas por Salmonella durante 2005-2014 en Salamanca, España / Changes in the epidemiology of gastroenteritis caused by Salmonella during 2005-2014 in Salamanca, Spain

Introducción. En España no abundan estudios poblacionales actualizados sobre salmonelosis, a pesar de ser una de las etiologías de gastroenteritis agudas (GEAs) bacterianas más habituales en el mundo. El objetivo fue conocer los rasgos epidemiológicos más relevantes de las GEAs producidas por Salmonella spp. entre 2005-2014 en Salamanca (España). Métodos. Estudio descriptivo transversal realizado a partir del archivo informático del Servicio de Microbiología del Complejo Asistencial Universitario de Salamanca. El cultivo, aislamiento, identificación y serotipificación se realizaron según la metodología habitual. Resultados. Salmonella se aisló en 1.477 pacientes, representando el 47,7% del total de coprocultivos positivos y el 53,3% de todos los ingresos por GEA bacteriana. La prevalencia media fue de 42,1 casos/100.000 habitantes y año. La media de edad fue de 23 ± 28 años y la mediana 7 años. El 40,2% de todos los aislamientos se produjo en menores de 5 años, con una prevalencia media de 45,1 casos/10.000 habitantes y año. Globalmente, el serotipo aislado con más frecuencia fue S. Typhimurium con un 57%, seguido por S. Enteritidis con un 35,8%. Conclusiones. La prevalencia de Salmonella disminuyó a lo largo del tiempo. El grupo entre 0-4 años presentó la tasa más alta durante todo el periodo. Sin embargo, produjo el mayor porcentaje de hospitalizaciones por GEA bacteriana. El serotipo S. Typhimurium ha reemplazado en los últimos años al serotipo S. Enteritidis y predomina en pacientes de menor edad. Se aprecia una infranotificación de los casos de salmonelosis producidos en Salamanca a pesar de ser obligatoria su declaración desde 2007 (AU)

Background. In Spain there are not many updated population studies about salmonellosis, despite being one of the most common etiologies of acute gastroenteritis (AGEs) caused by bacteria in the world. The aim of the study was to know the most relevant epidemiological features of AGEs produced by Salmonella spp. between 2005 and 2014 in Salamanca (Spain). Methods. Descriptive cross-sectional study carried out through review of the clinical microbiologic records at Complejo Asistencial Universitario de Salamanca. Culture, isolation, identification and serotyping were performed according to standard methodology. Results. Salmonella was isolated in 1,477 patients, representing 47.7% of all positive stool cultures and 53.3% of all income bacterial AGE. The average prevalence was 42.1 cases/100,000 people per year. The mean age was 23 ± 28 years and the median 7 years. 40.2% of all isolates occurred in children under 5 years, with an average prevalence of 45.1 cases/ 10,000 people per year. Overall, the most frequently isolated serotype was S. Typhimurium with 57%, followed by S. Enteritidis with 35.8%. Conclusions. The prevalence of Salmonella decreased over time. The group aged 0-4 years had the highest rate throughout the period. However, Salmonella produced the highest percentage of hospitalizations for bacterial AGE. In recent years, S. Typhimurium serotype has replaced S. Enteritidis serotype and predominates in younger patients. It is observed under-reporting of cases of salmonellosis produced in Salamanca despite being mandatory notification of these since 2007 (AU)

[Changes in the epidemiology of gastroenteritis caused by Salmonella during 2005-2014 in Salamanca, Spain]. / Cambios en la epidemiología de las gastroenteritis causadas por Salmonella durante 2005-2014 en Salamanca, España.

