Lysozyme, hydrochloride

Toxicity to aquatic algae and cyanobacteria

Description of key information

Inconclusive

Additional information

No specific test about the toxicity to aquatic algae and cyanobacteria of lysozyme exists. The available information about the lysozyme activity has been taken into account, within the available data about other enzymes, i.e. amilases, cellulases, lipases and Subtilisins (Protease). Like lysozyme, they are all hydrolases enzymes, acting on glycosidic bonds in the cases of amilase and cellulase, acting on ester bonds in the case of lipase and acting on the amino acid esters, amides and peptide bonds in the case of subtilisins.

Lysozyme can be regarded as the precursor of lysozyme hydrochloride; the transformation into the salt form does not significantly impact the aquatic toxicity potential and it is expected that lysozyme and lysozyme hydrochloride share the same potential breakdown products via physical and biological process.

 

LYSOZYME INFORMATION

A growth inhibition screening demonstrated that lysozyme had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to lysozymes, which degrades polymers containing N-acetylglucosamine.

This layer must be established late in the growth or maturing stage of individual cells as growing cells are readily susceptible to a variety of enzymes causing slow or a complete block in growth. Lysozyme alone does not have a huge impact in the mature cells, removing the wall cell protective layer; nevertheless, it may favourite other substrates become available to a wide range of different enzymatic activities. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibres protruding from the surface. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme (Gerken et al., 2010).

 

Furthermore, a new method involving lysozyme was recently developed for the production of axenic cultures of Anabaena flos-aquae De Brebisson and Aphanothece nidulans P. Richter. In the experiments performed, cyanobacterial growth was not inhibited at concentrations up to 1.2 g/l of lysozyme, whereas the growth of heterotrophic bacteria was suppressed. At concentrations up to 0.8 g/l of lysozyme, ampicillin caused a reduction of heterotrophic bacteria. These cyanobacteria resisted digestion by lysozyme at the experimental concentrations, whereas bacteria were digested selectively (Kim et al., 1999).

 

OTHER ENZYMES

Amilases (HERA, 2005)

An acute algae toxicity study with α-amylase was carried out according to guideline OECD 201 (test species: Scenedesmus subspicatus). The EC50 value was 112 (mg aep/l) after an exposure time of 72 h.

Cellulases (HERA, 2005)

Alkaline cellulase was tested for algae (Scenedesmus subspicatus) toxicity in a 72 hour static test, with a concentration range of 31.25 to 1000 mg aep/l. The EC50 resulted higher than 1000 mg aep/l.

Lipases (Greenough and Everett, 1991)

EC50 for inhibition of growth at 72-h: 97 mg active enzyme protein (aep)/l.

EC50 for inhibition of maximum growth rates at 24 and 72-h = 99 mg aep/l.

NOEL = 40 mg aep/l

Subtilisins (HERA, 2007)

All the available data are based on tests with standard test species Desmodesmus subspicatus (syn. Scenedesmus subspicatus) or Raphidocelis subcapitata (syn. Selenastrum capricornutum) which exhibit a broad range of effect concentrations ranging between 0.3 - 200 mg aep/l. A systematic difference between the effect values of wild type and protein engineered Subtilisin specimens was not recognisable.

 

DISCUSSION and CONCLUSION

The assessment of the potential toxicity of lysozyme to aquatic algae and cyanobacteria is of difficult interpretation because there are few data available and because of lysozyme specific enzymatic activity.

Lysozyme is capable to damage bacterial cell walls by catalysing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins.

The result of the lytic action of lysozyme is dissolution of the bacterial wall with consequent microbial disruption. Acting alone lysozyme lyses and kills several Gram-positive microorganisms by damaging their surface-exposed peptidoglycan. In contrast, most Gram-negative organisms (as cyanobacteria and many micro-algae) are resistant. In these organisms an outer membrane shields the peptidoglycan murein sacculus from the external environment (Leive, 1974; Nikaido and Vaara, 1985). However, Lysozyme also demonstrates activity against some gram negative organisms as well. The resistance of gram negative organisms to lysozyme is due to the lipopolysaccharide (LPS) outer layer that protects the cell wall. The research has indicated lysozyme alone can have antimicrobial action, in a dose dependent fashion; this activity was independent of lysozyme’s enzymatic activity and appeared related to hydrophobic binding interaction with lipopolysaccharides (LPS).

Taken into account the suitable test organisms indicated into the OECD guideline 201, literature data on lysozyme are available about Selenastrum capricornutum and Anabaena flos-aquae (green algae and cyanobacteria, respectively). Based on the Gereken results (Gereken et al., 2012), lysozyme has an ambiguous impact on the Selenastrum capricornutum (reported in tables as +/-; please not that the strain assayed is not one of those suggested in the OECD guideline). Furthermore, Kim et al. (1999) reported that cyanobacterial growth was not inhibited at concentrations up to 1.2 g/l of lysozyme.

Algae typically possess wall cells made of glycoproteins and polysaccharides. Diatom cells are enclosed within a cell wall called frustule, which is composed almost purely of silica, made from silicic acid, and is coated with a layer of organic substance, which was referred to in the early literature on diatoms as pectin, a fibre most commonly found in cell walls of plants. Thus, it is expected that lysozyme may give different response in algae/cyanobacteria test depending to the organisms tested, in particular on the basis of the cell wall.

Furthermore, the test system response can be impacted by the presence of other co-factors. As abovementioned, lysozyme alone does not have a huge impact in the mature cells, removing the wall cell protective layer; nevertheless, it may favourite other substrates become available to a wide range of different enzymatic activities

Despite they interact with different sites, amylase and cellulase act on glycosidic bonds as lysozyme, thus they are expected to have a similar behaviour. Both were tested on Scenedesmus subspicatus. The Effect Concentration-50 for amylase was stated as higher than 1000 mg aep/l, while the EC50 for cellulose was 99 mg aep/l. Despite it can be noted a great difference in the values recorded, in both cases the effects were related to concentrations sufficient high to not give rise of reasons of concern.

 

Although it is expected that lysozyme HCl would not impact significantly the aquatic compartment, a definitive and robust conclusion cannot be reached about toxicity to algae and cyanobacteria.

REFERENCE

Gerken G.H, Donohoe B., Knoshaug E.P. (2010). Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production. Planta. 2013 Jan;237(1):239-53

Greenough RJ, Everett DJ (1991) Safety evaluation of alkaline cellulase. Food and Chemical Toxicology 29: 781-785.

HERA (2005). Human and environmental risk assessment on ingredients of household cleaning products - alpha-amylases, cellulases and lipases.

HERA (2007). Human and environmental risk assessment on ingredients of household cleaning products - Subtilisins (Proteases). Edition 2.0. 2007.

Kim J.-S., Y-H Park, B.-D. Yoon, H.-M. Oh (1999). Establishment of axenic cultures of Anabeana flos-aquae and Aphanothece nidulans (cyanobacteria) by lysozyme treatment, J Phycol, 35, pp. 865-869.

Leive, L. (1974). The barrier function of the Gram-negative envelope. Ann. NYAcad. Sci. 235:109-127.

Nikaido, H., and M. Vaara. (1985). Molecularbasis ofbacterial outer membrane permeability. Microbiol. Rev. 49:1-32.

Oystein Lie, Oystein Evensen, Anita Sorensen, Ellen Froysadal (1989). Study on lysozyme activity in some fish species. Dis. aquat. Org. 6: 1-5.