WO2010072892A1

WO2010072892A1 - Method and product for degrading antibiotic residues in aqueous waste material

Info

Publication number: WO2010072892A1
Application number: PCT/FI2009/051010
Authority: WO
Inventors: Tuula Heinonen
Original assignee: Tuula Heinonen
Priority date: 2008-12-22
Filing date: 2009-12-17
Publication date: 2010-07-01
Also published as: FI20086225A0; FI20086225A

Abstract

The present invention relates to a method and an enzyme preparation for treating aqueous waste material comprising antibiotic residues. According to the method the material is contacted with an effective amount of hydrolyzing enzymes capable of degrading antibiotic residues. The method can be used to degrade antibiotic residues in waste material from industrial processes, hazardous waste, waste from hospital tanks, sewage plants, farm house urine or feces collection tanks. The method of the invention can be used to prevent the emergence of active antibiotics into the environment and to prevent spread of antibiotic resistance in bacteria, animals or man.

Description

METHOD AND PRODUCT FOR DEGRADING ANTIBIOTIC RESIDUES IN AQUEOUS WASTE MATERIAL

Field of the Invention

The present invention relates to a method for treating aqueous waste material comprising antibiotic residues, to prevent emergence of antibiotic resistance in bacteria, environment, animals or man. In addition, the invention relates to enzyme preparations, which can be used in the method.

Description of Related Art

Antibiotics are one of the most commonly prescribed classes of drugs. In USA they form the second most commonly prescribed class of drugs (Monroe and Polk, 2000). Extensive and uncontrolled use of antibiotics in medication in man and animals, in animal growth promoters and ^'pestisides^', has led an exponentially growing problem of emergence of antibiotic resistant bacteria with serious consequences. The health care system has been greatly impacted by the emergence of antibiotic resistant bacterial infections (Levy and Marshall, 2004). Additional costs due to antibiotic resistance are remarkable. In U.S. only, 2 million patient per year get a hospital associated infection, of these 90 000 die each year. The annual costs due to hospital associated infections are estimated to be between 35 and 45 billion U.S. dollars. 70 % of the bacteria causing hospital associated infections are resistant to at least one of the antibiotics most commonly used to treat them (CDC statistics, AMR Hospital Antibiotics Report 2005). Only a few new antibiotics are coming into the market leading to the situation that we have super bacteria but no drugs.

Antibiotic resistance is the result of the use and in particular, of the uncontrolled use of antibiotics. Antibiotics are used to treat infections in man and animals but they are also used in high amounts as "pesticides" to honeybees, plants, trees, etc. In 1998, some 50 million pounds of antibiotics were produced in the United States, of which about half went to people in hospitals and homes. Of the remainder, about 80% was given to animals for various infections (Lewy, 2001 ). In 2004 13 300 kg of antibiotics were used for medication of animals in Finland. Of these, the amount of β-lactams was 66% (FINRES-Vet-2004). Antibiotics are also used as a growth promoter in farming animals, such as in pigs.

Probably the most important cause for increasing of antibiotic resistance is the exposure of human and animal gut flora to antibiotic also in a situation where the infection is not in the gastrointestinal tract, which in fact is the most common situa- tion (Monroe and Polk, 2000; Nord and Heimdahl, 1986; Christiaens et al., 2006). Antibiotics are excreted via urine or faeces either as intact active form or as metabolites. The exposure of gut flora to parent form of antibiotic is highest after oral intake of antibiotic. It is calculated that as much as up to 95 % of antibiotics taken by humans and animals are excreted unaltered and thus seep into environment and encourage antibiotic resistance there (Choi, 2006).

The intestinal microflora of humans and animals form a complex but relatively stable ecosystem. Antimicrobials that are present in the intestinal tract may cause profound disruption of the indigenous microflora, resulting in a number of adverse effects, such as antibiotic-associated diarrhea, Clostridium perfringens colitis and acquisition and overgrowth of antimicrobial-resistant pathogens, including vancomycin resistant enterococci (VRE), Candida species, and multiresistant gram- negative rods (Edlund and Nord, 2000). Overgrowth of these microorganisms may facilitate resistance gene transfer and increase the risk of person-to person trans- mission of resistance genes and also to the environment. Several studies have demonstrated that intestinal colonization with resistant bacteria precedes the onset of infection in man (Christiaens et al., 2006; Nord et. al., 1984). The prevalence of infections caused by extended-spectrum β-lactamase (ESPL)-producing Entero- bacteriaceae is increasing worldwide. Enterobacteria, such as E. coli, are typical causes for nosomical infections, especially urinary tract infections, sepsis/meningitis, intraabdominal infections and entehc/diarrhoeal disease. It is shown that in a single strain of bacteria after it has developed resistance to one antibiotic, the resistance is also at the same time developed to several other antibiotics and even to different classes of antibiotics.

Residues of antibiotics have been detected in sewage plants and raw water sources in many European countries. The removal rates of compounds through wastewater treatment are variable. The techniques in use are not capable of eliminating these compounds, which leads to a situation where rivers and even ground waters in some countries are also contaminated (Press release from European Commission DG Environment, 2003). Once the resistant bacteria have spread in the environment the resistance gene can be transferred to some other bacteria leading to spread of resistance (Salyers et. al., 2004; Choi, 2006). The low temperature in nature has been shown even to facilitate the transfer of plasmids. Gene transfer has been shown to occur between gram-positive and gram-negative bacteria, such that a resistance determinant occurring in any organism can eventually reach any other organism (Salyers et. al., 2004).

