AU2015203891A1 - Food Additive Comprising An Amidase For Detoxifying Ochratoxin - Google Patents

Abstract

The present invention relates to amidase enzymes and a feed or food additive comprising the amidase enzyme capable of degrading ochratoxin.

Description

WO 2012/032472 PCT/IB2011/053901 1 ADDITIVE Field of invention The present invention relates to methods of detoxifying mycotoxins. More specifically, the invention relates to amidase enzymes and feed and or food additives comprising 5 at least one amidase for detoxifying ochratoxin, particularly ochratoxin A. Background Mycotoxins are toxic secondary metabolites of fungi belonging, essentially, to the 10 Aspergillus, Penicilium and Fusarium genera. They can be produced on a wide range of agricultural commodities and under a diverse range of agronomic, ecological and post harvest conditions worldwide. Mycotoxins can enter the food chain in the field, during storage of a feed or food material, or at later points in the food chain. Their accumulation in foods and feeds 15 represents a major threat to human and animal health since consumption of a mycotoxin-contaminated diet may result in teratogenic, carcinogenic and oestrogenic or immunosuppressive effects. In 1985 the World Health Organization estimated that approximately 25% of the world's grains were contaminated by mycotoxins (Jelinek et al., 1989). This figure has 20 likely grown since then due to an increase in global import and export of grains and cereals and the changing environmental and weather patterns. Currently there are more than 400 mycotoxins documented but the mycotoxins of greatest concern and consequently the most studied include: aflatoxin, deoxynivalenol, zearalenone, fumonisin and ochratoxin. 25 Although there are many species of toxigenic moulds, only a few mycotoxins are considered to be significant for humans. Three genera of fungi, Aspergillus, Penici//ium, and Fusarium are most frequently 30 involved with cases of mycotoxin contamination. Fungal colonization, growth and mycotoxin production are generally influenced by a variety of factors. The most important of which are temperature and water activity.

WO 2012/032472 PCT/IB2011/053901 2 Generally, in warm regions aflatoxins are of major concern. This is because the Aspergillus species that produce these toxins find optimum conditions present in tropical regions. In contrast, Fusarium and Penicillium species have lower optimum temperatures and as a result are adapted to a more moderate climate. Ochratoxins, 5 fumonisins and zearalenone are consequently produced in regions providing these conditions. Ochratoxins are a group of mycotoxins produced as secondary metabolites by several fungi of the Aspergillus or Penicillium families and are weak organic acids consisting 10 of a derivative of an isocoumarin. There are three generally recognized ochratoxins, designated A, B and C. Ochratoxin A is the most abundant member of the ochratoxin family and hence the most commonly detected, but is also the most toxic. Ochratoxin A (ochratoxin A) is a nephrotoxic, teratogenic, hepatotoxic, and carcinogenic mycotoxin present in cereals and other starch rich foods. Besides cereals and cereal 15 products, ochratoxin A is also found in a range of other food commodities, including coffee, cocoa, wine, beer, pulses, spices, dried fruits, grape juice, pig kidney and other meat and meat products of non-ruminant animals exposed to feedstuffs contaminated with this mycotoxin. Many countries have set limits on ochratoxin A level in food, typically between 1 and 10 ppb (parts per billion) depending on the type 20 and quality of the foodstuffs. 0 OH OH 0 H H 0~ N H Y CH3 C1 Molecular structure of ochratoxin A 25 ochratoxin A production is due to a fungal infection in crops, in the field during growth, at harvest, in storage and in shipment under favourable environmental conditions, especially when they are not properly dried. Ochratoxin A is a stable compound that can be hydrolysed into ochratoxin a (OTU) 30 and L-phenylalanine by heating under reflux for 48h in 6M hydrochloric acid (Van der Merwe et al., 1965) or with the carboxypeptidase A (Pitout, 1969). The conversion of WO 2012/032472 PCT/IB2011/053901 OTA into ochratoxin a. is considered to be a way to reduce its toxicity since OTa is commonly reported to be much less toxic than OTA. Moreover, ochratoxin a, elimination half-time in the body (9.6h) is shorter than that of OTA (103h) (Li et al., 1997). 5 j + Phe CHH In order to ensure food safety, different approaches to prevent mycotoxin intake are developed at several stages along the food production chain. 10 It has been known since the 1970s that the mammalian digestive enzyme called carboxypeptidase A is cable of degrading OTA but that the efficiency of this enzyme is low. In fact in animals having carboxypeptidase A, such as pig, OTA toxicity due to its presence in feed is a problem. Furthermore, it has been found that OTA can to some 15 extent inhibit carboxypeptidase A activity. Prior to the present invention, there has been no disclosure of efficient enzyme solutions to degrade ochratoxins including ochratoxin A (OTA). Enzyme activities other than carboxypeptidase have been reported for OTA degradation, but until now 20 no one has been able to identify a protein showing OTA degrading activity, It is known that a commercial lipase product called "AmanoTM lipase" which is a crude lipase produced from A. niger (Amano TM company, Japan) has ochratoxin degrading activity. 25 The OTA degrading activity in this lipase product has previously been attributed to a lipase or protease activity. For example, Maria A. Stander (J. Agric. Food Chem. 2000, 48, 5736-5739) concluded that the OTA degrading activity of Amano TM lipase resulted from a lipase. 30 WO 2012/032472 PCT/IB2011/053901 4 Abrunhosa et al., (Biotechnology Lett., 2007, 29, 1909-1914) describe an enzyme preparation isolated from A. niger having OTA degrading activity. However, this enzyme preparation was not purified to an extent where the sequence of the active component could be determined at the amino acid or DNA level. Similar problems 5 have been reported in other cases (Abrunhosa et al., TOXINS, [2010], 2, 1078-1099) Summary The present invention is based on work undertaken by the inventors to identifiy and 10 isolate an enzyme which is capable of efficiently degrading ochratoxin, more specifically ochratoxin A (OTA). The inventors have discovered that contrary to their initial hypothesis formamidase from A. niger and A. nidulans and also the formamidase that exists in the AmanoTM 15 lipase product have no activity towards OTA but as expected do have activity towards formamide. The inventors have surprisingly found that a 480 amino acid hypothetical protein, hereinafter referred to as amidase 2, which is encoded by an A. niger open reading 20 frame has ochratoxin degrading activity, and particularly ochratoxin A degrading activity. Furthermore, the inventors have also surprisingly found that the full length 480 amino acid amidase 2 comprising the N-terminal or signal sequence and having a MW of 25 about 51 kDa (referred to as amidase 2 sig) has ochratoxin A degrading activity as well as the secreted mature 438 amino acid amidase 2 having a MW of about 47 kDa (referred to as amidase 2 mat). It has been found that in amidase 2 mat, the N-terminal 42 amino acids are cleaved, 30 that is, when amidase 2 is secreted into the culture medium its N-terminal 42 aa is cleaved to form the mature amidase 2 (i.e., amidase 2 mat) by an A. niger peptidase. The inventors have isolated and cloned the A. niger gene encoding the amidase enzyme responsible for the degradation of ochratoxin A and have identified its crystal 35 structure. They have identified that this gene encodes a polypeptide having the WO 2012/032472 PCT/IB2011/053901 5 sequence of SEQ ID NO: 1 (amidase 2 sig). They have further identified that this enzyme is post translationally modified by cleavage of an N-terminal 42 amino acid sequence the mature amidase 2 shown as SEQ ID NO: 3 5 According to a first aspect of the present invention there is provided an isolated amidase enzyme capable of degrading ochratoxin. More particularly ochratoxin A According to a second aspect of the present invention there is provided a food or feed additive comprising an amidase enzyme capable of degrading ochratoxin. More 10 particularly ochratoxin A According to a third aspect of the present invention there is provided a food or feed material comprising the feed additive of the present invention. 15 According to a Fourth aspect of the present invention there is provided a foodstuff or feedstuff comprising a feed material of the present invention. According to a fifth aspect of the present invention there is provided a method for making a food or feed additive comprising admixing an amidase enzyme capable of 20 degrading ochratoxin with at least one physiologically acceptable carrier. According to a sixth aspect of the present invention there is provided a method of reducing ochratoxin contamination in a material comprising adding to said material an amidase enzyme capable of degrading ochratoxin. 25 According to a seventh aspect of the present invention there is provided a composition comprising an ochratoxin contaminated material and an amidase enzyme capable of degrading ochratoxin. 30 According to a eighth aspect of the present invention there is provided a method of making a foodstuff or feedstuff comprising adding to a food or feed material a food or feed additive according to the present invention.

WO 2012/032472 PCT/IB2011/053901 6 According to a ninth aspect of the present invention there is provided a method of increasing the growth rate and/or health of an animal comprising feeding the animal an effective amount of a feedstuff according to the present invention. 5 According to an tenth aspect of the present invention there is provided the use of an amidase which degrades ochratoxin in the manufacture of a foodstuff or feedstuff. According to a eleventh aspect of the present invention there is provided a foodstuff or feedstuff obtainable by the methods of the present invention. 10 According to a twelfth aspect of the present invention there is provided a recombinant cell comprising an amidase enzyme capable of degrading ochratoxin. In a thirteenth aspect of the present invention there is provided an amidase enzyme 15 comprising a polypeptide sequence having at least 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:1 or SEQ ID NO: 3, or a polypeptide which differs from SEQ ID NO: 1 or SEQ ID NO: 3 by one or several amino acid additions, deletions and/or substitutions; or a polypeptide encoded by a polynucleotide having at least 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 20 98%, 99% identity to SEQ ID NO:2, or a polynucleotide which differs from SEQ ID NO: 2 by one or several nucleotide additions, deletions and/or substitutions; and wherein said polypeptide is not SEQ ID NO:1. According to a fourteenth aspect of the present invention there is provided a food or 25 feed additive comprising the peptidase enzyme PEPAd (SEQ ID NO: 12) capable of degrading ochratoxin A. According to a fifteenth aspect of the present invention there is provided a food or feed material comprising the feed additive of the thirteenth aspect. 30 According to a sixteenth aspect of the present invention there is provided a foodstuff or feedstuff comprising a feed material of the fourteenth aspect.

