Surface associations of enzymes and of organic matter: Consequences for hydrolytic activity and organic matter remineralization in marine systems

Abstract

The extent to which marine organic matter is associated with surfaces and the consequences of such associations for organic matter remineralization are the focus of considerable attention. Since extracellular enzymes operating outside microbial cells are required to hydrolyze organic macromolecules to sizes sufficiently small for substrate uptake, the effects of surface interactions–on enzymes as well as on substrates–for hydrolytic activity also require investigation. We used a simplified laboratory system consisting of a free (dissolved) polysaccharide (pullulan) and the same polysaccharide tethered to agarose beads to restrict mobility, plus the corresponding free enzyme (pullulanase) and the same enzyme sorbed to montmorillonite (Mte), to investigate systematically the consequences of surface associations of enzymes and of substrates on hydrolytic activity. Changes in substrate molecular weight were monitored with time to measure the course of enzymatic hydrolysis. Although hydrolysis of free substrate was nearly complete after 2 min incubation with the free enzymes, the sorbed enzymes also effectively hydrolyzed free substrate, and the data suggest that they retained activity longer in solution compared to the free enzymes. Sorbed enzymes progressively hydrolyzed the free substrate from > 50 kD to lower molecular weights during a 24 h incubation, with a final product distribution on average showing only 1.4% and 10.3% of substrate still in the > 50 kD and 10 kD size classes, while 46.6%, 29.3%, and 12.5% of substrate was in the 4 kD, monomer, and free tag size classes, respectively. This product distribution was very similar to that of the free substrate/free enzyme experiment. Tethering the substrate to agarose beads led to lower substrate release (2–3% of total substrate after 98 h incubation) into solution compared to the free substrate case. For tethered substrates, the state of the enzyme (free or sorbed) measurably affected the molecular weight distribution of the hydrolysis products, with free enzymes producing a higher fraction of high molecular weight hydrolysis products (28.7 ± 5.4% of substrate > 50 kD at the end of the incubation) compared to sorbed enzymes (11.6 ± 2.8% of substrate > 50 kD at the end of the incubation.) Tethered substrates were also hydrolyzed in a sediment slurry from surface sediments from Cape Lookout Bight, North Carolina; 0.1% of total substrate was released by enzymes naturally present in 1 cm 3 of sediment after 144 h incubation, demonstrating that the enzymes naturally present in marine sediments are also capable of accessing tethered substrates. These investigations suggest that surface associations of enzymes in marine systems may extend the active lifetime of such enzymes, providing an opportunity for hydrolysis over longer periods of time and producing a different size spectrum of hydrolysis products relative to free enzymes. Furthermore, in well-mixed systems, surface-associated enzymes can hydrolyze substrates whose mobility is restricted, highlighting the importance of processes such as resuspension and bioturbation on organic matter remineralization.