The antibiotic residues in food have also been identified by the regulatory authorities as a potential source for development of antimicrobial-resistant bacteria, which could spread via the food chain, or via zoonotic and spread to humans (Cerniglia and Kotarski, 2005; Sunde et al., 2008). In Finland the amount of pharmaceuticals in drinking water is typically below 10 ng/L (Vieno 2007).

In the prior art has been suggested methods to degrade β-lactam antibiotics in milk (WO 01/67879) to make milk acceptable as feed for animals, and the elimina- tion of residual antibiotic for example from animal or medicinal products (CN 101089178).

Literature has also shown that β-lactamase enzyme given orally with intravenously administered β-lactam antibiotic, ampicillin or piperacillin, breaks down antibiotic residues in canine jejunum preserving the microflora and preventing emergence of bacterial resistance (Harmoinen et al., 2003, Harmoinen et al., 2004, Stiefel et al., 2005). WO 2008/065247 suggests the use of β-lactamase for inactivating residual β-lactam in the intestine in connection with antibiotic treatment with a combination of β-lactam antibiotic and β-lactamase inhibitor. The orally administered pharmaceutical composition of β-lactamase was intended to reduce the effects of a β- lactam/ β-lactamase inhibitor combination on the major intestinal microbiota in the distal part of ileum and in the colon and to maintain the ecological balance of the instestinal microbiota. Furthermore, Stiefel and coworkers (Stiefel et al., 2005) have recently shown that recombinant β-lactamase prevents the breakage of colonization barrier and overgrowth of vancomycin-resistant enterococci (VRE).

If there were a method for hindering the antibiotic to go into the ecosystem, in ad- dition to the unwanted effects of antibiotics, the life span of several antibiotics could also be prolonged by preventing the emergence of resistant bacteria or spread of resistant genes.

There is thus a need to find a solution to the problem how to hinder or retard the emergence of antibiotic residues and the spread of antibiotic resistance to new microorganisms and thereby hinder or decrease the spread of antimicrobial- resistant bacteria to environment, humans and animals.

Summary of the Invention

It is an aim of the present invention to provide a method for hindering the spread of antibiotics into the environment.

In particular, it is an aim of the invention to inactivate antibiotic residues in waste material.

These and other objects, together with the advantages thereof over known solutions, are achieved by the present invention, as hereinafter described and claimed.

The present invention provides a method by which the emergence of active anti- biotics and bacterial resistance into the environment and food chain can be decreased. The invention is based on the idea that antibiotics in waste material, for example process residues from industrial processes, such as antibiotic production, hazardous waste comprising antibiotic residues, such as out-of-date pharmaceuti- cals, or waste from storage reservoirs, such as hospital tanks or sewage plants or farm house urine or faeces collection tanks, can be inactivated. In particular, the invention is based on the finding that antibiotics can be inactivated before they are emerged to the environment by using hydrolytic enzymes.

More specifically, the method of the present invention is mainly characterized by what is stated in the characterizing part of claim 1

The enzyme preparation of the present invention is mainly characterized by what is stated in the characterizing part of claim 11.

Hydrolytic enzymes and in particular β-lactamase enzyme are very efficient and also very active at low temperature. Therefore, only moderate amounts of enzyme are needed to treat remarkable amounts of waste material. The treatment can be carried out in places and under conditions where the waste material, such as collection tanks, are situated, since there is no need to elevate the temperature. Hence, the treatment is very cost effective.

By means of the invention, the amount of antibiotics delivered into the environment and food chain can be significantly reduced, which leads to a decreased emergence of bacterial resistance to humans and animals.

Next the preferred embodiment of the invention will be examined in greater detail with the aid of the following detailed description and with reference to a working example.

Detailed Description of Preferred Embodiments

A prior art waste treatment method generally comprises the following steps - the waste material is collected;

- the waste material is treated;and

- the waste material is released into the environment. Waste material comprising antibiotic residues is usually collected into reservoirs or containers. Such containers are for example

- containers for waste material from industrial processes, in particular washing waters from industrial antibiotic production process, or from food industry comprising waste from animal slaughter and processing;

- hospital tanks comprising urine and/or faeces;

- sewage plants;

- farmhouse urine and/or faeces collection tanks; or

- hazardous waste material, for example out-of-date medicaments.

Sewage may comprise waste material from residences, health care systems (e.g. hospitals, community homes), institutions, and commercial and industrial establishments. Raw sewage may include household waste liquid from toilets, showers, kitchen etc. Sewage may comprise also liquid waste from industry or commerce, or it may comprise surface waters (e.g. stormwater).

The treatment may comprise one or more treatment steps. The treatment may comprise physical, chemical, and biological processes to remove physical, chemical and biological contaminants.

Conventional sewage treatment may comprise three stages: primary, secondary and tertiary treatment, and optionally a pretreatment. In pretreatment bigger materials which can be easily collected are removed from the sewage. In primary treatment heavy solids can be sedimented to the bottom by holding the sewage in quiescence, whereas, oil, greese and lighter solids float to the surface. The settled and floating materials can be removed and the remaining liquid discharged or subjected to secondary treatment. Secondary treatment may remove dissolved and suspended biological matter, typically by indigenous water-borne microorganisms. Tertiary treatment may comprise a separation process to remove the microor- ganisnms from the treated water. Tertiary treatment may comprise for example disinfection, prior to discharge into environment (stream, river, wetland etc.) . In the present description, the term "antibiotic residues" or "antibiotics" stands for the unaltered and typically active form of the antibiotic. By "metabolized antibiotic" is meant an altered, degraded and typically inactive form of the antibiotic.

