Ontogeny of digestive enzymes in larvae of the Clown Anemonefish, Amphiprion ocellaris (Perciformes: Pomacentridae)

Introduction: The Clown anemonefish (Amphiprion ocellaris) is the most popular fish species in the marine aquarium trade; however, there is a lack of information on their digestive physiology during larval ontogeny, valuable information needed for diet design and management protocols. Objective: To characterize the early digestive enzymes of A. ocellaris larvae. Methods: We used three pools (10 larvae each) and extracted 10 samples per tank, from just before hatching to the 38th day. We analyzed the specific activity of acid and alkaline proteases, trypsin, chymotrypsin, leucine aminopeptidase and lipase; and did acid and alkaline protease zymograms. Results: We detected all measured enzymes at hatching. Acid proteases increased in activity until the 38th day. Alkaline proteases, trypsin, chymotrypsin, and leucine aminopeptidase had the same pattern, and maximum activity on the 8th day, decreasing at the 38th day. Lipase activity peaked on the 8th and 30th day. The acid zymogram had a single band, appearing on the 8th day. A total of eight alkaline proteases were revealed (154.2, 128.1, 104.0, 59.8, 53.5, 41.9, 36.5 and 25.1 KDa), with seven bands on the 1st day and all bands from the 3rd to 8th day, decreasing at two bands (41.9 and 25.1 KDa) in the 38th day. Conclusion: A. ocellaris has a functional stomach on the 8th day, and, on the 38th day, a digestive omnivore pattern with a tendency to carnivory.

Introducción: El pez payaso (Amphiprion ocellaris) es la especie de pez más popular en el comercio de acuarios marinos; sin embargo, falta información sobre su fisiología digestiva durante la ontogenia larval, información valiosa necesaria para protocolos de diseño y manejo dietético. Objetivo: Caracterizar las enzimas digestivas tempranas de larvas de A. ocellaris. Métodos: Usamos tres homogenados (con 10 larvas cada uno) y extrajimos 10 muestras por tanque, justo antes de la eclosión hasta el día 38. Analizamos la actividad específica de proteasas ácidas y alcalinas, tripsina, quimotripsina, leucina aminopeptidasa y lipasa; e hicimos zimogramas de proteasas ácidas y alcalinas. Resultados: Detectamos todas las enzimas medidas en la eclosión. La actividad de proteasas ácidas incrementó hasta el día 38. Proteasas alcalinas, tripsina, quimotripsina, y leucina aminopeptidasa tuvieron el mismo patrón, con actividad máxima en el octavo día, decreciendo en el día 38. Hubo picos en la actividad lipasa a los ocho y 30 días. El zimograma ácido tuvo una banda única, apareciendo al octavo día. Se hallaron ocho proteasas alcalinas (154.2, 128.1, 104.0, 59.8, 53.5, 41.9, 36.5 y 25.1 KDa), con siete bandas al primer día, y todas las bandas entre el tercer y octavo día, bajando a dos bandas (41.9 y 25.1 KDa) al día 38. Conclusión: A. ocellaris tiene un estómago funcional al octavo día, y, al día 38, un patrón digestivo omnívoro con tendencias carnívoras.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Isla Arena está localizada na coordenada 20°70’N - 90°45’W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70N - 90°45W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Identification by molecular techniques of halophilic bacteria producing important enzymes from pristine area in Campeche, Mexico / Identificação por técnicas moleculares de bactérias halofílicas que produzem enzimas importantes em área intocada de Campeche, México

Abstract Isla Arena is located in the coordinate 20° 70´ N - 90° 45´ W, from Campeche, Mexico. In these estuaries, the ocean mixes with fresh water, and ecosystems are concentrated where petenes and pink flamingos proliferate. Crustaceans and mollusks abound in the sea. Despite its enormous marine wealth, there are no studies carried out on which halophilic microorganisms are present in these waters. In this work, the diversity and structure of the microbial community was investigated through a metagenomics approach and corroborated for sequencing of 16S rRNA genes. It was found that the phylum Fimicutes predominates with more than 50%, in almost the same proportion of the class Bacilli and with almost 41% of relative abundance of the order Bacillales. The sequencing results showed that one of the samples presented a high percentage of similarity (99.75%) using the Nucleotide BLAST program with a peculiar microorganism: Bacillus subtilis. This microorganism is one of the best characterized bacteria among the gram-positive ones. Our results demonstrate that B. subtilis can be an efficient source of proteases, lipases and cellulases, from halophilic microbial communities located in poorly explored areas.

