Nylon nose

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Nylonase is the popular scientific name for a class of three enzymes that are able to catalyze the hydrolysis of oligomers of 6-aminohexanoic acid (Ahx) . The enzymes could be isolated from several types of bacteria . The scientifically correct name for these enzymes is 6-aminohexanoate oligomer hydrolases . They are counted among the amidases , which in turn form a subgroup of hydrolases . The substrates for these enzymes, the oligomers of 6-aminohexanoic acid, have only existed on earth since 1938. They arise as by-products in the large-scale production of polyamide 6 ( Perlon ) and thus end up in the environment. According to the prevailing scientific consensus , the bacteria equipped with these enzymes acquired the ability to digest the polyamide-6 by-products through evolutionary processes. For evolutionary biologists , the development of nylonases by bacteria is a prime example of observable evolution.

Polyamide 6

Polyamide 6 (PA6), also known as nylon 6 and better known under the trademark Perlon , was first produced on January 29, 1938 by the German chemist Paul Schlack in the laboratories of the research department of Aceta GmbH in Berlin-Lichtenberg . For the synthesis of PA6, Schlack took the ε-caprolactam, which is still used today, as the starting material . The polymerization starts from 6-aminohexanoic acid (Ahx), which is formed from ε-caprolactam and water.

Formation of 6-aminohexanoic acid by hydrolysis of ε-caprolactams

Formation of 6-aminohexanoic acid by hydrolysis of ε-caprolactam

The 6-aminohexanoic acid then reacts with amide formation with a molecule of ε-caprolactam to form a dimer , the N - (6-aminohexanoyl) -6-aminohexanoate, called (Ahx 2 ) for short .

Reaction of 6-aminohexanoic acid with ε-caprolactam to form Ahx2

Reaction of 6-aminohexanoic acid with ε-caprolactam to Ahx 2

This in turn can react with another molecule of ε-caprolactam to form a trimer . The polymerization reaction can proceed through the addition of further ε-caprolactam molecules. Polyamide 6 essentially consists of over 100 aminohexanoic acid units linked to one another.

The structure of polyamide 6

The structure of polyamide 6. The letter n stands for the number of units of 6-aminohexanoic acid linked to one another. The average degree of polymerization is n = 100.

However, the polymerization is a largely uncontrolled process that results in a relatively broad distribution of the molecular weights . Low molecular weight compounds (oligomers) such as dimers (Ahx 2 ), trimers (Ahx 3 ), etc., of 6-aminohexanoic acid are also found in the polymer . Cyclic oligomers such as 1,8-diazacyclotetradecane-2,9-dione can also be formed by head-to-tail linkage . These oligomers are undesirable by-products of polyamide 6 production.

1,8-Diazacyclotetradecane-2,9-dione, a cyclic dimer of aminohexanoic acid, is one of the by-products of polyamide-6 production.

In addition to ε-caprolactam and 6-aminohexanoic acid, the wastewater from the production facilities for polyamide 6 also contains the linear and cyclic oligomers of polyamide 6. The solubility of the linear oligomers decreases with increasing chain length. The trimer Ahx 3 is 1.8%, the tetramer Ahx 4 0.3% and the pentamer Ahx 5 only 0.01% soluble in water. The Hexamer Ahx 6 is practically insoluble in water. During the production process, the oligomers are separated from the desired product with hot water and thus also end up in the wastewater. When the wash water cools down, a large part of the by-products precipitate as solids. Depending on the process management and the size of the production plant, between 20 and 180 tonnes of oligomers and ε-caprolactam are produced for one company each year. This production waste is often disposed of by burying it in landfills. Around 4 million tons of polyamide 6 are produced worldwide every year (as of 2010).

"Normal" microorganisms are not able to break down the by-products of polyamide 6 production. You can essentially only cleave peptidic bonds of α- amino acids with the help of peptidases . Amide bonds in the ε position have only been known since the experiments by Schlack, or in the case of polyamide 6.6 since February 28, 1935 by Wallace Hume Carothers .

