Glyceric Prodrug of Ursodeoxycholic Acid (UDCA): Novozym 435-Catalyzed Synthesis of UDCA-Monoglyceride

Bile acids (BAs) are a family of steroids synthesized from cholesterol in the liver. Among bile acids, ursodeoxycholic acid (UDCA) is the drug of choice for treating primary biliary cirrhosis and dissolving cholesterol gallstones. The clinical effectiveness of UDCA includes its choleretic activity, the capability to inhibit hydrophobic bile acid absorption by the intestine under cholestatic conditions, reducing cholangiocyte injury, stimulation of impaired biliary output, and inhibition of hepatocyte apoptosis. Despite its clinical effectiveness, UDCA is poorly soluble in the gastro-duodeno-jejunal contents, and pharmacological doses of UDCA are not readily soluble in the stomach and intestine, resulting in incomplete absorption. Indeed, the solubility of 20 mg/L greatly limits the bioavailability of UDCA. Since the bioavailability of drug products plays a critical role in the design of oral administration dosages, we investigated the enzymatic esterification of UDCA as a strategy of hydrophilization. Therefore, we decided to enzymatically synthesize a glyceric ester of UDCA bile acid to produce a more water-soluble molecule. The esterification reactions between UDCA and glycerol were performed with an immobilized lipase B from Candida antarctica (Novozym 435) in solvent-free and solvent-assisted systems. The characterization of the UDCA-monoglyceride, enzymatically synthesized, has been performed by 1H-NMR, 13C-NMR, COSY, HSQC, HMBC, IR, and MS spectroscopy.

Evaluation of Consortium Enzyme towards Kojic Acid Synthesis

Consortium enzyme is a new approach in enzymatic esterification reaction to solve Kojic acid esters' low production. Combining a non-specific enzyme (Novozym 435) and a specific enzyme (Lipozyme RM IM) led to an improvement of enzymatic activity in the enzymatic routes. The findings proved that a significant percentage conversion of ester (29.2 %) was successfully achieved at the enzyme ratio (1:1) of Lipozyme RMIM and Novozym 435, the temperature at 50 ?C, amount of enzyme 0.12 g and 8 hours of reaction time. Moreover, the enzyme showed high stability against heat at 500C and can be used repeatedly up to the 5th cycle, as demonstrated by the high percentage conversion of kojic acid ester. This finding confirmed that consortium enzymes have significantly improved the reaction performance by promoting better catalytic efficiency due to the synergistic effect between combined enzymes.

Sustainable Synthesis of Omega-3 Fatty Acid Ethyl Esters from Monkfish Liver Oil

The search for economic and sustainable sources of polyunsaturated fatty acids (PUFAs) within the framework of the circular economy is encouraged by their proven beneficial effects on health. The extraction of monkfish liver oil (MLO) for the synthesis of omega-3 ethyl esters was performed to evaluate two blending systems and four green solvents in this work. Moreover, the potential solubility of the MLO in green solvents was studied using the predictive simulation software COnductor-like Screening MOdel for Realistic Solvents (COSMO-RS). The production of ethyl esters was performed by one or two-step reactions. Novozym 435, two resting cells (Aspergillus flavus and Rhizopus oryzae) obtained in our laboratory and a mix of them were used as biocatalysts in a solvent-free system. The yields for Novozym 435, R. oryzae and A. flavus in the one-step esterification were 63, 61 and 46%, respectively. The hydrolysis step in the two-step reaction led to 83, 88 and 93% of free fatty acids (FFA) for Novozym 435, R. oryzae and A. flavus, respectively. However, Novozym 435 showed the highest yield in the esterification step (85%), followed by R. oryzae (65%) and A. flavus (41%). Moreover, selectivity of polyunsaturated fatty acids of R. oryzae lipase was evidenced as it slightly esterified docosahexaenoic acid (DHA) in all the esterification reactions tested.

Development of zero-trans shortenings with high thermo-oxidative stability by enzymatic transesterification

Novel zero-trans frying shortenings were formed by enzymatic transesterification by exploring a palm stearin and canola oil mixture and stearic acid as substrates. Both immobilized (Novozym 435, Lipase PS “Amano” IM) and non-immobilized (Lipomod TM 34P) enzymes were applied as biocatalysts. Palmitic acid, the fatty acid which defines the proper type of crystal formation, was present at the 15% level in the reaction mixtures. The novel structured lipids had comparable physical properties and offered similar frying performance to those of commercial shortening. Needle-shaped crystals were predominant both in the transesterification products and the commercial frying shortening. Furthermore, solid fat content profiles of the zero-trans structured lipids produced by Novozym 435 and Lipase PS “Amano” IM were close to those of the commercial shortening.

