Meta genomics, a field fast becoming open to me, has many applications. Park et al. present it as an efficient method for isolating novel and useful genes from uncultured micro-organisms from deep sea sediments. In their research a gene encoding a newly (in 2007) discovered esterase enzyme (Em2L8) was cloned and characterised from a DNA library of deep sea sediment (which was isolated from the metagenome of a deep sea clam bed microbial community). The gene was expressed in Escherichia Coli at 18◦c. The expressed gene consisted of 804bp and encoded for a polypeptide of 267 amino acids. Furthering their research they explore the physical properties of this discovered enzyme, they expected the enzyme to exhibit novel properties due to its unique niche origin.
Lipases/esterases catalyse the hydrolysis and synthesis of ester compounds. Certain enzymes exhibit a narrow substrate specificity, position selectivity, and stereoselectivity. Due to these properties they serve as useful biocatalysts which can be utilised by the pharmaceutical and fine chemical industries (Jaeger 2002 and Gupta et al. 2004). Thus the sequencing in properties of unique enzymes could contribute to industry, and potentially have medical benefits. Bearing this in mind there are increasing incentives for the discovery of novel enzymatic macromolecules. Em2L8 post analysis revealed it could hydrolyse tributyrin substrate, it was not clear to me what the significance of this finding was. Park et al characterised it as an esterase enzyme due to its ability to hydrolyse P-nitrophenyl; from pNPA (C2) → pNPL (C12).
The general characteristic properties of Em2L8 were found to be: 1. Activation in the range of 10◦C- 40◦C (8.34Kcal/mol), this is an indicator it is a cold adapted enzyme. 2. It was found to have an optimum pH of 10-11, demonstrating it is an alkaline enzyme. 3. The enzyme had phylogenetic similarities to enzymes from Kurthia, Haloarcula and Thermoanaerobacter spp.
In my opinion a key thing to be further tackled would be to assign this enzyme to a host micro-organism. Promoting further identification of further novel enzymes could have a range of benefits i.e play potential roles in environmental management and other biochemical industry applications (e.g. P.C.R. was aided by the discovery of polymerase and its unique properties) I also suggest that metagenomic analysis of metabolic enzymes may reveal more about the physiology in symbiotic relationships. Therefore further research into the nature of unique properties of enzymes may deepen the understanding of the potential diversity in physiology, which enables deep sea micro-organisms to inhabit extreme conditions. Eventually such enzyme property discoveries could contribute to environmental management i.e. further understanding of extra cellular enzymes in carbon cycling could be harnessed as a powerful climate change management tool. To conclude Park et al. used a variety of molecular techniques to investigate and discover a new enzyme, its attributes were described from their findings.
A review of (Park et al. 2007):
Park, HJ, Jeon, JH, Kang, SG, Lee, JH, et al. 2007, “Functional expression and refolding of new alkaline esterase, EM2L8 from deep-sea sediment metagenome.” Protein expression and purification, vol. 52, no. 2, pp. 340-7. Retrieved August 8, 2011, from http://www.ncbi.nlm.nih.gov/pubmed/17126562
4 comments:
Corin - an interesting review from a journal that's new to me. I am assuming that this research is largely to prove the principle of how an enzyme can be produced based only on metagenomic information. In that case, I'm not sure why you think it important to "assign the microorganism to a host function". It's not clear why this particular gene was chosen. I think if a very abundant gene with previously unknown function was identified, it would be good to link it to a particular microbe in order to understand its ecological role. However, the point of the metagenomic approach used by Park et al seems to be to simply clone and express a gene to produce a recombinant protein, and then see if that protein has interesting properties. That seems quite a hit and miss approach, or am I missing something?
Hi Colin, I just read the paper again and realized that one of the motives for discovering and producing unique target specific enzymes is their potential commercial uses i.e. for making drugs and organic materials. They do state it is difficult to find suitable ones, but the construction of lipases/esterase ‘toolbox’ is important for the synthesis of various ester compounds. They think that only a few novel enzymes have been discovered and therefore the hunt for unique enzymes has only just began! I think this highlights why this gene was chosen. There development of the expression of proteins from metagenomes sequences is a complex and impressive technique fraught with pitfalls, but may have commercial value.
As for assigning it to a microbe, my thinking was that if the genes ecological context and function was understood it may indicate its potential as a biocatalyitc tool. Might be erroneous thinking, but it may demonstrate its potential in action. Park et al. did carry out some basic test with metal ions and detergents to investigate their effect, the conclusion was that the physiological function of Em2L8 could not be addressed.
So yes mainly they demonstrated that they cloned and expressed a protein documented its biochemical properties, but they did not discover its true substrate physiological function. If it was understood they recommend it could be used to carry out hydrolysis and synthesis reactions within the pharmaceutical and fine chemical industries. This conclusion got me thinking perhaps rather whimsically; but if enzyme functions can be harnessed as a catalytic tool then the potential is there; to use them for environmental management. I’m sure this has been considered before. I am going to research it further. My point is that enzymes on their own are probably not the key, but within a host microorganism whose ecological context is understood its biogeochemical cycling potential might be harnessed.
Perhaps under more controlled conditions then just fertilizing the oceans with iron.
This caught my eye: Extract from (Cunha et al., 2010):
Cunha, A., Almeida, A., Coelho, F. J. R. C., Gomes, N. C. M., Oliveira, V., & Santos, A. L. (2010). Bacterial Extracellular Enzymatic Activity in Globally Changing Aquatic Ecosystems. Applied Microbiology, 124-135.
Microorganisms have an enormous catabolic potential and molecular tools have been used to
characterize relevant groups or strains and their involvement in pollutant degradation processes [145-146]. Nonetheless,
the application of this knowledge into effective microbial bioremediation protocols is still on a preliminary phase [147].
Because enzymes are simpler systems than the whole microorganism, in the past years enzymatic bioremediation has
been seen as a possible alternative [148-149]. Some advantages in using enzymes instead of microorganisms or
chemicals have been pointed out. The degradation of pollutants does not generate toxic or bio-hazardous products, the
enzymes are themselves biodegradable by the indigenous microorganisms and the efficiency of the process can be
improved by recombinant-DNA technology [148, 150].
The role of bacterial extracellular enzymes in the degradation of organic matter and their broad range of substrates
makes them suitable candidates for remediation of pollutants from contaminated environments [151]. Bacterial
hydrolases are a class of enzymes that are able to degrade several pollutants, including recalcitrant plastic polymers. For
example, an extracellular esterase involved in the degradation of polyester polyurethanes was isolated from Comamonas
acidovorans TB-35 [152].
Specific or extreme environments, such as the surface microlayer, are potential natural reservoirs of extracellular
hydrolases with unusual properties, worth to explore for biotechnological and bioremediation applications. Higher rates
of peptide and polysaccharide hydrolysis were found in the surface microlayer in relation to subsurface waters [153-
154]. High rates of polymer degradation have also been found in the rhizospheres of salt marsh vegetation [51-52].
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