Resumo Isla Arena está localizada na coordenada 20°70'N - 90°45'W, de Campeche, México. Nesses estuários, o oceano se mistura com a água doce e os ecossistemas se concentram onde proliferam petenos e flamingos rosa. Crustáceos e moluscos abundam no mar. Apesar de sua enorme riqueza marinha, não há estudos realizados sobre a presença de microrganismos halofílicos nessas águas. Neste trabalho, a diversidade e estrutura da comunidade microbiana foram investigadas através de uma abordagem metagenômica e corroboradas para o sequenciamento de genes 16S rRNA. Verificou-se que o filo Fimicutes predomina com mais de 50%, quase na mesma proporção da classe Bacilli e com quase 41% de abundância relativa da ordem Bacillales. Os resultados do sequenciamento mostraram que uma das amostras apresentou alto percentual de similaridade (99,75%) pelo programa Nucleotide BLAST com um microrganismo peculiar: Bacillus subtilis. Nossos resultados demonstram que B. subtilis pode ser uma fonte eficiente de proteases, lipases e celulases, provenientes de comunidades microbianas halofílicas localizadas em áreas pouco exploradas.

Isolation and identification of proteolytic, amylolytic, lipolytic, and chitinolytic bacteria from shrimp waste / Aislamiento e identificación de bacterias proteolíticas, amilolíticas, lipolíticas y quitinolíticas de residuos de langostinos

Abstract Bacteria and microbial enzymes are biocatalysts and can be used as an alternative to industrial chemical processes. The present study focused on isolating and identifying bacterial strains from shrimp waste, that produce amylases, lipases, proteases and chitinases with potential use on shrimp waste treatment. Thirtytwo bacterial strains were isolated, phenotypically characterized, and identified by the API system and the molecular analysis of the 16S rDNA. It was found that 28.13% of the isolated bacterial strains had amylolytic capacity, 87.50% lipolytic, 96.88% proteolytic and 28.13% chitinolytic capacity on agar plates with specific substrates. The genera Bacillus, Burkholderia, Ochrobactrum, Vibrio, Pseudomonas and Shewanella were identified. Bacteria with enzymatic capacities isolated in the present study, could be used to obtain by-products from shrimp waste as well as other industrial applications.

Resumen Las bacterias y enzimas microbianas son biocatalizadores y pueden ser usadas como alternativa en los procesos químicos industriales. El presente estudio se centró en aislar e identificar cepas bacterianas a partir de desechos de langostinos, capaces de producir amilasas, lipasas, proteasas y quitinasas, que tuvieran potencial aplicación en el tratamiento de residuos de langostinos. Se aisló treinta y dos cepas bacterianas, caracterizadas fenotípicamente e identificadas mediante el sistema API 20 y mediante análisis molecular basado en el ADNr 16S. Se encontró que el 28.13% de las cepas bacterianas aisladas tenían capacidad amilolítica, 87.50% lipolítica, 96.88% proteolítica y 28.13% capacidad quitinolítica en placas de agar con sustratos específicos. Los géneros identificados fueron Bacillus, Burkholderia, Ochrobactrum, Vibrio, Pseudomonas y Shewanella. Las bacterias con capacidades enzimáticas aisladas en el presente estudio, podrían ser usadas para obtener subproductos de los desechos de langostinos, así como en otras aplicaciones industriales.

