Kinetic study of a commercial lipase for hydrolysis of semi-refined oil of anchovy (Engraulis ringens) [Estudio cinético de una lipasa comercial para la hidrólisis de aceite semirrefinado de anchoa (Engraulis ringens)]

Lipases due to their ecological nature and catalytic versatility, are ideal for their application in the fish oil hydrolysis industry due to their selective property, which allows the preservation of polyunsaturated fatty acids (PUFAs) in the lipid structure. The objective of this research was to determine the activity and kinetic parameters of a commercial AY AMANO "30SD" lipase, as well as the temperature and time values ​​to achieve an optimal degree of hydrolysis in semi-refined anchovy oil. The experiments were carried out in a jacketed minireactor with a working volume of 400 mL (oil-water-enzyme) with temperature control and pH 7.00, enzyme concentration 350 U/mL and stirring 160 rpm. A 3x3 factorial design and the response surface methodology were used. The results obtained from the study of the enzyme were: activity = 37 384.55 ± 395.07 U/g and kinetic parameters: Km = 7.98 g/L and Vmax. = 0.038887 g/Lxmin. Correspondingly, the following optimal parameters were obtained: Degree of hydrolysis 4.01%, temperature 46.86 °C and hydrolysis time 90 minutes, with a confidence level of 95% (p <0.05). Conclusions: The study allowed us to kinetically characterize the commercial lipase and determine the optimum degree of hydrolysis of the semi-refined anchovy oil.

Synthesis of Linoleic Acid 13-Hydroperoxides from Safflower Oil Utilizing Lipoxygenase in a Coupled Enzyme System with In-Situ Oxygen Generation

Linoleic acid hydroperoxides are versatile intermediates for the production of green note aroma compounds and bifunctional ω-oxo-acids. An enzyme cascade consisting of lipoxygenase, lipase and catalase was developed for one-pot synthesis of 13-hydroperoxyoctadecadienoic acid starting from safflower oil. Reaction conditions were optimized for hydroperoxidation using lipoxygenase 1 from Glycine max (LOX-1) in a solvent-free system. The addition of green surfactant Triton CG-110 improved the reaction more than two-fold and yields of >50% were obtained at linoleic acid concentrations up to 100 mM. To combine hydroperoxidation and oil hydrolysis, 12 lipases were screened for safflower oil hydrolysis under the reaction conditions optimized for LOX-1. Lipases from Candida rugosa and Pseudomonas fluorescens were able to hydrolyze safflower oil to >75% within 5 h at a pH of 8.0. In contrast to C. rugosa lipase, the enzyme from P. fluorescens did not exhibit a lag phase. Combination of P. fluorescens lipase and LOX-1 worked well upon LOX-1 dosage and a synergistic effect was observed leading to >80% of hydroperoxides. Catalase from Micrococcus lysodeikticus was used for in-situ oxygen production with continuous H2O2 dosage in the LOX-1/lipase reaction system. Foam generation was significantly reduced in the 3-enzyme cascade in comparison to the aerated reaction system. Safflower oil concentration was increased up to 300 mM linoleic acid equivalent and 13-hydroperoxides could be produced in a yield of 70 g/L and a regioselectivity of 90% within 7 h.

The Activation Energies and Optimum Temperatures of Olive Oil Hydrolysis By Lipase Porcine Pancreas

Lipase activity is a perfect indicator for the monitoring of processes of bioremediation of degraded soils. Lipase is also used in the processes of oil hydrolysis in wastewater treatment. To be able to predict and model processes with used lipase in environmental operations, knowledge of the kinetic parameters of the process are required. The paper presents the determined values of activation energies and optimum temperatures for porcine pancreas lipase. The parameters were estimated based on the literature of the activity curves vs. temperature for hydrolysis of olive oil by lipase. It was noticed that concentration of gum arabic added as an emulsifier during lipase activity measurements influences on the obtained values of determined parameters. A mathematical model describing the effect of temperature on porcine pancreas lipase activity was used. Based on the comparison analysis, the optimum temperature Topt were obtained in the range from 313.30 ±0.56 to 319.62 ±0.96 K, activation energies Ea were from 51 ±10 to 82.6 ±9.9 kJ/mol, and values of deactivation energies Ed were in the range from 122.7 ±4.0 to 150.9 ±5.8 kJ/mol.