WO 2012/032472 PCT/IB2011/053901 7 Detailed Description of the Invention In the description which follows, it will be understood that any of the preferred features described are applicable to any aspect of the present invention unless explicitly stated otherwise. It will be further understood that any of the preferred features are 5 envisaged as being used in combination where appropriate. It will be further understood that the terms ochratoxin or an amidase enzyme capable of degrading ochratoxin in preferred embodiments refers to ochratoxin A or an amidase enzyme capable of degrading ochratoxin A. 10 As used herein the term amidase (amidohydrolase) refers to an enzyme of the amidohydrolase superfamily which can hydrolyse an amide. The inventors have isolated and cloned the amidase 2 enzyme from A. niger 15 responsible for ochratoxin degradation. This enzyme is particularly effective at degrading ochratoxin A. They have shown that this enzyme is expressed by an ORF encoding a hypothetical 480 amino acid A. niger protein (An14g02080, Acc No. XP_001400834) which shows -40% identity to certain bacterial amidases which are uncharacterised biochemically especially with respect to their substrate specificity. 20 Database searching indicates that this putative amidohydrolase (amidase 2) has 36% amino acid sequence identity with certain dipeptidases, such as the carboxypeptidase Sgx9355e encoded by an environmental DNA sequence isolated from the Sargasso Sea (Biochemistry 48(2009): 4567-4576). The 3D structure of Sgx9355e is known and 25 it is a member of the amidohydrolase superfamily (AHS). Sequence alignment of amidase 2 with Sgx9355e suggests that amidase 2 is as a member of the AHS. This is a group of enzymes which have a remarkable substrate diversity with a (P3la)3 barrel (Tim barrel) structural fold. Most of the enzymes within this superfamily catalyse 30 the hydrolysis of C-0, C-N, or P-O bonds. No members of the superfamily have been linked to the hydrolysis of OTA.

WO 2012/032472 PCT/IB2011/053901 0 0 It will be understood that the terms amidase (i.e amidohydrolase) and amidase which can degrade ochratoxin are used interchangeably herein unless specifically stated otherwise or it is obvious from the context that a different enzyme is being discussed. 5 The terms amidase and amidase which can degrade ochratoxin A refer to an enzyme which can break down at least 5%, 10%,15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100% of the ochratoxin A present in a sample to ochratoxin a. 10 It will be apparent to the skilled person that any amidase which can degrade ochratoxin will be suitable for use in the present invention. Preferably, the amidase for use in any aspect of the present invention is an amidase which degrades at least ochratoxin A. 15 Preferably, the amidase enzymes according to the present invention comprises at least one, preferably, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, more preferably nine of the amino acid sequence motifs: 1) x-P-G-x-x-D-x-H-x-H-x-xG, where the two His residues are in the active site; 20 2) G-x-T; 3) G-x-x-x-G-P; 4) G-H-x-D where the His residue is in the active site; 5) D-G-x-x-x-C-x-x-x-x-R-x-x-x-R-x-x-A-x-x-l-K, where the Lys residue is in the active site 25 6) G-G-V-x-S-x-x-D-x-P, where the Val residue is in the active site; 7) V-x-A-H-x-x-G-x-x-G, where the two His residues are in the active site; 8) H-x-x-x-x-D, where the His residue is in the active site; 9) G-V-x-I-x-x-G-T-D, where Asp residue is in the active site. 30 Prefrably, the motifs are: 1) xl-P-G-x-x-D-x 4 -H-x 5 -H-x 6

__

7 -G, where the two His residues are in the active site; 2) G-x 8 -T; 3) G-x 9 -x 0 -x 1 4-G-P; 4) G-H-x 12 -D where the His residue is in the active site; WO 2012/032472 PCT/IB2011/053901 9 5) D-G-x x 1 4 -xC-x x x9 x-R- where the Lys residue is in the active site 6) G-G-V-x 27 -S-x 2 x 29 -D-x 30 -P, where the Val residue is in the active site; 7) V-x"-A-H-x 32 x 33 -G-x 3 4 -x 3 5 -G, where the two His residues are in the active site; 5 8) H-x 3 -xx x 38

X

3 9 -D, where the His residue is in the active site; 9) G-V-x 40 -l-x 41

-X

4 2 -G-T-D, where Asp residue is in the active site; wherein: x'= I/m; x 2 = I/m; x 3 = w/i; x 4 = c/v/s/a; x 5 = any amino acid; x 6 = f/y/; x 7 = any amino acid; x 8 = y/f; x9= t/a/s/v/r; x' 1 = i/f/a; x"=- any amino acid; x 1 = g/s; x 1 = v/e; x 1 4 = any amino 10 acid; x 1 = e/d/g; x'6= any amino acid; x 7 = any amino acid; x' 8 = a/g/t; x 19 = v/a; x 20 = any amino acid; x 2 = q/m/a; x 2 = 1/i/v; x 23 r/h/c; x 24 g/n; x 25 = k/r/t/e/d; x 2 = any amino acid; x^=l/m/v/g; x 2 = any amino acid; x 2 = any amino acid; x 3 a= any amino acid; x 3 = a/s/h; x 32 = c/v/a; x 33 =h/q; x 34 =k/r; x 35 = any amino acid; x 36 = g/v/a; x 37 = s/t/i; x 38 = y/f/e; x 3 9 = I/a/i; x4 0 = any amino acid; x 41 = a/v; x 42 =-l/a. 15 It will be understood that the term any amino acid refers to any one of amino acids G, A, V, L, I, M, F, W, P, S, T, C, Y, N, 0, D, E, K, R or H, or an unnatural amino acid or amino acid derivative. 20 In a more preferred embodiment the motifs are: 1) I/m-P-G-l/m-w-D-c-H-x-H-f-x-G, where the two His residues are in the active site; 2) G-y/f-T; 3) G-t-i-x-G-P; 25 4) G-H-g-D where the His residue is in the active site; 5) D-G-v-x-e-C-x-x-a-v-R-x-q--R-r-g-A-k-x-1-K, where the Lys residue is in the active site 6) G-G-V-l/-S-x-x-D-x-P, where the Val residue is in the active site; 7) V-a-A-H-c-h-G-k-x-G, where the two His residues are in the active site; 30 8) H-g-s-y-l-D, where the His residue is in the active site; 9) G-V-x-l-a-l-G-T-D, where Asp residue is in the active site. It will be readily apparent to the skilled person that the carboxypeptidase enzymes of the prior art which are capable of degrading ochratoxin do not comprise any of the 35 recited motifs and show no sequence homology to amidases of the present invention.

WO 2012/032472 PCT/IB2011/053901 10 In one preferred embodiment the amidase is an isolated amidase substantially free from other components in the culture media in which it is produced. 5 In a further preferred embodiment the amidase is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% pure. In a further preferred embodiment, the amidase is an amidase comprising a distorted Tim-like barrel structure including an active site comprising 6 histidine residues, 1 10 lysine residue and 1 aspartic acid residue, wherein the amino acids residues in the active site corresponding to positions H111, H113, H191, K246, H287, H289, H307 and D378 of SEQ ID NO:1 when the tertiary structure of the amidase and SEQ ID NO:1 are compared. 15 It will be understood by the skilled person that a Tim barrel is a conserved protein fold consisting of eight a-helices and eight parallel P-strands that alternate along the peptide backbone. Tim barrels are considered a/P protein folds because they include an alternating pattern of a-helices and p-strands in a single domain. In a Tim barrel the helices and strands (usually 8 of each) form a solenoid that curves around to 20 close on itself in a doughnut shape, topologically known as a toroid. The parallel p strands form the inner wall of the doughnut, whereas the a-helices form the outer wall of the doughnut. Each p-strand connects to the next adjacent strand in the barrel through a long right-handed loop that includes one of the helices. 25 The structure of amidase 2 can be divided into two domains, a core catalytic domain and a smaller p-sandwich domain. The catalytic domain comprises residues 107-425, which form a rather distorted TIM-like barrel of eight parallel p-strands (P5-7, 9-13) flanked on the outer face by a-helices. A kink in the middle of strand P7 occurring at residue 182 divides it into two separate strands, p7a and p7b. This kink represents 30 the features that cause the barrel distortion. While p7a has only one neighbouring barrel strand, P6, strand @7b has two neighbours to which it is hydrogen-bonded, P8 and $9. The former represents a secondary structure element that is attached to the barrel core without formally being a part of it. The second cause for the barrel distortion is the absence of a canonical hydrogen-bonding interaction of residues 375 35 378 directly downstream of p13 with the first barrel strand 15 that would close the WO 2012/032472 PCT/IB2011/053901 11 circle. Thus, the overall appearance of the domain core rather resembles a sandwich of parallel p-sheets, one consisting of three and the other of seven strands. Of them in total 13 helices (12 a and I g) of the catalytic domain those directly following the barrels strands and P8 can be assigned as barrel helices, while the remaining four 5 helices represent additional secondary structure elements lining the top and bottom of the barrel. The f-sandwich domain comprises residues of both the N- and the C-terminus (43 106, 426-480). The three strands formed by C-terminal residues (P14-16) are part of 10 the larger of the two sheets. From the four strands formed by N-terminal residues P3 belongs to the smaller sheet, while the long and sharply bent strands P1, P2 and P4 contribute to the formation of both sheets. Only P1 and P4 are parallel to each other. Helical turns y16 and y17 are inserted between p14 and p15 and pack against the catalytic domain. Helices yl and a2 inserted between P3 and P4 are pointing in the 15 opposite direction and are solvent exposed. It will be understood by the skilled person that the tertiary structure of a given amidase can be readily compared to the tertiary structure of SEQ ID NO:1 having the co ordinates shown in Figure 25 using standard software packages with their default 20 parameters, for example, PyMOL (www. Pymol.org). It will be further readily apparent to the skilled person that the carboxypeptidase enzymes of the prior art which are capable of degrading ochratoxin do not comprise a Tim barrel-like structure including the specified residues in the active site. 25 In one embodiment, the amidase is an enzyme of EC 3.5.1.X, according to the Recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) where X is a designation number provided by the committee. 30 It will be understood by the skilled person that carboxypeptidase A and carboxypeptidase Y of the prior art are designated in EC 3.4.17.1 and 3.4.16.5 respectively.