Caracterización, clasificación y usos de las enzimas lipasas en la producción industrial / Characterization, classification and uses of lipase enzymes in industrial production

Introducción: La bioquímica, como ciencia particular dentro de las ciencias médicas, ha tenido un gran desarrollo. Las enzimas lipasas se obtienen de organismos vivos que abundan en la naturaleza y han sido utilizadas en la producción de alimentos, jabones, detergentes, aceites y otros productos industriales. Actualmente se han logrado nuevas clasificaciones de estas, subdivididas en grupos y subgrupos. Se aprecia además interés de utilizarlas en la producción de biodiesel y en la biotecnología y genética médica. Objetivo: Recopilar las principales consideraciones teóricas actualizadas acerca la caracterización, clasificación y usos de las enzimas lipasas. Método: La búsqueda y análisis de la información se realizó desde el primero de septiembre al 23 de diciembre de 2019, con un total de 50 artículos publicados en las bases de datos PubMed, Hinari, SciELO y Medline, mediante el gestor de búsqueda y administrador de referencias EndNote. se utilizaron 42 citas seleccionadas para realizar la revisión, de ellas 38 de los últimos cinco años. Conclusiones: Las enzimas lipasas son proteínas que catalizan procesos biológicos. son activas en un amplio rango de sustrato, realizan reacciones de síntesis, hidrólisis o de intercambio de grupos. Poseen diversas actividades catalíticas, son menos costosas y menos contaminantes, se obtienen en gran cantidad, se producen de forma regular. Son estables y su proceso de producción es más factible y seguro. Se caracterizan por su capacidad de catalizar reacciones de acidólisis, alcohólisis, aminólisis, esterificación, interesterificación y transesterificación, entre otras características(AU)

Introduction: Biochemistry has experienced great development as a particular medical science. Lipase enzymes are obtained from living organisms which are abundant in nature, and have been used in the manufacture of foods, soap, detergents, oils and other industrial products. New classifications are now available of lipase enzymes, and they have been subdivided into groups and subgroups. An interest is also noticed in using them for biodiesel production and in biotechnology and medical genetics. Objective: Collect the main updated theoretical considerations about the characterization, classification and uses of lipase enzymes. Method: The search for and analysis of the information extended from 1 September to 23 December 2019, for a total 50 papers published in the databases PubMed, Hinari, SciELO and Medline, using the search engine and reference manager EndNote. Forty-two citations were selected for the review, 38 of which were from the last five years. Conclusions: Lipase enzymes are proteins that catalyze biological processes. They are active in a wide range of substrates, performing synthesis reactions, hydrolysis or group exchanges. They display a variety of catalytic activities, are less costly and less contaminating, are obtained in large quantities and are produced in a regular manner. They are stable and their production process is more feasible and safer. They are characterized by their ability to catalyze reactions of acidolysis, alcoholysis, aminolysis, esterification, interesterification and transesterification, among other characteristics(AU)

Enzymatic synthesis of 4-methoxycinnamoylglycerol Uv filter mediated by immobilized-lipase and nanoparticle formation on N-succinylchitosan / Síntesis enzimática del filtro UV 4-metoxicinamoilglicerol mediado por lipasa inmovilizada y formación de nanopartículas N-succinilquitosano

ABSTRACT The synthesis of 4-methoxycinnamoylglycerol takes advantage of the biodiesel subproduct for obtaining a hydrophilic UV cinnamate derivate filter, useful in sunscreen formulations. The objective here was to demonstrate that esterification of 4-methoxycinnamic acid and glycerol mediated by immobilized-lipase from Thermomyces lanuginosus is selective towards 4-methoxycinnamoylglycerol monoester UV filter, whose chemical characteristics favor the nanoparticles formation by ionotropic gelation on N-Succinyl chitosan. A cinnamic acid conversion (34% in hexane is higher than in other reports, without the presence of other sub-products or degradation products. This eases the purification process by liquid-liquid extraction. The free glyceryl entities favour its incorporation on N-Succinyl chitosan nanoparticles with size around 185(77nm, which are promissory for sunblock products.

RESUMEN En la síntesis de 4-metoxicinamoilglicerol, se aprovecha el subproducto de biodiesel para obtener un filtro UV hidrofílico, derivado de cinamato, útil en formulaciones de bloqueadores solares. El objetivo de este trabajo fue demostrar que la esterificación del ácido 4-metoxicinámico y el glicerol, mediado por la lipasa inmovilizada de Thermomyces lanuginosus, es selectiva hacia el monoester del filtro UV 4-metoxicinamoilglicerol, cuyas características químicas favorecen la formación de nanopartículas, por gelificación ionotrópica en N-succinil-quitosano. Una conversión de ácido cinámico (34% en hexano es mayor que los valores ya reportados, sin la presencia de otros subproductos o productos de degradación. Esto facilita, el proceso de purificación por extracción líquido-líquido. Las entidades de glicerilo libre favorecen su incorporación en nanopartículas de N-succinil-quitosano, con un tamaño de alrededor de 185±77nm, que son promisorias para los productos de protección solar.

