Rajni Kaul

Extractive bioconversion in aqueous two-phase systems

SummaryThe transformation of hydrocortisone to prednisolone was studied in aqueous two-phase systems, as a model for the extractive bioconversion of fine chemicals. The bacterium, Arthrobacter simplex, was able to grow in the two-phase system and the cells could be revitalized after a period of use. Use of aqueous two-phase systems made it possible to operate the reaction at higher substrate concentrations than in pure buffer solution. An adsorptive method to remove the product from the top phase was tested and shown to be both efficient and compatible with the overall process. In order to reduce the costs of operation in aqueous two-phase systems, a cheaper starch-based polymer, Reppal-PES, was successfully used as a substitute for dextran.

Extractive lactic acid fermentation in poly (ethyleneimine)-based aqueous two-phase system.

The potential of an aqueous two‐phase system composed of a polycation, poly(ethyleneimine) (PEI), and an uncharged polymer, (hydroxyethyl) cellulose (HEC), for extractive lactic acid fermentation was tested. Batch fermentation with 20 g/L glucose in two‐phase medium using Lactococcus lactis without external pH control resulted in 3–4 times higher amount of lactate and biomass produced as compared to that in a conventional one‐phase medium. Lactic acid was preferentially partitioned to the PEI‐rich bottom phase. However, the cells which favored the HEC‐rich top phase in a fresh two‐phase medium were partitioned to a significant extent to the bottom phase after fermentation. Addition of phosphate buffer or pH adjustment to 6.5 after fermentation caused fewer cells to move to the bottom phase. With external pH control, fermentation in normal and two‐phase medium showed no marked differences in glucose consumption and lactic acid yield, except that about 1.3 times higher cell density was obtained in the two‐phase broth, especially at initial glucose concentrations of 50–100 g/L. Use of higher concentration of phosphate during batch fermentation in the two‐phase medium with 50 g/L sugar provided a 15% higher yield of lactic acid, but the growth rate of cells was nearly half of the normal, thus affecting the productivity. Continuous fermentation with twice the normal phosphate concentration resulted in higher cell density, product yield, and productivity in two‐phase medium than in monophasic medium.

Affinity precipitation of proteins.

Affinity precipitation is being studied as a technique to be introduced at an early stage of downstream processing for the selective isolation of proteins. The technique utilizes a heterobifunctional ligand, which, in addition to having affinity for the target protein(s), possesses another function for controlling precipitation. The latter component is comprised of a polymer which can be made reversibly soluble and insoluble by altering a specific parameter such as pH or temperature. Different polymers of natural and synthetic origin have been used for this purpose. The soluble form of the ligand is used for the affinity binding step and precipitation is induced for obtaining separation of the affinity complex. Some of the polymers used in this laboratory include chitosan, alginate, Eudragit S‐100 (copolymer of methacrylic acid and methyl methacrylate) and polyethyleneimine. Chitosan and alginate served as natural ligands for wheat germ agglutinin and pectinase, respectively. The aromatic dye Cibacron Blue 3GA coupled to Eudragit S 100 and polyethyleneimine way used for the affinity precipitation of some model enzymes such as lactate dehydrogenase and alcohol dehydrogenase. As prior removal of cell debris, etc., is essential for affinity precipitation, the possibility of integration of the technique with extraction in aqueous two‐phase systems was also demonstrated.

A model study on Eudragit and polyethyleneimine as soluble carriers of α-amylase for repeated hydrolysis of starch

Eudragit and polyethyleneimine have been evaluated for their potential as soluble carriers of enzymes acting on macromolecular substrates. The present study is based on coupling of α-amylase (Termamyl) to these polymers for the degradation of starch. The enzyme was bound to both the polymers by carbodiimide coupling. Using soluble starch as substrate, about 96% of the enzyme activity was found to be retained on binding to Eudragit, while the binding to polyethyleneimine resulted in activation of the enzyme. Kinetic properties and optimal conditions for activity and stability of the soluble-immobilized enzyme preparations were compared with that of the free enzyme. The polymer-enzyme complexes were also used for the repeated hydrolysis of starch, the biocatalyst being recovered in between runs by precipitation. The enzyme in the immobilized form exhibited good stability during repeated runs of starch liquefaction.

