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

Summary

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.

Abstract

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.

Introduction

Reductive whole-cell biotransformation using microorganisms has become a widespread method for the chemo-enzymatic synthesis of commercially and therapeutically important biomolecules^1-3. 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 lifetime^4-7. 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-substrate....

Protocol

1. Whole-cell Biocatalysts Preparation  

NOTE: The recombinant E. coli cells harboring pET28a-SpNox³¹ or pET28a-HjLAD²⁸ are hereafter referred to as E. coli_SpNox and E. coli_HjLAD, respectively.

1. Inoculate a single colony of E. coli_HjLAD in 3 ml of Luria-Bertani (LB) medium supplemented with kanamycin (50 µg/ml) and incubate in an incubator shaker O/N at 37 ^oC, 250 rpm.
2. Dilute the culture by 1:100 in 200 ml of fresh LB containing 50 µg/ml kanamycin and incubate at 37 ^oC, 250....

Results

To enable cofactor regeneration, L-xylulose synthesis was carried out in a coupled whole-cell biocatalytic system containing E. coli_HjLAD and E. coli_SpNox 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 .......

Discussion

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 .......

Materials

Name	Company	Catalog Number	Comments
LB broth	Sigma Aldrich	L3022-6X1KG
Kanamycin	Fisher	BP906-5
Isopropyl β-D-thiogalactopyranoside (IPTG)	Sigma Aldrich	I6758-10G
Tris base	Fisher	BP1521
B-Nicotinamide adenine dinucleotide hydrate	Sigma Aldrich	N7004-1G
L-Arabinitol	Sigma Aldrich	A3506-10G
L-Cysteine	Sigma Aldrich	168149
Sulfuric acid	Sigma Aldrich	320501-500ML
Carbazole	Sigma Aldrich	C5132
Ethanol	Fisher	BP2818-4
Sodium alginate	Sigma Aldrich	W201502
Calcium chloride dihydrate	Sigma Aldrich	223506-500G
Excella E24 shaker incubator	New Brunswick Scientific
Cary 60 UV-Vis Spectrophotometer	Agilent Technologies
Centrifuge 5810R	Eppendrof
Beakers	Fisher
Syringe	Fisher
Needle	Fisher
Pioneer Analytical and Precision Weighing Balance	Ohaus