Caixia Cui

Immobilization of Yarrowia lipolytica lipase Ylip2 for the biocatalytic synthesis of phytosterol ester in a water activity controlled reactor.

In this work, phytosterol ester was synthesized using Yarrowia lipolytica lipase Ylip2 that had been immobilized on inorganic support in a solvent-free system and reacted in a computer-aided water activity controlled bioreactor. The immobilization of Ylip2 on celite led to a remarkable increase in the phytosterol conversion compared to that of free lipase. An investigation of the reaction conditions were oleic acid as the fatty acid variety, 10,000U/g substrate, and a temperature of 50°C for phytosterol ester synthesis. Controlling of the water activity at a set point was accomplished by the introduction of dry air through the reaction medium at a digital feedback controlled flow rate. For the esterification of phytosterol ester, a low (15%) water activity resulted in a considerable improvement in phytosterol conversion (91.1%) as well as a decreased reaction time (78h). Furthermore, Ylip2 lipase immobilized on celite retained 90% esterification activity for the synthesis of phytosterol oleate after reused 8 cycles, while free lipase was only viable for 5 batches with 90% esterification activity remained. Finally, the phytosterol oleate space time yield increased from 1.65g/L/h with free lipase to 2.53g/L/h with immobilized lipase. These results illustrate that the immobilized Yarrowia lipolytica lipase Ylip2 in a water activity controlled reactor has great potential for the application in phytosterol esters synthesis.

Synergistic effects of amine and protein modified epoxy-support on immobilized lipase activity

We have developed an improved and effective method to immobilize Yarrowia lipolytica lipase Lip2 (YLIP2) on an epoxy poly-(glycidylmethacrylate-triallyisocyanurate-ethyleneglycoldimethacrylate) (PGMA-TAIC-EGDMA) support structure with or without amine or/and protein modifications. Our results show that there is an increase in the activity of the immobilized lipase on n-butylamine (BA) modified support (420U/g support) and the biocompatible gelatin modified support (600U/g support) when compared to the support without modification (240U/g support). To further study the influences of BA and gelatin modification on the activity of the immobilized lipase, gelatin and BA were concurrently used to decorate the support structure. Lipase immobilized on 2% BA/gelatin (1:1) modified support obtained the highest activity (1180U/g support), which was five-fold higher than that on a native support structure. These results suggest that the activity of a support-immobilized lipase depends on the support surface properties and a moderate support surface micro-environment was crucial for elevated activity. Collectively, these data show that a combined gelatin and BA modification regulates the support surface more suitable for immobilizing YLIP2.

Enhancing trimethylolpropane esters synthesis through lipase immobilized on surface hydrophobic modified support and appropriate substrate feeding methods.

Candida sp. 99-125 lipase immobilized on surface hydrophobic modified support and appropriate substrate feeding methods were used to improve the synthesis of tri-substituted trimethylolpropane (TMP) esters, which can be used as raw materials for biodegradable lubricants. The proposed novel production method is environmentally friendly. Lipase was adsorbed on surface hydrophobic silk fibers that were pretreated by amino-modified polydimethylsiloxane. A 5-level-4-factors central composite model, including reaction time, temperature, enzyme amount, and molar ratio of fatty acid to TMP, was designed to evaluate the interaction of process variables in the enzymatic esterification. The water activity was kept constant using a LiCl-saturated salt solution. Under the optimum conditions with 30% enzyme amount and substrates molar ratio 8.4 at 45°C for 47h, the total conversion of caprylic acid is 97.3% and the yield of tri-substituted TMP esters is 95.5%. The surface hydrophobic treatment resulted in less cluster water accumulated on the surface immobilized lipase, which was demonstrated by near-infrared spectra. Consequently, the optimum temperature and water tolerance of immobilized lipase were increased. Two TMP-feeding methods were used to maintain high molar ratio of fatty acid to TMP, and increase the final tri-substituted TMP esters content exceeding 85% (w/w) in reactant.

Synthesis of biosafe isosorbide dicaprylate ester plasticizer by lipase in a solvent-free system and its sub-chronic toxicity in mice

In this study, biosafe isosorbide dicaprylate ester based plasticizer was prepared using renewable feedstock with lipase in a solvent-free system. Different kinds of water removal methods and some important factors including molar ratio of the substrates, reaction temperature and catalyst loading were investigated. Bubbling dried air was determined to be the most effective water removal method. The activation energy (E) of hydrolysis and esterification to form di-substituted isosorbide were examined to determine the limiting step in the synthesis of isosorbide dicaprylate ester. The Ehydrolysis (25.51 kJ mol−1) was found to be higher than Esynthesis (35.65 kJ mol−1), demonstrating that the formation of diester from monoester is the critical step in the process. The lipase can be recycled up to 16 times while 80% diester was maintained. Additionally, the properties of poly(vinyl chloride) PVC blends plasticized with isosorbide ester as secondary plasticizer was studied. The results indicated that the thermal stability of plasticized PVC blends was improved, and the tensile strength was increased. Furthermore, the sub-chronic toxicity study in mice showed that the isosorbide ester was safe, indicating its great potential in industrial applications as a plasticizer.

