WO2018151649A1

WO2018151649A1 - Identification of enzymes degrading recalcitrant environmentally hazardous organic compounds

Info

Publication number: WO2018151649A1
Application number: PCT/SE2018/050138
Authority: WO
Inventors: Martin RYEN
Original assignee: Pharem Biotech Ab
Priority date: 2017-02-15
Filing date: 2018-02-14
Publication date: 2018-08-23
Also published as: SE1750143A1

Abstract

The present invention provides for a method for identification, selection and modification of enzymes that may be used to eliminate environmentally hazardous organic substances such as drug molecules, from water within the environment of water treatment plants or systems, or toilets.

Description

IDENTIFICATION OF ENZYMES DEGRADING RECALCITRANT ENVIRONMENTALLY HAZARDOUS ORGANIC COMPOUNDS

TECHNICAL FIELD

The invention relates to the field of enzyme identification for the purpose of water purification.

BACKGROUND ART

The pollution of water is a serious environmental problem. Today, the water treatment of sewage water, after a passage through a mechanical filter, generally comprises use of activated sludge, comprising sedimentation of gross solids, followed by aerobic degradation of any organic matter and a subsequent sedimentation to remove biomass. The water may thereafter go through a chemical treatment. None of the existing treatments can fully eliminate environmentally hazardous organic substances, such as for instance drug molecules, plasticizers, flame retardants, herbicides, pesticides, fungicides, cosmetics, fabrics additives, etc., from the water, since not all of the environmentally hazardous organic substances are cleaved or otherwise disintegrated. Thus, there is still quite a substantial amount of such substances in the effluent from the water treatment plants, which is an environmental problem as well as a health problem. Many environmentally hazardous organic substances remain in the water, enter the environment, and have an environmentally negative impact, and in particular exert negative effects on aquatic life. WO 2009/076711 Al discloses using polypeptides for degrading chlorinated s-triazines, which are pest control agents. Polypeptides with enhanced degradation ability, compared to AtzA are identified, expressed and brought into contact with for instance water, to eliminate the s- triazines present therein.

WO 2015/145222 A2 discloses the production of recombinant enzymes for the purpose of hydrolysing organophosphates that are used as pesticides, fungicides, insecticides, herbicides and petroleum additives. WO 2012/007536 Al discloses methods using enzymes for inactivation of antibiotics in the environment, and in particular inactivating antibiotics from waste and waste water effluents before they reach the environment.

SUMMARY OF THE INVENTION

There is a need to improve water purification methods in order to eliminate most of, if not all, environmentally hazardous organic substances present in the sewage and toilet water to a lower cost and with a larger efficiency. The present invention attains to solve this problem by providing a method for identification, selection and modification of enzymes that may be used to eliminate environmentally hazardous organic substances from water within the environment of water treatment plants or systems, or toilets.

Thus, in a first aspect, the present invention relates to a method of identifying one or more enzymes having the ability to eliminate one or more environmentally hazardous organic substances, such as drug molecules, plasticizers, flame retardants, herbicides, pesticides, fungicides, cosmetics, fabrics additives, etc., comprising the steps of: a) providing the environmentally hazardous organic substance to be eliminated; b) screening an enzyme library for one or more enzymes having the capacity to eliminate the environmentally hazardous organic substance, based on structural similarity between the environmentally hazardous organic substance and a known substrate for the enzyme(s); c) selecting one or more enzymes having the capacity to eliminate the environmentally hazardous organic substance; d) modifying a polynucleotide sequence corresponding to selected enzyme(s) by mutagenesis; e) selecting the mutated variant(s) of the enzyme(s) having the highest ability to eliminate the environmentally hazardous organic substance. According to a second aspect the present invention further attains to solve the problem above by providing enzymes identified, selected and modified according to the method above, for the elimination of drug molecules, wherein the enzyme is chosen from the group comprising of epoxide hydrolase, Acetoacetate decarboxylase, Melamine Deaminase and dehalogenase LinB.