According to the present invention the antibiotics in industrial process residues or in hospital tanks or in sewage plants or in farmhouse urine/faeces collection tanks can be inactivated which will decrease the amount of antibiotics delivered into the environment and food chain leading to decreased emergence of bacterial resistance.

In the method, the material is contacted with an effective amount of hydrolyzing enzymes capable of degrading antibiotic residues.

The enzyme can be in liquid form, in lyophilized form or formulated into formulation, e.g. coated pellets, capsules, granules or flour, which will release the enzyme in a controlled manner.

The present invention provides a method for treating aqueous waste material comprising antibiotic residues. Typical reservoirs for such waste are process residues from industrial processes, for example washing waters from antibiotic pro- duction industry, hazardous waste comprising antibiotics, hospital tanks or sewage plants or farmhouse urine or faeces collection tanks. By "sewage" is meant the wastewater released by residences, businesses and industries in a community. It comprises mainly water and only about 0.06 % dissolved and suspended solid material.

This type of waste material comprises antibiotic residues in amounts of about 3-27 000 ng/liter (Huang et al. 2001 )

By aqueous waste material is heat meant in particular material, which has no use as food or feed. According to a preferred embodiment, aqueous waste material to be treated is contacted with an effective amount of one or more hydrolytic enzymes. Hydrolytic enzymes can be added to the container comprising the waste material or they can be added when transferring the waste material from one container to another dur- ing the waste treatment procedure. Hydrolytic enzymes can be added to the waste material at any stage of the treatment procedure before the waste material is released (after purification) into the environment. For example in sewage treatment hydrolytic enzymes can be added after pretreatment or after primary treatment of the sewage. Hydrolytic enzymes can be added to treat urine or faeces in hospital or farmhouse tanks for example during or after clarification or sedimentation step.

Homogenous presence of the hydrolytic enzymes in the waste material should be ensured in order to confirm that hydrolytic enzymes are capable of degrading the antibiotic residues in the waste material. This can be achieved by mixing the en- zyme with the waste material. The treatment can be carried out also for example during transfer of the waste material, the transfer of the container by car for example causing the material to become evenly mixed with the enzyme. According to one preferred embodiment of the invention the enzyme may be formulated to deliver hydrolytic enzymes in a retarded manner. The enzyme prepara- tion can be added to the waste reservoir before filling the container, during the filling of the container or after the filling of the container, or at various stages of filling the container with waste material.

Hydrolytic enxymes may be continuously added or the treatment may be a batch type treatment.

By an "effective amount" of hydrolytic enzymes is meant an amount capable of degrading the antibiotic residues present in the volume of the waste material to be treated. The amount varies depending on the amount (concentration) of antibiotic residues in the waste material. This depends for example on the origin of the waste material, the water content of the waste material and on the waste treatment methods, which have been used to treat the waste material (and which may have decreased the amount of antibiotic residues in the material). The amount of antibi- otic residues in the material can be measured before the treatment by using methods well known for a person skilled in the art. The effect of the treatment on the amount of antibiotic residues in the material can be monitored at a suitable frequency, for example in the beginning of a new batch of waste material, and/or every week, every second week or once a month during the treatment process.

The present invention can be used to degrade all antibiotics, which are degradable by enzymatic hydrolysis. One big group are β-lactams comprising β-lactam (azeti- din-2-one) ring. The comprise penicillins, cephalosporins, cephamycins, oxa-beta- lactams, carbapenems, carpacephems and monobactams. They are among the most widely used classes of antimicrobials.

The enzyme can be any hydrolytic enzyme, which can break down any antibiotic. It can be β-lactamases or amidases that hydrolyse penicillins, cephalosporins or carbapenems or esterases that break down ester bond in an antibiotic. The enzyme can be a natural enzyme or it can be a recombinant enzyme. According to a preferred embodiment of the invention the enzyme is β-lactamase.

The activity (amount) of β-lactamase in the preparation must be suitable for treat- ment of product batches of tens or even hundreds or thousands liters of waste material in practical conditions so that very low level or no β-lactam is left in the treated material. On the other hand the activity of the product should not be too high so that the amount of unreacted β-lactamase residues in the waste material does not become too large. Being a natural type of enzyme, protein toxicity of β- lactamase to environment is negligible.

The amount of hydrolytic enzymes used in the treatment is preferably 0.0001 - 100 000 pmol/liter, 0.0001 - 10 000 pmol/liter, 0.0001 - 1000 pmol/liter, or 0.0001 - 100 pmol/liter, more preferably 0.005 - 50 pmol, still more preferably 0.0005 - 5 pmol/liter, of the waste material. If expressed as enzyme units, the method comprises that the amount of active hydrolytic enzyme is used 100 - 10 000 000 U/ml or 100 - 10 000 000 U/ g, preferably 100 - 1 000 000, or 100 - 100 000, or 100 - 10 000 U/ml or U/g of the waste material to be treated The treatment may be carried out at the temperature of 10 to 40 ^°C, preferably of 15 to 30 ⁰C typically 20 to 25 ⁰C. The degradation is more effective in higher temperatures and shorter incubation (treatment) times are needed, if the temperature is higher.