OBJECTIVE: In Spain there are not many updated population studies about salmonellosis, despite being one of the most common etiologies of acute gastroenteritis (AGEs) caused by bacteria in the world. The aim of the study was to know the most relevant epidemiological features of AGEs produced by Salmonella spp. between 2005 and 2014 in Salamanca (Spain). METHODS: Descriptive cross-sectional study carried out through review of the clinical microbiologic records at Complejo Asistencial Universitario de Salamanca. Culture, isolation, identification and serotyping were performed according to standard methodology. RESULTS: Salmonella was isolated in 1,477 patients, representing 47.7% of all positive stool cultures and 53.3% of all income bacterial AGE. The average prevalence was 42.1 cases/100,000 people per year. The mean age was 23 ± 28 years and the median 7 years. 40.2% of all isolates occurred in children under 5 years, with an average prevalence of 45.1 cases/ 10,000 people per year. Overall, the most frequently isolated serotype was S. Typhimurium with 57%, followed by S. Enteritidis with 35.8%. CONCLUSIONS: The prevalence of Salmonella decreased over time. The group aged 0-4 years had the highest rate throughout the period. However, Salmonella produced the highest percentage of hospitalizations for bacterial AGE. In recent years, S. Typhimurium serotype has replaced S. Enteritidis serotype and predominates in younger patients. It is observed under-reporting of cases of salmonellosis produced in Salamanca despite being mandatory notification of these since 2007.

Comparative study of bacterial translocation control with nitric oxide donors and COX2 inhibitor.

OBJECTIVE AND DESIGN: To evaluate the beneficial effects of exogenous NO and an inhibitor of the COX2, and their action levels in a model of SIRS/bacterial translocation (BT) induced by Zymosan A(®). MATERIAL AND METHODS: Ninety Wistar rats were submitted to different treatments, and after 12h and 24h they were anaesthetized in order to collect blood, mesenteric lymph nodes, and kidney for subsequent biochemical analyses and microbiological examinations. TREATMENTS: A nitric oxide donor, Molsidomine(®), was compared with a COX2 inhibitor, Celecoxib(®). METHODS: Zymosan A(®) was administered to Wistar rats. The animals were divided into 6 groups: one group for survival study, Group (1) No manipulation (BASAL); Group (2) vehicle of Zymosan A(®) given intraperitoneally (SHAM); Group I (control), with Zymosan A(®) (0.6g/kg) intraperitoneally; Group II (Molsidomine), with Molsidomine(®) (4mg/kg) through the penis dorsal vein, 30min prior to administration of the Zy(®) (0.6g/kg); Group III (Celecoxib), with Celecoxib(®) (400mg/kg) orally through a stomach tube, 6h prior to administration of the Zy (0.6g/kg). DETERMINATIONS: The parameters survival, bacterial translocation, renal function, neutrophil accumulation, oxygen free radicals (OFR), detoxifying enzymes, and cytokines were measured at different times after Zymosan administration. RESULTS: The model established induced a mortality rate of 100% and generated BT and systemic inflammatory response syndrome (SIRS) in all samples. It also significantly increased all variables, with p<.001 for MPO and all pro-inflammatory cytokines, and p<.01 for all OFR. Treatment with Molsidomine reduced mortality to 0%, decreased BT, MPO, pro-inflammatory cytokines and OFR (p<.001) significantly and increased IL-10 and IL-6 production. Moreover, the Celecoxib(®) showed a lower capacity for SIRS regulation. CONCLUSIONS: The exogenous administration of NO prevented BT and controlled SIRS. Therefore these results suggest that Molsidomine could be used as a therapeutic strategy to protect against BT.

Molecular characterization of a new N-acetylneuraminate synthase (NeuB1) from Idiomarina loihiensis.

N-Acetylneuraminate lyase synthase (NeuB; E.C. 2.5.1.56) is a key enzyme in pathogenic microorganisms for producing N-acetylneuraminic acid through the irreversible condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). However, nothing is known about this enzyme in non-pathogenic bacteria. This paper describes, for the first time, one of the two putative N-acetylneuraminate synthases from the halophilic non-pathogenic gamma-proteobacterium Idiomarina loihiensis NeuB1 (IlNeuB1). The obtained 95-kDa dimeric enzyme showed maximal activity at pH 7.0 and 40°C and was more stable at pH 8.0 (8 h half-life) than the previously described NeuB. Its catalytic efficiency towards ManNAc and PEP was 10- and 40-fold higher, respectively, than that determined for Campylobacter jejuni NeuB, but only half that found for Neisseria meningitidis NeuB towards PEP. The phylogenetic and structural analyses of NeuB enzymes revealed the new domain architecture 4 has no cystathionine-ß-synthase domain (cystathionine-ß-synthetase domain), unlike domain architecture 3. In addition, 10 conserved blocks (I-X) were found, and surprisingly, this study showed that the arginine essential for catalysis that is present in antifreeze-like domain (block X) was not fully conserved in NeuB, but is replaced by a serine in a long sequence (>700 residues) NeuB, such as that existing in domain architectures 3 and 4.