Preferably the drug molecules are chosen from the group comprising of Ciprofloxacin, Citalopram, Clarithromycin, Diclofenac, Erythromycin, Estradiol, Etinylestradiol, Fluconazole, Ibuprofen, Karbamazepin, Ketoconazol, Levonorgestrel, Losartan, Metoprolol, Metotrexat, Naproxen, Oxazepam, Sertralin, Sulfametoxazol, Tramadol, Trimetroprim, Zolpidem, Ketoprofen, Risperidone, Fluoxetine, Venlafaxine, Ofloxacine, Doxycycline, Norfloxacine and Tetracycline.

According to one embodiment of the second aspect, the present invention relates to the use of lignin peroxidase identified by the method above for eliminating environmentally hazardous drug molecules, wherein the environmentally hazardous molecules are chosen from a group comprising Metoprolol, Propranolol, Clarithrymocin, Doxycycline and Trimethoprim.

According to a second embodiment of the second aspect, the present invention relates to the use of epoxide hydrolase identified by the method above for eliminating environmentally hazardous drug moleculeswherein the environmentally hazardous molecule is Carbamazepine and/or Trimethoprim.

According to a third embodiment of the second aspect, the present invention relates to the use of laccase identified by the method above for eliminating Doxycycline, Levonorgestrel and Oxazepam. According to a fourth embodiment of the second aspect, the present invention relates to the use of P450 BM3 identified by the method above for eliminating environmentally hazardous drug molecules wherein the environmentally hazardous molecule is Metoprolol.

According to a fifth embodiment of the second aspect, the present invention relates to the use of Acetoacetate decarboxylase identified by the method above for eliminating environmentally hazardous drug molecules.

More specifically, the present invention relates to the use of Acetoacetate decarboxylase according to the above, wherein the environmentally hazardous molecule is chosen from the group comprising Ibuprofen, Methotrexate, Naproxen, Ketoprofen and Ofloxacin. According to a sixth embodiment of the second aspect, the present invention relates to the use of Melamine Deaminase identified by the method above for eliminating environmentally hazardous drug molecules.

More specifically, the present invention relates to the use of Melamine Deaminase according to the above, wherein the environmentally hazardous molecule is chosen from the group comprising Sertraline, Sulfametoxazole and Trimethoprim.

According to a seventh embodiment of the second aspect, the present invention relates to the use of Dehalogenase LinB identified by the method above for eliminating environmentally hazardous drug moleculeswherein the environmentally hazardous molecule is chosen from the group comprising Citalopram, Fluconazole, Ketoconazole and Losartan.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 - Flowchart of the process according to the invention

DETAILED DESCRIPTION

Environmentally hazardous organic substances are present in sewage water due to the utilisation thereof by our society. It may relate to organic substances used within the manufacturing of consumer products, such as for instance flame retardants, plasticizers, fabrics additives, or to organic substances used within agriculture, such as herbicides, pesticides, fungicides, etc. It may also relate to drug molecules that are excreted by the users into the sewage water, via bodily fluids, faeces etc. It may further relate to anti-bacterial agents used within cosmetics, or hygiene products such as soap, shampoo etc., which may enter the water either from the manufacturing or from the usage thereof. Regardless of the origin of the environmentally hazardous organic molecule, the environmentally hazardous organic molecules of interest within the present invention are molecules that have been identified to pose a threat to the environment, to health and/or to aquatic life, and that are difficult to eliminate from sewage water using present techniques.

The environmentally hazardous organic substances used within the manufacturing of consumer products that are of interest for elimination from sewage water may for instance be nonylphenol, Bisphenol A or PFAS (Per- and polyfluoroalkyl substances).

Nonylphenol is a not easily biodegradable substance, used in manufacturing antioxidants, lubricating oil additives, laundry and dish detergents, emulsifiers, and solubilizers, and may also be present in for instance textiles. It is banned within the EL⁾, but imported items from outside of the EL⁾ may still contain the substance. Nonylphenol plays a potential role as an endocrine disruptor and xenoestrogen, due to its ability to act with estrogen-like activity. Nonylphenol is easily bioaccumulated in organisms coming in contact with the substance.