Application of Partial Hydrolysis of Virgin Coconut oil (VCO) on Carrageenan-based Edible Coatinng as (Sphyraena barracuda) Fishball Preservative during Room Storage

Food spoilage during storage occurs physically, chemically and biologically which is related to the activity of bacteria. One of the natural preservation that is currently developing is the application of edible coating on perishable food, such as fishball. Addition of antibacterial agent is important to improve edible coating. Virgin coconut oil contains medium chain fatty acids which have antimicrobial properties, particularly monoglycerides and free fatty acids that produced by hydrolyzing partially triglycerides at the Sn-1 and Sn-3 position using Lipozyme. The aim of this research was to test the effect of edible coating carrageenan enriched with virgin coconut oil hydrolysis (HVCO) (concentrations 1%,3%,5%) on fishball quality.The method was an experimental with factorial completely randomized design.The samples were analyzed for sensorial assessments, Total plate Count, Total Volatile Base-Nitrogen, Water content, and pH for 5 days at room temperature. The study demonstrated that fish meatball coated with carrageenan based-edible coating fortified with HVCO showed the best result compared to controls (Uncoated fishball, coated without HVCO). Sensory attributes were still accepted by panelists until 3 days. Meanwhile, the same pattern depicted by TPC and TVB-N parameters. HVCO5% had inhibited microbial growth and retarded the increase of TVB-N number on fishball, the results were Log 5,08cfu/gr, 29,69 mg/100 gr respectively.

High Epoxidation Yields of Vegetable Oil Hydrolyzates and Methyl Esters by Selected Fungal Peroxygenases

Epoxides of vegetable oils and free and methylated fatty acids are of interest for several industrial applications. In the present work, refined rapeseed, sunflower, soybean, and linseed oils, with very different profiles of mono- and poly-unsaturated fatty acids, were saponified and transesterified, and the products treated with wild unspecific peroxygenases (UPOs, EC 1.11.2.1) from the ascomycete Chaetomium globosum (CglUPO) and the basidiomycete Marasmius rotula (MroUPO), as well as with recombinant UPO of the ascomycete Humicola insolens (rHinUPO), as an alternative to chemical epoxidation that is non-selective and requires strongly acidic conditions. The three enzymes were able of converting the free fatty acids and the methyl esters from the oils into epoxide derivatives, although significant differences in the oxygenation selectivities were observed between them. While CglUPO selectively produced “pure” epoxides (monoepoxides and/or diepoxides), MroUPO formed also hydroxylated derivatives of these epoxides, especially in the case of the oil hydrolyzates. Hydroxylated derivatives of non-epoxidized unsaturated fatty acids were practically absent in all cases, due to the preference of the three UPOs selected for this study to form the epoxides. Moreover, rHinUPO, in addition to forming monoepoxides and diepoxides of oleic and linoleic acid (and their methyl esters), respectively, like the other two UPOs, was capable of yielding the triepoxides of α-linolenic acid and its methyl ester. These enzymes appear as promising biocatalysts for the environmentally friendly production of reactive fatty-acid epoxides given their self-sufficient monooxygenase activity with selectivity toward epoxidation, and the ability to epoxidize, not only isolated pure fatty acids, but also complex mixtures from oil hydrolysis or transesterification containing different combinations of unsaturated (and saturated) fatty acids.

Thermostable acidic lipase of Bacillus glycinifermentans-MK840989 isolated from contaminated environment; its optimization, purification and exploring potential applications

This study is the first report about isolation, purification and optimization of lipase from Bacillus glycinifermentans. In this study, Bacillus glycinifermentansMK-840989 was isolated from a local petrol pump. The bacterium showed lipolytic zones of 0.19cm, 0.044cm, and 0.28cm on peptone yeast agar, olive oil hydrolysis agar and chromogenic plate agar, respectively. B. glycinifermentans also produced an extracellular lipase (55.1µmol/ml). This bacterium preferred acidic environment (pH 5) for growing optimally at 80˚C when the medium was supplemented with 1% olive oil. The olive oil induced its growth up to 9h. The protein content of the purified lipase was estimated about 75mg/ml as compared to its crude form, i.e. 350mg/ml. The purified lipase was found to be thermostable acidic in nature as its optimum activity was observed at 90˚C (0.08U/ml) and pH 5 (0.02U/ml). Other optimization factors included 1% olive oil (0.065U/ml), 0.1mM maltose (0.023U/ml), 0.1mM Ca (0.025U/ml), 1% yeast extract (16.8U/ml), 1% wheat waste (0.019U/ml), 1% commercial detergent (0.016U/ml) and 1% tween-20 (0.015 U/ml). The purified lipase showed a polypeptide of 26.7kDa on SDS-PAGE. These features such as thermostability, acidic nature, ability to show activity in wheat waste and tolerance to detergents render the lipase of B.glycinifermentans MK-840989 as an attractive choice for biotechnologists to employ it at industrial level. The purified lipase of B.glycinifermentans MK-840989 can be a potential candidate for detergent and oil-remediation industry. It can help to replace conventional synthetic detergent as it is cost-effective and eco-friendly.