Arthrographis curvata and Rhodosporidium babjevae as New Potential Fungal Lipase Producers for Biotechnological Applications

Abstract Fungi have always attracted a lot of attention as they are able to produce a vast repertoire of enzymes that find a broad spectrum of uses in biotechnological and industrial fields. Undoubtedly, one of the most promising biocatalysts is the lipase, which has been widely used for the biotransformation of a number of commercial products due to its high stability, high catalytic efficiency, versatility and selectivity, making it one of the most attractive and best-studied enzymes. In this study we report the isolation and molecular identification of new lipase-producing fungi from different environmental samples from Morocco. The production and activity of extracellular lipases, at different parameters, was evaluated using the Rhodamine B agar, submerged fermentation and biochemical methods. Two fungal strains Arthrographis curvata and Rhodosporidium babjevae, were isolated and found to produce large amounts of lipases. The optimal activity of the extracellular lipase was detected at 40°C and pH 9.0 for A. curvata and at 40 °C and pH 8.0 for R. babjevae. This study add new information at the growing list of fungal species producing lipases with improved physicochemical proprieties which could constitute a new line of research for further studies and to be exploited for industrial or bioremediation purposes.

Lipasas en síntesis de polímeros: avances y contribución a la química verde de polímeros / Lipases in polymer synthesis: advantages and its contribution to green polymer chemistry

RESUMEN Las lipasas han sido usadas en las últimas décadas como catalizadores eficientes en la síntesis enzimática de polímeros y gracias a características como alta selectividad, reciclabilidad, inocuidad y fácil separación/purificación se han convertido en una herramienta importante en el campo de los polímeros. En este trabajo se recopilan los desarrollos más importantes en el área y a su vez se muestra la tendencia actual de este campo de investigación.

ABSTRACT Lipases have been used in last decades as efficient catalysts in enzimatic polymer chemistry, thanks to its high selectivity, recyclabil-ity, safety and easy recovery/ separation, lipases have become in an important tool in polymer field. In this paper we compile the most important achievements in the enzimatic polymer chemistry and, at the same, time we show the current trend in this field of research.

Bioprospection of proteases from Halobacillus andaensis for bioactive peptide production from fish muscle protein

BACKGROUND: Biologically active peptides produced from fish wastes are gaining attention because their health benefits. Proteases produced by halophilic microorganisms are considered as a source of active enzymes in high salt systems like fish residues. Hence, the aim of this study was the bioprospection of halophilic microorganisms for the production of proteases to prove their application for peptide production. RESULTS: Halophilic microorganisms were isolated from saline soils of Mexico and Bolivia. An enzymatic screening was carried out for the detection of lipases, esterases, pHB depolymerases, chitinases, and proteases. Most of the strains were able to produce lipases, esterases, and proteases, and larger hydrolysis halos were detected for protease activity. Halobacillus andaensis was selected to be studied for proteolytic activity production; the microorganism was able to grow on gelatin, yeast extract, skim milk, casein, peptone, fish muscle (Cyprinus carpio), and soy flour as protein sources, and among these sources, fish muscle protein was the best inducer of proteolytic activity, achieving a protease production of 571 U/mL. The extracellular protease was active at 50°C, pH 8, and 1.4 M NaCl and was inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of H. andaensis was used to hydrolyze fish muscle protein for peptide production. The peptides obtained showed a MW of 5.3 kDa and a radical scavenging ability of 10 to 30% on 2,2-diphenyl-1-picrylhydrazyl and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and a ferric reducing ability of plasma. Conclusion: The use of noncommercial extracellular protease produced by H. andaensis for biologically active peptide production using fish muscle as the protein source presents a great opportunity for high-value peptide production.

