Efficient Biodiesel Production Catalyzed by Nanobioconjugate of Lipase from Pseudomonas fluorescens.
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ABSTRACT:
SUBMITTER: Bartha-Vari JH
PROVIDER: S-EPMC7037990 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:As a kind of important biocatalysts, Pseudomonas lipases are commonly applied in various industrial fields. Pflip1, a new extracellular lipase gene from Pseudomonas. fluorescens Pf0-1, was first cloned and respectively expressed in Escherichia coli BL21(DE3) and Pichia pastoris KM71, the recombinant proteins Pflip1a and Pflip1b were later purified separately. Then Pflip1a was further characterized. The optimum pH of Pflip1a was 8.0 and its optimal temperature was 70 °C. After incubation at 70 °C for 12 h, Pflip1a could retain over 95% of its original activity. It showed the highest activity towards p-nitrophenyl caprylate. Moreover, its activity was profoundly affected by metal ion, ionic surfactants and organic solvents. Furthermore, the two obtained recombinant lipases were immobilized on the magnetic nanoparticles for biodiesel preparation. The GC analysis showed that for the immobilized lipases Pflip1b and Pflip1a, the biodiesel yield within 24 h respectively attained 68.5% and 80.5% at 70 °C. The activities of the two immobilized lipases still remained 70% and 82% after 10 cycles of operations in non-solvent system. These characteristics and transesterification capacity of the recombinant protein indicated its great potential for organic synthesis, especially for biodiesel production.
| S-EPMC5691200 | biostudies-literature
Project description:BackgroundWe have recently developed a one-step, genetically encoded immobilization approach based on fusion of a target enzyme to the self-crystallizing protein Cry3Aa, followed by direct production and isolation of the fusion crystals from Bacillus thuringiensis. Using this approach, Bacillus subtilis lipase A was genetically fused to Cry3Aa to produce a Cry3Aa-lipA catalyst capable of the facile conversion of coconut oil into biodiesel over 10 reaction cycles. Here, we investigate the fusion of another lipase to Cry3Aa with the goal of producing a catalyst suitable for the conversion of waste cooking oil into biodiesel.ResultsGenetic fusion of the Proteus mirabilis lipase (PML) to Cry3Aa allowed for the production of immobilized lipase crystals (Cry3Aa-PML) directly in bacterial cells. The fusion resulted in the loss of PML activity, however, and so taking advantage of its genetically encoded immobilization, directed evolution was performed on Cry3Aa-PML directly in its immobilized state in vivo. This novel strategy allowed for the selection of an immobilized PML mutant with 4.3-fold higher catalytic efficiency and improved stability. The resulting improved Cry3Aa-PML catalyst could be used to catalyze the conversion of waste cooking oil into biodiesel for at least 15 cycles with minimal loss in conversion efficiency.ConclusionsThe genetically encoded nature of our Cry3Aa-fusion immobilization platform makes it possible to perform both directed evolution and screening of immobilized enzymes directly in vivo. This work is the first example of the use of directed evolution to optimize an enzyme in its immobilized state allowing for identification of a mutant that would unlikely have been identified from screening of its soluble form. We demonstrate that the resulting Cry3Aa-PML catalyst is suitable for the recyclable conversion of waste cooking oil into biodiesel.
| S-EPMC6598307 | biostudies-literature
Project description:Cyclolipopeptides (CLPs) are biosurfactants produced by numerous Pseudomonas fluorescens strains. CLP production is known to be regulated at least by the GacA/GacS two-component pathway, but the full regulatory network is yet largely unknown. In the clinical strain MFN1032, CLP production is abolished by a mutation in the phospholipase C gene (plcC) and not restored by plcC complementation. Their production is also subject to phenotypic variation. We used a modelling approach with Boolean networks, which takes into account all these observations concerning CLP production without any assumption on the topology of the considered network. Intensive computation yielded numerous models that satisfy these properties. All models minimizing the number of components point to a bistability in CLP production, which requires the presence of a yet unknown key self-inducible regulator. Furthermore, all suggest that a set of yet unexplained phenotypic variants might also be due to this epigenetic switch. The simplest of these Boolean networks was used to propose a biological regulatory network for CLP production. This modelling approach has allowed a possible regulation to be unravelled and an unusual behaviour of CLP production in P. fluorescens to be explained.