Improving performance of Yarrowia lipolytica lipase lip2-catalyzed kinetic resolution of (R, S)-1-phenylethanol by solvent engineering

Abstract Extracellular Yarrowia lipolytica lipase Lip2 (YLIP2) demonstrated an (R)-enantiopreference for efficient resolution of (R,S)-1-phenylethanol by solvent engineering with different kinds of binary solvent. The enantioselectivity was significantly improved by the addition of 1, 4-dioxane. The reaction parameters including co-solvent concentration, reaction temperature, and the reaction time were optimized. When the reaction was carried out with n-hexane in the presence of 0.8% 1,4-dioxane at 50°C for 72 h, the enantiomeric excess of product markedly increased to 99.1% from 66% in pure n-hexane; the enantiomeric ratio was higher than 200, which was 500-fold compared with that in pure n-hexane. The results indicated that it is very important to design the proper co-solvents, especially to create appropriate micro-environment for YLIP2 for catalyzing the resolution of (R,S)-1-phenylethanol.

Environmentally-friendly strategy for separation of 1,3-propanediol using biocatalytic conversion

Glycerol waste from the biodiesel production can be used as a carbon source in the production of 1,3-propanediol (1,3-PD) through microbial fermentation. However, downstream processing is a major bottleneck that restricts its biological production. Here, we investigated an environmentally-friendly method to enzymatically separate 1,3-PD. The transformation of 1,3-PD to an ester was achieved by exploiting the esterification reaction with fatty acids under lipase catalysis. The reaction efficiency was optimized using different poly-alcohols that were existed in the fermentation broth reacted with a fatty acid. Whereas the 1,3-PD conversion reached 62%, only a 0.06% and 0.08% conversion was reached for 2,3-butanediol and glycerol, illustrating the formers more efficient separation. The recovery efficiency of 1,3-PD was 96%. Finally, 1,3-PD was obtained by lipase-directed ester hydrolysis. Taken together, the bio-catalyzed separation process presented here is a novel and promising method for recovering 1,3-PD.

Insight into the synthesis of isosorbide diester plasticizer using immobilized lipases

A bio-plasticizer isosorbide diester was synthesized using three immobilized lipases to select a suitable catalyst. These three immobilized lipases demonstrated an (R)-enantio-preference for synthesizing isosorbide monoesters with different MS : MR values. The S-monoester content was close to zero when the reaction was catalyzed by Yarrowia lipolytica lipase lip2 (Ylip2) or RM IM. Thus, the MS synthesis rate was the limiting step for diester production. To overcome this constraint, the catalysis condition was optimized by using mixture of Novo 435 & Ylip2, or Novo 435 & RM IM, or Ylip2 and Novo 435 sequentially, respectively. When the reaction was sequentially catalyzed by Ylip2 and Novo 435, the highest diester content was obtained (77.4%), which was 2.1- and 1.7-fold greater than the content obtained using either Ylip2 or Novo 435 alone, respectively.

A water-dependent kinetics guide for complex lipase-mediated synthesis of biolubricants in a water activity control reactor

A water-dependent kinetic model for a lipase-mediated reaction with multiple substrates and products in a water activity control reactor was developed. Solvent-free esterification of trimethylolpropane (TMP), the products of which can be used as biolubricants, was investigated using a lipase from Candida sp. 99–125 as catalyst under variable water activities in a 5 L batch stirred tank reactor. Water activity control was accomplished at the set point by introduction of dry air through the reaction medium at a digital feedback-controlled flow rate. For the cases of esterification of TMP, long-term (>72 h) control of water activity resulted in a considerable improvement of yield. By introducing progressive water removal and combining principles with an intrinsic kinetic model of the Ping-Pong Bi-Bi mechanism, the integrated model can predict both the forward and reverse rates for TMP reactions. Kinetic parameters depending on the water activity were estimated by nonlinear regression fitting of experimental data. The proposed approach not only enables optimization of the reaction under defined conditions, but also provides a solution for industrial upscaling of similar environmentally friendly biocatalytic processes.

Synthesis of 2-ethyl hexanol fatty acid esters in a packed bed bioreactor using a lipase immobilized on a textile membrane

Abstract An enzymatic process using a packed bed bioreactor with recirculation was developed for the scale-up synthesis of 2-ethylhexyl palmitate with a lipase from Candida sp. 99–125 immobilized on a fabric membrane by natural attachment to the membrane surface. Esterification was effectively performed by circulating the reaction mixture between a packed bed column and a substrate container. A maximum esterification yield of 98% was obtained. Adding molecular sieves and drying the immobilized lipase both decreased the water content at the reactor outlet and around the enzyme, which led to an increase in the rate of esterification. The long-term stability of the reactor was tested by continuing the reaction for 30 batches (over 300 h) with an average esterification yield of about 95%. This immobilized lipase bioreactor is scalable and is thus suitable for industrial production of 2-ethylhexyl palmitate.