WO 2012/032472 PCT/IB2011/053901 12 in one preferred embodiment, the amidase is one which degrades ochratoxin A when incubated at pH3-9 and 21-401C. It will be apparent that the ability of an amidase enzyme to degrade ochratoxin A can 5 be determined using the OTA degradation assay described in the method section below. Suitable amidases comprise those from Aspergillus niger, Aspergillus flavus, Talaromyces stipitatus, Neurospora crassa, Streptomyces, for example, S. tO roseosporus, Thermotoga lettingae, Salinispora arenicola, Glomerella graminicola, Metarhizium anisopliae and Aspergillus oryzae shown as SEQ ID NO: 1, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14 and 15 respectively or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 99% identity to any one of SEQ ID NOs:1, 3, 5 to 11, 13, 14 or 15. 15 In preferred embodiments of the present invention the amidase enzyme for use in the additives, foods, feeds, uses and methods of the present invention comprises a polypeptide having the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 20 99% identity to SEQ ID NO: 1 or SEQ ID NO: 3, or a polypeptide which differs from SEQ ID NO: 1 or SEQ ID NO 3 by one or several amino acid additions, deletions and/or substitutions; or a polypeptide produced by expression of a polynucleotide comprising SEQ ID NO: 2 or a polynucleotide having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO:2, or a 25 polynucleotide which differs from SEQ ID NO: 2 by one or several nucleotide additions, deletions and/or substitutions; or a sequence which hybridises under stringent conditions to the complement of SEQ ID NO:2, or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98% or 99% identity thereto. 30 In further preferred embodiments, the enzyme according to the present invention catalyzes the chemical bond breakage of C-N, C-0 or P-O, more preferably C-N. It will be apparent to the skilled person that full length amidase 2 variants can be made using any well known technique in the art. More stable variants resistant to WO 2012/032472 PCT/IB2011/053901 13 cleavage of the N-terminal or signal sequence can be produced by mutating the protease liable peptide bonds in the peptide sequence. For example, mutations can be made at one or more of the following 71 sites of the 5 480aa amidase 2 sequence shown in SEQ ID NO: 1 these may alter the sensitivity to hydrolysis by signal peptidases, peptidases secreted by the host organism into its medium and pepsin: 18 31 32 33 34 42 55 56 56 64 69 70 78 79 107 114 127 128 139 146 147 156 184 10 185 194 195 197 202 203 220 236 262 264 269 270 304 317 318 325 335 336 336 337 343 344 351 352 354 355 359 360 374 375 386 387 388 389 390 391 400 412 420 424 425 435 436 440 446 461 462 462 480; mutations at the following 44 sites may alter trypsin resistance: 15 343648667588899299 138 141 155 166 231 232 235 239 240 243 246 256 271 279 282 291 299 303 320 322 330 346 351 361 368 395 405 426 445 451 458 461 464 476; mutations at the following 90 sites may alter chymotrypsin resistance: 20 1 11 16 19 23 32 34 39 53 56 57 65 70 79 90 91 98 103 107 108 112 113 114 120 121 124 128 139 144 147 151 154 157 160 179 185 191 195 203 206 209 215 216 221 236 248 254 262 265 270 287 289 295 305 307 310 316 318 319 325 326 336 337 344 349 352 355 359 360 363 375 387 389 391 398 401 413 421 425 429 436 441 447 455 457 462 463 471 479 480. 25 It will be further understood that the 480 aa amidase 2 polypeptide or its variants may be N- and /or 0-glycosylated at any of its asparagine (Asn),- serine (Ser) and threonine (Thr) residues in order to improve solubility and heat-stability. 30 It will be understood that as defined herein, the term stringent conditions refers to washing at. 500C and 0.2xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na 3 citrate pH 7.0}.

WO 2012/032472 PCT/IB2011/053901 14 It will be understood that these conditions may also be high stringent conditions which are defined herein as washing at 650C and 0.1xSSC {1xSSC = 0.15 M NaCl, 0.015 M Na 3 citrate pH 7.0}. 5 In another preferred embodiment, the amidase enzyme is produced by expression of a polynucleotide encoding a polypeptide having an amino acid sequence comprising SEQ ID NO: 1, 3, 5-11, 13, 14 or 15, or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99% identity thereto, or a polypeptide which differs from SEQ ID NO: 1, 3, 5-11, 13, 14 or 15 by one or several 10 amino acid additions, deletions and/or substitutions. More preferably, the amidase is encoded by a polynucleotide selected from: a) a polynucleotide comprising SEQ ID NO: 2 or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity 15 thereto, or a polynucleotide which differs from SEQ ID NO: 2 by one or several nucleotide additions, deletions and/or substitutions; b) a polynucleotide which differs from SEQ ID NO: 2 due to the degeneracy of the genetic code; c) a polynucleotide which hybridises under stringent conditions to the 20 complement of SEQ ID NO: 2 or a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% identity thereto, or a polynucleotide which differs from the complement of SEQ ID NO: 2 by one or several nucleotide additions, deletions and/or substitutions. 25 In another preferred embodiment, there is provided a vector comprising a polynucleotide encoding the amidase enzyme which degrades ochratoxin A for use in the present invention. It will be apparent to the skilled person that the vector can be any suitable expression 30 vector and that the choice of vector may vary depending upon the type of cell into which the vector is to be inserted. Suitable vectors include pGAPT-PG, pRAX1, pGAMD and pGPT-pyrGl.

WO 2012/032472 PCT/IB2011/053901 15 In a further preferred embodiment, the vector is comprised in a cell. In a further embodiment, the cell is a spore. It will be understood that as used herein the term spore refers to a fungal or bacterial 5 spore, endospore or exospore. The cell according to the present invention may be any suitable cell. More preferably, any suitable bacterial, fungal or plant cell. Even more preferably the cell is selected from E. coli, Streptomyces, Hansenula, Trichoderma (particularly T. reesei), Bacillus, 10 Lactobacillus, Aspergilus (particularly A. niger), a plant cell and/or spores of Bacillus, Trichoderma, or Aspergillus. In a more preferred embodiment there is provided a recombinant cell or spore comprising a polynucleotide encoding an amidase enzyme which degrades 15 ochratoxin, preferably ochratoxin A for use in the present invention. In further preferred embodiments, the amidase enzyme for use in the present invention is recombinant. 20 In a preferred aspect of the present invention there is provided a food or feed additive comprising an amidase enzyme which is capable of degrading ochratoxin. Preferably, the amidase enzyme will degrade at least ochratoxin A. 25 More preferably, the amidase enzyme will also degrade at least one other ochratoxin, more preferably, at least ochratoxin B. In a further embodiment, the enzyme will also degrade at least one ochratoxin derivatives beside ochratoxin B and ochratoxin C, and/or at least one ergot alkaloid. 30 It will be understood that ergot alkaloids are compounds containing amide bonds and include, for example, ergocornine, ergocorninine, ergocristine, ergocristinine, ergocryptine, ergocryptinine, ergometrine, ergosine, ergotamine and ergotaminine. These compounds are toxic to living organisms including humans and farm animals. 35 WO 2012/032472 PCT/IB2011/053901 16 It will be apparent to the skilled person that this food or feed additive may be added to a food or feed material contaminated with ochratoxin in order to reduce the level of the toxin present in the food or feedstuff consumed by an animal. Ochratoxin A is known to be an important contaminant of cereals and other starch rich foods as well 5 as, for example, coffee, cocoa, wine, beer, pulses, spices, dried fruit, grape juice, milk and meat products from non ruminant animals. Ochratoxin A is a nephrotoxic, teratogenic, hepatotoxic and carcinogenic compound which if present even at low levels can be harmful. 10 In a more preferred embodiment the additive is a feed additive. In preferred embodiments the food or feed additive comprises the amidase at a level of at least 0.001 g/kg, at least 0.01 g/kg, at least 0.1 g/kg, at least 1 g/kg, at least 5 g/kg , at least 7.5 g/kg , at least 10.0 g/kg , at least 15.0 g/kg , at least 20.0 g/kg , at 15 least 25.0 g/kg. Preferably, the food or feed additive comprises the amidase at a level such that when added to a food or feed material, the feed material comprises the amidase in a range of 1-500mg/kg, 1-100mg/kg, more preferably 2-50mg/kg or 2 10mg/kg. 20 In preferred embodiments of the present invention the amidase can hydrolyse at least 10, 20, 50, 100, 200, 300, 500, 700, 900, 1000 nanomoles OTA per min per mg protein at pH 7.0 and 40 0 C when the OTA is present at a concentration of 1 pg/ml. More preferably, the food or feed additive of the present invention comprises a 25 recombinant cell capable of expressing an amidase enzyme capable of degrading ochratoxin. More preferably, the amidase enzyme is capable of degrading at least ochratoxin A. Even more preferably, the amidase enzyme is also capable of degrading at least one 30 further ochratoxin or ochratoxin derivative and/or at least one ergot alkaloid, more preferably, ochratoxin B. Most preferably, the amidase comprises SEQ ID NO: 1, SEQ ID NO: 3 or any one of SEQ ID NO: 5-11, 13, 14 or 15 or a sequence having at least 10%, 20%, 30%, 40%, WO 2012/032472 PCT/IB2011/053901 17 50%, 60%, 70%, 75%, 80%, 90%, 95%, 98%, 99% identity to any one of SEQ ID NOs:1, 3, 5 to 11, 13, 14 or 15. In another preferred embodiment the cell is an Aspergillus cell or spore. It will be 5 apparent to the skilled person that Aspergillus cell may be a living cell, a dead cell or a disrupted cell. In preferred embodiments, the recombinant cell is an A. niger cell. More preferably, the cell has increased OTA degrading activity when compared to a non recombinant 10 cell of the same species. It will be understood by the skilled person that the amidase enzyme capable of degrading ochratoxin A for use in the present invention can be provided independently as either liquid or solid/granulated compositions. 15 Preferably, when said enzyme is in liquid form, said enzyme is secreted into the medium following culturing of a cell comprising said enzyme. Preferably said medium is cell-free (i.e. the cell(s) have been separated from the medium). Preferably said medium is concentrated. It will be understood that the medium can be granulated to 20 provide a solid enzyme composition. It will be further understood that the food or feed additive according to the present invention may be provided in the form of a solution or as a solid - depending on the use and/or the mode of application and/or the mode of administration. 25 In a further embodiment, the additive may be used to pre-treat a material which will be used as a food or feed. For example, the amidase may be used for treating liquids such as those produced as by-products from ethanol plants. In this case, the additive will be added to a liquid 30 fermentation broth in order to degrade ochratoxin A contaminants found in the medium used to grow yeast or microbes for ethanol production. It will be understood that the broth may be subsequently dried and fed to animals.