| S-EPMC3269426 | biostudies-literature
Project description:Pseudomonas fluorescens, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of P. fluorescens SIK W1 was cloned and expressed in Escherichia coli, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter, tliD, tliE, and tliF, upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transporters of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi protease, Serratia marcescens lipase, and Pseudomonas fluorescens CY091 protease. tliDEF and the lipase structural gene in a single operon were sufficient for E. coli cells to secrete the lipase. In addition, E. coli harboring the lipase gene secreted the lipase by complementation of tliDEF in a different plasmid. The ABC transporter of P. fluorescens was optimally functional at 20 and 25 degrees C, while the ABC transporter, aprD, aprE, and aprF, of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37 degrees C. These results demonstrated that the lipase is secreted by the P. fluorescens SIK W1 ABC transporter, which is organized as an operon with tliA, and that its secretory function is temperature dependent.
| S-EPMC93584 | biostudies-literature
Project description:BackgroundRhizopus species is among the most well-known lipase producers, and its enzyme is suitable for use in many industrial applications. Our research focuses on the production of lipase utilizing waste besides evaluating its applications.ResultsAn extracellular lipase was partially purified from the culture broth of Rhizopus oryzae R1 isolate to apparent homogeneity using ammonium sulfate precipitation followed by desalting via dialysis. The partially purified enzyme was non-specific lipase and the utmost activity was recorded at pH?6, 40?°C with high stability for 30 min. The constants Km and Vmax, calculated from the Lineweaver-Burk plot, are 0.3?mg/mL and 208.3?U/mL, respectively. Monovalent metal ions such as Na+ (1 and 5?mM) and K+ (5?mM) were promoters of the lipase to enhance its activity with 110, 105.5, and 106.5%, respectively. Chitosan was used as a perfect support for immobilization via both adsorption and cross-linking in which the latter method attained immobilization efficiency of 99.1% and reusability of 12?cycles. The partially purified enzyme proved its ability in forming methyl oleate (biodiesel) through the esterification of oleic acid and transesterification of olive oil.ConclusionThe partially purified and immobilized lipase from Rhizopus oryzae R1 approved excellent efficiency, reusability, and a remarkable role in detergents and biodiesel production.
| S-EPMC7785608 | biostudies-literature
Project description:BackgroundEnzymatic biodiesel is becoming an increasingly popular topic in bioenergy literature because of its potential to overcome the problems posed by chemical processes. However, the high cost of the enzymatic process still remains the main drawback for its industrial application, mostly because of the high price of refined oils. Unfortunately, low cost substrates, such as crude soybean oil, often release a product that hardly accomplishes the final required biodiesel specifications and need an additional pretreatment for gums removal. In order to reduce costs and to make the enzymatic process more efficient, we developed an innovative system for enzymatic biodiesel production involving a combination of a lipase and two phospholipases. This allows performing the enzymatic degumming and transesterification in a single step, using crude soybean oil as feedstock, and converting part of the phospholipids into biodiesel. Since the two processes have never been studied together, an accurate analysis of the different reaction components and conditions was carried out.ResultsCrude soybean oil, used as low cost feedstock, is characterized by a high content of phospholipids (900 ppm of phosphorus). However, after the combined activity of different phospholipases and liquid lipase Callera Trans L, a complete transformation into fatty acid methyl esters (FAMEs >95%) and a good reduction of phosphorus (P <5 ppm) was achieved. The combination of enzymes allowed avoidance of the acid treatment required for gums removal, the consequent caustic neutralization, and the high temperature commonly used in degumming systems, making the overall process more eco-friendly and with higher yield. Once the conditions were established, the process was also tested with different vegetable oils with variable phosphorus contents.ConclusionsUse of liquid lipase Callera Trans L in biodiesel production can provide numerous and sustainable benefits. Besides reducing the costs derived from enzyme immobilization, the lipase can be used in combination with other enzymes such as phospholipases for gums removal, thus allowing the use of much cheaper, non-refined oils. The possibility to perform degumming and transesterification in a single tank involves a great efficiency increase in the new era of enzymatic biodiesel production at industrial scale.
