OXIDISED FORM DIHYDRODIOL

REDUCTASE (xylZ)

+

xyDCY

BENZOATE

NAD _{REDUCED FORM}

FIGURE 1.8:

The role o f benzoate 1,2-dioxygenase

respectively. The enzyme is stable at pH 8 and a 50 % drop in activity was observed at pH 7. Considerable enzyme inhibition was noted in the presence of N a2S0 4,

particularly below pH 8.

1.4.4.3 X y l T Gene Product

The xylTgene product is the least known of the four enzymes, but it is thought to be a chloroplast-type ferrodoxin. Studies involving the expansion of substrate specificity described the role of the xylT gene product as being regulatory with respect to the enzyme C 230 (Polissi et a i, 1993). It was suggested that the xylT gene product is involved in the reduction of the oxidised iron cofactors of C 230 when inactivated by agents such as 4MC. Subsequent reactivation of the xylT product following reduction of the C 2 30 cofactors is therefore necessary, and it was suggested that a non-specific electron donor such as the xylZ gene product may be responsible. The proposed scheme for the reactivation of this enzyme by xylZ is shown in Figure 1.9.

1.4.4.4 C atechol 2,3-D ioxygenase

Catechol 2,3-dioxygenase encoded by the xylE gene is a homotetramer with a total molecular weight of 140 kDa. Each subunit has a weight of 35 kDa and contains a non-heme ferrous ion molecule. Due to the current inability to isolate the complete homoenzyme, research has not advanced as far as research on T 12 0 . Using a cloned

xylEgene from P. putida mt-2 and an E. coli host, the almost complete homoenzyme was isolated in crystalline form and the accumulation of gramme quantities of the enzyme was achieved for the first time. This allowed a detailed characterisation of this enzyme as well as the development of a protocol for the purification o f this enzyme resulting in a 40% yield (Kobayashi et a l, 1995). The total iron content associated with this enzyme was found to be 45% of the total iron present in the crude extract and the enzyme exhibited a specific activity of 536 U .m g'T This enzyme also has a broad substrate specificity which has been used for the synthesis of picolinic acids from catechols (Asano e t a l , 1994).

Picolinic acid is an industrially important chemical used as a raw material for the production of pyridines used to produce herbicides and dyes. Production using the mgra-cleavage pathway enzymes is discussed in the next section together with some other industrially important bioprocesses.

Fiona Vanier PhD Thesis Introduction xylZ xylT R E D U C E D F O R M O X ID IS E D F O R M N A D xylZ I O X ID IS E D F O R M x y /T / R E D U C E D F O R M + N A D H + H

FIGURE 1.9:

Proposed scheme for the reactivation o f the xy/T gene product (Polissi et a l, 1993).

1.4.5 TOL Plasmid & Biochemical Engineering 1.4.5.1 Ci5, cis-M uconic Acid Production

Cis, cis muconic acid is an unsaturated dicarboxylic acid which is produced on the orr/zo-cleavage of catechol by C 120 (see Figure 1.5). The structure of ccMA suggests that it has a potential use as a raw material for new functional resins, pharmaceuticals and agrochemicals. This compound can also be easily converted to adipic acid which is a commodity chemical used for nylon production (see Figure 1.10). It is difficult to synthesise ccMA by chemical means and therefore industrial use of this chemical was underdeveloped. An attempt to develop a bioreactor process which synthesises ccMA from benzoic acid for industrial production was undertaken by Yoshikawa et a i, (1993). Six strains able to produce ccMA with a yield of 0.5 g.L"^ or above were selected using a suitable screening procedure. The treatment of all 6 strains with a mutagen resulted in the selection of a colony originally from Arthrobacter sp (T8626) which had a stable capacity for over accumulating ccMA. The biotransformation was carried out in a 30 L stirred-tank containing a medium supplemented with benzoic acid which was intermittently supplied to avoid substrate inhibition. The mutant accumulated 44 g.L‘1 product.

On examination of the metabolic pathways shown in Figure 1.10 one can see that substrates such as toluene and catechol as well as benzoic acid can be used for this bioconversion. However, on further evaluation of alternative substrates catechol was found to be particularly expensive, toluene was a flammable and volatile solvent and both compounds were toxic (Yoshikawa et ah, 1990). Benzoic acid caused less problems when used as a substrate since the toxic effects associated with this compound were minimal. In addition, benzoic acid is cheap, non-volatile and can form a highly water soluble salt.

1.4.5.2 Picolinic Acid Production

Picolinic acid can be produced by the microbial synthesis of 2-HMSA followed by nitrogenation. The chemical decarboxylation of picolinic acid results in the production of pyridine as shown in Figure 1.10, and nitrogen-containing heteroaromatics such as pyridines are important starting materials for the production of herbicides and dyes. A mutant strain of P. putida was developed which was able to metabolise aromatic substrates such as toluene or catechol via the mcfa-cleavage pathway, but lacked any 2-HMSA degrading enzymes (Hagedorn et a l, 1989). The

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