Morphological, biochemical and kinetic properties of lipase from Candida rugosa immobilized in zirconium phosphate (original) (raw)
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Engineering Journal IJOER, 2020
The current preference of costumers for natural and healthy products is increasing the employment of biotechnological processes that use enzymes, and the synthesis of esters is an example of this change. However, enzymes are high-cost product, which stimulates research in finding solutions that make them more economically attractive, like immobilization. This work aimed to use different protocols for immobilizing lipase and its application in ester synthesis. The results showed that Pseudomonas fluorences lipase (AKL) was the most efficient for immobilization among other studied lipases (Pseudomonas fluorences lipase (AKL), Pseudomonas cepacia lipase (PSL), Hog Pancreas lipase (PHL), Pancreas Porcinas lipase (PPL), and Mucor Javanicus lipase (MJL)), with hydrolytic activity of 3323.6 U/g. Both immobilization methods (physical adsorption and entrapment) showed promising results towards hydrolytic activity. The best immobilization by adsorption was obtained using AKL onto PHB (polyhydroxybutyrate), with 698.61 U/g of hydrolytic activity. For entrapment, AKL also presented the best result, with 247.30 U/g of hydrolytic activity. For the synthesis of ester, after a 60 h-reaction using the immobilized derivatives by physical adsorption, the esterification yield was 74.26 %. In terms of hydrolytic activity, the employed protocols were very promising and encourage the continuity of this study towards the optimization of processes using industrial lipases.
Immobilization of Lipases – A Review. Part I: Enzyme Immobilization
ChemBioEng Reviews, 2019
Free enzymes employed as biological catalysts have many advantages such as low reaction time, involving low energy and less waste output when compared to conventional chemical catalysts. However, commercial utilization of free enzymes is often hampered by the lack of operational stability, high cost, and non‐reusability. Immobilization of enzyme is an option to overcome these obstacles. Immobilized enzyme expresses stable performance in organic solvents even in adverse pH, which makes the biomolecules reusable and prosperous as a biological catalyst. Biological catalysis with immobilized enzymes is found to be an alternative method instead of chemical catalysis for chemical reactions in the foreseeable future. Sources of lipase, techniques in immobilization and cross‐linkers are dealt with in this paper.
Effective immobilisation of lipase to enhance esterification potential and reusability
Chemical Papers, 2013
A commercial lipase, "Lipolase T100", was immobilised onto silica by means of physical adsorption. The silica-bound lipase was subsequently exposed to 1 vol. % glutaraldehyde (pentane-1,5dial). The silica was loaded repeatedly with the Lipolase T100 in 0.05 M Tris buffer (pH 8.5) until saturation was achieved. During the 1st, 2nd, 3rd, 4th, and 5th cycles of loading of silica with the enzyme, the protein-binding on the silica achieved 51.73 %, 48.27 %, 26.92 %, 10.73 %, and 4.29 %, respectively. The synthesis of methyl salicylate (methyl 2-hydroxybenzoate) and linalyl ferulate (3,7-dimethylocta-1,6-dien-3-yl 4-hydroxy-3-methoxycinnamate) carried out at 45 • C under shaking with mole ratios of 200 mM of acid and 500 mM alcohol in DMSO using 15 mg mL −1 of hyper-activated biocatalyst resulted in yield(s) of 77.2 % of methyl salicylate and 65.3 % of linalyl ferulate in the presence of molecular sieves. The hyper-activated biocatalyst was more efficient than the previously reported silica-bound lipase with minimum leaching of the enzyme from the reaction mixture. The Km and Vmax of the free (0.142 mM and 38.31 µmol min −1 mL −1 , respectively) and silica-bound lipase (0.043 mM and 26.32 µmol min −1 mg −1 , respectively) were determined for the hydrolysis of p-NPP. During repeated esterification studies using silica-bound lipase, yields of 50.1 % of methyl salicylate after the 5th cycle, and 53.9 % of linalyl ferulate after the 7th cycle of esterification were recorded. In the presence of molecular sieves (30 mg mL −1 ) in the reaction mixture, the maximum syntheses of methyl salicylate (77.2 %) and linalyl ferulate (65.3 %) were also observed. In a volumetric batch scale-up, when the reaction volume was increased to 50 mL, 44.9 % and 31.4 % yields of methyl salicylate and linalyl ferulate, respectively, were achieved.
Biochemical Engineering Journal, 2015
Lipase from Candida rugosa was immobilized by adsorption onto laboratory prepared supports, silica SBA-15 and zirconia. The adsorption process was studied as a function of pH in terms of percent of adsorbed lipase, enzyme activity and zeta potential of support and enzyme. Several analytical approaches such as laser Doppler electrophoresis, Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) showed that the lipase was successfully immobilized onto both supports. The -potential data suggest that the adsorption efficiency does not depends on the sign but on the magnitude of the surface charge of adsorption partners, and therefore underline the importance of their dispersion stability. Adsorption to material surface altered enzyme characteristics. v max for the lipase immobilized onto silica and zirconia were 4.8-fold and 3.6-fold lower than that of the free lipase, respectively. The K m showed no alteration of enzyme-substrate affinity on zirconia support, whereas the enzyme immobilized on silica had 3.6 times lower affinity. Thermostability at 60 • C of the lipase was improved 12-fold on zirconia and 4-fold on silica. Finally, in examining reusability, the immobilized lipase retained more than 90% of initial activity after eight reuses on both supports. Immobilization methods can be divided into three categories: cross-linking, entrapment and binding to a support . Each method can influence the enzyme and its properties: activity, stability and selectivity . Cross linking offers high stability but it can cause steric hindrance by the substrate [5], while enzyme immobilization by entrapment in microspheres or membranes is often followed by inefficient contact between enzyme and substrate, and the exit of products . Finally, the method based on physical adsorption of enzyme onto the surface of water-insoluble carriers [8] causes little or no conformational change of the enzyme or destruction of its active site. This method is usually simple and inexpensive if a suitable carrier is used. As a result of the weak binding force between the enzyme and the carrier, this method, however, has a disadvantage, in that the adsorbed enzyme leaks from the carrier during the repeated use. Considering that the underlying reason for enzyme immobilization, besides improvement in stability, is its recyclability [3], it seems that the adsorption has been somewhat neglected in comparison to other methods.