WO 2012/032472 PCT/IB2011/053901 18 In an alternative embodiment, the additive may be added to milk, for example, cows milk, contaminated with OTA. In one embodiment the food or feed additive according to the present invention is in a 5 liquid formulation suitable for consumption, preferably such liquid composition further comprises at least one of a buffer, a salt, sorbitol and/ or glycerol. Preferably, the food or feed additive further comprises at least one physiologically acceptable carrier. 10 The physiologically acceptable carrier is preferably selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose, starch, Na 2

SO

4 , Talc, PVA and mixtures thereof, 15 In a further embodiment, the food or feed additive may further comprise a metal ion chelator. The metal ion chelator may be selected from EDTA or citric acid. In one embodiment the amidase enzyme is dried on the physiologically acceptable carrier. 20 In one embodiment the food or feed additive is granulated or co-granulated with other enzymes. In preferred embodiments, the amidase for use in the present invention may be used 25 is combination with one or more further enzyme. In preferred embodiments, the one or more further enzyme is selected from the group consisting of those involved in protein degradation including carboxypeptidases preferably carboxypeptidase A, carboxypeptidase Y, A. niger aspartic acid proteases of PEPAa, PEPAb, PEPAc and PEPAd, elastase, amino peptidases, pepsin or pepsin-like, trypsin or trypsin -like 30 proteases and bacterial proteases including subtilisin and its variants, and of those involved in starch metabolism, fibre degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism, enzymes which degrade other contaminants, acetyl esterases, amylases, arabinases, arabinofuranosidases, exo and endo-peptidases , catalases, cellulases, chitinases, chymosin, cutinase, 35 deoxyribonucleases, epimerases, esterases, formamidase, -galactosidases, for WO 2012/032472 PCT/IB2011/053901 19 example a or P -galactosidases, exo-glucanases, glucan lysases, endo-glucanases, glucoamylases, glucose oxidases, -glucosidases, for example a or p - glucosidases, glucuronidases, hemicellulases, hydrolases, invertases, isomerases, laccases, phenol oxidases, lipase, lyases, mannosidases, oxidases, oxidoreductases, 5 pectinase, pectate lyases, pectin acetyl esterases, pectin depolymerases, peptidase, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytase, polygalacturonases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: 02 oxidoreductase, EC 1.1.3.5), acid phosphatases and/or others or combinations 10 thereof. These include enzymes that, for example, modulate the viscosity of the composition or feed. In a particularly preferred embodiment, the enzyme is used in combination with carboxypeptidase A. 15 In a further preferred embodiment, the enzyme is used in combination with PEPAd protease. In a further embodiment the amidase may be used in combination with at least one of 20 a detoxifying enzyme selected from the group consisting of a mycotoxin degrading enzyme, for example, aflatoxin detoxizyme, zearalenone esterases, zearalenone lactonases, fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyol amine oxidases, deoxynivalenol expoxide hydrolases, a mycotoxin degrading microorganism, for example, Bacillus subtilis, B. licheniformis, Lactobacillus or an 25 absorbent (i.e., myctoxin binders) including at least one polymer, for example, microbial cell walls or an inorganic material such as bentonite. It will be understood that the feed additive may be for any suitable animal. In one preferred embodiment the animal is a monogastric animal, for example, poultry, 30 swine, fish, shellfish and crustacea, for example, shrimps, pet animals such as, for example, cats or dogs. In an alternative preferred example the animal is a ruminant selected from, for example, cows or other bovines, sheep, goats, camels, deer, llama, antelope, alpacas or wildebeest.

WO 2012/032472 PCT/IB2011/053901 20 It will be obvious to the skilled person that the food or feed additive according to the present invention may also comprise other components such as stabilising agents and/or bulking agents and/or other enzymes. 5 Preferably, the food or feed additive according to the present invention will be thermally stable to heat treatment up to about 70 0C; up to about 800C; or up to about 950C. The heat treatment may be performed for up to about 0.5 minute; up to about 5 minutes; up to about 10 minutes; up to about 30 minutes; up to about 60 minutes. The term thermally stable means that at least about 75% of the enzyme components 10 that were present/active in the additive before heating to the specified temperature are still present/active after it cools to room temperature. Preferably, at least about 80% of the enzyme components that were present and active in the additive before heating to the specified temperature are still present and active after it cools to room temperature. 15 The food or feed additive according to the present invention may have a shelf-life of greater than about 30 weeks; greater than about 40 weeks; greater than about 50 weeks; greater than about 1 year; greater than about 1.5 years. The shelf-life means that at least about 80% of the enzyme components that were present and active in the 20 additive when it was prepared are still present and active. Preferably, the method of preparing a food or feed additive according to the present invention comprises a mixing step that comprises admixing the amidase enzyme which degrades ochratoxin, preferably ochratoxin A, optionally with at least one 25 physiologically acceptable carrier. More preferably, the amidase enzyme is also capable of degrading at least one further ochratoxin or ochratoxin derivative and/or at least one ergot alkaloid, more preferably, ochratoxin B. 30 In a particularly preferred embodiment the food or feed additive is homogenized to produce a powder WO 2012/032472 PCT/IB2011/053901 21 in an alternative preferred embodiment, the food or feed additive is formulated in to granules as described in W02007/044968 (referred to as TPT granules) incorporated herein by reference. 5 In another preferred embodiment when the food or feed additive is formulated into granules the granules comprises a hydrated barrier salt coated over the protein core. The advantage of such salt coating is improved thermo-tolerance, improved storage stability and protection against other food or feed additives otherwise having adverse effect on the enzyme. 10 Preferably, the salt used for the salt coating has a water activity less than 0.25 or constant humidity greater than 60% at 20 C. Preferably, the salt coating comprises a Na 2

SO

4 15 The method of preparing a food or feed additive may also comprise the further step of pelleting the powder. The powder may be mixed with other components known in the art. The powder, or mixture comprising the powder, may be forced through a die and the resulting strands are cut into suitable pellets of variable length. 20 Optionally, the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets. The mixture comprising the powder may be placed in a conditioner, e.g. a mixer with steam injection. The mixture is heated in the conditioner up to a specified temperature, such as from 60-1000C, typical 25 temperatures would be 700C, 850C, 90'C or 950C. The residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minutes 2 minutes., 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour. 30 In a further aspect there is provided a food or feed material comprising the food or feed additive of the present invention. It will be understood that the food or feed additive of the present invention is suitable for addition to any appropriate food or feed material.

