Abstract:An extra cellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa SRT 9 to apparent homogeneity using ammonium sulfate precipitation followed by chromatographic techniques on phenyl Sepharose CL-4B and Mono Q HR 5/5 column, resulting in a purification factor of 98 fold with specific activity of 12307.8 U/mg. The molecular weight of the purified lipase was estimated by SDS-PAGE to be 29 kDa with isoelectric point of 4.5. Maximum lipase activity was observed in a wide range of t… Show more
“…similar to the observed activity increase of FS02 in the current work, enzymatic activity of an alkaline lipase was shown to be increased in the presence of e.g. methanol [31], and high solvent tolerance of a Pseudomonas lipase has been shown [30]. Hence enzyme activity increase in the presence of solvents observed for FS02 is not unique; however, despite of some uncertainties concerning the absolute activity levels from high standard deviations in the analyses, the increases in activity observed for enzymes in previous studies are rarely as high as observed for FS02, rendering the newly discovered enzyme with distinct features compared to other similar enzymes.…”
Section: Production Of Fs02 Enzyme and Enzyme Characterizationsupporting
confidence: 88%
“…Lipase activity in the presence of different solvents has been analyzed in several previous studies, mainly showing no or negative effects; e.g. unaffected activity with methanol, ethanol and acetone, and a decreased lipase activity in the presence of isopropanol [30], or reduced lipase activity in the presence of isopropanol, methanol, acetone, acetonitrile and/or ethanol [27] [29]. On the other hand, Figure 2.…”
Section: Production Of Fs02 Enzyme and Enzyme Characterizationmentioning
With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90˚C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.
“…similar to the observed activity increase of FS02 in the current work, enzymatic activity of an alkaline lipase was shown to be increased in the presence of e.g. methanol [31], and high solvent tolerance of a Pseudomonas lipase has been shown [30]. Hence enzyme activity increase in the presence of solvents observed for FS02 is not unique; however, despite of some uncertainties concerning the absolute activity levels from high standard deviations in the analyses, the increases in activity observed for enzymes in previous studies are rarely as high as observed for FS02, rendering the newly discovered enzyme with distinct features compared to other similar enzymes.…”
Section: Production Of Fs02 Enzyme and Enzyme Characterizationsupporting
confidence: 88%
“…Lipase activity in the presence of different solvents has been analyzed in several previous studies, mainly showing no or negative effects; e.g. unaffected activity with methanol, ethanol and acetone, and a decreased lipase activity in the presence of isopropanol [30], or reduced lipase activity in the presence of isopropanol, methanol, acetone, acetonitrile and/or ethanol [27] [29]. On the other hand, Figure 2.…”
Section: Production Of Fs02 Enzyme and Enzyme Characterizationmentioning
With the aim of identifying novel thermostable esterases, comprehensive sequence databases and cloned fosmid libraries of metagenomes derived from an offshore oil reservoir on the Norwegian Continental Shelf were screened for enzyme candidates using both sequence-and function-based screening. From several candidates identified in both approaches, one enzyme discovered by the functional approach was verified as a novel esterase and subjected to a deeper characterization. The enzyme was successfully over-produced in Escherichia coli and was shown to be thermostable up to 90˚C, with the highest esterase activity on short-chain ester substrates and with tolerance to solvents and metal ions. The fact that the thermostable enzyme was solely found by functional screening of the oil reservoir metagenomes illustrates the importance of this approach as a complement to purely sequence-based screening, in which the enzyme candidate was not detected. In addition, this example indicates the large potential of deep-sub-surface oil reservoir metagenomes as a source of novel, thermostable enzymes of potential relevance for industrial applications.
“…Reported reaction times varied from 8 hours for immobilized enzymes to 90 hours for the same free enzymes, depending on the feedstock and alcohol [25]. Moreover, temperature of 55°C as the working temperature for lipases from P. aeruginosa has been reported [28,29]. This temperature showed, for all the cases, the best balance between stability and reaction rate.…”
Section: Discussionmentioning
confidence: 98%
“…We carried out a partial purification process that must be improved further according the results. Other researches had reached lipase purification factors of approximately 300, improving their activity approximately 1000-fold compared to the enzyme supernatant [15,28,47]. Also, protein engineering is widely used to improve the lipase production yields and enzymatic activity [48,49].…”
A partially purified lipase from the Pseudomonas aeruginosa strain (PSA-01) isolated from the palm oil fruit Elaeis guineensis was used as biocatalyst to produce fatty acid methyl esters (FAME). Lyophilized lipase supernatant (LLS) was used during the first step to screen the main variables (pH, temperature, stoichiometric oil:methanol ratio, water content and type of oil). Other variables which were identified during the screening assays (scale of reaction recipient, LLS amount and use of hexane to solubilize methanol forming a methanol-oil microemulsion) were tested during a second step. The response variable was % molar yield of FAME. It was quantified by GC. Additionally, the LLS work parameters were optimized and compared to a partially purified lipase (PPL) during a final assay. The first-order interactions between the analyzed factors were significant (p<0.05). The highest yield was 4.16% w/w (respect to oil) using a partially purified lipase (PPL) with pH 8, refined, bleached, and deodorized oil (RBD), 5% water (by volume) in oil and 10% hexane (by volume), and a stoichiometric ratio of 1:170 oil:methanol. The final assay was carried out at 54°C and 200 rpm for 48 hours. It resulted in a 34.68% conversion using PPL. It also showed a 13-fold improvement versus the initial yield with LLS, suggesting the need for a better purification process. During this research, the lipase was partially purified and used at an alkaline pH. It showed resistance to organic compounds such as methanol and hexane. This implies great potential to act as an effective biocatalyst in the implementation of biodiesel production processes.
“…3c). Lipase activity of Pseudomonas aeruginosa MTCC 2488 was drastically reduced in the presence of 2-propanol, hexane and butanol (Borkar et al 2009). Also, the lipase activity of Pseudomonas aeruginosa SRT 9 was drastically reduced in the presence of ethanol, butanol and isopropanol (Chouhan and Dawande 2010).…”
Section: Effect Of Organic Solvents On Lipase Activitymentioning
Enterococcus durans NCIM5427 (ED-27), capable of producing an intracellular acid stable lipase, was isolated from fish processing waste. Its growth and subsequent lipase production was optimized by Box Behneken design (optimized conditions: 5 % v/v fish waste oil (FWO), 0.10 mg/ml fish waste protein hydrolysates (FWPH) at 48 h of fermentation time). Under optimized conditions, ED-27 showed a 3.0 fold increase (207.6 U/ml to 612.53 U/ml) in lipase production, as compared to un-optimized conditions. Cell growth and lipase production was modeled using Logistic and LuedekingPiret model, respectively; and lipase production by ED-27 was found to be growth-associated. Lipase produced by ED-27 showed stability at low pH ranges from 2 to 5 with its optimal activity at 30°C , pH 4.6; showed metal ion dependent activity wherein its catalytic activity was activated by barium, sodium, lithium and potassium (10 mM); reduced by calcium and magnesium (10 mM). However, iron and mercury (5 mM) completely inactivated the enzyme. In addition, modifying agents like SDS, DTT, β-ME (1%v/v) increased activity of lipase of ED-27; while, PMSF, DEPC and ascorbic acid resulted in a marked decrease. ED-27 had maximum cell growth of 9.90309 log CFU/ml under optimized conditions as compared to 13 log CFU/ml in MRS. The lipase produced has potential application in poultry and slaughterhouse waste management.
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