Publication:
Partial purification of agarases from halophilic microorganisms isolated from marine and hypersaline environments in Puerto Rico
Partial purification of agarases from halophilic microorganisms isolated from marine and hypersaline environments in Puerto Rico
Authors
Oyola Rivera, Miguel X.
Embargoed Until
Advisor
Montalvo Rodríguez, Rafael R.
College
College of Arts and Sciences - Sciences
Department
Department of Biology
Degree Level
M.S.
Publisher
Date
2017
Abstract
Las agarasas son enzimas que son capaces de catalizar la hidrólisis de agar. Se clasifican en dos grupos, que son α-agarasa y β-agarasa, de acuerdo con el patrón de escisión. Varias agarasas se han aislado de diferentes géneros de bacterias que se encuentran en agua de mar y sedimentos marinos, así como microorganismos modificados genéticamente. El agar es un producto ficocoloide extraído de la pared celular de un grupo de algas rojas (Rhodophyceae) que incluye especies de Gelidium y Gracilaria. Estudios recientes han demostrado el gen de agarasa (AgaV) puede ser útil en dos aspectos: en primer lugar, como una enzima agarolítica, el AgaV recombinante purificada podría ser empleada en la recuperación de ADN a partir de geles de agarosa; en segundo lugar, como una proteína de secreción, AgaV se exploró en el nivel genético y se utilizó como un reportero en la construcción de una trampa de señal de secreción, que resultó ser una herramienta molecular simple y eficiente para la selección de genes que codifican proteínas de secreción de bacterias tanto en Gram-positivas y Gram-negativas. Según nuestro conocimiento, no se han publicado estudios sobre agarasas en Puerto Rico y debido a esto, el propósito de este estudio es examinar las muestras de agua que fueron tomadas de ambientes marinos (3.5% - 5.0% w / v de NaCl) y halófilicos (6% -30% w / v de NaCl) de Puerto Rico. Las muestras fueron recolectadas en bolsas estériles Whirl-pak® y fueron transportadas al laboratorio para ser procesadas. Las muestras de agua fueron concentradas por filtración con una membrana de 0.45 μm. Los aislados fueron crecidos en medio con extracto de levadura, medio sin extracto de levadura, medio con extracto de levadura y agarosa, y medio sin extracto de levadura con agarosa molecular, demostrando que pueden degradar el agar en medios con o sin nutrientes. El medio de crecimiento es una versión modificada del medio “Synthetic Crenarcheota” (Könneke et al., 2005). Un total de 55 aislados fueron obtenidos del muestreo, 17 de éstos demostraron la capacidad de degradar agar. Los aislados fueron caracterizados utilizando propiedades físicas, moleculares y morfológicas. Se realizó un análisis del gen de ARN ribosomal 16S, luego se analizó el ADN metagenómico utilizando la reacción de polimerasa en cadena, lo cual demostró la presencia de bacterias y arqueas halofílicas. Los resultados preliminares indican que tenemos 17 aislados que pertenecen al dominio Bacteria y tienen actividad de agarasa. Este experimento puede contribuir al esclarecimiento sobre la diversidad y función de la enzima agarasa en ambientes halófilicos en Puerto Rico. El aislado MD25A produce una agarasa tipo beta, la cual exhibe una salinidad óptima de 1.5 M NaCl, una temperatura óptima de 37⁰C, un pH de 8, es termoestable y tiene una vida media por 60 min a 50⁰C. La producción inducida de la agarasa se hizo en el medio HA-I que contenía una solución al 0.2% de agarosa. La enzima purificada resultó ser homogénea y su peso molecular es de aproximadamente 80 kDa, como se observó en el “SDS-PAGE”. Los oligosacáridos producidos por la degradación del agar fueron analizados por cromatografía de capa fina. La agarasa generó los productos de degradación en el siguiente orden: neoagarohexaosa, neoagarotetraosa y una pequeña cantidad de neoagarobiosa. Estos resultados sugieren que nuestra enzima es una β-agarasa.