WO 2012/032472 PCT/IB2011/053901 22 It will be obvious to the skilled person that the food or feed additive can be added to any food or feed material as a precautionary step. Alternatively, the food or feed additive can be added to food or feed materials which are known to be prone to ochratoxin, preferably ochratoxin A, contamination or to food or feed materials which 5 have been shown to be contaminated with ochratoxin, preferably ochratoxin A. It will be further apparent to the skilled person that the presence of ochratoxin, preferably ochratoxin A can be identified by any suitable means, for example, HPLC, ELISA or through the use of commercially available ochratoxin detection strips (Helica Biosystems, Inc., Fullerton CA). 10 As used herein, the term feed material refers to the basic feed material to be consumed by an animal. It will be further understood that this may comprise, for example, at least one or more unprocessed grains, and/or processed plant and/or animal material such as soybean meal or bone meal. 15 In some embodiments, the feed material will comprise one or more of the following components: a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and 20 citrus pulp; c) silage such as maize silage; d) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; e) oils and fats obtained from vegetable and animal sources; f) minerals and vitamins. As used herein, the term feedstuff refers to a feed material to which one or more feed 25 additives have been added. According to another aspect there is provided a feedstuff comprising the feed material of the present invention. It will be understood by the skilled person that different animals require different feedstuffs, and even the same animal may require different feedstuffs, depending 30 upon the purpose for which the animal is reared. Preferably, the feedstuff may comprise feed materials comprising maize or corn, wheat, barley, triticale, rye, rice, tapioca, sorghum, and/ or any of the by-products, as WO 2012/032472 PCT/IB2011/053901 23 well as protein rich components like soybean mean, rape seed meal, canola meal, cotton seed meal, sunflower seed mean, animal-by-product meals and mixtures thereof. More preferably, the feedstuff may comprise animal fats and / or vegetable oils. 5 Optionally, the feedstuff may also contain additional minerals such as, for example, calcium and/or additional vitamins. As used herein, the term food material refers to a basic food to be consumed by a 10 human. It will be understood that this may comprise grain, plant or animal material. In some embodiments, the food material may comprise one or more of a cereal or cereal product, coffee, cocoa, wine, beer, pulses, spices, dried fruits, grape juice, milk, meat or meat products. 15 As used herein, the term foodstuff refers to a food material to which one or more food additives have been added. According to another aspect there is provided a foodstuff comprising the food material of the present invention. 20 In preferred embodiments, the foodstuff or feedstuff comprises amidase at a level of about 0.001mg - 10g/kg, 0.01mg-10g/kg, 0.1mg-10g/kg, 0.lmg-5g/kg, lmg-5g/kg, 0.5g-1g/kg food/feedstuff. It will be readily apparent to the skilled person that in order for the food or feed additive of the present invention to provide the claimed advantages the food/feedstuff 25 must be a food/feedstuff contaminated with ochratoxin, preferably ochratoxin A. In another aspect there is provided a method for producing a feedstuff. Feedstuff is typically produced in feed mills in which raw materials are first ground to a suitable particle size and then mixed with appropriate additives. The feedstuff may then be produced as a mash or pellets; the later typically involves a method by which the 30 temperature is raised to a target level and then the feed is passed through a die to produce pellets of a particular size. The pellets are allowed to cool. Subsequently liquid additives such as fat and enzyme may be added. Production of feedstuff may WO 2012/032472 PCT/IB2011/053901 24 also involve an additional step that includes extrusion or expansion prior to pelleting in particular by suitable techniques that may include at least the use of steam. The feedstuff may be a feedstuff for any suitable animal. Preferably, the feedstuff is for domestic or farm animals. 5 In one embodiment the animal is a monogastric animal, such as poultry (for example, broiler, layer, broiler breeders, turkey, duck, geese, water fowl), swine (all age categories), fish, shell fish including crustaceans such as shrimps, a pet (for example dogs, cats). 10 In a further embodiment the animal is a ruminant, such as a bovine (for example, cow water buffalo, bison, yak), sheep, goats, camels, deer, llamas, antelope, alpacas or wildebeest. The feedstuff may comprise at least 0.0001% by weight of the feed additive. Suitably, 15 the feedstuff may comprise at least 0.0005%; at least 0.0010%; at least 0.0020%; at least 0.0025%; at least 0.0050%; at least 0.0100%; at least 0.020%; at least 0.100% at least 0.200%; at least 0.250%; at least 0.500% by weight of the feed additive. In a further aspect there is provided the use of at least one amidase enzyme capable 20 of degrading ochratoxin, preferably ochratoxin A, in the manufacture of a foodstuff or feedstuff for reducing the level of mycotoxin contamination in the foodstuff or feedstuff. Preferably, the amidase enzyme is also capable of degrading at least one further 25 ochratoxin or ochratoxin derivative and/or at least one ergot alkaloid, more preferably, ochratoxin B. Preferably, the at least one amidase enzyme is formulated as a food or feed additive. More preferably, the food or feed additive is the food or feed additive according to the 30 present invention. In a further aspect of the present invention there is provided a composition comprising an ochratoxin, preferably ochratoxin A, contaminated material and an amidase which degrades ochratoxin, preferably ochratoxin A as described herein.

WO 2012/032472 PCT/IB2011/053901 25 In a preferred embodiment of the present invention, the composition comprises a recombinant cell capable of expressing the ochratoxin A degrading amidase. 5 It will be understood that said composition can comprise any suitable ochratoxin A contaminated material. In preferred embodiments the composition comprises a food or feed material, a fermentation broth or waste product of the fermentation process such as DDGS, waste water or contaminated soil. 10 The skilled person will understand that ochratoxin A contamination of compositions other than food and feed materials can be problematical. For example, waste water containing ochratoxin A from industrial processes can result in contamination of water ways. 15 Also provided by the present invention, is a method of reducing ochratoxin contamination in a material, comprising adding to the contaminated material an amidase enzyme capable of degrading ochratoxin, preferably ochratoxin A. Also provided by the present invention is a food or feed material obtainable by the 20 methods of the present invention. In a further aspect of the present invention there is provided a method of increasing the growth rate and/or health of an animal comprising feeding the animal an effective amount of a feedstuff according to the present invention. 25 As used in the present context, the term health refers to a reduction in the detrimental effects on an animal caused by ochratoxin toxicity resulting from levels of ochratoxin present in the feedstuff. 30 Examples The invention will be further described with reference to the examples and figures in which:- WO 2012/032472 PCT/IB2011/053901 26 Figure 1 shows the ability of PEPAd (a Danisco aspartic acid protease cloned from A, niger), AmanoTM lipase and bovine Carboxypeptidase A at the same protein concentrations to breakdown OTA. Activity was expressed in % OT-a (product of OTA) area: the area corresponding to the product divided by the total area (peak area 5 of OTA plus OT-a). Figure 2 shows the percentage of OT-a area and relative lipase activity (%) in fractions from (A): hydrophobic interaction chromatography (HIC)/ affinity chromatography with a Phenyl Sepharose packed column (2.6cm x 10cm) and 10 gradient from 1M ammonium sulfate to 0. (B): Anion exchange chromatography (AEX) Q30 column with gradient from 0 to 1 M NaCl after HIC. Figure 3 shows SDS-PAGE analysis of the concentrated AEX fraction having highest OTA degrading activity. The indicated bands A and B were in-gel digested with trypsin 15 and the digested peptides were separated on reverse phase HPLC and analyzed with MS for peptide identification. Figure 4 shows graphically the separation of OTA active fractions by HIC/affinity chromatography after ammonium sulfate fractionation. Figure 4A shows HIC or affinity 20 chromatography on Phenyl Sepharose column; Figure 4B shows HIC on Buty Sepharose column. Figure 4C shows the lipase and OTA activity distribution in the collected fractions 2-28 from the Butyl Sepharose column. Figure 5 shows SDS-PAGE analysis of concentrated OTA active fractions after the 25 purification steps of ammonium fractionation, HIC/affinity on Phenyl Sepharose, HIC on Butyl Sepharose, AEX on Source Q30 and membrane separation/concentration on Amicon centriprep 10 concentrator (molecular cutoff of 1OkDa). Figure 6 shows the activities present in the membrane separated and concentrated 30 AEX fractions of Figure 5 on formamide (Figure 6A) and OTA (Figure 6B), protein peaks assayed at 280nm. Figure 7 shows the construction of the amidase 2 expression cassette.

WO 2012/032472 PCT/IB2011/053901 27 Figure 8 shows SDS-PAGE analysis of intracellular proteins of A. niger transformants harboring the Amidase 2 gene. The protein band of amidase 2 is indicated by an arrow bar. 5 Figure 9 shows SDS-PAGE analysis of extracellular proteins of A. niger transformants harboring the Amidase 2 gene. The protein band of amidase 2 is indicated by an arrow bar. Figure 10 shows the screening of intracellular and extracellular (fermentation broth) 10 fractions of Aspergillus niger transformants (1-15) harboring the amidase 2 gene for their ability to break down OTA. 10pl of the intracellular fractions or the broth was mixed with 25pl 1pg/ml OTA, 100pl sodium phosphate (67mM, pH7.0) and incubated at 40 'C for 35 min. At the end of the reaction 130pl acetonitrile containing 0.2% acetic acid was added to stop the reaction. The reaction mixture was filtered and 5pl 15 analysed by RP-HPLC analysis of remaining OTA. wt refers to the parent strain used for the transformation; ctrl, refers to the reaction in the absence of A. niger transformants. Figure 11 shows the pH optimum of the amidase 2 from transgenic A. niger 20 intracellular preparation in degrading OTA. The reaction mixture contained 5pl amidase 2 mixture (a mixture of the intracellular fractions of transformant 3, 4, 7-8,10 11 and 13-15) with a protein concentration of 155pg protein/mI), 0.1 ml 67mM phosphate buffer (pH3, 4, 5, 6, 7, 8 and 9), 25pil OTA (1pg/ml). The reaction was performed at 30'C for 60min and stopped by the addition of 0.13 ml acetonitrile 25 containing 0.2% HAC. An injection volume of 10pl was used for RP-HPLC analysis of remaining OTA after filtering. Figure 12 shows the heat stability of amidase 2 from transgenic A. niger intracellular preparation. The pre-incubation mixture in a 1.5ml ependorpf tube contained 5pl of 30 the amidase 2 mixture (155pg protein/ml), and 45pl 67mM phosphate buffer pH7.0. The samples were pre-incubated at 80'C for 0, 5, 10, 15, 20, 25 and 30 min. After cooling to 15'C, 50pl 67mM phosphate buffer and 2 5pl OTA (lpg/ml) were added to start the reaction this was incubated at 30'C for 60min, the reaction was stopped by the addition of 130pl acetonitrile containing 0.2% HAC. An injection volume of 10pl 35 was used for RP-HPLC analysis of remaining OTA after filtering.

WO 2012/032472 PCT/IB2011/053901 28 Figure 13 shows pH and temperature stability of amidase 2 from transgenic A. niger inrtracellular preparation at incubated at 70 0C at pH 3-9 in 67 mM sodium phosphate. The reaction mixture contained 5pl amidase 2 mixture (155pg protein/ml), 4 5 pl 67mM 5 phosphate buffer at either pH3, 4, 5, 6, 7, 8 or 9. The reaction mixes were incubated at 700C for 30min, before 2pl 0.5mg/mi OTA in ethanol and 80pl 0.2M Mops pH 7.0 were added and the samples incubated at 30 0C for 60min. The reaction was stopped by adding 130pl acetonitrile containing 0.2% HAC, filtered and analysed by RP-HPLC. 10 Figure 14 shows analysis of recombinant amidase 2 purified from the broth of A. niger transformant 13 by SDS-PAGE on AEX fractions (A) and by mass-spectrometry (B) on fraction 13 after AEX. Figure 15 shows OTA degradation as a function of amidase protein concentration. 15 The amidase used was purified from the broth of transformant 13 (438aa protein referred to as amidase 2 mat). Figure 16 shows the effect of calcium ion concentration on the activity of amidase 2 mat purified from the transgenic A. niger and the AmanoTM lipase product. 20 Figure 17. Degradation of OTA by the amidase 2 extracted from the intracellular fraction (Amidase 2 sig) of A. niger. No inhibition by CaCl 2 or EDTA to amidase 2 sig is seen. 25 Figures 18A shows the amidase 2 amino acid sequence (480aa) (SEQ ID NO: 1) from Aspergillus niger in one letter format. Single underlined sequences are those obtained from Figure 5; double underlined are those obtained from Figure 8. Figure 18B shows the amidase 2 amino acid sequence (480aa) plus 6 histidine 30 residues at its C-terminal from Aspergillus niger in one letter format (SEQ ID NO. 4). Figure 19 shows the amidase 2 amino acid sequence (480aa) from Aspergillus niger in three letter format. Features characteristic of OTA-degrading amidases are indicated as bold and underlined amino acid residues: conserved residues (bold) and 35 nine sequence motifs (underlined).

