Jingbo Li

Liquid lipases for enzymatic concentration of n-3 polyunsaturated fatty acids in monoacylglycerols via ethanolysis: Catalytic specificity and parameterization

This work examined catalytic specificity and fatty acid selectivity of five liquid lipases C. antarctica lipase A and B (CAL-A/B), and lipase TL (T. lanuginosus), Eversa Transfrom and NS in ethanolysis of fish oil with the aim to concentrate n-3 PUFAs into monoacylglycerols (MAGs) products. Lipase TL, Eversa Transform & NS entail a much faster reaction and produce higher MAGs yield (>30%); whereas CAL-A obtains the highest concentration of n-3 PUFAs/DHA/EPA into MAGs products (88.30%); followed by lipase NS (81.02%). 13C NMR analysis indicates that CAL-B and lipase TL are sn-1,3 specific; but CAL-A and lipase Eversa Transform are non-regiospecific or weak sn-2 specific; which plausibly explains high enrichment effect of the latter two lipases. All liquid lipases are observed reusable for a certain times (lipase Eversa Transform up to 12 times), demonstrating their competitive advantage over immobilized form for industrial application because of their higher activity and cheaper operation cost.

The near-ideal catalytic property of Candida antarctica lipase A to highly concentrate n-3 polyunsaturated fatty acids in monoacylglycerols via one-step ethanolysis of triacylglycerols.

Declining quantity/quality of available n-3 polyunsaturated fatty acids (n-3 PUFAs) resources demand innovative technology to concentrate n-3 PUFAs from low quality oils into value-added products/health-beneficial ingredients rich in n-3 PUFAs. This work proposed the catalytic property and specificity of an ideal enzyme required to tackle this task and identified Candida antarctica lipase A (CAL-A) is such a near-ideal enzyme in practice, which concentrates n-3 PUFAs from 25% to 27% in oils to a theoretically closer value 90% in monoacylglycerols (MAGs) via one-step enzymatic ethanolysis. Non-regiospecificity and high non-n-3 PUFAs preference of CAL-A are the catalytic feature to selectively cleave non-n-3 PUFAs in all 3 positions of triacylglycerols (TAGs); while high ethanol/TAGs ratio, low operation temperature and high tolerance to polar ethanol are essential conditions beyond biocatalyst itself. C-13 Nuclear magnetic resonance ((13)C NMR) analysis and competitive factor estimation verified the hypothesis and confirmed the plausible suggestion of catalytic mechanism of CAL-A.

Catalytic Biodiesel Production Mediated by Amino Acid-Based Protic Salts

Hetero- and homogeneous acid catalysts are effective catalysts for the production of biodiesel from oils containing high free fatty acids. The protic salts synthesized from natural amino acids were examined for catalytic activity and efficiency for the esterification of oleic acid after structural identification and characterization. In the esterification reaction of oleic acid with methanol, [Asp][NO3 ] was the best catalyst, and its high activity correlated to its high Hammett acidity. The optimal reaction conditions for the esterification of oleic acid to achieve 97u2009% biodiesel yield were: 70u2009°C, 10u2009% catalyst loading (w/w, on oleic acid basis), methanol/oleic acid ratio 7.5:1, and 5u2005h. Generally, [Asp][NO3 ] could be a good catalyst for the esterification of oleic acid with alcohols with chain lengths of up to six. The biodiesel yield of 93.86u2009% obtained from palm fatty acid distillate implies that the catalyst has potential for industrial application. A study of the kinetics indicated that the reaction followed pseudo-first-order kinetics with an activation energy and pre-exponential of 57.36u2005kJu2009mol-1 and 44.24×105 u2005min-1 , respectively. The aspartic acid-derived protic salt is a promising, operationally simply, sustainable, renewable, and possibly biodegradable catalyst for the conversion of free fatty acids into biodiesel.

β-Glucosidase from Thermotoga naphthophila RKU-10 for exclusive synthesis of galactotrisaccharides: Kinetics and thermodynamics insight into reaction mechanism

This work reports a novel thermophilic β-glucosidase (TN0602) from Thermotoga naphthophila RKU-10, demonstrating exceptionally high catalytic selectivity (100%) for the exclusive synthesis of prebiotic galactotrisaccharides (GOS3) in a high volumetric production yield of 23.28gL-1h-1 (higher than the highest value ever reported) at pH 6.5 and 75°C, with milk processing waste lactose as both the galactosyl donor and acceptor. A comparative study with commercial β-galactosidase from Aspergillus oryzae (AO) with respect to reaction kinetics, enzyme-substrate thermodynamic binding (substrate induced fluorescence quenching) and molecular docking simulation studies showed that β-glucosidase TN0602 has a deep catalytic pocket with a narrow entrance that prevents simultaneous access of lactose and GOS3 to the catalytic site, explaining its distinct catalytic specificity and reaction kinetics. The findings revealed in this work offer an improved understanding of how enzyme protein structure determines catalytic specificity, which serves as new knowledge to engineer β-glucosidase for the biosynthesis of designer GOS.