The treatment time can vary in the range from 5 minutes to 12 hours, preferably it is about 10 min to 2 hours.

The function or efficacy of the treatment can be studied by various methods. For example the enzymatic reaction can be followed kinetically by photometer, typically at 235 nm, or the degradation of the antibiotic can be followed by any suitable analytical method, such as HPLC or capillary zone electrophoresis, typically with UV-diode array detection (Bailon-Berez et al. 2008 )

In the treatment of waste material according to the invention an enzyme preparation can be used which comprises a sufficient amount of hydrolytic enzymes. In liquid form, the composition comprises hydrolytic enzymes, for example β- lactamase, about 100 - 1 000 000 U/ml, or 100 - 100 000 U/ml, or 100 - 10,000 U/ml, most preferably 15,000 - 50,000 U/ml, typically 20,000 - 30,000 U/ml. If the preparation is in solid form, corresponding amounts of enzyme units are present per g of the preparation.

The preparation comprises advantageously hydrolytic enzymes 0.0001 - 100 or 0.001 - 500 or 0.01 - 1000 or 0.01 - 2 000 or 0.1 - 5000 pmol per g or ml of the formulation or per litre of the waste material to be treated.

Preferably it comprises also additives suitable for stabilizing and protecting the activity of the enzyme

The enzyme is preferably produced by an industrial process. This means here that the enzyme needs not to be purified to the same level as in pharmaceutical or foodstuff or feed products. The enzyme is preferably of industrial chemical quality. The enzyme may comprise unidentified impurities from 0.1 to 1 w %, typically 0.5 to 1 w-%. Also the final enzyme preparation, which may comprise various additives, may comprise unidentified impurities from 0.1 to 1 w %, typically 0.5 to 1 w- %.

The β-lactamase activity units indicated above mean the enzyme amounts obtained using nitrocefin as the substrate for lactamase (O^'Callaghen et al. 1972).

According to a preferred embodiment of the invention, the enzyme is β-lactamase. Preferably the enzyme is produced by Bacillus strain bacteria, preferably as a re- combinant protein. The host strain may be B. amyloliquefaciens, B. pumilis, or B. subtilis. The enzyme may originate from B. licheniformis. B. licheniformis can also be used for producing the enzyme as homologous protein.

The enzyme composition may be in liquid or in solid form. A liquid enzyme compo- sition may comprise a buffering component, a stabilizing agent and/or preservatives. The composition may comprise also an agent retarding the delivery of the enzyme into the waste material to be treated.

The buffer used can be, for example, a phosphate compound, such as potassium or sodium phosphate, the function of which is to stabilize, activate and protect the β lactamase. By means of the buffer the pH can be set at a value of 6 - 8, typically about 7.0, which is suitable in terms of the preservation and action of the lactamase enzyme. The concentration of the phosphate buffer may vary within the range of 1 mM - 100 mM. An example that can be mentioned of a suitable calcium phosphate buffer is a K2HPO4 - KH2PO4 -buffer. This may, however, be replaced with some other non-toxic buffer or another buffer suitable for use in waste material.

A stabilizing agent can be also incorporated into the enzyme preparation. The function of the preparation is to stabilize the lactamase so that it will not lose its activity even during long storage. The stabilizing agent should also prevent the precipitation of the enzyme during many freezing -melting cycles. It is necessary that the product can be stored both in a freezer and in a refrigerator. Preferably, the stabilizing agent used is a polyol, such as glycerol, or an amino acid base preparation, such as gelatin. Other suitable stabilizing agents include ion-free detergents, such as polyozyethylene-based detergents, e.g. polyozyethylene sorbi- tane (polysorbate 20), polyozyethylene lauryl ethyl (laureth 4), and polyozyethyl- ene, polyoxypropylene block polymer (poloxamer 188).

Glycerol is regarded as especially advantageous, since it cold properties are suitable. Other polyols are also suitable. Glycerol may be added in an amount of at least 10 % of the volume, preferably the preparation contains glycerol approxi- mately 20 - 60 %, typically but one half (50 %). This glycerol amount prevents the product from freezing. The concentrations of the other stabilizing agents vary within wide limits. Thus, gelatine can be used at bout 0.1 - 4 % of the volume of the preparation. The amounts used of ion-free detergents are about 1 - 20 % by volume.

In addition to buffers and stabilizing agents the preparation may comprise also preservatives. Benzoates, such as alkylparahydroxybenzoat.es can be used as such a preservative. Methyl hydroxybenzoate, and propyl parahydroybenzoate can be mentioned as advantageous examples.

The concentration of a benzoate preservative in the enzyme preparation may be 0.01 - 100 mg/ml, preferably approximately 0.05 - 50 mg/ml, the minimum concentration of methylparahydroxybenzoate being about 1.4 mg/ml and that propyl- parahydroxybenzoate about 0.2 mg/ml.

In the treatment of aqueous waste material can be used similar type of enzyme preparations as described in WO 01/67879. The enzyme composition preferably comprises a buffer in a sufficient amount to buffer the pH value of the composition to 6-8 and a stabilizing agent for inhibiting the inactivation and/or precipitation of β- lactamase during cold storage of the composition. According to another preferred embodiment the enzyme preparation may be in lyophilized form. It can be dissolved into a small amount of solution, typically buffer solution or water, before adding to the material to be treated.