WO 2012/032472 PCT/IB2011/053901 29 Figure 20a shows the structure of the amidase 2 (SEQ ID NO:1) protein tetramer forming the distorted TIM-like barrel structure; b shows the structure of 1 of the 8 subunits. 5 Figure 21 shows the structure of the amidase 2 active site environment. Figure 22 shows the crystal co-ordinates of a monomer of amidase 2. 10 Figure 23 shows a schematic representation of the expression plasmid pTTT-pyrG13 Amidase G. graninicola. Figure 24 shows a schematic representation of the expression plasmid pTTT-pyrGl 3 Amidase M. anisopliae. 15 Figure 25 shows a map of the expression plasmid pRAX-Amidase A. oryzae. Figure 26 shows an aligment of 11 amidase sequences showing the 9 sequence motifs that are essential for amidase activity degrading ochratoxin. 20 Figure 27 shows an aligment of SEQ ID NO:1 and carboxypeptidases A and Y which are known to have low ochratoxin degrading activity. Unless defined otherwise, all technical and scientific terms used herein have the 25 same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et aL., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in 30 this disclosure.

WO 2012/032472 PCT/IB2011/053901 30 This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, 5 nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of this disclosure which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by 10 reference to the specification as a whole. Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation. As used herein, the term "identity" means an entity having a certain homology with the amino acid sequences and the nucleotide sequences. The term "identity" in this 15 context refers to the percentage of sequence identity between two enzymes after aligning their sequences using alignment algorithms as described in more detail below. In the present context, a homologous amino acid sequence is taken to include an 20 amino acid sequence which may be at least 30, 40, 50, 60, 70, 75, 80, 85 or 90% identical, preferably at least 95, 96, 97, 98 or 99% identical to the sequence. Typically, the homologues will comprise the same active sites etc. - e.g. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions A), in 25 the context of the present invention it is preferred to express homology in terms of sequence identity. For amino acid sequences and nucleotide sequences, homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence 30 comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.

WO 2012/032472 PCT/IB2011/053901 31 % homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments 5 are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus 10 potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local homology. 15 However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - will achieve a higher score than one with 20 many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default 25 values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension. Calculation of maximum % homology therefore firstly requires the production of an 30 optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et a! 1984 Nuc. Acids Research 12 p387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4* Ed 35 Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) and the WO 2012/032472 PCT/IB2011/053901 32 GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). 5 However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and tatiana@ncbi.nlm.nih.gov). 10 Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the 15 BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62. 20 Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASISTM (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244). 25 Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. In a preferable aspect of the present invention the following software and settings for 30 calculating percentage sequence homology/identity are used. For amino acid sequences percentage of identities (homology) or "positives" are calculated by the AlignX VectorNTi (Vector NTI Advance 9.1 from Invitrogen Corporation, Carlsbad, California, USA.) for each possible pair of amino acid sequences. Settings are default parameters (Gap opening penalty - 10, Gap extension penalty 0.1).

WO 2012/032472 PCT/IB2011/053901 33 For nucleic acid sequences percentage of identities (homology) or "positives" are calculated by the AlignX VectorNTI programme from Informax Inc. (USA) for each possible pair of nucleic acid sequences. Settings are default settings for DNA are : 5 Gap opening penalty: 15 and Gap extension penalty: 6.66 (same settings for multiple alignments). Preferably the amino acid identity homologyy) is calculated across the full-length amino acid sequence or for nucleic acid to a corresponding polynucleotide which 10 encodes the respective the full-length amino acid sequence. The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity 15 in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids can be grouped together based on the properties of their side chain alone. However it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be 20 conserved for structural reasons. These sets can be described in the form of a Venn diagram (Livingstone C.D. and Barton G.J. (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput.Appl Biosci. 9: 745-756)(Taylor W.R. (1986) "The classification of amino acid conservation" J.Theor.Biol. 119; 205-218). Conservative substitutions may be made, for example 25 according to the table below which describes a generally accepted Venn diagram grouping of amino acids. SET SUB-SET Hydrophobic F W Y H K M I L V A G C Aromatic FWYH Aliphatic I L V Polar WYHKREDCSTNQ Charged H K R E D Positively H K R charged WO 2012/032472 PCT/IB2011/053901 34 Negatively E D charged Small VCAGSPTND Tiny A G S The present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e. like-for-like substitution such 5 as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine 10 (hereinafter referred to as 0), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine. Replacements may also be made by unnatural amino acids. The term "protein", as used herein, includes proteins, polypeptides, and peptides. The terms "amino acid residue equivalent to", "amino acid corresponding to" and 15 grammatical equivalents thereof are used herein to refer to an amino acid residue of a protein having the similar position and effect as that indicated in a particular amino acid sequence of a particular protein. The person of skill in the art will recognize the equivalence of specified residues in comparable proteins. The term "property" or grammatical equivalents thereof in the context of a 20 polypeptide, as used herein, refer to any characteristic or attribute of a polypeptide that can be selected or detected. These properties include, but are not limited to oxidative stability, substrate specificity, catalytic activity, thermal stability, temperature and/or pH activity profile, feed processing stability, and ability to be secreted. As used herein, the term "amino acid sequence" is synonymous with the term 25 "polypeptide" and/or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme".

WO 2012/032472 PCT/IB2011/053901 35 The amino acid sequence may be prepared/isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant IDNA techniques. The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino 5 acid residues are used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. The term "signal sequence" or "signal peptide" refers to any sequence of nucleotides 10 and/or amino acids which may participate in the secretion of the mature or precursor forms of the protein. This definition of signal sequence is a functional one, meant to include all those amino acid sequences encoded by the N-terminal portion of the protein gene, which participate in the effectuation of the secretion of protein. They are often, but not universally, bound to the N-terminal portion of a protein or to the N 15 terminal portion of a precursor protein. By "functional fragment" is meant a fragment of the polypeptide that retains that characteristic properties of that polypeptide. In the context of the present invention, a functional fragment of an amidase enzyme is a fragment that retains the amidase enzyme cleavage capability of the whole protein. 20 The term "isolated", "recovered" or "purified" refers to a material that is removed from its original environment. The term "substantially purified" means that the material has been purified to at least a substantial degree. In one aspect, preferably the nucleotide or amino acid sequence is in an isolated form. The term "isolated" means that the sequence is at least substantially free from 25 at least one other component with which the sequence is naturally associated in nature and as found in nature. Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to understand that this disclosure is not limited to particular embodiments described, as such may, of course, 30 vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

WO 2012/032472 PCT/IB2011/053901 36 Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and 5 any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes 10 one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure. It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" includes a plurality of such 15 candidate agents and reference to "the cell" includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. 20 The enzymes for use in the present invention can be produced either by solid or submerged culture, including batch, fed-batch and continuous-flow processes. Culturing is accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, the carbon and energy source material, molecular oxygen, and, of course, a starting inoculum of one or more particular microorganism 25 species to be employed. In addition to the carbon and energy source, oxygen, assimilable nitrogen, and an inoculum of the microorganism, it is necessary to supply suitable amounts in proper proportions of mineral nutrients to assure proper microorganism growth, maximize the 30 assimilation of the carbon and energy source by the cells in the microbial conversion process, and achieve maximum cellular yields with maximum cell density in the fermentation media.

WO 2012/032472 PCT/IB2011/053901 37 The composition of the aqueous mineral medium can vary over a wide range, depending in part on the microorganism and substrate employed, as is known in the art. The mineral media should include, in addition to nitrogen, suitable amounts of phosphorus, magnesium, calcium, potassium, sulphur, and sodium, in suitable soluble 5 assimilable ionic and combined forms, and also present preferably should be certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine, and others, again in suitable soluble assimilable form, all as known in the art. The fermentation reaction is an aerobic process in which the molecular oxygen 10 needed is supplied by a molecular oxygen-containing gas such as air, oxygen enriched air, or even substantially pure molecular oxygen, provided to maintain the contents of the fermentation vessel with a suitable oxygen partial pressure effective in assisting the microorganism species to grow in a thriving fashion. In effect, by using an oxygenated hydrocarbon substrate, the oxygen requirement for growth of the 15 microorganism is reduced. Nevertheless, molecular oxygen must be supplied for growth, since the assimilation of the substrate and corresponding growth of the microorganisms, is, in part, a combustion process. Although the aeration rate can vary over a considerable range, aeration generally is 20 conducted at a rate which is in the range of about 0.5 to 10, preferably about 0.5 to 7, - volumes (at the pressure employed and at 250C.) of oxygen-containing gas per liquid volume in the fermentor per minute. This amount is based on air of normal oxygen content being supplied to the reactor, and in terms of pure oxygen the respective ranges would be about 0.1 to 1.7, or preferably about 0.1 to 1.3, volumes 25 (at the pressure employed and at 250C.) of oxygen per liquid volume in the fermentor per minute. The pressure employed for the microbial conversion process can range widely. Pressures generally are within the range of about 0 to 50 psig, presently preferably 30 about 0 to 30 psig, more preferably at least slightly over atmospheric pressure, as a balance of equipment and operating cost versus oxygen solubility achieved. Greater than atmospheric pressures are advantageous in that such pressures do tend to increase a dissolved oxygen concentration in the aqueous ferment, which in turn can help increase cellular growth rates. At the same time this is balanced by the fact that 35 high atmospheric pressures do increase equipment and operating costs.

