Lipolytic activity from bacteria prospected in polluted portuary sites
Enzymes are the most remarkable and highly specialized proteins, presenting in most cases a catalytic efficiency much higher than the majority of synthetic catalyzers, being their industrial production mainly ensured by some species of microorganisms. Marine bacteria present in extreme environments...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2014-06-01
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| Series: | Frontiers in Marine Science |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/conf.fmars.2014.02.00012/full |
| _version_ | 1811273162984259584 |
|---|---|
| author | Kaori Levy Fonseca |
| author_facet | Kaori Levy Fonseca |
| author_sort | Kaori Levy Fonseca |
| collection | DOAJ |
| description | Enzymes are the most remarkable and highly specialized proteins, presenting in most cases a catalytic efficiency much higher than the majority of synthetic catalyzers, being their industrial production mainly ensured by some species of microorganisms.
Marine bacteria present in extreme environments tend to develop adaptation mechanisms to cope with adverse physico-chemical conditions or contaminated scenarios (Haller et al., 2011) which may confer the potential to produce relatively more active and/or stable enzymes under those conditions, hence being able to produce extracellular enzymes with high biotechnological and industrial potential. The singularity of these organisms outcome from specific properties of adaptation, providing them the ability to develop in extreme conditions, as elevated salinity and pressure, low temperatures and irregular illuminating conditions (Zhang & Kim, 2010).
Lipases, triacylglycerol acylhydrolases, are ubiquitous enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol under aqueous conditions. Besides this, lipases are also efficient in various reactions such as esterification and transesterification aminolysis and can exhibit chemo-, regio- and enantioselectivity. Preferably, lipases are enzymes capable of hydrolyzing carboxyl esters (≥ 10 carbon atoms), while esterases hydrolyze carboxyl esters of short-chain acylglycerol. Lipases are by and large produced from microbes and are mostly extracellular. These multifaceted properties make lipases one of the most used biocatalysts for industrial purposes, in particular in oils and fats industry, digestive enzymes production, food additives, reagents for clinical analysis, detergents, in biodegradation of plastics, as PHA and PCL, in wastewater treatment, and more recently in biodiesel production (Lailaja & Chandrasekaran, 2013), replacing many of the chemical agents used and elevating industry to another ecological step in order to energetic and contaminating costs.
Tributyltin (TBT) despite the recent ban of its usage has been considered to be one of the most toxic substances ever introduced into the marine environment. TBT is a tin compound that has been widely used in wood preservation, as an antifungal in textiles in industrial wastewater systems, the paper industry and as anti-fouling in boat painting, the latter being the major input of marine contamination. It is toxic to many aquatic organisms, including bacteria, algae, zooplankton and mollusks, affecting growth, development, reproduction (imposex - phenotypic appearance of male characteristics in female gastropods) and survival of many marine species.
The aim of this study was to isolate and identify the TBT resistant marine bacteria capable to produce extracellular lipases and then test the lipolytic activity of their extracts. For this purpose, TBT resistant bacteria (able to grow at 3 mM TBT) from 7 Portuguese harbors were collected (Póvoa de Varzim (V; 41.376120,-8.766945), Leixões (L; 41.195238,- 8.684177), Aveiro (A; 40.645899,-8.727098), Figueira da Foz (F; 40.146848,-8.849176), Peniche (P; 39.355422,-9.375479), Setúbal (St; 38.521228,-8.887277) e Sines (S; 37.950219,-8.864599)), isolated and then REP-PCR characterized. Their extracellular lipase activity was assayed by the method of Rhodamine B in solid culture medium. Rhodamine is a dye which together with fatty acids released by the hydrolysis of triacylglycerols, forms a fluorescent complex when exposed to ultraviolet light. The use of this test was due to its sensitivity in detecting lipase activity even in organisms with low production of extracellular lipases. Lipolytic extracts activities were estimated using p-nitrophenyl palmitate method for optimization of activity conditions. This highly sensitive spectrophotometric method estimates the amount of p-nitrophenol (p-NP) released during the hydrolysis of the substrate p-nitrophenyl palmitate (p-NPP). Isolates producing extracellular lipases were then identified by MALDI-TOF-MS.
The concentration of TBT resistant isolates varied between 0.08% (in Setúbal harbor) and 7.67% (Peniche) (Monteiro et al, 2011). From a total of 111 different isolates, 10 were able to produce extracellular lipases - belonging to <i>Serratia</i> and <i>Pseudomonas</i> genus. The F3, A2, L2 and V25 isolates were those with higher potential in the production of lipolytic enzymes. Two operational conditions (temperature and pH) were studied to optimize lipase activity for these isolates. Temperature was varied between 8.9ºC and 51.2ºC. However, only isolates F3, L2 and V25 presented activity and only in the range of 30ºC.