According to a preferred embodiment of the invention the enzyme composition is in solid form. It may be formulated by well-known methods to pellets, granules, tablets, capsules, powder etc. The enzyme product may comprise particles with a diameter between 0.1 to 5 mm. The enzyme product may contain 1 to 5 w-% enzyme protein. Granules for example can be produced in an agglomeration process or by spraying the enzyme onto a core.

Suitable methods for granule production are described for example in US 7,419,947 and in US 7,425,528. The enzyme product may be also in immobilized form. The preparations can be added to the material to be treated directly or they can be dissolved to a solution before adding into the material to be treated.

In the example below it has been shown that β-lactamase is very efficient in degrading β-lactamase antibiotic, ampicillin, in sewage plant water under the natural temperature. The efficacy was studied by two ways; following the enzymatic reac- tion kinetically by photometer at 235 nm and by analysing the incubation time- dependent degradation of ampicillin by HPLC. Based on quantification of ampicillin by HPLC it was found that at as low temperature as 20 ⁰C one mol of β-lactamase degraded over 500 moles of ampicillin in a second. The efficacy was shown to be at least 2 times greater at 30 ⁰C. There one mole of β-lactamase degraded over 1000 moles of ampicillin.

It can be concluded that one efficient mean to prevent emergence of antibiotic residues in environment and to prevent emergence of bacterial resistance into environment, humans and animals is to prevent the transfer of residual antibiotics into the nature or drinking water by inactivating the antibiotic residues e.g. by enzymatic hydrolysis of industrial process residues, sewage plant water or hospital or farm waste tanks. This is a new mean to protect environment, animals and man from emergence of bacterial resistance and to prevent unwanted exposure of environment, animals and man to antibiotics.

EXAMPLE

The aim of this example was to show enzymatic inactivation of antibiotic residues in waste water of sewage plant. As an example the efficacy of commercial β- lactamase in degrading a β-lactam antibiotic, ampicillin, in waste water of sewage plant was studied. The efficacy of the β-lactamase was studied in two tempera- tures of around 20 ⁰C and 30 ⁰C to mimic the actual temperature in sewage plants. The efficacy was studied by two ways: following the enzymatic reaction kinetically by photometer at 235 nm and by analysing the incubation time-dependent degradation of ampicillin by HPLC.

Test materials

β-lactamase was commercially purchased from Finnzymes, Finland. Molecular weight of the B. licheniformis β-lactamase was 31 500 KD (SDS/PAGE) (Matagne et al.; 1991 ). β-lactamase activity was 1 .44 x 10⁶U/ml. The specific activity was 217 000 U/mg. The enzyme was dissolved in 10 mM K₂HPO₄ - KH₂PO₄ buffer, pH 7.0, 50 % glycerol.

Ampicillin (D-(-)-α-Aminobenzylpenicillin sodium salt) was commercially purchased from Fluka BioChemika, Finland. The molecular formula of the compound was Ci₆Hi₈N₃NaO₄S and the molecular weight 371.39. CAS Number was 69-52-3 and Beilstein Registry Number 411921.

Other chemical used in the example: 10 mM Na-phosphate buffer, pH 7.0.

The chemicals used in the HPLC-analysis were as follows:

Ampicillin sodium salt (Fluka BioChemica) Penicillin G procaine salt hydrate (Sigma) Acetonithle (Labscan) Perchloric acid (Merck)

2.7 M perchloric acid: 23.3 ml perchloric acid add water to 100 ml Citrate acid monohydrate (Merck)

1 M citric acid: 21 g citrate acid monohydrate add water to 100 ml Potassium dihydrogen phosphate (Merck) Disodium phosphate dihydrate (Merck) o-Phosphoric acid 85% (Merck) Citrate buffer: mix 19.9 g Na2HPO4-2H2O and 40 ml 1 M citric acid and add water to 250 ml

Perchloric acid-citrate buffer: 1.2 ml of 2.7 M perchloric acid and 8.8 ml of citrate buffer

Sewage plant waste water

Sewage plant waste water was obtained from Suomenoja plant, Espoo, owned by Espoo city. Sewage water entering into the plant was collected for 24 hours in a tank the temperature of which was around 8 ⁰C (primary sedimentation stage). From this tank one litre was taken in a sewage water collection bottle in the morning of the day to be used in the tests immediately after collection. Sewage water was stored at refrigerator in the laboratory.

Equipments

Incubations for all main kinetic assays were made in an incubation room, the temperature of which was adjusted to the desired temperature (around 20 ⁰C and 30 ⁰C). In the incubation room β -lactamase and ampicillin dilutions were kept in an ice bath. The sewage plant water was adjusted into the desired temperature. Pho- tometer (Wallac, Turku) was located in the incubation room. Plastibrand ® ISO9001 -14001 certified UV-cuvettes were used. As pipettes Finn-pipettes with sterile tips (Thermolab, Finland) were used. For weighting analytical balance was used (Sartorius). A Hewlett Packard 1100 HPLC with a diode-array detector was used and the data acquisition and peak integration were performed with Agilent ChemStation data system. The column used was Phenomenex Luna C18(2), 25 cm x 4.6 mm, 5 μm.

Analysed parameters

1. Km (mM). Km is (roughly) an inverse measure of the affinity or strength of binding between the enzyme and its substrate. The lower the Km, the greater the affin- ity (so the lower the concentration of substrate needed to achieve a given rate).