First functional and mutational analysis of group 3 N-acetylneuraminate lyases from Lactobacillus antri and Lactobacillus sakei 23K.

N-acetyl neuraminate lyases (NALs) catalyze the reversible aldol cleavage of N-acetyl neuraminic acid (Neu5Ac) to pyruvate and N-acetyl-D-mannosamine (ManNAc). Previous phylogenetic studies divided NALs into four different groups. Groups 1 and 2 have been well characterized at both kinetic and molecular levels, but no NAL from group 3 has been studied to date. In this work, a functional characterization of two group 3 members was performed using the recombinant NALs from Lactobacillus antri and Lactobacillus sakei 23K, revealing an optimal pH of between 6.0 and 7.0, low stability at basic pHs (>8.0), low optimal temperatures and, especially, low catalytic efficiency compared with their counterparts in group 1 and 2. The mutational analysis carried out showed that a plausible molecular reason for the low activity shown by Lactobacillus antri and Lactobacillus sakei 23k NALs compared with group 1 and 2 NALs could be the relatively small sugar-binding pocket they contain. A functional divergence analysis concluding that group 3 is more closely related to group 2 than to group 1.

[Antimicrobial susceptibility testing of anaerobic bacteria]. / Estudios de sensibilidad en bacterias anaerobias.

The anaerobic bacteria resistance to antibiotics is increasing, and even has appeared against the most active of those, like metronidazol and carbapenems. This fact forces to make and periodical sensibility tests -at least in the most aggressive and virulent species, in cases that they are isolated from life locations and in the absence of therapeutic response- to check the local sensibility and to establish suitable empiric therapies, all based on multicentric studies carried out in order to this or well to check the activity of new antibiotics. For the laboratory routine, the easiest sensibility method is the E-test/MIC evaluator. Another alternative is microdilution, that's only normalized for Bacteroides. There are preliminary facts that allow the use of disc diffusion method in some species of Bacteroides and Clostridium. For the temporal and multicentric studies, the procedure is dilution in agar plate, the reference method.

Estudios de sensibilidad en bacterias anaerobias / Antimicrobial susceptibility testing of anaerobic bacteria

La resistencia de las bacterias anaerobias a los antimicrobianos es creciente e incluso ha aparecido frente a los más activos, como metronidazol y carbapenémicos. Este hecho obliga a realizar pruebas de sensibilidad rutinarias -al menos en los casos en que se aíslan de infecciones graves en las especies más agresivas y virulentas y ante la falta de respuesta terapéutica- de forma periódica para comprobar la sensibilidad local y establecer terapias empíricas adecuadas, con independencia de los estudios multicéntricos realizados con estos fines o para comprobar la actividad de nuevos antimicrobianos. Para la rutina de laboratorio, el método de sensibilidad más sencillo es el de difusión a partir de tiras con gradientes exponenciales de antimicrobianos; una alternativa es la microdilución, particularmente a partir de placas comerciales, pero solo está normalizada para Bacteroides. Hay datos preliminares que permiten el uso del método de difusión disco placa en algunas especies de Bacteroides y Clostridium. Para los estudios periódicos y multicéntricos, el procedimiento es la dilución en agar, que es el método de referencia

The anaerobic bacteria resistance to antibiotics is increasing, and even has appeared against the most active of those, like metronidazol and carbapenems. This fact forces to make and periodical sensibility tests -at least in the most aggressive and virulent species, in cases that they are isolated from life locations and in the absence of therapeutic response- to check the local sensibility and to establish suitable empiric therapies, all based on multicentric studies carried out in order to this or well to check the activity of new antibiotics. For the laboratory routine, the easiest sensibility method is the E-test/MIC evaluator. Another alternative is microdilution, that's only normalized for Bacteroides. There are preliminary facts that allow the use of disc diffusion method in some species of Bacteroides and Clostridium. For the temporal and multicentric studies, the procedure is dilution in agar plate, the reference method

Estudios de sensibilidad en bacterias anaerobias / Antimicrobial susceptibility testing of anaerobic bacteria

