Name: immobilization of multi-biocatalysts in alginate beads for cofactor regeneration and improved reusability
Uploaded: 2016-04-22T15:00:00
Description: Watch this Scientific Journal Video about Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability at JoVE.com

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2012159 and NRF-2013R1A1A2007561), Konkuk University, and the Department of Chemical Engineering and MCubed Program at the University of Michigan.

1. Whole-cell Biocatalysts Preparation &#160;
NOTE: The recombinant E. coli cells harboring pET28a-SpNox31 or pET28a-HjLAD28 are hereafter referred to as E. coliSpNox and E. coliHjLAD, respectively.
<ol>
	<li>Inoculate a single colony of E. coliHjLAD in 3 ml&#160;of Luria-Bertani (LB) medium supplemented with kanamycin (50 &#181;g/ml) and incubate in an incubator shaker O/N at 37 oC, 2...

Recent technological advancements have enabled a surge in the commercialization of recombinant biotherapeutics, resulting in a gradual rise in their market value in the biotechnology industry. One such advancement is the advent of metabolic engineering in recombinant microorganisms, which has shown a great promise in establishing scalable industrial systems38. As with most processes, the successful commercialization of recombinant biomolecules produced by genetically engineered microbes is highly dependent on ...

Reductive whole-cell biotransformation using microorganisms has become a widespread method for the chemo-enzymatic synthesis of commercially and therapeutically important biomolecules1-3. It presents several advantages over the use of isolated enzymes, especially the elimination of cost-intensive downstream purification processes and the demonstration of an extended lifetime4-7. For biocatalytic pathways where cofactors are required for product formation, whole-cell systems have the potential to provide in situ cofactor regeneration via the addition of inexpensive electron-donating co-substrates5,8,9. However, this capacity is diminished for reactions that require a stoichiometric concentration of rare or expensive co-substrates10-13. Together with poor reusability of whole cells, this impedes the establishment of a scalable and continuous production system. Strategic modifications of whole-cell systems for these cofactor-dependent biotransformations are required to overcome the aforementioned limitations. Specifically, the combination of whole-cell biocatalysts that work cooperatively have been shown to significantly enhance the productivity and stability of the harbored enzymes14. These factors, which are often critical for enabling large-scale production of commercially viable products, can be optimized further by co-immobilizing biocatalytic microbes15. We have recently developed a simple and reusable whole-cell biocatalytic system that allows both cofactor regeneration and biocatalyst immobilization for the L-xylulose production16. In this study, this system was utilized as an example to illustrate the experimental procedures of applying these two strategies for improved biotransformation production yield and biocatalyst reusability.
L-xylulose belongs to a class of biologically useful molecules named rare sugars. Rare sugars are unique monosaccharides or sugar derivatives that occur very rarely in nature, but play crucial roles as recognition elements in bioactive molecules17,18. They have a variety of applications ranging from sweeteners, functional foods to potential therapeutics19. L-xylulose can be used as a potential inhibitor of multiple &#945;-glucosidases, and may also be used as an indicator of hepatitis or liver cirrhosis17,20. High efficiency conversion of xylitol to L-xylulose in whole-cell systems has been reported previously in Pantoea ananatis21,22, Alcaligenes sp. 701B23, Bacillus pallidus Y2524,25 and Escherichia coli26. In E. coli, however, it was achieved only using low (&#60;67 mM) xylitol concentrations26 due to potential inhibitory effects of an initial xylitol concentration higher than 100 mM on xylitol-4-dehydrogenase activity21,26. The thermodynamic equilibrium between xylulose and xylitol has been shown to strongly favor the formation of xylitol25,27. Additionally, xylulose yield is limited by the amount of expensive cofactors that have to be supplied in the absence of an in situ cofactor regeneration system. Together, these factors limit the potential for scaling into sustainable systems for L-xylulose biosynthesis.
To overcome these limitations and improve the L-xylulose biotransformation yield, the strategy of cofactor regeneration was employed first by establishing a coupled whole-cell biocatalytic system. Specifically, L-Arabinitol 4-dehydrogenase (EC 1.1.1.12) from Hypocrea jecorina (HjLAD), an enzyme in the L-arabinose catabolic pathway of fungi, was selected to catalyze the conversion of L-arabinitol into L-xylulose28,29. Like many biosynthetic enzymes, a major limitation of HjLAD is that it requires a stoichiometric amount of the expensive nicotinamide adenine dinucleotide cofactor (NAD+, the oxidized form of NADH) to carry out this conversion. NADH oxidase found in Streptococcus pyogenes (SpNox) has been shown to display high cofactor-regeneration activity30,31. Taking advantage of this attribute of SpNox, E. coli cells expressing HjLAD for the production of L-xylulose were coupled with E. coli cells expressing SpNox for the regeneration of NAD+ to boost the L-xylulose production depicted by the coupled reaction shown in Figure 1A. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, making this system much more efficient than those reported in literature.