Rationale behind the near-ideal catalysis of Candida antarctica lipase A (CAL-A) for highly concentrating ω-3 polyunsaturated fatty acids into monoacylglycerols.

Dramatic decline in the quality and quantity of ω-3 PUFAs from marine resource demands new environmental-friendly technology to produce high quality ω-3 PUFAs concentrates in a better bioavailable form. Accordingly this work demonstrated an exceptionally highly efficient non-aqueous approach that non-regiospecific and non ω-3 PUFAs preferential Candida antarctica lipase A (CAL-A), functioning as a near-ideal biocatalyst, is capable to directly concentrate ω-3 PUFAs from 20% to 30% in oils to up to >90% in monoacylglycerols form through one step reaction. The rationale behind the experimental observation is justified and the catalytic property and specificity of an ideal enzyme tackling this task are defined. High selectivity and efficiency, excellent reusability of biocatalyst, general applicability for concentrating ω-3 PUFAs from both fish and microalgae oils, simple process for product recovery (e.g. by short path distillation), make this novel approach a highly industrially relevant and with potential application in food and drug industries.

Valorizing dairy waste: thermophilic biosynthesis of a novel ascorbic acid derivative

l-Ascorbic acid (l-AA) is an essential nutrient that is extremely unstable and cannot be synthesized by the human body. Therefore, attempts have been performed to develop biologically active l-AA derivatives with improved stability. This work presents a facile, scalable, and efficient enzymatic transgalactosylation of lactose to l-AA using β-glucosidase (TN0602) from Thermotoga naphthophila RKU-10. β-Glucosidase TN0602 displays high transgalactosylation activity at pH 5.0, 75 °C, and l-AA/lactose ratio of 2:1 to form a novel l-AA derivative [2-O-β-d-galactopyranosyl-l-ascorbic acid (l-AA-Gal)] with a maximal productivity of 138.88 mmol L-1 in 12 h, which is higher than most reports of enzymatic synthesis of l-AA-α-glucoside. Synthetic l-AA-Gal retains most l-AA antioxidant capability and presents dramatically higher stability than l-AA in an oxidative environment (Cu2+). In conclusion, this work reports a new way to valorize dairy waste lactose into a novel molecule l-AA-Gal, which could be a promising l-AA derivative to be used in a wide range of applications.

Biodiesel Production Using Lipases

Abstract Compared to chemical processes of biodiesel production, enzymatic processes have a range of advantages such as being environmentally friendly, offering low energy consumption, and following a simple process setup. In this chapter, biodiesel production using lipases as catalysts is reviewed. For comparison, the current chemical technology for biodiesel production is introduced, covering transesterification reactions with acid and alkali catalysts as well as two-step transesterifications. For the enzymatic approach, reactions and systems for the enzymatic biodiesel production are elaborated in the first place so that a basic understanding of the system can be built. This is followed by the analysis of the mechanism and kinetics of enzymatic biodiesel synthesis based on the Ping-Pong Bi Bi mechanism, and system parameters are further elucidated for the kinetic understanding. Furthermore, the effect of reaction conditions is evaluated for the enzyme-catalyzed biodiesel production process, including reaction temperature, alcohol donor, water content, lipase loading, reaction time, and (co)solvent systems. In the last part, industrial processes for enzymatic biodiesel production technology are covered.

Enhanced fish oil-in-water emulsions enabled by rapeseed lecithins obtained under different processing conditions

It is hypothesized that rapeseed lecithins may have different emulsifying and antioxidant properties in delivering fish oil compared to soy lecithin based on previous studies. The results showed that in vitro antioxidant activities of rapeseed lecithins were stronger than those of soy lecithin. Emulsions stabilized by rapeseed based lecithins and DATEM were stable over 3u202fmonths at 4u202f°C, whereas the creaming of emulsions containing soy lecithin started immediately after its preparation. Zeta-potential of rapeseed lecithins was higher than soy lecithin and DATEM, which partially contributed to the emulsion stability. Although the particle sizes of emulsions prepared by rapeseed lecithins increased after 14u202fdays storage, no creaming was observed. Lipid oxidation as indicated by TBARS values suggested that DATEM was the most unfavorable, followed by soy lecithin. It is concluded that rapeseed lecithins are better than soy lecithin and DATEM in terms of emulsion stability and antioxidant capability, respectively.