2. Vmax = maximum velocity

3. Kcat (1/s) = Vmax (delta Abs/second)/n enzyme (nmol)

4. Total turnover time of ampicillin in various ampicillin and enzyme concentrations.

For enzyme kinetics the Linewear-Burk plot was used as follows:

Vmax was determined by the point where the line crosses the 1/Vi = = axis (so the (S) is infinite) Km = Michaelis constant (substrate concentration when initial velocity is 50 % of maximum)

Vi = initial velocity. Because optical density (absorbance) is directly proportional to the concentration of the product, absorbance can be used as a measure of the rate or velocity of the reaction (Vi).

Series of initial rates were measured at various substrate and enzyme concentrations. Kinetic parameters of Km and V_maχ were estimated by using the Michaelis- Menten equation.

Test procedure

Concentrations of ampicillin and β -lactamase 1 g of ampicillin (Mr 371.4.4 g/mol) in vial is equivalent to 2.69 mmol. 50 mg of ampicillin sodium salt was dissolved into 4.0 ml of 10 mM Na-phosphate buffer, pH 7.0 giving stock concentration of 0.0337 mmol/ml (33.7 μmol/ml = 12.5 mg/ml, Stock 1 ). From this a dilution of 1 :4 (2 ml + 6 ml) was made into the Na-phosphate buffer giving a stock 2 concentration of 8.4 μmol/ml (= 3.125 mg/ml), and from this stock concentration of 3 of 4.2 μmol/ml (= 1.56 mg/ml) was made in Na-phosphate buffer by diluting 1 :2 (2.0 ml+2.0 ml). Stock 4 (2.1 μmol/ml = 0.78 mg/ml) was prepared by diluting Stock 3 1 :2 (1 ml + 1 ml). The stock solutions were kept in an ice bath.

β -lactamase was dissolved in 10 mM Na-phosphate buffer, pH 7.0. 2.13 pmol (= 67 ng) (Stock 6), 1 .06 pmol (Stock 7), 0.53 (Stock 8) or 0.27 pmol (Stock 9) was added in a 10 μl^'s proportions into the reaction mixture of 1 ml. The β -lactamase solution was prepared as follows: From the original solution (6.7 mg/ml) 1 :10 dilu- tion (0.1 ml + 0.9 ml) was made in 10 mM Na-phosphate buffer, pH 7.0 giving stock concentration of 0.67mg/ml (Stock 2). From this a dilution of 1 :10 (0.5 ml + 4.5 ml) was made into the phosphate buffer giving Stock 3 (67 μg/ml), and from this stock concentration a dilution of 1 :2 was made (Stock 4), from Stock 4 dilution of 1 :2 (Stock 5). From Stock 3 a dilution of 1 :10 was made (3.0 ml + 3.0 ml) (Stock 6, 6.7 μg/ml) and from Stock 6 a dilution of 1 :2 was made (3.0 ml + 3.0 ml) (Stock 7, 3.35 μg/ml) and from 7 a dilution of 1 :2 (3.0 ml + 3.0 ml) (Stock 8, 1.68 μg/ml) and from 8 a dilution of 1 :2 (3.0 ml + 3.0 ml) (Stock 9, 0.84 μg/ml) was made in Na-phosphate buffer. The stock solutions were kept in an ice bath.

Concentrations of β -lactamase to be used in the main test were selected by the preliminary tests. In the main test Stock concentrations of 6, 7, 8 and 9 were used in Kinetic assay at 20^°C and 6, 7 and 8 at 30^°C. In the HPLC-assay Stock concentration of 9 was used at both temperatures. Table 1 : Summary of the incubation conditions in the photometer cuvette of 1 ml in the Kinetic assay used in experiments performed at 30⁰C and 20⁰C.

Stock concentration Volume Waste Final ampicillin β -lactamase added of ampicillin taken water concentration /ml Stock 6 (2.1 added pmol) or stock 7 (1.1 pmol) or Stock 8 (0.53 pmol) or Stock 9 (0.27 pmol)

33.6 μmol/ml, Stock 1 24 μl 965 μl 10 μl

12 μl 980 300 10 μl

8.4 μmol/ml, Stock 2 96 895 μl 800 μM (3OC ) μg/ml) 10 μl

48 μl 940 μl 400 μM (15C ) /ml) 10 μl

24 μl 965 μl 200 μM (75 μg/ml) 10 μl

12 μl 980 μl 100 μM (37. 5 μg/ml) 10 μl

4.2 μmol/ml, Stock 3 18 μl 970 μl 75 μM (27.8 μg/ml) 10 μl

12 μl 980 μl 50 μM (18.7 μg/ml) 10 μl

2.1 μmol/ml, Stock 4 18 μl 970 μl 37.5 μM (13 .9 μg/ml) 10 μl

12 μl 980 μl 25 μM (9.37 μg/ml) 10 μl

Table 2: Summary of the incubation conditions used in the HPLC-assay at 30°C and 20⁰C.

