BASICS OF NYLON AND NYLONASES
Nylon. Nylon is a generic designation for a family of synthetic silky plastic material that can be melt-processed into fibers, films or shapes. Several different molecules can be called nylon. This fact can create some confusion.
Nylonase. A nylonase is a protein/enzyme that can break apart a nylon molecule. There are different kinds of nylonases, which can break apart different kinds of nylons. There are a variety of molecules that can act as nylonases.
Nylon-6. Nylon-6 is the family of nylons on which most nylonases can act. A nylon-6 molecule is a chain (oligomer or polymer) of nylon-6 monomers.
The nylon-6 monomer C6H11NO is a dehydrated 6-amino hexanoic acid molecule C6H13NO2:
A hydrated nylon-6 monomer (a 6-amino hexanoic acid) strongly resembles a common biological molecule found in proteins known as lysine, so much so it 6-amino hexanoic acids are called “lysine analogs”:
A nylon-6 dimer is an oligomer composed of 2 nylon-6 monomers (2 modified 6-aminohexanoic acids):
A nylon-6 trimer is an oligomer composed of 3 nylon-6 monomers (3 modified 6-aminohexanoic acids):
Extending the above ideas, one can construct nylon-6 tetramers, pentamers, etc. A commercial grade nylon-6 polymer has 100 nylon-6 monomers (modified 6-aminohexoic acids).
The bonds joining the nylon-6 monomers are peptide bonds similar to those found in biological proteins, thus it should not be surprising that certain biological enzymes known as proteases, which can break apart proteins, can also break apart nylons, and thus can act as nylonases.
The Nylonase Story Timeline –
1935. The first nylon, known as nylon-6,6 was invented. On January 29, 1938, another nylon known as nylon-6 was invented.
1959. Ebata and Morita discovered the first nylonase. It was a well-known enzyme in vertebrates called trypsin, which is formally classified as a protease (an enzyme that can break apart proteins) but was discovered to also act as a nylonase. Because vertebrate trypsin enzymes have been around long before 1935, it is obvious that the nylonase activity of trypsin and other proteases existed long before the invention of nylon.
1966. Fukumura is the first to demonstrated a bacteria, known as Corynebacterium aurantiacum B-2could eat nylon. This implied the organism had nylonase enzymes.
1977-1981. Kinoshita isolated two nylonase enzymes (eventually named NylA and NylB) in a bacterial organism designated as KI72. Late the genes NylC and NylB’ were found on the same plasmid. Kinosita claimed the ability for KI72 to eat nylon was due to nylonase genes which were “newly evolved” only after 1935 (because nylon didn’t exist prior to 1935).
Based on the fact Kinoshita referenced Fukumura’s 1966 experiment which referenced Ebata’s 1959 experiment, it is reasonable to speculate that Kinoshita was aware of the fact other nylonases like trypsin existed long before 1935. Nevertheless, Kinoshita was convinced the nylonases which he discovered in bacteria must have evolved only after 1935. He made this claim despite the fact he had no clear basis for this..\
1983. Okada et. al sequence the nylB and nylB’ genes in KI72 and reaffirm the Kinosihita’s claim that nylB is a newly evolved gene. They suggest nylB evolved through gene duplication of nylB’ and 47 subsequent amino acid alterations.
1984. Susumu Ohno speculated that one of the nylonases (NylB), which Kinoshita discovered arose de novo, via a single DNA frame-shift insertion. He hypothesized that this mutation changed more than 400 out of 427 amino acids on a hypothetical pre-existing enzyme. This was purely speculation, since neither Ohno nor Kinoshita had samples of pre-1935 bacteria to actually prove what what happened. Ohno declared that the post-1935 emergence of the NylB enzyme was the “birth of a unique enzyme”. Ohno’s theory is sometimes referred to as “the frame-shift hypothesis” of the origin of nylonase NylB.
1985. William Thwaites of the National Center for Science Education published an essay presenting Ohno’s speculation as if it were an incontestable historical fact Thwaites was apparently unware of Ebata and Morita’s experiments which showed that other nylonases had pre-existed in the biosophere prior to 1935.
1992. Yomo suggests that NylB in KI72 and NylB in Pseudomonas NK87 descended from a common ancestor 140 million years earlier. Ohno is credited in the paper with making the paper’s publication possible in PNAS even though it could be argued Yomo’s paper conflicted with the claims in Ohno’s 1984 paper. Yomo reports a perplexing property of DNA in the genes (absence of stop codons in the antisense strand).
2008. Like Thwiates, biology professor Ken Miller very actively promoted the idea it is not difficult to evolve stable proteins with novel functions from random amino acid sequences, based upon Ohno’s speculations.. A more detailed critique of Ken Miller’s claims can be found in Ken Miller’s Misleading Promotion of Ohno’s Frame-Shift Hypothesis. It is not unusual in the blogsphere to hear people argue that Ohno’s frame shift was directly observed in laboratory experiments. This seems to trace back to Miller’s confounding of Ohno’s speculations and the lab experiments conducted by the Japanese group.
2016. Dennis Venema repeats Ken Miller’s claims in his book Adam and the Genome. Venema misrepresents experiments by Negoro in 2005as confirming Ohno’s frame shift hypothesis, when in fact Negoro’s experiments refute it.
August 25, 2017. Cordova and Sanford release a draft copy of a comprehensive review of nylonases showing Ohno’s hypothesis has been effectively falsified based upon data that has emerged after 1984. This new paper brings to light Ebata and Morita’s forgotten experiment. The authors list 193 organisms with a variety of NylB nylonases and highlight the geographic distribution of over 1800 organisms with nylonases spread over the globe from the Indian Ocean to an isolated island in the Arctic.
For reasons stated in the paper, the presence of these 193 organisms each with a slightly different version of NylB, make Ohno’s frame-shift hypothesis infeasible, because at best it can explain only one version of NylB (in Arthrobacter KI72) and not in other versions of NylB (such as in Pseudomonas sp. NK87, and Bacillus cereus, etc.). Furthermore, no searches of present day databases since 1984 show any traces that would confirm Ohno’s speculation.
Because of the widespread geographic distribution of over 1800 organisms nylonases with sufficient differences to make it astronomically improbable for them to have descended from a common ancestor only after 1935, these nylonases, including NylB, existed long before 1935. So contrary to Ohno hypothesis, NylB in Arthrobacter KI72 was not the result of evolution of a unique enzyme after 1935, it is clear that NylB is an enzyme that is much more widely distributed than previously thought, and has existed long before 1935.
Therefore, Ohno’s speculation that NylB arose via an instant change of over 400 out of 427 amino acids from a pre-existing protein has been effectively falsified. Therefore, the claims of Thwaites, Miller, and Venema are also falsified, since they are derived directly from Ohno’s falsified speculations.
Cordova and Sanford summarize their findings as follows:
Our analyses indicate that nylonase genes are actually abundant, come in many diverse forms, are found is a great number of organisms, and such organisms are found in a great number of natural environments. We show there is no reason to think that any of these nylonases emerged since 1935, and so there is no basis for invoking any de novo genes arising since 1935. Furthermore, there are numerous glaring problems with the specific de novo speculations of Okada and Ohno. The early claims of de novo nylonase genes were unsupported and speculative, and in light of new data these hypotheses now appear to be unwarranted and essentially falsified.
The discovery of numerous naturally-occurring genes having nylonase activity, along with a multitude of homologous genes and proteins that provisionally have similar activities, opens the door to further exploration of nylonases and their functions.