| S-EPMC4015511 | biostudies-literature
Project description:Recently, ionic liquids (ILs) have been regarded as ideal media for non-aqueous bio-catalysis. In this work, the synthesis of warfarin by the lipase-catalyzed Michael addition in IL media and the parameters that affected the warfarin yield were investigated. Experimental results demonstrated that the chemical structures of the ILs were a major factor for influencing the warfarin yield. The ILs containing the NTf?? anion were suitable reaction media due to the high chemical stability of this anion. The incorporation of the hydroxyl group on the IL cation significantly improved the lipase activity due to the H?O-mimicking property of this group. The lipase activity decreased by increasing the alkyl chain length on the IL cation due to the non-polar domain formation of the IL cation at the active site entrance of lipase. The ILs and lipase could be reused no less than five times without reduction in the warfarin yield.
| S-EPMC6225191 | biostudies-literature
Project description:BackgroundBiodiesels are methyl esters of fatty acids that are usually produced by base catalyzed transesterification of triacylglyerol with methanol. Some lipase enzymes are effective catalysts for biodiesel synthesis and have many potential advantages over traditional base or acid catalyzed transesterification. Natural lipases are often rapidly inactivated by the high methanol concentrations used for biodiesel synthesis, however, limiting their practical use. The lipase from Proteus mirabilis is a particularly promising catalyst for biodiesel synthesis as it produces high yields of methyl esters even in the presence of large amounts of water and expresses very well in Escherichia coli. However, since the Proteus mirabilis lipase is only moderately stable and methanol tolerant, these properties need to be improved before the enzyme can be used industrially.ResultsWe employed directed evolution, resulting in a Proteus mirabilis lipase variant with 13 mutations, which we call Dieselzyme 4. Dieselzyme 4 has greatly improved thermal stability, with a 30-fold increase in the half-inactivation time at 50°C relative to the wild-type enzyme. The evolved enzyme also has dramatically increased methanol tolerance, showing a 50-fold longer half-inactivation time in 50% aqueous methanol. The immobilized Dieselzyme 4 enzyme retains the ability to synthesize biodiesel and has improved longevity over wild-type or the industrially used Brukholderia cepacia lipase during many cycles of biodiesel synthesis. A crystal structure of Dieselzyme 4 reveals additional hydrogen bonds and salt bridges in Dieselzyme 4 compared to the wild-type enzyme, suggesting that polar interactions may become particularly stabilizing in the reduced dielectric environment of the oil and methanol mixture used for biodiesel synthesis.ConclusionsDirected evolution was used to produce a stable lipase, Dieselzyme 4, which could be immobilized and re-used for biodiesel synthesis. Dieselzyme 4 outperforms the industrially used lipase from Burkholderia cepacia and provides a platform for still further evolution of desirable biodiesel production properties.
| S-EPMC3670234 | biostudies-literature
Project description:BackgroundProduction of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called "China wood oil" is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications.ResultsThe prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C.ConclusionsThis is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw material and a chimeric lipase from R. oryzae as an economic catalyst make this study a promising one for biodiesel applications.
| S-EPMC3674748 | biostudies-literature
Project description:Lipases catalyze the hydrolysis of fats and oils, and have been widely used in various industrial fields. However, bacterial lipases have a lower thermostability in industrial processes, which was a limiting factor in their industrial application. In this study, we obtained an improve variant of Pseudomonas fluorescens lipase (PFL) with enhanced thermostability using classical error-prone PCR. Wild-type PFL showed an optimal temperature and pH of 50°C and pH 7.5, respectively. Due to the low thermostability of PFL, a library containing over 3000 individual mutants as constructed using error-prone PCR. Screening for thermotolerance yielded the mutants L218P and P184C/M243C with T m values of 62.5 and 66.0°C, which was 2.5 and 6°C higher than that of the WT, respectively. The combination of the two mutants (P184C/M243C/L218P) resulted in an approximately additive effect with a T m value of 68.0°C. Although the increase of T m was not substantial, the mutant also had dramatically increased methanol tolerance. Structural analysis revealed that the introduction of a disulfide bond between P184C and M243C and the substitution of Pro to reduce the flexibility of a loop increased the thermostability of PFL, which provides a theoretical foundation for improving the thermostability and methanol tolerance of lipase family I.1 to resist the harsh conditions of industrial processes.
| S-EPMC7492553 | biostudies-literature