Stock concentration VoI urn Waste Final ampicillin β -lactamase added of ampicillin e taken water concentration Stock 8 (0.53 added pmol/ml) or Stock 9 (0.27 pmol/ml)

33.7 μmol/m , Stock 1 240 μl 10 ml 800 μM (=300 μg/ml) 100 μl

8.4 μmol/ml Stock 2 240 μl 10 ml 200 μM (= 75 μg/ml) 100 μl

120 μl 10 ml 100 μM (= 37.5 100 μl μg/ml)

4.2 μmol/ml, Stock 3 120 μl 10 ml 50 μM (= 18.8 μg/ml) 100 μl

2.1 μmol/ml, Stock 4 120 μl 10 ml 25 μM (=9.4 μg/ml) 100 μl

8.4 μmol/ml stock 2 240 μl 10 ml 200 μM (=75 μg/ml) - Incubations

One portion of waste water from sewage plant was stabilized into RT (appr 20⁰C) and another into 30⁰C. Different ampicillin solutions and β -lactamase solutions were prepared as described above just before use in the 10 mM Na-phosphate buffer (Table 1 and 2). These solutions were kept in an ice bath. Absorbance change at 235 nm to measure enzyme kinetics

Baseline absorbances against distilled water was measured for stabilised waste water of desired temperature alone and for waste water containing highest enzyme concentration. Waste water stabilized into the desired temperature (20°C or 30⁰C) was pipetted into the photometer cuvette (UV) followed by adding ampicillin and β -lactamase dilutions kept in an ice bath (see Table 1 ). After mixing the reaction was followed at 235 nm for maximum of 5 minutes. Reaction for each ampicil- Nn concentration was performed in triplicate. Photometer and sewage water were kept in the temperature room stabilised into the desired temperature (20°C or 30°C). In addition to that, actual temperature in the cuvette was then measured in the end of the kinetic reaction by using an electronic thermometer.

The reaction rate was determined from the linear part of the absorbance curve (delta Abs/min). When the substrate concentration is low with the standard enzyme concentration, will the linear reaction time be shorter compared to the high substrate concentration. In the assay the lowest substrate concentration was the one where linear part will stay for at least 30 seconds, the highest will be maxi- mum of 5 minutes.

HPLC- experiment

Based on the results from the kinetic experiments both enzyme and ampicillin concentrations were selected for the HPLC experiment (Table 2). Samples of reaction mixture were taken in an incubation-time dependent manner and total amount of residual ampicillin was measured. The reaction mixtures were made as follows: 10 ml of sewage waste water was added in a 50 ml Falcon tube followed by adding β -lactamase and ampicillin as described in the Table 2. A sample of 0.2 ml is taken into 0.1 ml of perchloric acid kept in an ice bath at following time points: 0, 5, 10, 20 and 40 minutes, 1 h 20 minutes and 2h. Two parallel incubations were performed at 30⁰C and 20⁰C. After the incubations perchloric acid tubes were stored at -20°C until analysed by HPLC at United Laboratories, Finland.

HPLC-analysis was started from the β-lactamase Stock 8 and from the highest concentration of ampicillin. In case no ampicillinwas found the lower concentrations were not analysed. Based on the results of analysis of Stock 8 β-lactamase it were decided whether the β-lactamase Stock 7 samples will be analysed, too.

In the HPLC-analysis ampicillin and the internal standard were separated using gradient elution. The mobile phases were mixtures of acetonitrile and 50 mM phosphate buffer, pH 3.5.

Eluent A contained 10 % acetonitrile and eluent B 50 % acetonitrile.

Eluents were delivered initially in a ratio of 90:10 (v/v) for 5 min, then changed to 20:80 to next 12 min, and finally equilibrated back to 90:10.

The detective wavelengths used were 225 nm for ampicillin and 300 nm for internal standard (penicillin G procaine).

Chromatography was carried out at ambient temperature and the mobile phase is pumped at a rate of 1.0 ml/min.

Sample preparation

Stock solutions of ampicillin were prepared in 10 mM sodium phosphate buffer pH 7.0. These solutions were used to spike sewage water for the calibration curve and for quality control samples. Calibration standards were made daily. Quality control samples were divided into portions and stored frozen at -65 ⁰C until needed for analysis. After thawing incubation samples were vortex-mixed, placed in an ice water bath and centhfuged at 2000 rpm for 7 min. Calibration standards and quality controls were mixed with perchloric acid-citrate buffer in the ratio of 2:1.

Calibrations standards, quality control samples and analytical samples were treated identically.

To 200 μl of sewage water containing perchloric acid-citrate buffer was added 10 μl of internal standard and vortex-mixed. 50 μl of the mixture was injected into the HPLC column.

Calculations

The chromatograms were integrated and analyzed with the HP ChemStation data system, revision A.08.03. Linear regression analysis with weighting factor of 1/x was used to calculate the concentration of ampicillin. Quantification was based on peak height ratios.

Linear calibration curves were obtained in the concentration range of 0.1 -80.0 μg/ml (as Na-ampicillin). Samples with concentration higher than 80 μg/ml were diluted with purified water (purified by MiIIi-Q reversed osmosis-system) and re- analyzed.

DATA HANDLING

Kinetic parameters were calculated according to the Section 4. From HPLC-test the efficacy of β-lactamase at 20^°C and 30^°C was calculated i.e. the velocity of enzymatic reaction (amount of ampicillin degraded pr amount of enzyme) was calculated at each temperature and at each β -lactamase concentration.

RESULTS

Kinetic parameters The activity of commercial β-lactamase to degrade ampicillin in sewage water was studied by monitoring the activity at 235 nm by a spectrophotometer. Activity was studies at four (20 ⁰C) and two (30 ⁰C) different β-lactamase concentrations by using five different ampicillin concentrations (1 :2 dilutions) at each β-lactamase concentrations. The concentrations were selected as based on the preliminary assays. . The main test was repeated twice (20 ⁰C with 0.5 pmol) or once (30 ⁰C and 20 ⁰C with 2.13, 1.06 and 0.27 pmol).