Nylonases and the bacteria that produce them

In 1965, the Japanese Takashi Fukumura isolated from the wastewater of a polyamide-6 production facility of Toyo Rayon Co., Ltd. (today Toray ) in Nagoya eleven different bacterial strains that were able to grow in a nutrient medium with 0.6% ε-caprolactam. A bacterial strain ( Corynebacterium aurantiacum ) was also able to metabolize (metabolize) linear and cyclic oligomers of 6-aminohexanoic acid - with the exception of the cyclic 6-aminohexanoic acid dimer . Four years after Fukumura, another Japanese research group isolated from the same environment a bacterium from the genus Pseudomonads , which is also able to metabolize cyclic oligomers of 6-aminohexanoic acid. In 1974, a working group led by Hirosuke Okada isolated the bacterial strain KI72 from Flavobacterium sp. From sewage sludge , which is able to thrive with the cyclic dimer of 6-aminohexanoic acid as the only source of carbon and nitrogen. The KF71 strain of the flavobacterium, isolated two years earlier by the same working group, could not do this. KI72 is able to metabolize ε-caprolactam, 6-aminohexanoic acid and the cyclic Ahx dimer as well as the linear di- to hexamers of 6-aminohexanoic acid. The researchers isolated two enzymes from the bacterium, one of which catalyzes the hydrolysis of the cyclic 6-aminohexanoic acid dimer to form Ahx 2 . The other enzyme catalyzes the hydrolysis of the linear oligomers. The enzyme, which can cleave the cyclic 6-aminohexanoic acid dimer, was tested for its hydrolytic properties against other, natural substrates. Surprisingly, it could not catalyze hydrolysis in any of the 50 dipeptides and 16 tripeptides . In subsequent experiments, the number of natural compounds with an amide bond, which do not form a substrate for this enzyme called NylA, increased to over 100. Even for the enzyme, which has the linear oligomers as a substrate and is now called NylB, no "natural" Substrate to be found.

The cell lysate obtained from cells whose nutrient medium did not contain a cyclic dimer was unable to catalyze the hydrolysis of the cyclic dimer. In contrast to the cell lysate obtained from cells with cyclic dimer as a nutrient medium. Okada and his co-authors concluded from this behavior that 6-aminohexanoic acid dimer hydrolase is an inducible enzyme , which means that the concentration of the enzyme is increased by the presence of an inducer - in this case the cyclic dimer.

There are two possible reasons why an enzyme might have activity against an unnatural substrate. In the first, simpler case, the unnatural substrate is an analogue of the natural substrate. In the second case, the catalysis is brought about by an evolutionarily developed enzyme that has lost its original activity towards a natural substrate through mutation . Since Okada and his co-authors did not find a natural substrate for the 6-aminohexanoic acid dimer hydrolase in their experiments and this enzyme has a comparatively low catalytic constant k cat ( > 35 s −1 ) compared to other cyclic amide hydrolases such as penicillinase (> 35 s −1 ) 8 s −1 ), they concluded that the 6-aminohexanoic acid dimer hydrolase was probably formed through evolutionary adaptation .

In 1992, Seiji Negoro and other colleagues from Okada's research group isolated a third enzyme, now called NylC, from the bacterial strain. NylC is an endohydrolase, which means that the enzyme catalyzes the hydrolysis inside ( Greek ἔνδον endon , 'inside') of the oligomer chain. It has no activity towards the cyclic dimer, but it does towards the cyclic tetramer and pentamer. Even with NylC, no activity towards “natural” amide bonds could be determined.

A total of three enzymes are responsible for the degradation of the 6-aminohexanoic acid oligomers:

  • cyclic 6-aminohexanoic acid dimer hydrolase (NylA) EC  3.5.2.12
  • 6-Aminohexanoic acid dimer hydrolase (NylB) EC  3.5.1.46
  • 6-aminohexanoate oligomer endohydrolase (NylC) EC  3.5.1.117
Schematic representation of the degradation of polyamide-6 oligomers by the three nylonases.