Bisphenol A is employed to make certain plastics and epoxy resins, and has been used in for instance many food and beverage cans. It has been shown that Bisphenol A exhibits estrogen mimicking, hormone-like properties, but also neurological effects and cancer have been indicated.

PFAS (Per- and polyfluoroalkyl substances) are chemicals that are used to produce smooth, water resistant, fat resistant surfaces, but is also present in for instance fire extinguishing foam. PFAS may be degraded to perflourated alkylic acids (PFAA), which in turn is not degradable and thus may accumulate in organisms. They have been shown to cause liver damage and may also influence fat metabolism, the immune system and the reproduction capability.

The drug molecules of interest to be eliminated from sewage water are numerous and comprise for instance antibiotics, anti-depressive agents, anti-inflammatory agents, hormones, antifungal agents, blood pressure agents, antineoplastic agents (anti-cancer), sedatives and painkillers. Antibiotics are of particular interest for elimination from sewage water as a part of the struggle to combat the development of antibiotic resistance. Examples of antibiotics that are of interest to be eliminated from sewage water are for instance Ciprofloxacin, Clarithromycin, Erytromycin, Sulfametoxazol, Trimetroprim, Ofloxacin, Doxycycline, Norfloxacin and Tetracycline. Examples of anti-depressive agents that are of interest to be eliminated from sewage water are for instance Citalopram, Sertralin, Fluoxetin and Venlafaxin.

Examples of anti-fungal agents that are of interest to be eliminated from sewage water are for instance Flukonazol and Ketoconazol. Examples of anti-inflammatory agents that are of interest to be eliminated from sewage water are for instance Diclofenac, Ibuprofen, Naproxen, Ketoprofen and Metotrexat.

Examples of bloodpressure agents that are of interest to be eliminated from sewage water are for instance Losartan and Metoprolol.

Examples of hormones that are of interest to be eliminated from sewage water are for instance Estradiol, Etinylestradiol and Levonorgestrel.

An example of painkiller that is of interest to be eliminated from sewage water is for instance Tramadol.

Examples of sedatives that are of interest to be eliminated from sewage water are for instance Zolpidem, Risperidon, Carbamazepin and Oxazepam.

By "enzymes" is meant a polypeptide or protein which can act as a biological catalyst. Enzymes work by binding molecules so that they are held in a particular geometric configuration that allows a reaction to occur. Enzymes are very specific in that few molecules closely fit a binding site on the enzyme. Each enzyme catalyses a specific type of chemical reaction between a few closely related compounds, substrates. According to the present invention, the studied catalytic action of the enzyme that is being identified and modified is the cleavage of the substrate at a specific structural site.

In order to screen an enzyme library in search for a suitable enzyme to be used in the elimination of an environmentally hazardous organic substance, the chemical structures involved in biological activity for the environmentally hazardous organic substances are identified. Then a database for enzyme activity is searched through for identifying enzymes with activity against similar structures as those chemical structures that have been identified to be involved in the biological activity above. Examples of databases to be used for such a search are for instance ExPaSy or BRENDA. In a first step in the identification of an enzyme, the chemical functional groups that are essential for the biological activity of the environmentally hazardous organic substance, such as alcohols, carboxyl acids, esters, halogens etc. are identified. In a second step, the type of enzymes that are able to digest such a chemical functional group is identified. It relates to digestion of -OH, -COOH, -C-O-C, etc. In a third step, the carbon backbone structure of the environmentally hazardous organic substance is compared to the carbon backbone structure of the substrates that are or known to be digested by the enzyme. This is to ensure that the environmentally hazardous organic substance may be able to fit into the binding site of the enzyme, and that for instance no sterical hindrance exists. Once identified, the enzymes are acquired for further testing of their activity against the correlating environmentally hazardous organic substance. The enzymes may be purchased in a produced, purified form. The enzyme may also be purchased as a polynucleotide in a plasmid or a vector, which will facilitate the mutagenesis of the polynucleotide and allows for a choice of expression system or expression organism. It is also possible to acquire the organism originally producing the enzyme, for in-house sequencing and subsequent expression in any expression system. There are a great number of commercial channels through which enzymes, polynucleotides encoding the enzymes, or organisms producing the enzymes may be purchased, which are known to the person skilled in the art.