La resistencia de las bacterias anaerobias a los antimicrobianos es creciente e incluso ha aparecido frente a los más activos, como metronidazol y carbapenémicos. Este hecho obliga a realizar pruebas de sensibilidad rutinarias -al menos en los casos en que se aíslan de infecciones graves en las especies más agresivas y virulentas y ante la falta de respuesta terapéutica- de forma periódica para comprobar la sensibilidad local y establecer terapias empíricas adecuadas, con independencia de los estudios multicéntricos realizados con estos fines o para comprobar la actividad de nuevos antimicrobianos. Para la rutina de laboratorio, el método de sensibilidad más sencillo es el de difusión a partir de tiras con gradientes exponenciales de antimicrobianos; una alternativa es la microdilución, particularmente a partir de placas comerciales, pero solo está normalizada para Bacteroides. Hay datos preliminares que permiten el uso del método de difusión disco placa en algunas especies de Bacteroides y Clostridium. Para los estudios periódicos y multicéntricos, el procedimiento es la dilución en agar, que es el método de referencia (AU)

The anaerobic bacteria resistance to antibiotics is increasing, and even has appeared against the most active of those, like metronidazol and carbapenems. This fact forces to make and periodical sensibility tests -at least in the most aggressive and virulent species, in cases that they are isolated from life locations and in the absence of therapeutic response- to check the local sensibility and to establish suitable empiric therapies, all based on multicentric studies carried out in order to this or well to check the activity of new antibiotics. For the laboratory routine, the easiest sensibility method is the E-test/MIC evaluator. Another alternative is microdilution, that's only normalized for Bacteroides. There are preliminary facts that allow the use of disc diffusion method in some species of Bacteroides and Clostridium. For the temporal and multicentric studies, the procedure is dilution in agar plate, the reference method (AU)

Kinetic study of hydroxytyrosol oxidation and its related compounds by Red Globe grape polyphenol oxidase.

Red Globe grape polyphenol oxidase, partially purified using phase partitioning with Triton-X114, was used to study the oxidation of hydroxytytosol (HT) and its related compounds tyrosol (TS), tyrosol acetate (TSA), and hydroxytyrosol acetate (HTA). The enzyme showed activity toward both monophenols (monophenolase activity) and o-diphenols (diphenolase activity) with a pH optimum (pH 6.5) that was independent of the phenol used. However, the optimal temperature for diphenolase activity was substrate-dependent, with a broad optimum of 25-65 °C for HT, compared with the maximum obtained for HTA (40 °C). Monophenolase activity showed the typical lag period, which was modulated by pH, substrate and enzyme concentrations, and the presence of catalytic amounts of o-diphenols. When the catalytic power (Vmax/K(M)) was determined for both activities, higher values were observed for o-diphenols than for monophenols: 9-fold higher for the HT/TS pair and 4-fold higher for HTA/TSA pair. Surprisingly, this ratio was equally higher for TSA (2.2-fold) compared with that of TS, whereas no such effect was observed for o-diphenols. This higher efficiency of TSA could be related to its greater hydrophobicity. Acetyl modification of these phenols not only changes the kinetic parameters of the enzyme but also affects their antioxidant activity (ORAC-FL assays), which is lower in HTA than in HT.

Biochemical and mutational analysis of a novel nicotinamidase from Oceanobacillus iheyensis HTE831.

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD(+) salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (kcat/Km  = 43.5 mM(-1)s(-1)) than pyrazinamidase (kcat/Km  = 3.2 mM(-1)s(-1)). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.

Insights into the evolution of sorbitol metabolism: phylogenetic analysis of SDR196C family.