WO 2012/032472 PCT/IB2011/053901 38 The fermentation temperature can vary somewhat, but for filamentous fungi such as Aspergillus niger or Trichoderma reesei the temperature generally will be within the range of about 20'C to 40'C, generally preferably in the range of about 250C to 34'C, 5 depending on the strain of microorganism chosen. The microorganisms also require a source of assimilable nitrogen. The source of assimilable nitrogen can be any nitrogen-containing compound or compounds capable of releasing nitrogen in a form suitable for metabolic utilization by the 10 microorganism. While a variety of organic nitrogen source compounds, such as protein hydrolysates, can be employed, usually cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea, and various ammonium salts such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride, or various other ammonium compounds can be utilized. Ammonia gas itself 15 is convenient for large scale operations, and can be employed by bubbling through the aqueous ferment (fermentation medium) in suitable amounts. At the same time, such ammonia can also be employed to assist in pH control. The pH range in the aqueous microbial ferment (fermentation admixture) should be in 20 the exemplary range of about 2.0 to 8.0. With filamentous fungi, the pH normally is within the range of about 2.5 to 8.0; with Aspergillius niger or Trichoderma reesei, the pH normally is within the range of about 3.0 to 7.0. pH range preferences for certain microorganisms are dependent on the media employed to some extent, as well as the particular microorganism, and thus change somewhat with change in media as can be 25 readily determined by those skilled in the art. While the average retention time of the fermentation admixture in the fermentor can vary considerably, depending in part on the fermentation temperature and culture employed, generally it will be within the range of about 24 to 500 hours, preferably 30 presently about 24 to 400 hours. Preferably, the fermentation is conducted in such a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing good conversion of the carbon-containing substrate to cells and avoiding contamination of the cells with a substantial amount of unconverted substrate. The latter is not a problem with water-soluble substrates, since any 35 remaining traces are readily washed off. It may be a problem, however, in the case of WO 2012/032472 PCT/IB2011/053901 39 non-water-soluble substrates, and require added product-treatment steps such as suitable washing steps. As described above, the time to reach this level is not critical and may vary with the particular microorganism and fermentation process being conducted. However, it is well known in the art how to determine the carbon source 5 concentration in the fermentation medium and whether or not the desired level of carbon source has been achieved. Although the fermentation can be conducted as a batch or continuous operation, fed batch operation is much to be preferred for ease of control, production of uniform 10 quantities of products, and most economical uses of all equipment. If desired, part or all of the carbon and energy source material and/or part of the assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium prior to feeding the aqueous mineral medium to the fermentor. Each of the streams introduced into the reactor preferably is controlled at a predetermined rate, or in 15 response to a need determinable by monitoring such as concentration of the carbon and energy substrate, pH, dissolved oxygen, oxygen or carbon dioxide in the off gases from the fermentor, cell density measurable by light transmittancy, or the like. The feed rates of the various materials can be varied so as to obtain as rapid a cell growth rate as possible, consistent with efficient utilization of the carbon and energy 20 source, to obtain as high a yield of microorganism cells relative to substrate charge as possible. In either a batch, or the preferred fed batch operation, all equipment, reactor, or fermentation means, vessel or container, piping, attendant circulating or cooling 25 devices, and the like, are initially sterilized, usually by employing steam such as at about 121 0C for at least about 15 minutes. The sterilized reactor then is inoculated with a culture of the selected microorganism in the presence of all the required nutrients, including oxygen, and the carbon-containing substrate. The type of fermentor employed is not critical, though presently preferred is operation under 15L 30 Biolafitte (Saint-Germain-en-Laye, France). The collection and purification of the enzymes of the present invention from the fermentation broth can also be done by procedures known per se in the art. The fermentation broth will generally contain cellular debris, including cells, various 35 suspended solids and other biomass contaminants, as well as the desired enzyme WO 2012/032472 PCT/IB2011/053901 40 product of the present invention, which are preferably removed from the fermentation broth by means known in the art. Suitable processes for such removal include conventional solid-liquid separation techniques such as, e.g., centrifugation, filtration, dialysis, microfiltration, rotary vacuum filtration, or other known processes, to produce 5 a cell-free filtrate. It may be preferable to further concentrate the fermentation broth or the cell-free filtrate using techniques such as ultrafiltration, evaporation or precipitation. Precipitating the proteinaceous components of the supernatant or filtrate may be accomplished by means of a salt, e.g., ammonium sulfate. Further purification may optionally be achieved by crystallization or by a variety of 10 chromatographic procedures, e.g., ion exchange chromatography, affinity chromatography or similar art recognized procedures. VARIANTS/DERIVATIVES 15 The present invention also encompasses the use of variants, homologues and derivatives of any amino acid sequence of an enzyme or of any nucleotide sequence encoding such an enzyme. Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups 20 such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or p-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, "the peptoid form" is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the 25 residue's nitrogen atom rather than the a-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134. OTHER COMPONENTS 30 The feed additives or compositions of the present invention may be used in combination with other components or carriers.

WO 2012/032472 PCT/IB2011/053901 41 Suitable carriers for feed enzymes include maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat, wheat bran or a wheat component, rice or rice bran, sucrose, starch, Na 2

SO

4 , Talc, PVA and mixtures thereof. In addition there are a number of encapsulation techniques including those based on fat/wax coverage, 5 adding plant gums etc. Examples of other components include one or more of: thickeners, gelling agents, emulsifiers, binders, crystal modifiers, sweeteners (including artificial sweeteners), rheology modifiers, stabilisers, anti-oxidants, dyes, enzymes, carriers, vehicles, 10 excipients, diluents, lubricating agents, flavouring agents, colouring matter, suspending agents, disintegrants, granulation binders etc. These other components may be natural. These other components may be prepared by use of chemical and/or enzymatic techniques. As used herein the term "thickener or gelling agent" as used herein refers to a product t5 that prevents separation by slowing or preventing the movement of particles, either droplets of immiscible liquids, air or insoluble solids. The term "stabiliser" as used here is defined as an ingredient or combination of ingredients that keeps a product (e.g. a food product) from changing over time. The term "emulsifier" as used herein refers to an ingredient (e.g. a food product 20 ingredient) that prevents the separation of emulsions. As used herein the term "binder" refers to an ingredient (e.g. a food ingredient) that binds the product together through a physical or chemical reaction. The term "crystal modifier" as used herein refers to an ingredient (e.g. a food ingredient) that affects the crystallisation of either fat or water. 25 "Carriers" or "vehicles" mean materials suitable for compound administration and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubiliser, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner. Examples of nutritionally acceptable carriers include, for example, grain, water, salt 30 solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, and the like.

WO 2012/032472 PCT/IB2011/053901 42 Examples of excipients include one or more of: microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols. Examples of disintegrants include one or more of: starch (preferably corn, potato or 5 tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates. Examples of granulation binders include one or more of: polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, maltose, gelatin and acacia. 10 Examples of lubricating agents include one or more of: magnesium stearate, stearic acid, glyceryl behenate and talc. Examples of diluents include one or more of: water, ethanol, propylene glycol and glycerin, and combinations thereof. The other components may be used simultaneously (e.g. when they are in admixture 15 together or even when they are delivered by different routes) or sequentially (e.g. they may be delivered by different routes). As used herein the term "component suitable for animal or human consumption" means a compound which is or can be added to the composition of the present invention as a additive which may be of nutritional benefit, a fibre substitute or have a 20 generally beneficial effect to the consumer. By way of example, the components may be prebiotics such as alginate, xanthan, pectin, locust bean gum (LBG), inulin, guar gum, galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS), lactosucrose, soybean oligosaccharides, palatinose, isomalto-oligosaccharides, gluco-oligosaccharides and xylo-oligosaccharides. 25 ISOLATED In one aspect, preferably the amidase enzyme for use in the present invention is in an isolated form. The term "isolated" means that the amidase enzyme is at least 30 substantially free from at least one other component with which the amidase enzyme is naturally associated in nature and as found in nature. The term "isolated" may WO 2012/032472 PCT/IB2011/053901 43 mean that the amidase enzyme is at least substantially free from at least one other component in the culture media in which it is produced. The amidase enzyme of the present invention may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated or with which 5 the enzyme may be produced. Thus, for example it may be substantially free of the cell(s) or one or more potentially contaminating polypeptides and/or nucleic acid molecules. The amidase enzyme may be isolated by separating the cell(s) from the broth during or after fermentation so that 10 the amidase enzyme remains in the broth. The amidase enzyme may be isolated by subjecting the fermentation broth to cell separation by vacuum filtration. In one embodiment the term isolated means that the amidase is isolated from the broth such that it substantially free from other components in the culture media in 15 which it is produced. PURIFIED In one aspect, preferably the amidase enzyme for use in the present invention is in a 20 purified form. The term "purified" means that the given component is present at a high level. The component is desirably the predominant component present in a composition. Preferably, it is present at a level of at least about 30%, 40%, 50%, 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least 25 about 97%, or at least about 99% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. For some embodiments the amount is at least about 85% said level being determined on a dry weight/dry weight basis with respect to the total composition under consideration. 30 CONCENTRATE In one aspect, preferably the amidase enzyme for use in the present invention is used as a concentrate. The concentrate may be a concentrated form of the medium into which the enzyme has been excreted. Preferably, the concentrate may be a WO 2012/032472 PCT/IB2011/053901 44 concentrated form of the medium into which the enzyme has been secreted and wherein the cell(s) have been removed. 5 Materials and Methods Ochratoxin A was from Fluka (cat. no. 32937-5MG). Carboxypeptidase A from bovine pancreas (Sigma, cat. No. C9268). All other enzymes including commercial available amidases and reagents were from Sigma, Roche, Merck and Danisco. AmanoTM lipase was from Amano Enzyme Inc. (Nagoya, Japan). All chromatgraphy medium 10 including Phenyl-, Octyl- Butyl-Sepharose, anion Exchange, gel filtration (PD10 column) and protein purification apparatus Akta explorer were from GE Healthcare. Microplates were from Nunc A/S (Denmark) and Corning (USA). Microplate reader was from Biotek (USA). Corn flour was obtained from local market while soy-corn based feed was obtained from The Danish Technological Institute at Kolding 15 (Denmark). Mycotoxin degradation assays: Method for measuring formamidase activity Solutions used: Megazyme formic acid assay kit (K-form 05/06) Bottle 1, add 4ml 20 milliQ water to produce potassium phosphate buffer pH7.6. Keep at 50C; Bottle 2, add 5.2 ml water to obtain the right NAD concentration (Kept at -20'C); Bottle 3, is FDH shake before use (Kept it at 5'C). Formamide is a liquid product at 99.80% from Sigma (F9037) (kept at 50C) and was used as it was without further dilution. 25 Assay procedure: to 96 well half area uv transparent microplate add: 10pl potassium phosphate buffer (pH 7.6) (Solution 1 from the kit), 1 OpI NAD (Solution 2 from the kit), 2 .5pl formic acid dehydrogenase (FDH)(Solution 3 from the kit), 1.6pl 100% (v/v) formamide solution (from Sigma, F9037), 30 100pl milliQ water. At 37 0C in a microplate reader, read OD increase at 340nm every min and shake before each reading for 1 5-60min. Reading interval: 1 min.