Evaluation of alginate-immobilized Lactobacillus casei for lactate production

SummaryLactate production by immobilized Lactobacillus casei has been studied. The cells were immobilized in alginate and the effect of variations in different parameters on product formation and productivity was investigated. The performance of the reaction was evaluated in stirred batch as well as in packed-bed conditions. pH control was a problem in the packed-bed reactor. In stirred batch experiments, nearly total glucose utilization was observed with a lactate yield of 90–99% and a total productivity of 1.6 g·l−1·h−1. Under standard conditions only a low percentage (3–4%) of the total lactate formed was the abetd-isomer. When immobilized cells were reused, increased formation of abetd-lactate took place, especially when the cell conditions were sub-optimal. After revitalization by exposure to growth nutrients the balance was restored.

Purification of xylanase from Trichoderma viride by precipitation with an anionic polymer Eudragit S 100

Endo-xylanase from T. viride has been purified 4.2 fold by precipitation with a commercially available enteric polymer Eudragit S-100. Electrophoretic analysis also indicated removal of contaminant proteins. The enzyme could be recovered in more than 89% yield. The binding of the enzyme to the polymer was predominantly by electrostatic interaction.

Integration of aqueous two-phase extraction and affinity precipitation for the purification of lactate dehydrogenase.

Integration of extraction in aqueous two-phase system and affinity precipitation was investigated as a technique for purification of lactate dehydrogenase (LDH) from porcine muscle extract. An enteric coating polymer, Eudragit S 100, which can be made reversibly soluble and insoluble by change in pH was used as the ligand carrier. The ligand used was Cibacron blue 3GA. The polymer is nearly totally partitioned to the top phase (> 98%) in PEG-dextran aqueous two-phase system. The enzyme, lactate dehydrogenase, was first spontaneously partitioned to the bottom phase in a 6% (w/w) PEG 8000-8% (w/w) dextran T250 phase system. New PEG phase and Eudragit-dye were then added to the bottom phase, which helped in extraction of LDH to the top phase. After a washing step with a fresh bottom phase, Eudragit-dye-target protein affinity complex was precipitated out from the top phase by lowering the pH to 5.1. The enzyme was recovered by treatment of the complex with 0.5 M NaCl with a yield of 54% and a specific activity of 245 units/mg. The purification of LDH by this procedure was better than that obtained by a single step of affinity partitioning.

Affinity thermoprecipitation of lactate dehydrogenase and pyruvate kinase from porcine muscle using Eudragit bound Cibacron blue

Abstract Cibacron blue coupled to Eudragit S 100 was used for isolation of lactate dehydrogenase and pyruvate kinase from crude porcine muscle extract by the technique of affinity thermoprecipitation. The polymer preparation with an immobilized dye content of about 195 mg g −1 dry weight was precipitated at 40°C in the presence of 50 mM Ca 2+ ions, and was made soluble at room temperature in the absence of the calcium ions. Isolation of the enzymes from the crude extract by co-precipitation with Eudragit-Cibacron blue was studied at two loadings of the extract. At a low load, both the enzymes were precipitated and were desorbed sequentially by treatment with increasing concentrations of salt. Of the two enzymes, mainly lactate dehydrogenase was precipitated from the high load of the crude extract and was recovered with a higher purity.

Immobilization of Lactobacillus casei cells to ceramic material pretreated with polyethylenimine

SummaryThe cells of Lactobacillus casei were adsorbed to Poraver, foam glass particles pretreated with polyethylenimine (PEI). Exposure of cells for a relatively short period to Poraver beads coated with a high concentration of PEI resulted in maximal adsorption with good retention of metabolic activity. The immobilized cells were tested in packed-bed and stirred-tank reactors for lactic acid production. Stirred-tank operations were more effective in terms of productivity but the support was sensitive to attrition. The beads exhibited good mechanical stability to withstand pressure in the packed-bed reactor.

Purification of the d-lactate dehydrogenase from Leuconostoc mesenteroides ssp. cremoris using a sequential precipitation procedure

Abstract Selective precipitation was used for the purification of d -lactate dehydrogenase from cell homogenate of Leuconostoc mesenteroides ssp. cremoris . This was facilitated by the use of a copolymer of methacrylic acid and methylmethacrylate with the tradename of Eudragit S 100, which is precipitated by a pH-shift towards acidic conditions. Eudragit modified with ethanolamine was used to precipitate contaminating proteins which were removed along with cell debris. d -LDH was recovered from the supernatant by adsorption to Eudragit-Cibacron blue. The dissociation of the specifically bound protein was carried out by treatment with salt. The enzyme was finally subjected to ion exchange chromatography. The enzyme was purified more than 33 times and the specific activity of the pure enzyme after the three-step process was 534 units per mg protein.