Improvement of the alkali stability of Penicillium cyclopium lipase by error-prone PCR

BACKGROUND: Lipases are extensively exploited in lots of industrial fields; cold-adapted lipases with alkali-resistance are especially desired in detergent industry. Penicillium cyclopium lipase I (PCL) might be suitable for applications of detergent industry due to its high catalytic efficiency at low temperature and relatively good alkali stability. In this study, to better meet the requirements, the alkali stability of PCL was further improved via directed evolution with error-prone PCR. RESULTS: The mutant PCL (N157F) with an improved alkali stability was selected based on a high-throughput activity assay. After incubating at pH 11.0 for 120 min, N157F retained 70% of its initial activity, which was 23% higher than that of wild type PCL. Combined with the three-dimensional structure analysis, N157F exhibited an improved alkali stability under the high pH condition due to the interactions of hydrophilicity and ß-strand propensity. Conclusions: This work provided the theoretical foundation and preliminary data for improving alkali stability of PCL to meet the industrial requirements, which is also beneficial to improving alkali-tolerance ability of other industrial enzymes via molecular modification.

Lipase Production by Aspergillus niger C by Submerged Fermentation

Abstract This study aimed to evaluate the effects of variables on the process of lipases production by Aspergillus niger C by submerged fermentation (SmF). The production assays were performed in shake flasks for 72 hours at 150 rpm and 32°C. First, a fractional factorial design 25-1 (FFD) was carried out to evaluate the effect of the following process variables: sucrose, ammonium sulphate, soybean oil, yeast extract concentration and pH. After the selection of the variables that significantly influenced the lipase production, a central composite rotational design 22 (CCRD) was used, aiming to find the most favorable operational conditions. The selected assay condition (15.0 g.L-1 sucrose, 4.0 g.L-1 ammonium sulphate, 4.0 g.L-1 soybean oil and 1.0 g.L-1 yeast extract at pH 5.0) was the one that presented a lipase activity of 27.46 U.mL-1. It was very close to that best assay (30.76 U.mL-1), but using half of the inducer concentration, consequently reducing process cost. The kinetics of lipase production showed that the highest specific activity was 57.17 U.mg-1. The pH and temperature effects on lipase activity produced in this study was investigated. The optimum activity was found in a more acidic pH (5.0-6.0) and 55°C.

Fruit Peel from Astrocaryum aculeatum G. Mey. AS Substrate for Lipases Production by Aspergillus niger A2B1

The fruit of Astrocaryum aculeatum G. Mey. are consumed by population in the Brazil Northern resulting fruit peels. These peels are rich in lignocellulose and fat. The present study investigated peels from the Astrocaryum aculeatum G. Mey. as a substrate for lipases production by solid state bioprocess. To reach this objective: 1) we isolated fungi from peels from the fruit of Astrocaryum aculeatum G. Mey; 2) we screened the isolates for lipase production (screening in petri dish and screening in submerged bioprocess), 3) we investigated the production of lipases by using peels from Astrocaryum aculeatum G. Mey as substrate. The isolates belonged to the genera Aspergillus (16), Penicillium (3) and Fusarium (1). These strains were submitted to petri dishes and submerged fermentation for lipases production, these experiments resulted in the selection of five strains belonging to the Aspergillus genera. The lipases produced by these five strains performed enzymatic transesterification; however, the lipases from the strain Aspergillus niger A2B1 produced the highest ester content. The utilisation of fruit peel from Astrocaryum aculeatum G. Mey. as the main substrate, fruit peel from Astrocaryum aculeatum G. Mey. oil (13%), moisture (70%) and 75 h of incubation were the optimal conditions identified for the production of lipases by Aspergillus niger A2B1(17.42 U/g) in solid state bioprocess (SSB).

Response Surface Methodology (RSM) for analysing culture conditions of Acidocella facilis strain USBA-GBX-505 and Partial Purification and Biochemical Characterization of Lipase 505 LIP / Metodología de Superficie de Respuesta (RSM) para el análisis de las condiciones de cultivo de Acidocella facilis cepa USBA-GBX-505 y la purificación parcial y caracterización bioquímica de la lipasa 505 LIP / Metodologia de Superfície de Resposta (MSR) para a análise das condições de cultivo de Acidocella facilis cepa USBA-GBX-505 e purificação parcial e caracterização bioquímica da lip ase 505 LIP

