Encapsulation of ß-amylase in water-oil-water enzyme emulsion liquid membrane (EELM) bioreactor for enzymatic conversion of starch to maltose.

The ß-amylase was encapsulated in emulsion liquid membrane (ELM), which acted as a reactor for conversion of starch to maltose. The membrane phase was consisted of surfactant (span 80), stabilizer (polystyrene), carrier for maltose transport (methyl cholate) and solvent (xylene). The substrate starch in feed phase entered into the internal phase by the process of diffusion and hydrolyzed to maltose by encapsulated ß-amylase. Methyl cholate present in the membrane acts as a carrier for the product maltose, which helps in transport of maltose to feed phase from internal aqueous phase. The residual activity of ß-amylase after the five-reaction cycle was found to decrease to ∼70%, which indicated possibility to recycle the components of the emulsion and enzyme. The pH and temperature of the encapsulated enzyme were found to be optimum at 5.5 and 60 °C, respectively. The novelty of the present work lies in the development of Enzyme Emulsion Liquid Membranes (EELM) bioreactor for the hydrolysis of starch into maltose mediated by encapsulated ß-amylase. The attempt has been made for the first time for the successful encapsulation of ß-amylase into EELM. The best results gave the highest residual enzyme activity (94.1%) and maltose production (29.13 mg/mL).

Selective extraction of lipase and amylase from enzyme mixture by employing liquid emulsion membrane.

This work deals with the extraction of lipase and amylase from enzyme mixture by employing liquid emulsion membranes (LEM). The electrostatic interaction between enzymes and reverse micellar surfactant polar head group plays an important role for selective extraction of two different enzymes having different isoelectric points. The optimized conditions for lipase extraction (pH 7.0) resulted in the purification fold and activity recovery of 5.43 fold and 89.53%, respectively, whereas, in case of amylase (pH 9.0) the purification fold and activity recovery were 6.58 and 94.32%, respectively. The results were compared with the control sample (containing individual enzymes) and mixture of enzymes lipase and amylase and it was shown that for optimum conditions the activity recovery and purification fold was higher for the individual enzymes as compared to their mixture. Downstream processing involving LEM was shown to be a feasible method for selective extraction of enzymes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:721-729, 2018.

Downstream processing of lactoperoxidase from milk whey by involving liquid emulsion membrane.

The current work deals with downstream processing of lactoperoxidase using liquid emulsion membrane from the bovine milk whey, which is a by-product from dairy industry. It is an alternate separation technique that can be used for the selective extraction of lactoperoxidase. The extraction of lactoperoxidase in liquid emulsion membrane takes place due to the electrostatic interaction between the enzyme and polar head group of reverse micellar surfactant. The optimum conditions resulted in 2.86 factor purity and activity recovery of 75.21%. Downstream processing involving liquid emulsion membrane is a potential technique for the extraction of lactoperoxidase from bovine whey.

Mixed reverse micelles facilitated downstream processing of lipase involving water-oil-water liquid emulsion membrane.

Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water-oil-water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0-fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1-17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream processing of enzymes/proteins.

ATPase inhibitor based luciferase assay for prolonged and enhanced ATP pool measurement as an efficient fish freshness indicator.

The nucleotide degradation pathway in somatic cells leads to the accumulation of products such as hypoxanthine and inosine, which are commonly used as fish and meat freshness indicators. Assays based on these molecules cannot differentiate the postmortem time over a short period of time (5-10 h). Further, quantification of these degradation products is cumbersome, costly and time-consuming. For the proposed assay, optimal concentrations of 30 and 2 mM, respectively, for the ATPase inhibitors sodium orthovanadate and EDTA were found. Further, it was observed that a firefly luciferase based assay could enhance the sensitivity levels up to 165-fold at 30 °C. In addition, it was observed that the sensitivity for ATP assay was enhanced up to 60-fold even after 12 h. The limit of detection for the ATP assay was 1 pM, unlike other conventional methods, which are sensitive only up to micromolar levels. Moreover, as little as 0.044 g fish fillet was required for the assay, and no time-consuming sample preparation was necessary. Luminescence of prolonged duration was observed in harvested fish kept at -20 °C in comparison with fish kept at 4 and 30 °C, which reflects the shelf life of fish preserved at lower temperatures.

Reverse micelles-mediated transport of lipase in liquid emulsion membrane for downstream processing.

This work deals with the downstream processing of lipase (EC 3.1.1.3, from Aspergillus niger) using liquid emulsion membrane (LEM) containing reverse micelles for the first time. The membrane phase consisted of surfactants [cetyltrimethylammonium bromide (CTAB) and Span 80] and cosolvents (isooctane and paraffin light oil). The various process parameters for the extraction of lipase from aqueous feed were optimized to maximize activity recovery and purification fold. The mechanism of lipase transport through LEM consisted of three steps namely solubilization of lipase in reverse micelles, transportation of reverse micelles loaded with lipase through the liquid membrane, and release of the lipase into internal aqueous phase. The results showed that the optimum conditions for activity recovery (78.6%) and purification (3.14-fold) were feed phase ionic strength 0.10 M NaCl and pH 9.0, surfactants concentration (Span 80 0.18 M and CTAB 0.1 M), volume ratio of organic phase to internal aqueous phase 0.9, ratio of membrane emulsion to feed volume 1.0, internal aqueous phase concentration 1.0 M KCl and pH 7.0, stirring speed 450 rpm, and contact time 15 min. This work indicated the feasibility of LEM for the downstream processing of lipase.