Comparison of the properties of the three polyamide-6-degrading enzymes from Flavobacterium sp. KI72:

properties enzyme
gene nylA nylB nylC
Molar mass 52 kDa 42 kDa 37 kDa
Number of amino acids 493 392 355
optimal pH 7.4 9.0 7.0
Temperature optimum 34 ° C 40 ° C 42 ° C
Specific activity towards
Ahx 2 (linear dimer) <0.001 0.94 0.00044
Ahx 3 (linear trimer) <0.001 0.75 0.11
Ahx 4 (linear tetramer) <0.001 0.57 0.42
Ahx 5 (linear pentamer) <0.001 0.24 0.47
cyclo (Ahx) 2 (cyclic dimer) 2.8 <0.0001 <0.0001
cyclo (Ahx) 4 (cyclic tetramer) <0.001 <0.0001 0.36
ε-caprolactam <0.001 <0.0001 <0.0001

The activity values ​​were obtained at a substrate concentration of 1 mM and are given in mol of NH 2 per minute and mg of purified protein (U / mg). The values ​​highlighted in green indicate a significant activity towards the respective substrate.

NylA

Ribbon model from NylA.

The cyclic 6-aminohexanoic acid dimer hydrolase (NylA) specifically catalyzes the hydrolysis of one of the two equivalent amide bonds in 1,8-diazacyclotetradecane-2,9-dione, the cyclic dimer of 6-aminohexanoic acid. This produces N - (6-aminohexanoyl) -6-aminohexanoate, the linear dimer of 6-aminohexanoic acid. NylA belongs to the amidase signature family . The members of this family of more than 200 enzymes have a conserved sequence region of 160 amino acids, the amidase signature. Compared to other polyamide-6-oligomers, NylA shows almost no activity.

NylB

Ribbon model of NylB with the D370Y mutation

The 6-aminohexanoic acid dimer hydrolase (NylB) has the catalytic triad Ser 112 / Lys 115 / Tyr 215 and an oxyanion hole in its active center . The catalytic triad corresponds to that of some other bacterial serine hydrolases, such as D-alanine transpeptidase , carboxylesterase 2 (estB) and class C serine-β-lactamases . An essential difference, however, is the amino acid tyrosine in position 170 (Tyr170), which is not found in any of the serine β-lactamases. Tyr170, through its interaction with the catalytic triad, plays a major role in NylB's ability to catalyze the hydrolysis of Ahx 2 . The substrate binding in the enzyme is increased by Tyr170. In addition, Tyr170 in the enzyme - by disrupting a tetrahedral transition state - promotes a conformational change of the substrate which is favorable for its protonation by Tyr215 of the catalytic triad. If the tyrosine in position 170 is replaced by the sterically very similar phenylalanine (Tyr170Phe), the activity of NylB against Ahx 2 drops to 1.4%.

The activity of NylB towards linear polyamide-6 oligomers decreases markedly with increasing chain length. With the hexamer (Ahx 6 ) the activity is only 8% of the value of the dimer (Ahx 2 ), with the eikosamer (Ahx 20 ) the value drops to 0.3%.

Like other serine proteases, NylB also shows properties of an esterase and is able to catalyze the hydrolysis of various carboxylic acid esters.

NylB ′

NylB has a homologue called NylB ′. Both enzymes consist of 392 amino acids, but they differ in 46 positions. NylB ′ has only about 0.5% of the activity of NylB. In 2009 a Japanese working group was able to use molecular design to show that the catalytic properties of NylB ′ can be increased by a factor of 160 through the targeted exchange of three amino acids.

NylC

Ribbon model from NylC

The 6-aminohexanoate oligomer endohydrolase (NylC) catalyzes the hydrolysis of linear and cyclic polyamide-6 oligomers with more than three 6-aminohexanoic acid units. The activity towards the linear and cyclic 6-aminohexanoic acid dimer is very low. The enzyme was isolated from cultures of Arthrobacter (NylC p2 ), Agromyces (NylC A ) and Kocuria (NylC K ). NYLC A and NYLC K differ by 5 to 15 amino acid substitutions of NYLC p2 and by 10 to 20 K thermostable . In aqueous solution, NylC forms oligomers with an average molar mass of approx. 93 kDa. X-ray structure analyzes also speak in favor of a dimeric or trimeric structure.

Evolution in the laboratory

Pseudomonas aeruginosa (colored against a blue background) in the scanning electron microscope .