Abstract Using Response Surface Methodology (RSM) we evaluated the culture conditions (nitrogen source, carbon source, pH and agitation rate) that increase the biomass of Acidocellafaalis strain USBA-GBX-505 and therefore enhance the production of its lipolytic enzyme, 505 LIP. RSM results revealed that yeast extract and agitation were key culture factors that increased the growth-associated lipolytic activity by 4.5-fold (from 0.13 U.mg-1 to 0.6 U.mg-1). The 505 LIP lipase was partially purified using size-exclusion chromatography and ion-exchange chromatography. Its molecular weight was >77 kDa. The enzyme shows its optimum catalytic activity at 55 °C and pH 7.5. EDTA, PMSF, 1-butanol and DMSO inhibited enzymatic activity, whereas Tween 20, acetone, glycerol and methanol increased it. Metallic ions are not required for the activity of 505 LIP, and even have an inhibitory effect on the enzyme. This study shows the potential use of A. facilis strain USBA- GBX-505 for the production of a newly identified lipolytic enzyme, 505 LIP, which is stable at moderate temperatures and in the presence of organic solvents. These are important characteristics for the synthesis of many useful products.

Resumen Por medio de la Metodología de Respuesta de Superficie (RSM) evaluamos las condiciones de cultivo (fuente de N, fuente de C, pH y tasa de agitación) que incrementan la biomasa de Acidocella facilis cepa USBA-GBX-505 y, como consecuencia, la producción de su enzima lipolítica, llamada 505 LIP. Los resultados de la RSM revelaron que el extracto de levadura y la agitación fueron factores de cultivo claves, que incrementaron de 4 a 5 veces la actividad lipolítica asociada al crecimiento (de 0.13 U.mg-1 a 0.6 U.mg-1). La lipasa 505 LIP se purificó parcialmente usando cromatografía de exclusión por tamaño y cromatografía de intercambio iónico. Su peso molecular fue > 77 kDa. La enzima muestra su actividad catalítica óptima a 55 °C y pH 7.5. El EDTA, el PMSF, el 1-butanol y el DMSO inhibieron la actividad enzimática, mientras que el Tween 20, la acetona, el glicerol y el metanol la incrementaron. La enzima 505 LIP no requiere iones metálicos para su actividad, e incluso se inhibe en presencia de ellos. Este estudio muestra el uso potencial de A. facilis cepa USBA-GBX-505 para la producción de una nueva enzima lipolítica, 505 LIP, que es estable a temperaturas moderadas y en la presencia de solventes orgánicos. Estas son características importantes en la síntesis de muchos productos útiles.

Resumo Utilizando a Metodologia de Superfície de Resposta (MSR) avaliamos as condições de cultivo (fontes de nitrogénio e carbono, pH e taxa de agitação) que aumentam a biomassa de Acidocella facilis cepa USBA-GBX-505, e, portanto, elevam a produção de sua enzima lipolítica 505 LIP. Os resultados da MSR revelaram que o extrato de levedura e a agitação foram fatores de cultivo chave que permitiram aumentar 4 a 5 vezes a atividade lipolítica associada ao crescimento (de 0,13 U.mg-1 a 0,6 U.mg-1). A lipase 505 LIP foi parcialmente purificada utilizando cromatografia por exclusão de tamanho e cromatografia de intercambio iónico. Seu peso molecular foi > 77 kDa. A enzima mostra sua atividade catalítica ótima a 55 °C e pH 7,5. EDTA, PMSF, 1-butanol e DMSO inibiram a atividade enzimática, enquanto que Tween 20, acetona, glicerol e metanol aumentaram esta atividade. Íons metálicos não são necessários para a atividade da 505 LIP, apresentando inclusive efeito inibitório da enzima. Este estudo demonstra o potencial uso de A. facilis cepa USBA-GBX-505 para a produção de uma nova enzima lipolítica, 505 LIP, a qual é estável a moderadas temperaturas e na presença de solventes orgánicos. Estas características são importantes para a síntese de diversos produtos úteis.

Enzyme Therapy: Current Perspectives.

