May, Oliver (Am Rebenborn 17A, Frankfurt, 60388, DE)
Osswald, Steffen (Landwehrstrasse 33, Rodenbach, 63517, DE)
May, Oliver (Am Rebenborn 17A, Frankfurt, 60388, DE)
| 1. | Biocatalyst for hydrolysing hydrocyanic acid to give ammonium formate, characterized in that the biocatalyst is a cell possessing a heterologously expressed cyanidase and the biocatalyst is used in the form of whole cells. |
| 2. | Biocatalyst according to Claim 1, characterized in that the host organism is E. coli. |
| 3. | Biocatalyst according to Claim 1 or 2, characterized in that the cyanidase is the Pseudomonas stutzeri cyanidase. |
| 4. | Biocatalyst according to one of Claims 13, characterized in that the gene which encodes the cyanidase has been optimized in regard to the codon usage of the host organism employed. |
| 5. | Biocatalyst according to one or more of the preceding claims, characterized in that the gene which encodes the cyanidase exhibits a sequence identity of > 70% with the sequence depicted in SEQ ID NO: 1. |
| 6. | Biocatalyst according to Claim 5, characterized in that the gene which encodes the cyanidase exhibits a sequence identity of > 90% with the DNA sequence depicted in SEQ ID NO: 1. |
| 7. | Biocatalyst according to one or more of the preceding claims, characterized in that the gene which encodes the cyanidase corresponds to the sequence depicted in SEQ ID NO: 1. |
| 8. | Method for hydrolysing hydrocyanic acid to give ammonium formate, characterized in that the method is carried out in the presence of a wholecell catalyst according to claim 1 which comprises a cyanidase. |
| 9. | Method according to Claim 8, characterized in that the biocatalyst concentration employed does not exceed 1000 mg/L. |
| 10. | Method according to Claim 9, characterized in that the biocatalyst concentration employed does not exceed 10 mg/L. |
| 11. | Method according to Claim 8 or 9, characterized in that use is made of a wholecell catalyst which comprises at least one cyanidase which is selected from the group consisting of a Pseudomonas stutzeri AK61 cyanidase. |
The present invention relates to a biocatalyst and to a method for hydrolyzing hydrocyanic acid to give ammonium formate in the presence of this biocatalyst, which comprises a cyanidase having a specific activity which is increased as compared with that of the isolated enzyme.
Prior art
A variety of chemical methods exist for detoxifying cyanide contaminations or cyanide-containing effluent waters which accrue in the mining industry and in association with chemical processes. However, these methods suffer from the disadvantage that substances which are likewise toxic, such as Caro's acid, peroxides and sulphur dioxide have to be used for this purpose (Zaidi, S.A.; Schmidt, J.W.; Simovic, L. Sciences et Techniques de l'Eau [Water sciences and techniques] (1985), 18(1), 43-9) .
Microbiological processes for degrading cyanide have been employed since middle of the 1980s. However, the slow growth and the low cyanide tolerance of the living microorganisms result in a very poor space-time yield (A. Akcil, T. Mudder; Biotechnology Letters 25 (2003) 445-450) . This might be remedied by spatially separating the growth of the microorganisms and their use for degrading the cyanide.
The heterologous expression of enzymes such as cyanidases (also known as cyanide dihydratases) in rapidly growing microorganisms offers particularly good prospects in this regard since the period required for culturing can be reduced and, at the same time, the quantity of cyanidase formed, based on the biomass, can be increased drastically, as has been demonstrated using, as an example, the expression of Pseudomonas stutzeri AK61 cyanidase in E. coli (A. Watanabe, K. Yano, I. Karube; Appl . Microbiol. Biotechnol. ; 50 (1998) 93-97) . This paper describes the hydrolysis of cyanide using the crude extract of the cells and using the enzyme which has been purified to homogeneity. In this connection, the specific activities are 14.4 units per milligram (U/mg) of total protein and, respectively, 59 U/mg of purified enzyme.
The use of whole-cell transformations (using whole cells to transform a substrate) , based on the activity of a cyanidase, for detoxifying cyanide contaminations or cyanide-containing effluent waters has not been disclosed.
Description of the invention
The object of the present invention was to develop a biocatalyst as a whole-cell catalyst, which is able to hydrolyse cyanide effectively, while avoiding elaborate cell disruption or isolation of the enzyme from the cell. At the same time, the whole-cell biocatalyst according to the invention offers the advantage that it can be readily separated off, after the biotransformation, by means of centrifugation or ultrafiltration.