Agarases are enzymes that can catalyze the hydrolysis of agar. They are classified in two groups, which are α-agarases and β-agarases, according to the cleavage pattern. Several agarases have been isolated from different bacterial genera found in seawater and marine sediments, as well as engineered microorganisms. Agar is a phycocolloid product extracted from the cell wall of a group of red algae (Rhodophyceae), including Gelidium and Gracilaria. Recent studies have demonstrated that the agarase gene (AgaV) is useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both Gram-positive and Gram-negative bacteria. To our knowledge, studies of agarases have not been conducted in Puerto Rico and because of that, the purpose of this study is to examine water samples that were taken from marine (3.5% - 5.0% w/v NaCl) and hypersaline (6.0% - 30.0% w/v NaCl) environments from Puerto Rico. The samples were collected in sterile bags Whirl-pak® and transported to the laboratory for processing. Additionally, water samples were concentrated by filtration using a 0.45 μm nitrocellulose membrane. The isolates were grown on media with yeast extract, media without yeast extract, media with yeast extract and molecular agarose, and media without yeast extract, but with molecular agarose to demonstrate agar hydrolisis even on media with or without nutrients. The culture medium is a modified version of Synthetic Crenarcheota (Könneke et al., 2005). A total of 55 isolates were obtained from these samplings, which 17 of these showed agarase activity. The isolates were characterized using physiological, molecular and morphological properties. PCR analysis of the 16S rRNA gene on the genomic DNA showed that isolates belong to the Bacteria domain. Preliminary results have shown that we have isolated 17 halophilic bacteria with agarase activity. This study can contribute in understanding the diversity and function of the enzyme agarase in saline to hypersaline environments from Puerto Rico. The isolate MD25A produces a β-type agarase that exhibits its best activity at 1.5 M NaCl, an optimal temperature of 37⁰C, an optimal pH of 8, and has thermostability (half life for 60 min at 50⁰C). The induced agarase production in the HA-I medium was made with a 0.2% agarose solution. The purified enzyme was homogeneous with a molecular weight of approximately 80 kDa, as presented by SDS-PAGE. The oligosaccharides produced by the degradation of agar were analyzed by Thin Layer Chromatography. The enzyme released its products in the following order: neoagarohexaose, neoagarotetraose and very small quantity of neoagarobiose. As a result, the data suggest that our enzyme is a β-type agarase.
Agarases are enzymes that can catalyze the hydrolysis of agar. They are classified in two groups, which are α-agarases and β-agarases, according to the cleavage pattern. Several agarases have been isolated from different bacterial genera found in seawater and marine sediments, as well as engineered microorganisms. Agar is a phycocolloid product extracted from the cell wall of a group of red algae (Rhodophyceae), including Gelidium and Gracilaria. Recent studies have demonstrated that the agarase gene (AgaV) is useful in two aspects: first, as an agarolytic enzyme, the purified recombinant AgaV could be employed in the recovery of DNA from agarose gels; second, as a secretion protein, AgaV was explored at the genetic level and used as a reporter in the construction of a secretion signal trap which proved to be a simple and efficient molecular tool for the selection of genes encoding secretion proteins from both Gram-positive and Gram-negative bacteria. To our knowledge, studies of agarases have not been conducted in Puerto Rico and because of that, the purpose of this study is to examine water samples that were taken from marine (3.5% - 5.0% w/v NaCl) and hypersaline (6.0% - 30.0% w/v NaCl) environments from Puerto Rico. The samples were collected in sterile bags Whirl-pak® and transported to the laboratory for processing. Additionally, water samples were concentrated by filtration using a 0.45 μm nitrocellulose membrane. The isolates were grown on media with yeast extract, media without yeast extract, media with yeast extract and molecular agarose, and media without yeast extract, but with molecular agarose to demonstrate agar hydrolisis even on media with or without nutrients. The culture medium is a modified version of Synthetic Crenarcheota (Könneke et al., 2005). A total of 55 isolates were obtained from these samplings, which 17 of these showed agarase activity. The isolates were characterized using physiological, molecular and morphological properties. PCR analysis of the 16S rRNA gene on the genomic DNA showed that isolates belong to the Bacteria domain. Preliminary results have shown that we have isolated 17 halophilic bacteria with agarase activity. This study can contribute in understanding the diversity and function of the enzyme agarase in saline to hypersaline environments from Puerto Rico. The isolate MD25A produces a β-type agarase that exhibits its best activity at 1.5 M NaCl, an optimal temperature of 37⁰C, an optimal pH of 8, and has thermostability (half life for 60 min at 50⁰C). The induced agarase production in the HA-I medium was made with a 0.2% agarose solution. The purified enzyme was homogeneous with a molecular weight of approximately 80 kDa, as presented by SDS-PAGE. The oligosaccharides produced by the degradation of agar were analyzed by Thin Layer Chromatography. The enzyme released its products in the following order: neoagarohexaose, neoagarotetraose and very small quantity of neoagarobiose. As a result, the data suggest that our enzyme is a β-type agarase.
Keywords
Agarases,
Halophilic microorganisms,
Hypersaline environments,
Puerto Rico
Halophilic microorganisms,
Hypersaline environments,
Puerto Rico
Usage Rights
Persistent URL
Cite
Oyola Rivera, M. X. (2017). Partial purification of agarases from halophilic microorganisms isolated from marine and hypersaline environments in Puerto Rico [Thesis]. Retrieved from https://hdl.handle.net/20.500.11801/727