Enzymes control all metabolic processes in human system from simple digestion of food to highly complex immune response. Physiological reactions occuring in healthy individuals are disturbed when enzymes are deficient or absent. Enzymes are administered for normalizing biological function in certain pathologies. Initially, crude proteolytic enzymes were used for the treatment of gastrointestinal disorders. Recent advances have enabled enzyme therapy as a promising tool in the treatment of cardiovascular, oncological and hereditary diseases. Now, a spectrum of other diseases are also covered under enzyme therapy. But, the available information on the use of enzymes as therapeutic agents for different diseases is scanty. This review details the enzymes which have been used to treat various diseases/disorders.

Effects of Angelica dahurica on obesity and fatty liver in mice / 中国天然药物

Angelica dahurica (A. dahurica) is a traditional Chinese medicinal plant being used in clinical practice. The present study demonstrated that A. dahurica could reduce white-fat weight in high-fat-diet hyperlipidemic mice, decrease total cholesterol and triglyceride concentrations in the livers of both high-fat-diet and Triton WR1339 induced hyperlipidemic mice, and enhance the total hepatic lipase activities of them. These findings were further supported by the results derived from the experiments with HepG2 cells in vitro. In addition, the proteins related to lipids metabolism were investigated using LC-MS/MS, indicating that genes of lipid metabolism and lipid transport were regulated by A. dhurica. The results from LC-MS/MS were further conformed by Western blot and real time PCR assays. A. dahurica could down-regulate the expression of catalase (CAT) and sterol carrier protein2 (SCP2) and up-regulate the expression of lipid metabolism related genes-lipase member C (LIPC) and peroxisome proliferator-activated receptor gamma (PPARγ). In the Triton WR1339 mouse liver and HepG2 cells in vitro, A. dahurica was able to increase the expression of LIPC and PPARγ, confirming the results from in vivo experiments. Imperatorin showed the same activity as A. dahurica, suggesting it was one of the major active ingredients of the herb. In conclusion, our work represented a first investigation demonstrating that A. dahurica was able to regulate lipid metabolism and could be developed as a novel approach to fighting against fatty liver and obesity.

The potential use of lipases in the production of fatty acid derivatives for the food and nutraceutical industries

The potential of lipases (E.C.3.1.1.3.) as biocatalysts for the production of fatty acid derivatives for the food and nutraceutical industries, such as flavouring esters, fatty acid esters of antioxidants and structured lipids, is enormous, mainly due to their high regio- and stereo-selectivities, in addition to the other well-known advantages of enzymatic processes. The replacement of chemical catalysts by lipases presents great benefits in terms of the nutritional properties of the obtained products and environmental care. The reactions performed for the production of these compounds, as well as the best operation conditions, the biocatalysts used, reactor types and operation mode, are addressed in this review.

Characterization of cellulase, hemicellulase and lipase and its use in deinking of laser printed paper

Aims: It is recognized that laser printed paper are difficult to deink using conventional method. This had lead to the suggestion of enzymatic approach to overcome the problem encountered by commonly employed deinking techniques. The present study aimed to investigate 7 commercially available enzymes for their suitability use in deinking of laser printed paper. Methodology and results: 3 cellulases, hemicellulases, xylanase and 2 lipases were used in enzymatic deinking of laser-printed wastepaper. Cellulase A “Amano”3 (C), Hemicellulase (H) and lipase (L) were selected for used in deinking because they possess either highest activity or broad pH stability compared to others enzymes. Different combination of enzymes was carried out to evaluate their effectiveness in deinking process. CH enzymes sequence was determined to be the most effective sequence in toner removal with 1.90% of brightness increment. However, only 0.95% of brightness increment was gained by enzyme sequence L. Highest deinking efficiency obtained was not proportional to the highest total reducing sugar produced. Conclusion, significance and impact of study: Enzyme (cellulase and hemicellulase) can be used to de-ink laserprinted wastepaper, which are difficult to be deinked by conventional chemical deinking process. Thus, enzyme deinking has high possibility as alternative method to current chemical deinking process which is not environmental friendly.