BACKGROUND: Short chain dehydrogenases/reductases (SDR) are NAD(P)(H)-dependent oxidoreductases with a highly conserved 3D structure and of an early origin, which has allowed them to diverge into several families and enzymatic activities. The SDR196C family (http://www.sdr-enzymes.org) groups bacterial sorbitol dehydrogenases (SDH), which are of great industrial interest. In this study, we examine the phylogenetic relationship between the members of this family, and based on the findings and some sequence conserved blocks, a new and a more accurate classification is proposed. RESULTS: The distribution of the 66 bacterial SDH species analyzed was limited to Gram-negative bacteria. Six different bacterial families were found, encompassing α-, ß- and γ-proteobacteria. This broad distribution in terms of bacteria and niches agrees with that of SDR, which are found in all forms of life. A cluster analysis of sorbitol dehydrogenase revealed different types of gene organization, although with a common pattern in which the SDH gene is surrounded by sugar ABC transporter proteins, another SDR, a kinase, and several gene regulators.According to the obtained trees, six different lineages and three sublineages can be discerned. The phylogenetic analysis also suggested two different origins for SDH in ß-proteobacteria and four origins for γ-proteobacteria.Finally, this subdivision was further confirmed by the differences observed in the sequence of the conserved blocks described for SDR and some specific blocks of SDH, and by a functional divergence analysis, which made it possible to establish new consensus sequences and specific fingerprints for the lineages and sub lineages. CONCLUSION: SDH distribution agrees with that observed for SDR, indicating the importance of the polyol metabolism, as an alternative source of carbon and energy. The phylogenetic analysis pointed to six clearly defined lineages and three sub lineages, and great variability in the origin of this gene, despite its well conserved 3D structure. This suggests that SDH are very old and emerged early during the evolution. This study also opens up a new and more accurate classification of SDR196C family, introducing two numbers at the end of the family name, which indicate the lineage and the sublineage of each member, i.e, SDR196C6.3.

Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: new insights into the bacterial SDR sorbitol dehydrogenases family.

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described sequences.

Characterization of a novel N-acetylneuraminate lyase from Staphylococcus carnosus TM300 and its application to N-acetylneuraminic acid production.

The possibility of incorporating N-acetylneuraminic acid (Neu5Ac) in infant formulas and other functional foods has opened up the need to synthesize N-acetylneuraminic acid using N-acetylneuraminate lyases (NALs) by reversible aldol condensation of pyruvate and N-acetyl-d-mannosamine. Until now, NALs have been cloned from pathogenic microorganisms; however, this Article describes the expression and characterization of an N-acetylneuraminate lyase from the Staphylococcus carnosus TM300, a GRAS microorganism used in fermented meat. ScNAL showed a high level of expression in E. coli (403 mg L(-1) culture). This, combined with its simple two-step purification procedure, the highest recovery described to date (86%), its kinetic parameters, which are in the same order of magnitude as best reported NALs, and its optimum pH and temperature, make ScNAL a promising and cheap biocatalyst. To confirm its biotechnological potential, the Neu5Ac was synthesized in 3 h in simple industrial working conditions with a high degree of conversion (94%).

Properties of BoAGE2, a second N-acetyl-D-glucosamine 2-epimerase from Bacteroides ovatus ATCC 8483.

N-Acyl-D-Glucosamine 2-epimerase (AGE) catalyzes the reversible epimerization between N-acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc). Bacteroides ovatus ATCC 8483 shows 3 putative genes for AGE activity (BACOVA_00274, BACOVA_01795 and BACOVA_01816). The BACOVA_00274 gene encodes an AGE (BoAGE1) with strong similarity to the AGE previously characterized in Bacteroides fragilis. Interestingly, the BACOVA_01816 gene (BoAGE2) shares 57% identity with Anabaena sp. CH1 AGE, but has an extra 27-amino acid tag sequence in the N-terminal. When cloned and expressed in Escherichia coli Rosetta (DE3)pLys, BACOVA_01816 was able to convert ManNAc into GlcNAc and vice versa. It was stable over a broad range of pHs and its activity was enhanced by ATP (20 µM). The incubation with ATP stabilized its structure, raising its melting temperature by about 8 °C. In addition, the catalytic efficiency for ManNAc synthesis was higher than that for GlcNAc synthesis. These characteristics make BoAGE2 a promising biocatalyst for sialic acid production using cheap GlcNAc as starting material. BoAGE2 could be considered a Renin-binding Protein and its interaction with renin was studied for the first time in a prokaryotic AGE. Surprisingly, renin activated BoAGE2, an effect which is contrary to that described for mammalian AGE and unrelated with the unique N-terminal tag, since a mutant without this tag was also activated by renin. When BoAGE2 sequence was compared with other related (real and putative) AGE described in the databases, it was seen that AGE enzymes can be divided in 3 different groups. The relationship between these groups is also discussed.