Enzymes that show an activity against the environmentally hazardous organic substance are selected to be used in the method according to the present invention. For an enzyme to show an activity against the environmentally hazardous organic substance, and thus having a capacity to eliminate said environmentally hazardous organic substance, said enzyme needs to digest the environmentally hazardous organic substance at the functional chemical groups identified according to the above. The presence of a substrate after reaction of the substrate with an enzyme is usually detected by means of mass spectrometry. However, any other means known to the skilled person for this purpose may also be used. In order to determine if an enzyme is eligible for the method according to the invention, an elimination capacity is thus determined. This elimination capacity is determined by measuring the degree of elimination of the substrate at a concentration of 1U enzyme, in relation to the substrate, and 100 μιηοΙ of environmentally hazardous organic substance, after 1 hour of reaction within a temperature range of +8 °C - 20 °C and within a pH range of 5,5 - 8,5. A reduction of 15% of the environmentally hazardous organic substance, and thus an elimination capacity of 15%, must be observed for the enzyme to be selected for the method according to the present invention.

The aim of the method according to the present invention is to provide selected enzymes that possess, or are modified through mutagenesis to possess, the ability to exert their activity against the environmentally hazardous organic substances during the following conditions: within a temperature range of +8 °C - 20 °C; within a pH range of 5,5 - 8,5; with an activity of at least 10³

Thus all three criteria of temperature, pH and enzymatic activity according to the above must be fulfilled. These are the preset criteria that must be met by an enzyme in order to be able to be used against the environmentally hazardous organic substances.

In particular, the enzyme needs to be selected for fulfilling, or be mutated to fulfil, all of the above mentioned criteria regarding pH, temperature and enzymatic activity in order to be functional within the environment of water treatment plants or systems. Failure of the enzyme to be able to be active within either the temperature range or the pH range, will render the enzyme not suitable to be used in such an environment. Furthermore, a lower enzymatic activity for the enzyme than the activity threshold indicated above, will render it not efficient enough for eliminating a sufficient amount of drug molecules from the sewage water.

An enzyme that shows an activity, and thus an elimination capacity of at least 15 %,against the correlating environmentally hazardous organic substance, but do not initially fulfil all of the three above mentioned criteria, will be subject to a mutagenesis process comprising the following steps:

1. The original gene for the identified enzyme is acquired through DNA synthesis, or by PCR starting from the origin species;

2. The enzyme gene then go through a mutagenesis for obtaining new mutated enzyme genes that may fulfil the above specified criteria;

3. The mutated enzyme genes are transformed into a E. coli host with vector DNA containing the mutated enzyme gene; 4. An appropriate aliquot of E.coli host cells is spread on LB (Lysogeny broth)-plate containing a suitable antibiotic to obtain well-isolated colonies and thus create a mutation library;

5. Single colonies of host cells are picked and inoculated into LB media in a 96-well plate and stored overnight in 30°C;

Aliquots of host cells culture are transferred into a new 96 well plate containing TB media (Terrific Broth) and inducer agent and cultivated over night at 30°C;

The host cells are centrifuged down and resuspended in buffer containing Lysozyme and DNase and centrifuged down again to obtain a supernatant comprising any enzyme produced;

The supernatant from step 7 is used for testing enzyme activity against the correlating environmentally hazardous organic substance within the criteria specified above;

Best performing mutations are selected and restarted from step 2 until all three criteria have been fulfilled. All of the material and reagents used in the method according to the invention are within common knowledge within the technical field, and may be obtained from commercial channels.