Table 3 shows that Km-value varied at 20 ⁰C between 0.4 and 0.12 mM at β- lactamase amounts of from 2.13 to 0.27. Kcat (1/s) varied between 0.086 and 1.43, the mean being 1.2.

Table 4 shows that Km-value was at 30 ⁰C 1 .0 and 0.83 mM at β-lactamase amounts of 1.06 and 0.53 pmol, respectively. Kcat was 4.5 and 10.5 the mean was 7.5.

When comparing the Kcat values at 20 ⁰C and 30 ⁰C to each other it can be found that catalytic activity i.e. Kcat values, is 6 times higher at 30 ⁰C than at 20 ⁰C.

Table 3: Measured kinetic parameters at different amounts of β-lactamase at 2O ⁰C

Table 4: Measured kinetic parameters at different amounts of β-lactamase at 3O ⁰C

Incubation-time dependent degradation of ampicill in by β-lactamase of 0.27 pmol/ml

Results showed that β-lactamase at concentrations of 0.53 pmol/ml degraded the ampicillin within ten minutes even at the highest ampicillin concentration level to the level below the detection limit of < 0.1 μg/ml. Table 5 shows the incubation- time dependent degradation of ampicillin by 0.27 pmol/ml of β-lactamase at different ampicillin concentrations in waste water at start of incubation (nominal concentration). Each value is a mean of two replicates.

Table 5: Incubation time dependent degradation of ampicillin by 0.27 pmol of β-lactamase/ml reaction mixture at 20 ⁰C and 30 ⁰C. The last column is otherwise the same as other columns but without β-lactamase.

Nominal cone (ug/ml) 300 150 75 37,5 75

Temperature Time Amount of residual ampicillin (ug/ml)

2OC 0 268 120 67 28 75

5 250 100 48 15 75

10 233 88 37 9 75

20 200 59 16 1 ,9 74

40 129 13 0,7 0 73

80 64 0,7 0 0 72

120 1 ,1 0 0 0 51

Nominal cone (ug/ml) 300 150 75 37,5 75

Temperature Time Amount of residual ampicillin (ug/ml)

3OC 0 300 136 81 33,5 82

5 242 102 47 12 81

10 199 70 21 3,3 82

20 142 27 3 0,25 82

40 36 0 0 0 82

80 0,1 0 0 0 81

120 0 0 0 0 81

Table 6 shows that one pmol of commercial β-lactamase was found to degrade 10— 14μg ampicillin in sewage water in a minute at 20 ⁰C. At 30 ⁰C the corresponding activity varied from 16 to 37 μg of ampicillin. The results show that there were not excess amount of ampicillin at 30 ⁰C because the velocity was dependent on the initial concentration of ampicillin. However, it can be concluded that β- lactamase is very efficient to degrade ampicillin in sewage water even at 20 ⁰C; one pmole of β-lactamase can degrade 30 000 pmoles of ampicillin in a minute. The corresponding Kcat value is 538 ± 82 i.e. one mole of β-lactamase degrades over 500 moles of ampicillin. The efficacy was found to be two times greater at 30 0C, the Kcat value being 1144 ± 388.

Table 6: Catalytic activity of β-lactamase to degrade ampicillin at 2O C and 30⁰C.

REFERENCES

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Claims

1. A method for treating aqueous waste material comprising antibiotic residues, c h a r a c t e r i z e d in that said waste material is treated before it is released into the environment by contacting it with an effective amount of hydrolyzing enzymes capable of degrading antibiotic residues thereby hindering the emergence of antibiotic residues into the environment.

2. The method according to claim 1 , wherein the waste material comprises proc- ess residues from industrial processes, such as antibiotic production, hazardous waste, waste from hospital tanks, sewage plants, farm house urine or faeces collection tanks.

3.The method according to claim 1 or 2, wherein the method comprises that hydro- lyzing enzymes are selected from the group comprising β-lactamases, amidases, cephalosporins, carbapenems and esterases.

4. The method according to any one of the preceding claims, wherein the enzyme is β-lactamase.

5. The method according to any one of the preceding claims, wherein the method comprises that the amount of active hydrolytic enzyme used is 100 - 10 000 000 U/ml or 100 - 10 000 000 U/g of the waste material to be treated.

6. The method according to any one of the preceding claims, wherein the method comprises that the treatment is carried out at the temperature of 10 to 40 ⁰C, preferably at 15 to 30 ⁰C.

7. The method according to any one of the preceding claims, wherein the method comprises that the treatment is carried out in 5 minutes to 12 hours, preferably in 10 min to 2 hours.

8. Use of hydrolyzing enzymes for treating aqueous waste material comprising antibiotic residues before said waste material is released into the environment thereby hindering the emergence of antibiotic residues into the environment.

9. The use according to claim 8, wherein the enzyme is β-lactamase.

10. The method or use according to any one of claims 1 to 9, wherein the enzyme preparation is of industrial quality.

11. An enzyme preparation for degrading antibiotic residues in waste material, which comprises β-lactamase enzyme of industrial chemical quality 100 - 1 000 000 U/ml or 100 - 1 000 000 U/g of the preparation, and additives suitable for stabilizing and protecting the activity of the enzyme.