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 an 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.

TIMELINE

February 28, 1935. The first nylon, known as nylon-6,6 was invented. On January 29, 1938, another nylon known as nylon-6 was invented.

December 20, 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.

The term that Ebata and Morita used for a two-component nylon-6 molecule that trypsin broke apart was ԑ-Aminocaproyl-ԑ-amino caproic acid, but the modern term for the same molecule is N-(6-aminohexanoyl)-6-aminohexanoate, also known as a nylon-6 dimer (shown above).

An example of a reaction involving the nylonase Ebata and Morita discovered is the catalysis of the following reaction where a N-(6-aminohexanoyl)-6-aminohexanoate (also known as a nylon-6 dimer) is broken apart into two modified 6-aminohexanoate molecules (also known as modified nylon-6 monomers, shown above).

N-(6-aminohexanoyl)-6-aminohexanoate + H2O ↔ 2 6-aminohexanoate

Although nylonases make it possible for some organisms to digest nylon, a nylonase doesn’t actually digest nylon, it breaks it apart.  The byproducts of the reaction may or may not be eventually digested by the organism depending on the organism’s capabilities.

Because vertebrates, and thus trypsin, have been around long before 1935, it is reasonable to say the nylonase capabilities of trypsin existed before 1935.

Unfortunately, in popular literature and in the blogsphere, a common misunderstanding floats around that goes something like this:

“Because nylon did not exist until 1935, the ability to digest nylon must have evolved suddenly only after nylon was invented.”

But such reasoning regarding nylonases could be somewhat likened to a hypothetical statement about hailstones that would go something like:

 “Hailstones could not have the capacity to break car windows until after car windows were invented by man, therefore hailstones evolved their physical properties (like density) so that after cars were invented, hailstones were able to break car windows.”

Granted, biological organism can adapt to new environments, and hence there is the possibility an organism can evolve to deal with a new environment, however that doesn’t preclude the possibility that nylonase ability pre-existed prior to 1935.  Ebata and Morita’s experiments demonstrated pre-1935 existence of nylonase capability in the biological world.

Ebata and Morita’s experiment went into obscurity, but had their experiment remained in the public consciousness, the common misunderstanding that nylonases must have evolved only after 1935 might not persist to this day.

1966. Fukumura is the first to demonstrate a bacteria, known as Corynebacterium aurantiacum B-2 could eat nylon.  This implied the organism had nylonase enzymes.

1977-1981. Kinoshita isolates two nylonase enzymes (eventually named NylA and NylB) in a bacterial organism designated as KI72.  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 pre-existed 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 pre-1935 samples of bacteria prior to their supposed evolution of nylonase genes.

To complicate matters, Kinoshita was not specific about what he meant by saying “newly evolved.” Did “newly evolved” mean a change of 1 amino acid out of 427 on a pre- existing enzyme, or did “newly evolved” mean a change of 400 amino acids out of 427 on a pre-existing protein? The former is well within possibility, the latter is astronomically improbable as far as biologically useful enzymes are concerned (as discussed below).

1984. Susumu Ohno argued that one of the nylonases which Kinoshita discovered, known as NylB, evolved through a change of over 400 out of 427 amino acids on a pre-existing enzyme, where 35 of those changes were deletions at the tail end. He argues this happened through what is known as a single DNA frame-shift insertion. It was a pure speculation since neither Ohno nor Kinoshita had samples of pre-1935 bacteria to actually prove what or if anything actually evolved in the first place! Ohno further claimed the supposed post-1935 emergence of the NylB enzyme was “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 publishes an essay presenting Ohno’s speculation as if it were an incontestable historical fact despite complete absence of direct evidence such as pre-1935 samples of the KI72 bacteria. Thwaites totally ignored or was unware of Ebata and Morita’s experiments which showed that other nylonases had pre-existed in the biosophere prior to 1935.

Thwaites used Ohno’s speculation to promote the now largely discredited idea that viable proteins can emerge from long strings of random amino acids.

2008. Like Thwiates, biology professor Ken Miller promotes the idea viable proteins are not difficult to evolve from random amino acid sequences and uses Ohno’s speculation as “evidence” to that effect.

Miller makes a misleading citation of an experiment that falsified Ohno’s frame-shift hypothesis as if the experiment confirmed it.  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 here commenters argue that Ohno’s frame shift was directly observed in laboratory experiments on bacteria evolving in a test tube or petri dish.  Miller’s misleading claims either originated or perpetuated this myth.

2016. Dennis Venema repeats Ken Miller’s claims in his book Adam and the Genome. Venema represents experiments by Negoro in 2005 as 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 from data that emerged after 1984. Their paper brings to light Ebata and Morita’s forgotten experiment.  They 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’s,  NylB in Arthrobacter KI72 was not the result of evolution of a unique enzyme after 1935, it is evident NylB is an enzyme more common than previously thought and which 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 prexisting protein had been effectively falsified and so also the claims by Thwaites, Miller, and Venema which relied heavily on Ohno’s now falsified speculations.

The conclusion of the paper:

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.