DNA synthesis or PCR processes as performed in step 1 are part of the common general knowledge within the field, and the person skilled in the art will be readily able to perform any of the methods currently used within the field. The mutagenesis used in step 2 may be according to any of the methods disclosed in Salazar, O. et. al., Evaluating a Screen and Analysis of Mutant Libraries, Methods in Molecular Biology, vol. 230: Directed Enzyme Evolution: Screening and Selection Methods. The skilled person is able to readily make use of the methods disclosed therein forthe purpose of the mutagenesis according to the invention. In step 3, the transformation of the E. coli with the vector comprising a mutated strain gene for the enzyme can be done according to any transformation commonly used within the field, and can be chosen from for instance electroporation, gene gun technique, agitation with glass beads, ultrasound and Shock waves. An enzyme that, after mutagenesis and subsequent testing according to the above, do not fulfil the requirement of meeting the criteria of temperature, pH and enzymatic activity against the substrate, may be subject to further mutagenesis until the requirement is fulfilled.

An enzyme that in the initial screening is shown to already have an activity of at least 10³ s ^_1M^" at the specified temperature and pH intervals, does not need to be subjected to the mutagenesis and can be directly used for the purpose of eliminating environmentally hazardous organic substances from water within water treatment plants or systems.

By the method of the present disclosure, enzymes are identified, selected, and optionally modified when necessary as disclosed above, in order to find a collection of as few enzymes as possible which eliminate as many drug molecules as possible. Thus, a main advantage of the present invention is that a single enzyme may be modified to be able to eliminate more than one, prefereably at least two or more, drug molecules within water treatment plants or systems. Thus an enzyme may be obtained which, when applied to a water treatment plant or system, may be more efficient in the elimination of the drug molecules that are prioritized to be removed therefrom. Furthermore, the majority of the drug molecules prioritized to be removed from the sewage water may be eliminated with a minimum number of enzymes, which will render a method for purification of water using said enzymes more cost efficient.

EMBODIMENTS OF THE INVENTION Enzyme list with compounds

The following enzymes have been identified and proven to have the capacity to eliminate the specified environmentally hazardous organic substance, or been modified to do so, according to the method above:

Enzyme Origin species Activity against following compounds

La cease Trametes Ochracea Bisphenol A, Diclofenac,

Estradiol, Ciprofloxacin, Doxycycline, Levonorgestrel,

Oxazepam

Lignin Peroxidase Trametes Vesicolorum Metoprolol, Propranolol,

Clarithrymocin,

Trimethoprim

Epoxide hydrolase Rhodococcus Rhodochrous Carbamazepine,

Trimethoprim

P450 BM3 E. coli DE10 Metorpolol, Propranolol

Melamine Deaminase Sertraline, Sulfametoxazol,

Trimethoprim

Acetoacetate decarboxylase Human Ibuprofen, Metotrexat,

Naproxen, Ketoprofen, Ofloxacin

Dehalogenase LinB Sphingobium japonicum Citalopram, Flukonazol,

Ketoconazol, Losartan, Norfloxacin

EXAMPLES

Example 1 - Selection of enzyme with activity towards Metoprolol

In finding an enzyme that may eliminate metoprolol, the following important functional chemical groups were identified:

• Methoxy group

• Ether group

• Hydroxyl group.

Enzymes that have activity against similar groups in chemicals with a similar structural carbon backbone to Metoprolol where searched. A database search on ExPaSy identified Lignin Peroxidase as a potential candidate. Lignin Peroxidase acts by breaking down lignin which consists of chains of repeated regions with structural similarities to Metoprolol. A Lignin Peroxidase gene based on the sequence from Trametes Versicolorum was ordered from a commercial provider. The gene was introduced to the plasmid vector Pet 30 (a)+ (AGILENT) and then transfected into E. coli strain DE3 Bcl21 (Sigma).

The enzyme was expressed through standard expression protocols. The cells where disrupted by cell lysis and the solution was tested against Metoprolol according to the general protocol above. The enzyme showed an optimal activity against Metoprolol of 1.411xl0³ s ^_1M ¹ within the pH and temperature specified above.