At constant temperature (30º) the pH was varied (7.5, 9 and 10.5). Figure 1 depicts the effect of pH variation in the lipolytic extracts activity. For the A2 extract, no activities, for any of the pH established, have been recorded, although it showed significant activity through Rhodamine B assay. This can be explained by probable loss of lipase activity during the study, demonstrating to be less stable over time. To maintain their activity and operational stability, many biocatalysts are immobilized in specific supports, preventing them from degradation and inactivation in the reactional medium. Free enzymes are more labile and vulnerable to degradation. On the other hand, at pH 9 the lipolytic extracts, F3 and L2, exhibited activity of 8U/mL and 4 U/mL, respectively, demonstrating that these lipases are preferentially alkaline, whereas V25 showed greater activity at pH 7.5 and is considered preferentially neutral lipases. Most microbial lipases have optimal activity in a pH range of from 7 to 9 (Ulker & Karaoğlu, 2012), which further supports the absence of activity at pH 10.5.
The identified species, from the <i>Pseudomonas</i> and <i>Serratia</i> genus are currently representative sources for the production of extracellular lipases (Hasan et al., 2006; Kuddus & Ramteke, 2012), with high applicability in the detergent industry. They are for the most pathogenic, being related to nosocomial infections and antibiotic resistance (Kurz et al., 2003), and it is known that the production of extracellular lipases by microorganisms is closely related to the infectious potential and some are important in these same processes.
This study demonstrates that these TBT resistant isolates have, at the same time, the capacity to produce enzymes with a large biotechnological potential but, nevertheless, their relationship is not well understood, representing a novel approach. It is expected for these organisms to produce highly biotechnological relevant biocatalysts, due to their severe adaptations (Suehiro et al., 2007). The fully characterization of these lipases, mostly for F3 with elevated lipolytic activity exhibited, presents also a future challenge. |
| first_indexed | 2024-04-12T22:53:05Z |
| format | Article |
| id | doaj.art-fcb3e8f0fdfc4fc7b38b56f4548f5118 |
| institution | Directory Open Access Journal |
| issn | 2296-7745 |
| language | English |
| last_indexed | 2024-04-12T22:53:05Z |
| publishDate | 2014-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Marine Science |
| spelling | doaj.art-fcb3e8f0fdfc4fc7b38b56f4548f51182022-12-22T03:13:17ZengFrontiers Media S.A.Frontiers in Marine Science2296-77452014-06-01110.3389/conf.fmars.2014.02.00012100154Lipolytic activity from bacteria prospected in polluted portuary sitesKaori Levy Fonseca0ESTM & GIRM, Polytechnic Institute of LeiriaEnzymes are the most remarkable and highly specialized proteins, presenting in most cases a catalytic efficiency much higher than the majority of synthetic catalyzers, being their industrial production mainly ensured by some species of microorganisms. Marine bacteria present in extreme environments tend to develop adaptation mechanisms to cope with adverse physico-chemical conditions or contaminated scenarios (Haller et al., 2011) which may confer the potential to produce relatively more active and/or stable enzymes under those conditions, hence being able to produce extracellular enzymes with high biotechnological and industrial potential. The singularity of these organisms outcome from specific properties of adaptation, providing them the ability to develop in extreme conditions, as elevated salinity and pressure, low temperatures and irregular illuminating conditions (Zhang & Kim, 2010). Lipases, triacylglycerol acylhydrolases, are ubiquitous enzymes that catalyze the breakdown of fats and oils with subsequent release of free fatty acids, diacylglycerols, monoglycerols and glycerol under aqueous conditions. Besides this, lipases are also efficient in various reactions such as esterification and transesterification aminolysis and can exhibit chemo-, regio- and enantioselectivity. Preferably, lipases are enzymes capable of hydrolyzing carboxyl esters (≥ 10 carbon atoms), while esterases hydrolyze carboxyl esters of short-chain acylglycerol. Lipases are by and large produced from microbes and are mostly extracellular. These multifaceted properties make lipases one of the most used biocatalysts for industrial purposes, in particular in oils and fats industry, digestive enzymes production, food additives, reagents for clinical analysis, detergents, in biodegradation of plastics, as PHA and PCL, in wastewater treatment, and more recently in biodiesel production (Lailaja & Chandrasekaran, 2013), replacing many of the chemical agents used and elevating industry to another ecological step in order to energetic and contaminating costs. Tributyltin (TBT) despite the recent ban of its usage has been considered to be one of the most toxic substances ever introduced into the marine environment. TBT is a tin compound that has been widely used in wood preservation, as an antifungal in textiles in industrial wastewater systems, the paper industry and as anti-fouling in boat painting, the latter being the major input of marine contamination. It is toxic to many aquatic organisms, including bacteria, algae, zooplankton and mollusks, affecting growth, development, reproduction (imposex - phenotypic appearance