Review : Immobilization and application of lipase
2017
The current demands of the world’s biotechnological industries are enhancement in enzyme productivity and development of novel techniques for increasing their shelf life. Immobilized enzymes are more robust and more resistant to environmental changes than free enzymes in solution. Enzyme immobilization provides an excellent base for increasing availability of enzyme to the substrate with greater turnover over a considerable period of time. Several natural and synthetic supports have been assessed for their efficiency for enzyme immobilization. Future investigations should adopt logistic and appropriate entrapment techniques along with innovatively modified supports to improve the state of enzyme immobilization, providing new perspectives to the industrial sector. This paper reviews recent literature on enzyme immobilization by various techniques, as well as immobilization strategies and their application.
Immobilization of Biotechnologically Important Candida rugosa Lipase onto Commercial Matrices
2019
The continual search for alternative environmentally cleaner technologies in industrial processes has led to an increase in the use of enzymatic processes globally. However, due to their physical characteristics they require immobilization in order to remain effective. The objective of this study was to investigate the immobilization of the biotechnologically important and commercially available Candida rugosa lipase (CRL) by physical interfacial adsorption onto a number of matrices to act as biocatalysts. Five different types of support were tested: i) macroporous silica (synthetic inorganic), ii) polyhydroxybutyrate (natural organic), iii) polypropylene (synthetic organic), iv) polymethacrylate (synthetic organic), and v) polystyrene-divinylbenzene (synthetic organic). Results generated during this study showed that from the group of materials tested, polystyrene-divinylbenzene gave the best results with the highest amount of immobilized protein (8.10 ± 0.31 mg/g) and a good immobilization yield (90.35% ± 1.53%). The efficiency of protein immobilization was found to be highest when carried out at pH4.5, which is close to the isoelectric point of the enzyme.
Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica
Applied Biochemistry and Biotechnology, 1999
Candida rugosa lipase was immobilized by covalent binding on controlled poresilica (CPS) using glutaraldehyde ascross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5–8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10°C higher than that for the free lipase. The thermal stability of the CPS lipase was alsogreater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.
Lipase immobilized by different techniques on various support materials applied in oil hydrolysis
Journal of the Serbian Chemical Society, 2005
Batch hydrolysis of olive oil was performed by Candida rugosa lipase immobilized on Amberlite IRC-50 and Al2O3. These two supports were selected out of 16 carriers: inorganic materials (sand, silica gel, infusorial earth Al2O3), inorganic salts (CaCO3, CaSO4), ion-exchange resins (Amberlite IRC-50 and IR-4B, Dowex 2X8), a natural resin (colophony), a natural biopolymer (sodium alginate), synthetic polymers (polypropylene, polyethylene) and zeolites. Lipase immobilization was carried out by simple adsorption adsorption followed by cross-linking, adsorption on ion-exchange resins combined adsorption and precipitation, pure precipitation and gel entrapment. The suitability of the supports and techniques for the immobilization of lipase was evaluated by estimating the enzyme activity, protein loading immobilization efficiency and reusability of the immobilizates. Most of the immobilizates exhibited either a low enzyme activity or difficulties during the hydrolytic reaction. Only those p...
Improving Lipase Activity by Immobilization and Post-immobilization Strategies
Methods in Molecular Biology, 2013
One important parameter for the application of lipase catalysts in chemical industries is the specifi c activity displayed towards natural or unnatural substrates. Different strategies to enhance the lipase activity have been described. The immobilization of lipases on hydrophobic supports by interfacial adsorption at low ionic strength permitted the hyper-activation of these enzymes by fi xing the open conformation of the lipase on the hydrophobic support. Improvements of activity from 1.2-up to 20-fold with respect to the initial one have been observed for lipases from different sources.
Lipase immobilized in organic-inorganic matrics
Journal of Sol-Gel Science and Technology, 1997
Commercial lipase was immobilized into an organic-inorganic matrix formed by hydrolysis of silicon alkoxides (tetraethoxysilane, dimethyldiethoxysilane and methyltriethoxysilane) with (3-aminopropyl)triethoxysilane, (3-thiopropyl)trimethoxysilane and chlorodiisopropyloctylsilane. Hydrolytic activity of lipase was tested after addition of the enzyme to a prepolymer solution, after gelation, in xerogel particles and in thin layers deposited on glass slides by dip-or spin-coating. The prepolymer containing NH groups showed the higher activity then the native enzyme.