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252 Part 2: Biotechnology and Enzymology

TMA is further discussed in the food microbiology literature

(e.g., Barrett and Kwan 1985, Dalgaard 2000).

The Trimethylamine-N-Oxide

Aldolase Reaction

TMAO is also the precursor of the formation of dimethylamine

(DMA) and formaldehyde:

CH 3 CH (^3) O
 
ONCH 3 → HNCH 3 HC

CH 3 H

=.

This reaction is catalyzed by trimethylamine-N-oxide aldolase

(TMAOase or TMAO demethylase, EC 4.1.2.32) but may also

to some extent be nonenzymatic, catalyzed by iron and various

reductants (Vaisey 1956, Spinelli and Koury 1981, Nitisewojo

and Hultin 1986, Kimura et al. 2002). In most cases in which

significant amounts of formaldehyde and DMA are accumu-

lated, however, species possessing TMAOase enzyme activity

are involved. The reaction leads to cleavage of a C-N bond and

the elimination of an aldehyde, resulting in the classification of

TMAOase as a lyase (EC 4.1.2.32) and the International Union

of Biochemistry and Molecular Biology name aldolase.

DMA is a reactive secondary amine with a milder odor than

TMA. Formaldehyde is highly reactive and strongly affects the

texture of fish meat by making it tougher, harder, more fibrous,

and less juicy, as well as increasing the drip loss in the thawed

products. The quality changes are associated with a loss in pro-

tein solubility and in particular the solubility of the myofibrillar

proteins. For reviews, see Sikorski and Kostuch (1982), Hultin

(1992), Mackie (1993), Sikorski and Kolakowska (1994), and

Sotelo et al. (1995).

Formaldehyde can react with a number of chemical groups,

including some protein amino acid residues and terminal amino

groups, resulting in denaturation and possibly in the cross-

linking of proteins, both of which are believed to be the cause

of the observed effects on seafood products. The formaldehyde

concentration in severely damaged seafood products may reach

240 μg/g (Nielsen and Jørgensen 2004). These concentrations

are generally considered nontoxic, but may still exceed various

national trade barrier limits.

Whereas TMAO is widespread, TMAOase is only found in a

limited number of animals, many of which belong to the order

of gadiform fish (pollock, cod, etc.). In gadiform species, the

highest levels of enzyme activity are found in the inner organs

(kidney, spleen, and intestine), while the enzyme activity in the

large white muscle is low. The formation of DMA and formalde-

hyde in various tissues of certain nongadiform fish, as well as of

crustaceans and mollusks, has also been reported, as reviewed

by Sotelo and Rehbein (2000), although the taxonomic distri-

bution of the enzyme has not been investigated systematically.

The TMAOase content of gadiform fish exhibits large individual

variation, probably due to the influence of biological factors that

have not yet been adequately studied (Nielsen and Jørgensen

2004).

The enzyme is stable and tolerates both high salt concentra-

tions and freezing. The accumulation of DMA and formalde-

hyde progresses only slowly, and most of the accumulation is

produced during prolonged frozen storage or cold storage of

the salted fish (e.g., salted cod and bacalao). During freezing,

the enzymatic reaction proceeds as long as liquid water and

substrate are available. In practice, the formation of DMA and

formaldehyde is found at temperatures down to approximately

− 30 ◦C (Sotelo and Rehbein 2000). At higher temperatures, the

rate of formation of formaldehyde is also higher; thus, freezing

of gadiform fish at insufficiently low temperatures may result in

dramatic changes in the solubility of myofibrillar proteins within

a few weeks (Nielsen and Jørgensen 2004).

Despite the TMAOase concentration of the white muscle be-

ing low, it is the enzyme activity of the white muscle that is

responsible for the accumulation of formaldehyde in whole fish

and in fillets (Nielsen and Jørgensen 2004). In the case of minced

products, even minor contamination by TMAOase-rich tissues

leads to a marked rise in the rate of accumulation (Dingle et al.

1977, Lundstrøm et al. 1982, Rehbein 1988, Rehbein et al. 1997).

Despite its dependency upon other factors, such as cofactors, the

rate of accumulation of formaldehyde can to a large extent be

predicted from the TMAOase enzyme activity of fish meat alone

(Nielsen and Jørgensen 2004).

The physiological function of TMAOase remains unknown.

The formation of formaldehyde could have a digestive func-

tion, yet this would not explain the high TMAOase content of

the kidney and spleen. Accordingly, it has been speculated that

TMAO may not even be its primary natural substrate (Sotelo

and Rehbein 2000).

The in vitro reaction rate is low without the presence of a num-

ber of redox-active cofactors. Although the overall TMAOase

reaction is not a redox reaction, its dependency on redox agents

shows that the reaction mechanism must include redox steps.

Little is known about the in vivo regulation of TMAOase,

but studies suggest the importance of nucleotide coenzymes

and iron (Hultin 1992). TMAOase appears to be membrane

bound and is only partly soluble in aqueous solutions. This has

complications for its purification and is one of the main reasons

why the complete characterization of the enzyme remains yet to

be done.

POSTMORTEM PROTEOLYSIS

IN FRESH FISH

Postmortem proteolysis is an important factor in many changes

in seafood quality. During cold storage, the postmortem prote-

olysis of myofibrillar and connective tissue proteins contributes

to deterioration in texture. Despite the natural tenderness of

seafood, texture is an important quality parameter, in fish

and shellfish alike. Deterioration in seafood quality through

proteolysis involves a softening of the muscle tissue. This re-

duces the cohesiveness of the muscle segments in fillets, which

promotes gaping, a formation of gaps and slits between muscle

segments. The negative character of this effect contrasts with

the effect of postmortem proteolysis on the meat of cattle and