Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

The overall goal of this experimental procedure is to demonstrate the use of a coupled whole cell biocatalytic system immobilized in alginate beads for improved production yield and reusability. This is my subject, I have answered key questions in the biocatalysis field such as biocatalysts immobilizing, stability, cofactor regenerating, and whole cell biocatalysts. The main advantage of this technique is that it provides a simple yet flexible approach for improved production of many important biomolecules by cofactor dependent pathways.

Two recombinant E coli strains are used in this protocol. SpNOx E coli, which expresses NADH oxidase, and HjLAD E coli, which expresses L-arabinitol dehydrogenase. Since this procedure is the same for both E coli strains, it will only be demonstrated for HjLAD E coli.

Inoculate a single colony of HjLAD E coli in three milliliters of LB medium, supplemented with 15 micrograms per milliliter of Ket-A-My-Sin, and incubate over night in an incubator shaker. On the following day, dilute the culture by one to 100 in 200 milliliters of fresh LB containing 15 micrograms per milliliter of Ket-A-My-Sin, and incubate at 37 degrees Celsius at 250 RPM until the OD 600 reaches about 0.6. Induce HjLAD protein expression by adding 0.1 millimolar IPTG to the culture medium, and incubating at 25 degrees Celsius, 200 RPM, for six hours.

Harvest the induced cells by centrifugation at 3200 times G and four degrees Celsius for 20 minutes. Discard the supernatant, and proceed with processing the cell pellet as demonstrated in the next segment. To begin this procedure, we suspend the cell pellets of HjLAD E coli and SpNox E coli separately in 50 millimolar of Tris-Hcl buffer, at a cell density of five grams dry cell weight per liter.

In a 14 milliliter round bottom tube, mix 600 microliters of five gram dry cell weight per liter HjLAD E coli, 600 microliters of five gram dry cell weight per liter SpNOx E coli, 100 microliters of 20 millimolar NAD plus, and 150 microliters of two molar L-aribinitol. Bring the reaction volume to two milliliters by adding 550 microliters of 50 millimolar Tris-HCl. Incubate the reaction mixture at 30 degrees Celsius and 200 RPM for eight hours.

After eight hours, centrifuge the mixture at 3200 times G and four degrees Celsius for 10 minutes, and collect the reaction supernatant. To quantify L-xylulose production by a colorimetric assay, aspirate 100 microliters of the collected supernatant into a 1.5 milliter tube. Add 50 microliters of 1.5%cysteine, 900 microliters of 70%sulfuric acid, and 50 microliters of 0.1%Kar-Piz-All dissolved in ethanol, and mix gently by inverting the tube three times.

Incubate the reaction mixture at 37 degrees Celsius and 200 RPM for 20 minutes. Measure the optical absorbance of the reaction mixture at 560 nanometers using a spectrophotometer. Begin the immobilization protocol by preparing a 4%alginate solution.

Add 0.8 grams of sodium alginate to 20 milliliters of distilled water, and heat the mixture to dissolve the sodium alginate. Add 600 microliters of five gram dry cell weight per liter HjLAD E coli, and 600 microliters of five gram dry cell weight per liter SpNOx E coli to 1.2 milliliters of the 4%alginate solution. Mix the cells and alginate by gentle pipetting to avoid bubble formation.

To ensure the formation of alginate beads with uniform size, morphology, and rigidity, it is critical to add the cell alginate suspension for an optimal distance in a slow, drop wise fashion, into a sufficient volume of calcium chloride solution to cover all beads. Aspirate the alginate cell suspension into a syringe using a needle, then add the mixture drop wise into a 0.3 molar calcium chloride solution in a 100 milliliter beaker with continuous stirring. Leave the beads in the calcium chloride solution for two to three hours at room temperature without stirring to allow cross linking to take place.

Decant the calcium chloride solution without disturbing the beads by carefully pouring the calcium chloride solution into a 15 milliliter conical tube. Transfer the remaining beads into another 50 milliliter conical tube. To remove excessive calcium chloride and unencapsulated cells, wash the beads with 50 millimolar Tris-HCl buffer.

Add 10 milliliters of buffer to the beads, and allow the suspension to stand undisturbed for three to five minutes. Once the beads settle at the bottom, decant the buffer into another container. In this manner, wash the beads three times.

Do not centrifuge the beads at any given step or they will rupture. Do not discard the used Tris-HCl wash buffer. Pull the 30 milliliters of used wash buffer with the used calcium chloride solution collected earlier.

Pellet the unimmobilized E coli cells by centrifugation of the pulled calcium chloride and Tris-HCl solution at 3200 times G for 20 minutes. Transfer the washed beads into a tube. Subsequently, evaluate L-xylulose biosynthesis following the procedure demonstrated for the cell pellets but using all of the washed beads in place of cell pellets.

To assess the stability of the immobilized biocatalysts for L-xylulose production, collect the beads after they are formed. And wash twice with 10 milliliters of 50 millimolar Tris-HCl buffer without centrifugation as shown earlier. Use all of the washed beads to perform the reaction for L-xylulose production as demonstrated earlier.

Quantify L-xylulose production by a colorimetric assay, also as demonstrated earlier. Repeat the L-xylulose production reaction for the desired number of production cycles, and measure the amount of L-xylulose produced in the reaction supernatant in each cycle. L-xylulose production is enhanced by this coupled whole cell biocatalytic system.

Starting with an initial concentration of 150 millimolar L-aribinitol, the use of HjLAD E coli biocatalyst alone produced a yield of 79%L-xylulose. In comparison, the use of the HjLAD E coli and SpNOx E coli cells at a one to one ratio produced a yield of 96%L-xylulose. An evaluation of the parameters influencing biocatalyst immobilization efficiency revealed that efficiency increases with increasing sodium alginate concentration in the range of one to three percent.

However, sodium alginate concentrations greater than 2%result in decreased L-xylulose yield. Using a sodium alginate concentration of 2%the optimal calcium chloride concentration for immobilization and L-xylulose yield was determined as 0.3 molar. Biocatalyst reusability was assessed.

In this graph, L-xylulose production for seven successive cycles of reuse of immobilized and free coupled whole cell biocatalysts are shown in dark and light grey, respectively. Although the maximal L-xylulose yield by immobilized coupled whole cell biocatalysts is lower than by a free coupled whole cell system, reusability is improved by immobilization. At the end of seven cycles of successive reuse, the immobilized biocatalysts maintained their stability, and retained 65%of the original L-xylulose yield.

After its development, this technique paved the way for researchers in the field of biocatalysis to explore the yields of full multi cell biocatalysts, and the main reactions that involve cofactors.