Interesterification of Egg-Yolk Phosphatidylcholine with p-Methoxycinnamic Acid Catalyzed by Immobilized Lipase B from Candida Antarctica

The p-methoxycinnamic acid (p-MCA) is one of the most popular phenylpropanoids, the beneficial impact of which on the human health is well documented in the literature. This compound has shown many valuable activities including anticancer, antidiabetic, and neuro- and hepatoprotective. However, its practical application is limited by its low bioavailability resulting from rapid metabolism in the human body. The latest strategy, aimed at overcoming these limitations, is based on the production of more stability in systemic circulation bioconjugates with phospholipids. Therefore, the aim of this research was to develop the biotechnological method for the synthesis of phospholipid derivatives of p-methoxycinnamic acid, which can play a role of new nutraceuticals. We developed and optimized enzymatic interesterification of phosphatidylcholine (PC) with ethyl p-methoxycinnamate (Ep-MCA). Novozym 435 and a binary solvent system of toluene/chloroform 9:1 (v/v) were found to be the effective biocatalyst and reaction medium for the synthesis of structured p-MCA phospholipids, respectively. The effects of the other reaction parameters, such as substrate molar ratio, enzyme dosage, and reaction time, on the degree of incorporation of p-MCA into PC were evaluated by use of an experimental factorial design method. The results showed that substrate molar ratio and biocatalyst load have significant effects on the synthesis of p-methoxycinnamoylated phospholipids. The optimum conditions were: Reaction time of three days, 30% (w/w) of Novozym 435, and 1/10 substrate molar ratio PC/Ep-MCA. Under these parameters, p-methoxycinnamoylated lysophosphatidylcholine (p-MCA-LPC) and p-methoxycinnamoylated phosphatidylcholine (p-MCA-PC) were obtained in isolated yields of 32% and 3% (w/w), respectively.

Sustainable synthesis of omega-3 fatty acid ethyl esters from monkfish liver oil

The search for economical and sustainable sources of PUFAs within the framework of the circular economy is encouraged by their proven beneficial effects on health. The extraction of monkfish liver oil (MLO) for the synthesis of omega-3 ethyl esters was performed evaluating two blending systems and four green solvents. Moreover, the potential solubility of the MLO in green solvents was studied using the predictive simulation software COSMO-RS. The production of the ethyl esters was performed by one or two step reactions. Novozym 435, two resting cells (Aspergillus flavus and Rhizopus oryzae) obtained in our laboratory and mix of them were used as biocatalysts in a solvent-free system. The yields for Novozym 435, R. oryzae and A. flavus in the one-step esterification were 63%, 61% and 46%, respectively. The hydrolysis step in the two-step reaction led to 83%, 88% and 93% of free fatty acids (FFA) for Novozym 435, R. oryzae and A. flavus respectively. However, Novozym 435 showed the highest yield in the esterification step (85%) followed by R. oryzae (65%) and A. flavus (41%). Moreover, selectivity in front of polyunsaturated fatty acids of R. oryzae lipase was evidenced, since it did slightly esterified docosahexaenoic acid (DHA) in all the esterification reactions tested.

Enzymatic Synthesis of Formate Ester through Immobilized Lipase and Its Reuse

Octyl formate is an important substance used in the perfume industry in products such as cosmetics, perfumes, and flavoring. Octyl formate is mostly produced by chemical catalysts. However, using enzymes as catalysts has gathered increasing interest due to their environment-friendly proprieties. In the present study, we aimed to identify the optimal conditions for the synthesis of octyl formate through immobilized enzyme-mediated esterification. We investigated the effects of enzymatic reaction parameters including the type of immobilized enzyme, enzyme concentration, molar ratio of reactants, reaction temperature, and type of solvent using the optimization method of one factor at a time (OFAT). The maximum conversion achieved was 96.51% with Novozym 435 (15 g/L), a 1:7 formic acid to octanol ratio, a reaction temperature of 40 °C, and with 1,2-dichloroethane as solvent. Moreover, we demonstrated that the Novozym 435 can be reused under the optimal conditions without affecting the octyl formate yield, which could help reduce the economic burden associated with enzymatic synthesis.

Lipase-Catalyzed Synthesis of Glycerol-Free Biodiesel from Rapeseed Oil and Dimethyl Carbonate

The enzymatic production of glycerol-free biodiesel by the transesterification of rapeseed oil was studied using lipase as catalyst. A series of co-solvents were employed in an attempt to improve reaction kinetics. The effects of the reaction conditions (molar ratio of dimethyl carbonate (DMC) and rapeseed oil, type of lipases, amount of lipases, solvent effects, reaction temperature and time) on the conversion and yield of biodiesel and glycerol carbonate (GC) were investigated. The optimal conditions for biodiesel and GC were 20% Novozym 435, 3:1 molar ratio of DMC to rapeseed oil, and with 20% [Omim][BF6] as solvent. Under these conditions, the conversions of 88.51% biodiesel and 73.87% GC have been achieved after 48 h. The biodiesel and GC conversions were eight times higher compared to the conventional solvent-free system, respectively. There was no obvious loss in the biodiesel and GC yield after Novozym 435 having been used for five recycling.