of male characteristics in female gastropods) and survival of many marine species. The aim of this study was to isolate and identify the TBT resistant marine bacteria capable to produce extracellular lipases and then test the lipolytic activity of their extracts. For this purpose, TBT resistant bacteria (able to grow at 3 mM TBT) from 7 Portuguese harbors were collected (Póvoa de Varzim (V; 41.376120,-8.766945), Leixões (L; 41.195238,- 8.684177), Aveiro (A; 40.645899,-8.727098), Figueira da Foz (F; 40.146848,-8.849176), Peniche (P; 39.355422,-9.375479), Setúbal (St; 38.521228,-8.887277) e Sines (S; 37.950219,-8.864599)), isolated and then REP-PCR characterized. Their extracellular lipase activity was assayed by the method of Rhodamine B in solid culture medium. Rhodamine is a dye which together with fatty acids released by the hydrolysis of triacylglycerols, forms a fluorescent complex when exposed to ultraviolet light. The use of this test was due to its sensitivity in detecting lipase activity even in organisms with low production of extracellular lipases. Lipolytic extracts activities were estimated using p-nitrophenyl palmitate method for optimization of activity conditions. This highly sensitive spectrophotometric method estimates the amount of p-nitrophenol (p-NP) released during the hydrolysis of the substrate p-nitrophenyl palmitate (p-NPP). Isolates producing extracellular lipases were then identified by MALDI-TOF-MS. The concentration of TBT resistant isolates varied between 0.08% (in Setúbal harbor) and 7.67% (Peniche) (Monteiro et al, 2011). From a total of 111 different isolates, 10 were able to produce extracellular lipases - belonging to <i>Serratia</i> and <i>Pseudomonas</i> genus. The F3, A2, L2 and V25 isolates were those with higher potential in the production of lipolytic enzymes. Two operational conditions (temperature and pH) were studied to optimize lipase activity for these isolates. Temperature was varied between 8.9ºC and 51.2ºC. However, only isolates F3, L2 and V25 presented activity and only in the range of 30ºC. At constant temperature (30º) the pH was varied (7.5, 9 and 10.5). Figure 1 depicts the effect of pH variation in the lipolytic extracts activity. For the A2 extract, no activities, for any of the pH established, have been recorded, although it showed significant activity through Rhodamine B assay. This can be explained by probable loss of lipase activity during the study, demonstrating to be less stable over time. To maintain their activity and operational stability, many biocatalysts are immobilized in specific supports, preventing them from degradation and inactivation in the reactional medium. Free enzymes are more labile and vulnerable to degradation. On the other hand, at pH 9 the lipolytic extracts, F3 and L2, exhibited activity of 8U/mL and 4 U/mL, respectively, demonstrating that these lipases are preferentially alkaline, whereas V25 showed greater activity at pH 7.5 and is considered preferentially neutral lipases. Most microbial lipases have optimal activity in a pH range of from 7 to 9 (Ulker & Karaoğlu, 2012), which further supports the absence of activity at pH 10.5. The identified species, from the <i>Pseudomonas</i> and <i>Serratia</i> genus are currently representative sources for the production of extracellular lipases (Hasan et al., 2006; Kuddus & Ramteke, 2012), with high applicability in the detergent industry. They are for the most pathogenic, being related to nosocomial infections and antibiotic resistance (Kurz et al., 2003), and it is known that the production of extracellular lipases by microorganisms is closely related to the infectious potential and some are important in these same processes. This study demonstrates that these TBT resistant isolates have, at the same time, the capacity to produce enzymes with a large biotechnological potential but, nevertheless, their relationship is not well understood, representing a novel approach. It is expected for these organisms to produce highly biotechnological relevant biocatalysts, due to their severe adaptations (Suehiro et al., 2007). The fully characterization of these lipases, mostly for F3 with elevated lipolytic activity exhibited, presents also a future challenge.http://journal.frontiersin.org/Journal/10.3389/conf.fmars.2014.02.00012/fullPseudomonasSerratiaExtracellular LipasesTBT-resistant bacterialipolytic activity |
| spellingShingle | Kaori Levy Fonseca Lipolytic activity from bacteria prospected in polluted portuary sites Frontiers in Marine Science Pseudomonas Serratia Extracellular Lipases TBT-resistant bacteria lipolytic activity |
| title | Lipolytic activity from bacteria prospected in polluted portuary sites |
| title_full | Lipolytic activity from bacteria prospected in polluted portuary sites |
| title_fullStr | Lipolytic activity from bacteria prospected in polluted portuary sites |
| title_full_unstemmed | Lipolytic activity from bacteria prospected in polluted portuary sites |
| title_short | Lipolytic activity from bacteria prospected in polluted portuary sites |
| title_sort | lipolytic activity from bacteria prospected in polluted portuary sites |
| topic | Pseudomonas Serratia Extracellular Lipases TBT-resistant bacteria lipolytic activity |
| url | http://journal.frontiersin.org/Journal/10.3389/conf.fmars.2014.02.00012/full |
| work_keys_str_mv | AT kaorilevyfonseca lipolyticactivityfrombacteriaprospectedinpollutedportuarysites |