The bacterial strain Pseudomonas aeruginosa PAO, which was originally isolated in New Zealand , has been well researched biochemically and genetically and is the standard strain of this bacterial species. Pseudomonas aeruginosa PAO shows no activity whatsoever towards the linear dimer Ahx 2 or other by-products of polyamide 6 production. In 1995 a working group at Osaka University cultivated Pseudomonas aeruginosa PAO in the minimal medium M9, which in the experiment consisted of 2 g / l glucose and 1 g / l ammonium chloride . They applied various dilutions of this colony to culture dishes which contained Ahx 2 in a concentration of 2 g / l as the only carbon and nitrogen source. The polyamide 6 oligomers are not toxic to this type of bacteria. After nine days of incubation at 30 ° C, the experimenters received colonies with a frequency of 0.1% with pronounced growth (“ hypergrowing colonies ”). During this time, the bacteria in these colonies had acquired the ability to use the linear dimer of 6-aminohexanoic acid as a nutrient. One of these colonies, named PAO5502, was able to metabolize the cyclic dimer of 6-aminohexanoic acid after three months of incubation. The growth rate was 0.1 h −1 in the linear and 0.03 h −1 in the cyclic dimer. In the first step, PAO5502 hydrolyzes the cyclic to the linear dimer and in the second step the linear dimer into two molecules of 6-aminohexanoic acid.

A lack of nutrients can increase the mutation rate in bacteria by factors in the range of 10,000. Since these conditions were present when Pseudomonas aeruginosa was incubated , it can be assumed that this was a decisive factor in the rapid acquisition of the new capabilities of Pseudomonas aeruginosa .

ε-caprolactam as a substrate

In 1998, Pseudomonas aeruginosa MCM B-407 , a bacterial strain that is able to break down ε-caprolactam, was isolated from the activated sludge of a sewage treatment plant of a company producing polyamide-6. In 2002 this property was also demonstrated in strains of Alcaligenes faecalis , Arthrobacter citrus , Bacillus sphaericus and Rhodococcus rhodochrous . The strain Alcaligenes faecalis G grows at concentrations of up to 15 g ε-caprolactam per liter and breaks it down to 95 to 97% within 24 to 48 hours. For efficient degradation it needs magnesium , potassium and phosphate ions as nutrient salts . In Proteus sp. and Bordetella sp., from landfills in the state of Lagos ( Nigeria were isolated) were 2013 similar degradation rates are measured. The large amounts of ε-caprolactam in the wastewater from the polyamide 6 production plants have a considerable impact on the environment. ε-caprolactam-degrading bacterial strains are therefore of particular importance in the treatment of such wastewater.

Origin of the enzymes

The schematic representation of the structure of the plasmid pOAD2, which is located in Flavobacterium sp. KI72 finds.

With the discovery of nylonases, the question immediately arose where these enzymes come from. Finally there were those from the Flavobacterium sp. KI72 had only been using 6-aminohexanoic acid oligomers for about 30 years and no other natural substrates for these enzymes were found.

Flavobacterium sp. KI72 has three types of plasmids : pOPAD1, pOAD2 and pOAD3. Plasmids are small, mostly circular, double-stranded DNA molecules that are not part of the bacteria chromosome . The three polyamide-6-degrading enzymes are produced in the plasmid pOAD2. This plasmid consists of 45,519 base pairs , of which guanine and cytosine make up about two thirds. The plasmid contains the four genes nylA , nylB , nylC and nylB ' , as well as five times the insertion sequence IS 6100 . The sequence of pOAD2 shows a significant similarity with that of the genes of penDE ( isopenicillin-N-acyltransferase ), rep ( plasmid incompatibility ), ftsX ( filamentation temperature sensitive ) and oppA-F ( oligopeptide-transporting ATPase ).