The present invention is a biocatalyst for hydrolysing hydrocyanic acid to give ammonium formate, characterized in that the biocatalyst is a cell possessing a heterologously expressed cyanidase and the biocatalyst is used in the form of whole cells. The biocatalyst according to the invention can, for example, be used for detoxifying cyanide contaminations or cyanide-containing effluent waters which accrue in the mining industry and in association with chemical processes .
A further aspect of this invention is a method for hydrolysing hydrocyanic acid to give ammonium formate, characterized in that use is made of a whole-cell biocatalyst which comprises a cell possessing a heterologously expressed cyanidase.
The cyanidase derived from Pseudomonas stutzeri AK61 was used for preparing the biocatalyst according to the invention. However, other known cyanidases, for example derived from Alcaligenes denitrificans DSM4009 (EP 0282351) or Bacillus pumilus Cl (Jandhyala, D.; Berman, M.; Meyers, P.; Benedik, M. (2003) , Applied and Environmental Microbiology 69, 4794-4805) can advantageously be used in a similar manner. E. coli DSM14459 was used as the host organism. However, other microorganisms known to the skilled person, for example yeasts such as Hansenula polymorphs, Pichia sp. or Saccharomyces cerevisiae, or prokaryotes, such as E. coli or Bacillus subtilis, can advantageously be used in a similar manner. E. coli strains are preferably to be used for this purpose. Very particular preference is given to: E. coli XLl Blue, W3110, DSM14459 (PCT/USOO/08159) , NM 522, JMlOl, JM109, JM105, RRl, DH5α, TOP 10" and HBlOl. The methods for cloning the genes, and the expression systems and host cells which are required, are sufficiently well known to the skilled person from the literature (Sambrook, J.; Fritsch, E.F. and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, Laboratory Press, New York; Balbas, P. and Bolivar, F. (1990), Design and construction of expression plasmid vectors in E. coli, Methods Enzymol. 185, 14-37; Rodriguez, R.L. and Denhardt, D. T (eds) (1988), Vectors: a survey of molecular cloning vectors and their uses, 205-225, Butterworth, Stoneham) .
In the case of the present invention, a gene for a cyanidase is one of the genes which is preferably to be selected. The skilled person is likewise free to select the genes which encode such a cyanidase. According to the present invention a Vλwhole-cell catalyst" is understood as being an intact cell in which is expressed at least one gene which is able to catalyse the transformation, according to the invention, of a substrate into a product. According to the invention, the intact cell is able to express a cyanidase. The whole-cell catalyst is preferably a microorganism which is recombinantly altered and which is adapted to the requirements of the desired transformation. A particularly suitable whole-cell catalyst which is preferred is the whole-cell catalyst which is described in the experimental section.
In designing the whole-cell catalyst, the skilled person is both free to choose the suitable genes, as long as the corresponding gene products are only able to catalyse the desired reaction, and to choose the host organism. Suitable host organisms are any known host organisms, with E. coli having proved to be particularly suitable.
Genes which are suitable for the biocatalysts according to the invention are not only genes which encode cyanidases but also include genes in accordance with SEQ ID No: 1, or a fragment prepared therefrom, and also those which are at least 70%, in particular up to 80%, preferably at least from 81% to 89%, particularly preferably at least 90%, and very particularly preferably at least 91%, 93%, 95%, 97% or 99% identical to the gene in accordance with SEQ ID No. 1, or a fragment which is prepared therefrom.
In the method according to the invention, the above- described biocatalyst is used for carrying out the hydrolysis of hydrocyanic acid to ammonium formate.
In the method according to the invention, the concentration of the biocatalyst is at most 1000 mg/L, in a preferred embodiment up to 100 mg/L, preferably up to 10 mg/L and, particularly preferably, up to 1 mg/L, with mg referring to mg of dry biomass (DBM) . The biocatalyst is to be understood, in particular, as being a whole-cell catalyst.
In the method according to the invention, the concentration of the cyanide is at most I M, in a preferred embodiment up to 0.5 M, and particularly preferably up to 0.25 M.
The method according to the invention can be carried out at any reaction temperatures which are suitable for the host organism employed. A particularly suitable reaction temperature is to be regarded as being a reaction temperature which is from 4 to 6O0C, preferably from 10 to 5O0C, and particularly preferably from 20 to 400C.