Bioprospection of cellulolytic and lipolytic South Atlantic deep-sea bacteria

Background: Cellulases and lipases have broad industrial application, which calls for an urgent exploration of microorganisms from extreme environments as valuable source of commercial enzyme. In this context, the present work describes the bioprospection and identification of deep-sea bacteria that produce cellulases and lipases, as well their optimal temperature of activity. Results: The first step of this study was the screening of cellulolytic and lipolytic deep-sea bacteria from sediment and water column, which was conducted with substrates linked with 4-Methylumbelliferyl. Among the 161 strains evaluated, 40 were cellulolytic, 23 were lipolytic and 5 exhibited both activities. Cellulolytic and lipolytic bacteria are more common in sediment than at the water column. Based on the ability to produce cellulases and lipases three isolates were selected and identified (16S rRNA sequencing) as Bacillus stratosphericus, B. aerophilus and B. pumilus. Lipases of strain B. aerophilus LAMA 582 exhibited activity at a wide temperature range (4º to 37ºC) and include psychrophilic behaviour. Strain Bacillus stratosphericus LAMA 585 can growth in a rich (Luria Bertani) and minimal (Marine Minimal) medium, and does not need an inducer to produce its mesophilic cellulases and lipases. Conclusions: Deep-sea sediments have great potential for bioprospection of cellulase and lipase-producing bacteria. The strains LAMA 582 and LAMA 585 with their special features, exhibit a great potential to application at many biotechnology process.

Penicillium species present in Uruguayan salami / Especies de Penicillium presentes en salamines uruguayos

The surface coverage of certain dry fermented sausages such as Italian salami by some species of Penicillium provides their characteristic flavor and other beneficial properties. One of them is the protective effect by means of a uniform film of white mold against undesirable microorganisms. The aim of this work was to identify and to isolate the fungal species present in mature Italian type of salami and to evaluate if it is possible to obtain some of them as starters. In addition, the effects of temperature (14 °C and 25 °C), water activity (a w) (0.90, 0.95 and 0.995) and 2.5 % sodium chloride (NaCl) on fungal growth were determined. Similarly, the proteolytic and lipolytic activity and the ability to produce toxic secondary metabolites were evaluated in order to characterize some possible starter strain. All species found belong to the genus Penicillium, including a performing starter as Penicillium nalgiovense and some potentially toxicogenic species. All the strains showed a higher growth rate at 25 °C. The production of extracellular proteases and lipases was significantly higher at 25 °C than at 14 °C with and without sodium chloride. Only Penicillium expansum produced patulin. On the other hand, Penicillium griseofulvum was the only species that produced ciclopiazonic acid but none of the strains produced penicillin. The species present on salami, Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum and Penicillium puberulum were unable to produce any of the evaluated toxins. These findings suggest that some fungal isolates from the surface of salami such as P. nalgiovense are potentially useful as starters in sausage manufacture.

La cobertura de la superficie de los embutidos fermentados secos -como el salamín tipo italiano- por algunas especies de Penicillium les proporciona un sabor característico y otras propiedades beneficiosas. Una de ellas es el efecto de protección contra microorganismos indeseables, al formarse una película blanca uniforme de mohos. El objetivo de este trabajo fue aislar e identificar los hongos filamentosos encontrados en la superficie de salamines tipo italiano y evaluar la posibilidad de obtener especies para utilizarse como cultivos iniciadores. Se determinó el efecto de la temperatura, la actividad de agua y del cloruro de sodio sobre el crecimiento fúngico. La actividad proteolítica y lipolítica y la capacidad de producir metabolitos secundarios tóxicos fueron evaluadas con el fin de caracterizar algunos posibles cultivos iniciadores. Todas las cepas fúngicas aisladas e identificadas correspondieron a especies del género Penicillium, algunas benéficas, como Penicillium nalgiovense, y otras potencialmente toxicogénicas. Estas cepas tuvieron diferentes tasas de crecimiento en respuesta a las diferentes condiciones de cultivo. Todas las cepas mostraron mayor crecimiento a 25 °C. La producción de proteasas y lipasas extracelulares fue significativamente mayor a 25 °C que a 14 °C. Penicillium expansum fue la única especie que produjo patulina y Penicillium griseofulvum fue la única que produjo ácido ciclopiazónico. Ninguna de las especies produjo penicilina. Penicillium nalgiovense, Penicillium minioluteum, Penicillium brevicompactum y Penicillium puberulum no produjeron ninguna de las toxinas evaluadas. Estos resultados sugieren que algunos aislamientos fúngicos, como P. nalgiovense, son potencialmente útiles como cultivos iniciadores en la fabricación de estos productos.