In 1984, Susumu Ohno postulated that nylonase B was caused by gene duplication and subsequent frameshift . The source for the unique protein is an unused open reading frame of the existing coding sequence. Only sequences that begin with several repetitions are likely to be able to maintain deviating, long open reading frames. From the base sequence of NylB in the pOAD2 plasmid of Flavobacterium sp. K172, he concluded that the 392 amino acid gene product (NylB) was derived from another pre-existing open reading frame that originally encoded a 472 amino acid arginine-rich protein. The American Kenneth Miller also assumes in his book Only a Theory , published in 2008 , that a frameshift in a gene was the place of origin for the nylon noses. The bacterial strains that are able to degrade polyamide-6 oligomers are said to have arisen via this mutation and subsequent selection via the selection advantage utilization of polyamide-6 oligomers. Since the activities of the nylonases are comparatively low, he assumes that the evolutionary process of these enzymes is still in full swing. Frameshifts are a radical source of genes with new properties, which is relatively common in bacteria. Even in the human genome , 470 frameshift events have been detected, leading to proteins with new properties.

The results of a working group led by Seiji Negoro from the Prefectural University of Hyōgo lead to a different result than the speculation about a frameshift . By analyzing the X-ray structure of NylB, she comes to the conclusion that this enzyme has its origin in an esterase with β-lactamase folding.

Nylonase and creationism

According to creationists such as John N. Moore of the Creation Research Society , gene mutations are incapable of producing new properties in an organism. In their opinion, gene mutations only change properties that already exist or are known. According to William Dembski , a representative of intelligent design , the complexity of proteins is too high for new information to be incorporated into the genome through mutation and selection. Mutations would only produce varieties, but not new abilities. In the world view of the intelligent design representatives, new properties of an organism can only be explained by an intelligent designer (“God”) and not by the contradicting evolution, with its tools of mutation and selection.

This is in stark contrast to the view of most evolutionary biologists, can lead to mutations new, advantageous properties and these characteristics to subsequent generations inherited can be. Acquired by mutations ability of different bacterial species "unnatural" substances to use nylon 6 oligomers as nutrients such as, for them - similar to the E. coli -Langzeitexperiment of Richard Lenski - a prime example of the observable evolution. As a result, a controversy arose between evolutionary biologists and representatives of intelligent design about nylon noses. In the opinion of the representatives of intelligent design, the ability to metabolize polyamide-6 oligomers is “not new information” (“ Nylonase isn't new information ”).

Web links

Remarks

  1. ↑ The bacterium was originally classified as Achromobacter guttatus by the authors .
  2. ↑ Quote: “ Any gene mutation results in no more than alteration of already existing or known traits. "Or" Mutations are sources only of differences of characteristic expressions of traits already in existence, and not a source of new traits. Mutations result only in changes within the existing genetic structure. "John N. Moore, Roger J. Cuffey: Paleontological evidence and organic evolution. In: Journal of the American Scientific Affiliation. Volume 24, 1972, pp. 160-176.

Individual evidence

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  2. ↑ a b c d e Shinichi Kinoshita, Sadao Kageyama, Kazuhiko Iba, Yasuhiro Yamada, Hirosuke Okada: Utilization of a Cyclic Dimer and Linear Oligomers of ε-Aminocaproic Acid by Achromobacter guttatus KI 72. In: Agricultural and Biological Chemistry. Volume 39, number 6, 1975, pp. 1219-1223, doi: 10.1271 / bbb1961.39.1219
  3. ↑ E. Tsuchida, I. Shinohara, Oligomer and Oligomerization. In: Journal of Synthetic Organic Chemistry Japan. Volume 22, Number 33, 1964, doi: 10.5059 / yukigoseikyokaishi.22.33 (article in Japanese)
  4. ^ NN Baxi, AK Shah: Biological treatment of the components of solid oligomeric waste from a nylon-6 production plant. In: World Journal of Microbiology and Biotechnology. Volume 16, 2000, pp. 835-840.
  5. ↑ Polyamide 6. (No longer available online.) In: pcinylon.com. Archived from the original on July 9, 2015 ; accessed on September 5, 2015 . Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice.  @1@ 2Template: Webachiv / IABot / pcinylon.com
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  22. ↑ PDB  3A2P
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  26. ↑ PDB  2ZLY
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  35. ↑ PDB  3AXG
  36. ↑ a b c ID Prijambada, S. Negoro u. a .: Emergence of nylon oligomer degradation enzymes in Pseudomonas aeruginosa PAO through experimental evolution. In: Applied and environmental microbiology. Volume 61, Number 5, 1995, pp. 2020-2022, PMID 7646041 , PMC 167468 (free full text).
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