# US 8,569,036 B2 · Immobilized TAL biocatalyst for production of para-hydroxycinnamic acid

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- Number: US 8,569,036 B2
- Document ID: US8569036B2
- Status: Granted
- Kind: B2
- Application number: 11/485,575
- Filed: 2006-07-12
- Published: 2008-02-07
- Granted: 2013-10-29
- Earliest claimed: 2006-07-12
- Assignees: E. I. Du Pont de Nemours and Company
- Applicants: Robert Dicosimo; Sharon L. Haynie; Lixuan Lisa Huang; Fateme Sima Sariaslani; Tina K. Van Dyk; Robert J. Trotman
- Inventors: Robert Dicosimo; Sharon L. Haynie; Lixuan Lisa Huang; Fateme Sima Sariaslani; Tina K. Van Dyk; Robert J. Trotman
- Web page: https://patentgrep.com/patent/US8569036B2

## CPC

- C12N 11/04: entrapped within the carrier, e.g. gel or hollow fibres
- C12N 9/88: Lyases (4.)
- C12N 11/06: attached to the carrier via a bridging agent
- C12N 11/10: the carrier being a carbohydrate
- C12P 7/42: Hydroxy-carboxylic acids
- Y02P 20/50: Improvements relating to the production of bulk chemicals
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

TAL cell biocatalyst was immobilized in alginate cross-linked beads using low concentrations of glutaraldehyde. The biocatalyst beads have highly stable TAL activity and mechanical strength such that they withstand prolonged recycling in production of pHCA.

## Claims

### Claim 1

An immobilized tyrosine ammonia lyase biocatalyst comprising:
a) a bacterial cell tolerant to para-hydroxycinnamic acid and alkaline pH and transformed with a genetic construct comprising a gene, the gene encoding a tyrosine ammonia lyase (TAL) enzyme selected from the group of TAL enzymes having TAL enzyme activity from Rhodotorula glutinis, Phanerochaete chrysosporium, Trichosporon cutaneum, Rhodobacter sphaeroides and mutagenized Rhodosporidium toruloides; and
b) a cross-linked alginate bead prepared by a process in which glutaraldehyde is used at a final concentration of between about 2.5×10−4 g and about 6.3×10−3 g glutaraldehyde per gram of bead;
wherein the bacterial cell is embedded in the alginate bead; and
wherein the tyrosine ammonia lyase enzyme has an activity about 1.9 to about 8.0 measured as g pHCA produced/L/h per g dcw bead catalyst.

### Claim 2 (depends on 1)

The immobilized tyrosine ammonia lyase biocatalyst of claim 1 wherein the cross-linked alginate bead is crosslinked with glutaraldehyde and polyethylenimine.

### Claim 3 (depends on 2)

The immobilized tyrosine ammonia lyase biocatalyst of claim 2 wherein the alginate bead is crosslinked by a process where glutaraldehyde is added at a rate that is less than about 1×10−4 g glutaraldehyde/g beads per minute, and wherein glutaraldehyde and polyethylenimine are added in either order.

### Claim 4 (depends on 1)

The immobilized tyrosine ammonia lyase biocatalyst of claim 1 wherein the bacterial host cell is a strain of E. coli.

### Claim 5 (depends on 4)

The immobilized tyrosine ammonia lyase biocatalyst of claim 4 wherein the E. coli strain is an E. coli K12 strain selected from the group consisting of RFM443, W3110 and BW25113.

### Claim 6 (depends on 1)

The immobilized tyrosine ammonia lyase biocatalyst of claim 1 wherein the bacterial cell is in the form of whole or permeabilized bacterial cells.

### Claim 7 (depends on 1)

The immobilized tyrosine ammonia lyase biocatalyst of claim 1 wherein the cross-linked alginate bead comprises calcium alginate.

### Claim 8 (depends on 1)

A method for producing para-hydroxycinnamic acid comprising:
a) providing an tyrosine ammonia lyase biocatalyst according to claim 1;
b) providing a source of tyrosine;
c) contacting the biocatalyst or (a) with the tyrosine of (b) a suitable aqueous reaction mixture whereby p-hydroxycinnamic acid is produced;
d) recovering the p-hydroxycinnamic acid; and
e) optionally repeating c) and d) at least one time.

### Claim 9 (depends on 8)

The method of claim 8 wherein the source of tyrosine is a bacterial host that is overproducing for tyrosine.

### Claim 10 (depends on 8)

The method of claim 8 wherein the contacting step (c) is a pH of about 8.0 to about 11.0.

### Claim 11 (depends on 10)

The method of claim 10 wherein the pH is from about 9.5 to about 9.9.

### Claim 12 (depends on 8)

The method of claim 8 wherein the aqueous reaction mixture comprises calcium at a concentration between about 5 mM and about 20 mM.