The strategy of whole-cell immobilization was employed next to further enhance the reusability of the coupled biocatalytic system. Commonly used methods for whole-cell immobilization include adsorption/covalent linking to solid matrices, cross-linking/entrapment and encapsulation in polymeric networks32. Among these approaches, the most suitable method for cell immobilization is encapsulation in calcium alginate beads. Their mild gelation properties, inert aqueous matrix and high porosity help preserve the physiological properties and functionality of the encapsulated biologicals33. Therefore, the coupled biocatalyst system containing both E. coli cells harboring HjLAD or SpNox was immobilized in calcium alginate beads to enable multiple cycles of L-xylulose production (Figure 2).The immobilized biocatalyst system demonstrated good operational stability, maintaining 65% of the conversion yield of the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost its catalytic activity.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">LB broth&#160;</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">L3022-6X1KG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Kanamycin</td>
			<td style="width:200px;">Fisher</td>
			<td style="width:119px;">BP906-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Isopropyl &#946;-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">I6758-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Tris base</td>
			<td style="width:200px;">Fisher</td>
			<td style="width:119px;">BP1521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">B-Nicotinamide adenine dinucleotide hydrate</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">N7004-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">L-Arabinitol</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">A3506-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">L-Cysteine</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">168149</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sulfuric acid</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">320501-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Carbazole</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">C5132</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Ethanol&#160;</td>
			<td style="width:200px;">Fisher</td>
			<td style="width:119px;">BP2818-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Sodium alginate</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">W201502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Calcium chloride dihydrate</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:119px;">223506-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Excella E24 shaker incubator</td>
			<td style="width:200px;">New Brunswick Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Cary 60 UV-Vis Spectrophotometer</td>
			<td style="width:200px;">Agilent Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge 5810R</td>
			<td style="width:200px;">Eppendrof</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Beakers</td>
			<td style="width:200px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Syringe</td>
			<td style="width:200px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Needle</td>
			<td style="width:200px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Pioneer Analytical and Precision Weighing Balance</td>
			<td style="width:200px;">Ohaus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

immobilization of multi-biocatalysts in alginate beads for cofactor regeneration and improved reusability

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

To enable cofactor regeneration, L-xylulose synthesis was carried out in a coupled whole-cell biocatalytic system containing E. coliHjLAD and E. coliSpNox cells. Following the optimization of various parameters, the reusability of this system was improved by immobilizing it in calcium alginate beads (Figure 2).
L-xylulose Production with Cofactor ...

Watch this Scientific Journal Video about Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability at JoVE.com

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

Presented is the protocol for co-immobilizing whole-cell biocatalysts for cofactor regeneration and improved reusability, using the production of L-xylulose as an example. The cofactor regeneration is achieved by coupling two Escherichia coli strains expressing functionally complementary enzymes; the whole-cell biocatalyst immobilization is achieved by cell encapsulation in calcium alginate beads.

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

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