Claims

1. A method of identifying one or more enzymes having the ability to eliminate one or more environmentally hazardous organic substances, such as drug molecules, plasticisers, flame retardants, herbicides, pesticides, fungicides, cosmetics, fabrics additives, etc., comprising the steps of: a) providing the environmentally hazardous organic substance to be eliminated; b) screening an enzyme library for an enzyme having the capacity to eliminate the environmentally hazardous organic substance, based on structural similarity between the environmentally hazardous organic substance and a known substrate for the enzyme(s); c) selecting one or more enzymes having the capacity to eliminate the environmentally hazardous organic substance; d) modifying a polynucleotide sequence encoding the selected one or more enzymes by mutagenesis; e) selecting the one or more mutated variants of the one or more enzymes having the highest ability to eliminate the one or more environmentally hazardous organic substances.

2. The method of claim 1, wherein steps d) and e) are repeated at least once to increase the ability of the enzyme to eliminate the one or more environmentally hazardous organic substances.

3. The method according to claim 1 or 2 wherein the selected enzyme(s) are modified to be active within a pH of 5.5 - 8.5 in order to optimize the enzyme activity under conditions present in a water treatment system/plant.

4. The method according to any of claims 1-3, wherein the selected enzyme(s) are modified to be active within a temperature of +8°C - 20°C in order to optimize the enzyme activity under conditions present in a water treatment system/plant.

5. The method according to any of claims 1-4, wherein the ability of the mutated variant(s) of the enzyme(s) to eliminate the environmentally hazardous organic substance(s) is at least 10³ s^~

6. The method according to any of claims 1-5, wherein in step e) the modified enzymes are selected by the further steps of: i) transforming a host cell with vector DNA containing a mutated enzyme gene; ii) spreading an appropriate aliquot of host cells on a culture plate containing a suitable antibiotic to obtain well-isolated colonies; iii) picking single colonies of host cells and inoculating into a culture medium and storing overnight at 30°C; iv) transferring aliquots of host cell cultures into a plate containing culture medium and inducer agent and cultivating over night at 30°C; v) centrifuging the host cells down, resuspending the host cells in a buffer comprising Lysozyme and DNase, and centrifuging the host cells down again to obtain a supernatant comprising any enzyme produced; vi) using the supernatant from v) for testing enzyme activity against the correlating environmentally hazardous organic substance.

7. The method of any of claims 1-6, wherein the activity of an enzyme is measured after 1 hour of reaction with a concentration of 1U enzyme, in relation to the substrate, and 100 μιηοΙ environmentally hazardous organic substance.

8. An enzyme capable of eliminating one or more environmentally hazardous organic substances, such as drug molecules, plasticisers, flame retardants, herbicides, pesticides, fungicides, cosmetics, fabrics additives etc. from water under conditions present in a water treatment system/plant, wherein the enzyme is identified and/or modified according to the method of any of the claims 1-7, wherein the enzyme is chosen from the group comprising of epoxide hydrolase, Acetoacetate decarboxylase, Melamine Deaminase and dehalogenase LinB.

9. Use of enzymes identified and/or modified according to the method of any of claims 1-7, or of an enzyme of claim 8, for use in a water treatment system/plant for eliminating environmentally hazardous drug molecules, wherein the enzyme is chosen from the group comprising epoxide hydrolase, Acetoacetate decarboxylase, Melamine Deaminase and dehalogenase LinB .

10. Use of lignin peroxidase for eliminating Metoprolol, Propranolol, Clarithrymycin, Trimethoprim and Doxycycline.

11. Use of epoxide hydrolase according to claim 9, wherein the environmentally hazardous molecule is Carbamazepine and/or Trimethoprim.

12. Use of P450 BM3 for eliminating Metoprolol.

13. Use of Acetoacetate decarboxylase according to claim 9, wherein the environmentally hazardous molecule is chosen from the group comprising Ibuprofen, Metotrexat, Naproxen, Ketoprofen and Ofloxacin.

14. Use of Melamine Deaminase according to claim 9, wherein the environmentally hazardous molecule is chosen from the group comprising Sertraline, Sulfametoxazol and Trimethoprim.

15. Use of Dehalogenase LinB according claim 9, wherein the environmentally hazardous molecule is chosen from the group comprising Citalopram, Flukonazol, Ketoconazol and Losartan.