An efficient and high-water-content enzymatic esterification method for the synthesis of ß-sitosterol conjugated linoleate via a sodium citrate-based three-liquid-phase system.

Three-liquid-phase systems (TLPSs) are novel interfacial enzymatic reaction systems that have been successfully applied in many valuable reactions. However, these systems are suitable only for hydrolysis reactions and not for more widely used esterification reactions. Surprisingly, our recent research revealed that two water-insoluble substrates (ß-sitosterol and conjugated linoleic acid) could be rapidly esterified in this system. The initial rate of the esterification reaction in the TLPS based on sodium citrate was enhanced by approximately 10-fold relative to that in a traditional water/n-hexane system. The special emulsion structure (S/W1/W2 emulsion) formed may be vital because it not only provides a larger reaction interface but also spontaneously generates a middle phase that might regulate water activity to facilitate esterification. Furthermore, the lipase-enriched phase could be reused at least 8 times without significant loss of catalytic efficiency. Therefore, this TLPS is an ideal enzymatic esterification platform for ester synthesis because it is efficient, convenient to use, and cost-effective.

A green and efficient two-step enzymatic esterification-hydrolysis method for enrichment of c9,t11-CLA isomer based on a three-liquid-phase system.

A novel two-step enzymatic esterification-hydrolysis method that generates high-purity conjugated linoleic acid (CLA) isomers was developed. CLA was first partially purified by enzymatic esterification and then further purified by efficient, selective enzymatic hydrolysis in a three-liquid-phase system (TLPS). Compared with traditional two-step selective enzymatic esterification, this novel method produced highly pure cis-9, trans-11 (c9,t11)-CLA (96%) with high conversion (approx. 36%) and avoided complicated rehydrolysis and reesterification steps. The catalytic efficiency and selectivity of CLA ester enzymatic hydrolysis was greatly improved with TLPSs, as high-speed stirring provided a larger interface area for the reaction and product inhibition was effectively reduced by extraction of the product into other phases. Furthermore, the enzyme-enriched phase (liquid immobilization support) was effectively and economically reused more than 8 times because it contained more than 90% of the concentrated enzyme. Therefore, this novel enzymatic esterification-hydrolysis method can be considered ideal to produce high-purity fatty acid monomers.

A Highly Efficient Three-Liquid-Phase-Based Enzymatic One-Pot Multistep Reaction System with Recoverable Enzymes for the Synthesis of Biodiesel.

A three-liquid-phase system (TLPS) was developed and used as a novel enzymatic one-pot multistep reaction (EOMR) system. In this system, lipase and phospholipase were enriched in a single liquid phase with a high recovery (ca. 98%) and then used for the simultaneous catalysis of mutually inhibiting and interfering reactions (hydrolysis of phospholipids and glyceride in crude oil). A novel emulsion containing the two dispersed droplets (W2/O/W2 and W1/W2 emulsion structures) could be the key reason for this phenomenon because the emulsion system not only provided a new catalytic interface but also relieved the product inhibition. As a result, the content of free fatty acid (main hydrolysate of the glyceride) and the removal of phospholipid from the crude oil could be increased to 96 and 95%, respectively, within 1 h. The product obtained from the EOMR was directly used in the production of biodiesel via enzymatic esterification, and the content of fatty acid methanol ester could be increased to 93% within 2 h. Furthermore, the enzymes in the middle phase could also be reused, at least for eight rounds without significant loss in catalytic efficiency. Therefore, the TLPS could be considered as an ideal catalytic platform for the EOMR.

An in vitro and in vivo study of the brain-targeting effects of an epidermal growth factor-functionalized cholera toxin-like chimeric protein.

The development of neuroprotective drugs has proven to be extremely difficult because of the blood-brain barrier. Intranasal administration is thought to transport the drug from the nasal cavity along the olfactory and trigeminal nerves to the brain, thus bypassing the blood-brain barrier. However, macromolecular protein drugs have low delivery efficiency via this route in general. We hypothesized that an innocuous cholera toxin-like chimeric protein could better enhance the efficiency of protein delivery through the intranasal route. To test this hypothesis, we designed an enhanced green fluorescent protein (EGFP) chimera to evaluate the effect of the cholera toxin (CT) as a carrier for drug delivery into the brain. Then, the EGFP was replaced with epidermal growth factor (EGF) in the chimeric protein, and the therapeutic effect of the new chimeric protein was studied in an LPS-induced neuritis mouse model. The results suggest that the CT-like chimeric protein can bypass the blood-brain barrier and enter the brain in approximately 30 min. This EGF chimeric protein can effectively protect the spatial cognitive ability of and confer anti-anxiety protection to mice. The results indicate that cholera toxin-like chimeric proteins are potential tools for effectively delivering macromodecular drugs into the brain through intranasal administration.