The skilled person is also free to select the pH of the reaction, with it being possible to carry out the reaction both at a fixed pH and also of varying the pH in a given pH range. The pH is selected, in particular, while taking into consideration the properties of the enzyme employed. The reaction is preferably carried out at a pH which is from 5 to 11, preferably from 6 to 10 and, particularly preferably, from 7 to 9.
The substrate which is used is transformed into the desired product while using a suitable whole-cell catalyst. A nutrient medium which is suitable, in dependence on the host organism employed, is used for growing the whole-cell catalyst. The media which are suitable for the host cells are well known and can be obtained commercially. Furthermore, customary additives, such as antibiotics and similar known additives, can be added to the cell cultures.
Surprisingly, the whole-cell biocatalyst of the present invention exhibited, at 78 U/mg., a markedly higher specific activity, based on the dry biomass, than did the purified enzyme according to Watanabe et al. , at 59 U/mg based on the protein which was purified to homogeneity. This is particularly surprising because, according to Watanabe et al . , there was no loss of activity during the purification procedure. If account is taken of the fact that the proportion of the soluble protein, based on the dry biomass, is only approx. 27% (D.S. Goodsell; Trends in Biochemical Sciences 16 (1991) 203-206) and the proportion of a heterologously expressed protein in the total soluble protein in E. coli cannot markedly exceed 50%, it must be assumed that the cyanidase is operating at an elevated activity in E. coli cells.
By means of the present invention, the biocatalytic detoxification of cyanide-containing effluent waters can be carried out using much lower quantities of the biocatalyst, and consequently less expensively, than in the case of the previously described systems based on crude extracts or isolated enzymes.
The present invention is explained in more detail below with the aid of exemplary embodiments. These latter only serve to illustrate the invention and are in no case to be regarded as limiting its nature and scope. Example 1
Preparing a cyanidase-positive strain
A DNA double strand containing the sequence of the Pseudomonas stutzeri AK61 cyanidase gene was synthesized. While the gene encodes the amino acid sequence described in the literature, the DNA sequence was optimized in regard to the E. coli codon usage. In addition, an Ndel restriction cleavage site and a BamHI restriction cleavage site are located upstream and, respectively, downstream of the gene (SEQ ID NO: 1) . These two restriction cleavage sites were used to clone the DNA fragment into the vector pOMl7c. The complete sequence of the plasmid is given in SEQ ID NO: 2. Chemically competent E. coli DSM14459 cells were transformed with 10 ng of the plasmid pOM17c. The transformed cells, which had been streaked out on ampicillin-containing agar plates, were then characterized as in example 2.
Example 2
Growing and characterizing the cyanidase-positive strain.
An overnight culture (OD6oo=4) of the cyanidase-positive strain was diluted 1:100 in 100 ml of rhamnose (2 g/1)- supplemented LB medium (5 g/1 yeast extract, 10 g/1 tryptone, 10 g/1 NaCl) , and the diluted culture was incubated at 300C and 250 rpm for 18 hours. The biomass was pelleted by means of centrifugation (10 min, 10 000 g) and the supernatant was discarded. The cell pellet was then diluted with a 50 itiM solution of potassium cyanide in 50 mM phosphate buffer, pH 8.0, such that the concentration of the cells was 4 mg per litre, based on the dry cell mass, and the whole was incubated at 300C while being shaken. The decrease in the concentration of the cyanide was monitored using an analytical ready-mixed test (Spektroquant cyanide test, Merck) . After 60 min, the cyanide concentration had decreased from 50 πiM down to 26.6 mM. A U was defined as being a decrease in the cyanide concentration of 1 μmol per minute. Accordingly, the specific activity of the cells was 78 U/mg of dry biomass (DBM) . When the experiment was repeated, a decrease in the cyanide concentration of from 50 mM down to 26.9 mM, corresponding to a specific activity of 77 U/mg of DBM, was obtained.
In another experiment, the cell pellet was diluted with a 250 mM solution of potassium cyanide in 50 mM phosphate buffer, pH 8.0, such that the concentration of the cells was 25 mg per litre, based on the dry cell mass, and the whole was then incubated at 300C while being shaken. In this experiment, the cyanide concentration decreased within one hour down from 250 mM to 134.7 mM, corresponding to a specific activity of 74 U/mg of DBM.