WO 2012/032472 PCT/IB2011/053901 45 After 5 min reading, add 5pl- 2 0pl A. niger preparation (depending on the activity). Get the reaction rate (maximum linear slope). Control: no formamidase was added but add buffer or water or cell-extract having no 5 formamidase activity instead. Determination of enzymatic degradation of OTA. The buffers employed in the assay were: 50mM sodium acetate buffer (pH 4, 4.5, 5 and 5.5); 50mM MES buffer (pH 6, 6.5 and 7); 0.2M Mops-NaOH ) pH7.0), 50mM Tris-HCI buffer (pH 7.5, 8 and 10 8.5). Ochratoxin A (OTA) solution (1 mg/mL) was made of 5mg ochratoxin A (Sigma, ref. 32937) dissolved in ethanol/water (60/40) and stored at -20*C. Screening assays were performed with 15pL OTA solution and different enzyme concentrations (for example, 0.1-5mg/ml) in a final volume of 300tLL. Enzymatic reactions were done at 370C for 20h ±1h under constant agitation. Samples were filtered using syringe filter 15 0.20 Lm for HPLC analysis. The HPLC apparatus consisted of a Dionex P580 pump and Dionex ASI-100 autosampler connected to a Dionex RF-2000 fluorescence detector (Aex=333nm; Aem=460nm) and a Dionex UVD340U UV detector at 257nm. Elution was through a nucleosil 100-5 C18 column (250x4.6mm, Slm particle size; Chrompack) with water/acetonitrile/acetic acid (100:100:1, v/v/v) at a 0.6mL/min flow 20 rate. The column was kept at 300C and was connected to guard column (C18, 1mm, Optimize technologies). The OTA and its degradation products were monitored both by uv at 235nm and fluorescence (excitation at 278nm emission at 440nm). Assay of A. niger preparations for OTA activity. The reactions mixture consisted of 25 245pL OTA ( 1 pg/mL) diluted in buffer pH7.0 of 50mM Mes-NaOH and 5 tL of sample (A. niger broth, cell-free extract, fractions after ammonium fractionation and chromatography or product from A. niger). The reaction was performed at 40'C for 30 min to 2 hours. Heating at 950C for 5 min was used to stop the reaction. Samples were filtered using syringe filter 0

.

2 0 gm and 5lL injected in the HPLC apparatus. 30 Lipase and esterase assay. Lipase and esterase activity were assayed using p nitrophenyl butyrate (pNPB) purchased from Sigma as the substrate. Five microlitres pNPB 40mM dissolved in acetonitrile was added to 165gL of 50mM Tris-HCI buffer pH WO 2012/032472 PCT/IB2011/053901 46 7.5 and 5VtL of enzyme solution and used to test OTA degradation. Optical density was followed at 41 Onm for 60 min. Protease activity assay (Petri dish method). Protease activity was confirmed using plates containing 1% casein, 1% agarose in 100mM di-sodium hydrogen phosphate 5 buffer pH 6. The plate was incubated overnight at 370C and the protease activity was revealed by the development of a whitish ring around the well containing enzyme. Protein concentration assay. Protein concentration was determined with Bio-rad protein assay reagent based on the method of Bradford according to the procedure provided by the manufacturer. Using BSA as standard, the OD was measured in 10 microplate at 595nm, each samples dosed in duplicate. Purification of OTA degrading activity from A. niger Sample preparation: Ammonium sulfate fractionation. Five grams of Amano lipase enzyme were dissolved in 1 OOmL of 50mM Tris/HCI buffer pH 7.5. The amount 15 of ammonium sulfate corresponding to 40% saturation was added and crystals dissolved. After 10min centrifugation at 3500rpm, the supernatant was collected and ammonium sulfate added to reach 60% saturation. Crystals were dissolved by stirring and the solution centrifuged again. The pellet corresponding to the precipitate from 40% to 60% ammonium sulfate saturation was dissolved in 4x4OmL of 50mM tricine, 20 1 M ammonium sulfate pH7 and filtered with 0.22pm filter. Hydrophobic interaction chromatography (HIC). The sample from ammonium sulfate fractionation was injected and applied to either Phenyl Sepharose FF, Butyl or Octyl-Sepharose CL4B column equilibrated with 50mM tricine containing 1 M 25 ammonium sulfate pH 7 (buffer A). The column, connected to Akta purifier system, was washed with buffer A (10mL/min) and the bound proteins were eluted with a linear gradient of 50mM tricine (Buffer B). Fractions of 1OmL were collected and used for OTA activity assay or formamidase assay. Anion exchange chromatography (AEX). Fractions with high conversion of OTA 30 were pooled and desalted on a PD10 column equilibrated in 20 mM Tris-HCI, pH 7.5 (buffer A). The desalted sample was applied to a Source Q30 column equilibrated in buffer A (10 ml/min). The column was washed with buffer A and the bound proteins WO 2012/032472 PCT/IB2011/053901 47 were eluted with a linear gradient of 0-1 M NaCl in buffer A (Buffer B is the same as Buffer A containing 1 M NaCi). During the gradient, fractions of 5 ml were collected. Concentration of protein. Fractions with OTA degrading activity obtained by anion exchange chromatography were concentrated using Amicon CentriprepTM 5 concentrators with regenerated cellulose membrane (molecular cutoff 10kDa). Optimum pH determination. Five microliters of enzyme solution were combined with 245 .tL OTA 1lg/mL diluted in different buffers: 50mM sodium acetate buffer (pH 4, 4.5, 5 and 5.5); 50mM MES-NaOH buffer (pH 6, 6.5 and 7); 50mM Tris-HCI buffer (pH 10 7.5, 8 and 8.5). Reactions were performed at 37'C for 30min and stopped by heating at 950C for 5min. Optimum temperature. Five microliters of enzyme solution were mixed with 245pL OTA 1p.g/mL at optimum pH and incubated at 20, 30, 40, 60, 700C for 30min. Reactions were stopped by heating at 950C for 5min. 15 Temperature stability. The enzyme solution was incubated at different temperature (30, 40, 60, 70, 80and 900C) for 1 h and 5rtL were mixed with 245gL OTA 1 Vig/mL for ochratoxin degradation reaction under optimum conditions. Reactions were stopped by heating at 950C for 5min. The control was not incubated. 20 SDS-PAGE. To separate and analyze the proteins sodium dodecyl sulfate polyacrylamide gel eletrophoresis, (SDS-PAGE) was used. Forty microliter samples were mixed with 10pL 5x loading buffer (containing DDT and SDS), and boiled for 5min. 20 tL were loaded on a NuPAGE 4-12% Bis-Tris gel (Invitrogen, USA). The electrophoresis was run according to the procedure from the manufacturer. The gel 25 was stained with Coomassie brilliant blue. Mass spectrometry. The protein of interest was cut from the gel (as above) using a scalpel and transferred to an Eppendorf tube. The protein was digested with an enzyme (primarily trypsin). After digestion peptides were extracted and analyzed by 30 HPLC-MS/MS. The MS/MS spectra were automatically searched against protein databases for the purpose of idenity and characterisation.

WO 2012/032472 PCT/IB2011/053901 48 Inhibition assays for formamidase and amidase 2. One protease inhibitor cocktail (PIC) tablet (Roche Applied Science, Mannhein, Germany) was dissolved in 1.5mL milliQ water to obtain a 7x stock solution. For formamidase inhibition assay, instead of 100pL milliQ water normally employed, 20pL of 7x PIC stock solution and 80tL of 5 milliQ water were mixed with other components listed in formamidase activity assay section. For OTA degrading activity, 35pL 7x PIC solution were mixed with 210pL OTA 1p-g/mL and 5gL enzyme solution. A 125mM EDTA stock solution was prepared and appropriate volumes to obtain 10mM and 50mM final concentration were mixed with OTA l 1 ig/mL and 5 L enzyme. For controls, inhibitor solution is replaced either 10 by milliQ water for formamidase activity assay or MES-NaOH pH7 buffer for OTA degrading activity. Results Example 1 Screening for OTA activity 15 Thirty enzymes including various lipases, proteases and amdiases were investigated for their ability to degrade ochratoxin A (Table 1). Only carboxypeptidase A and lipase from A. niger (Amano TM A) were able to breakdown OTA as previously reported (Pitout, 1969; Stander et al., 2000). Moreover, aspartic acid protease pepAd2 (Danisco) from A. niger showed an OTA converting activity although this was less 20 efficient. To facilitate the comparison of these three enzymes, the protein concentration of PEPAd was determined and enzyme solutions of carboxypeptidase A and AmanoTM lipase A were prepared at the same concentration (1mg/ml). A kinetic of the degradation was carried out (Figure 1). Figure 1 shows that the OTA degradation kinetics of AmanoTM lipase A and carboxypeptidase A were similar. After 25 7h reaction at 37*C, no OTA peak was visible, meaning that full conversion of OTA was obtained. For PEPAd, full conversion was not reached even after 1 day reaction. Amano TM lipase A product was a mixture of different enzymes with the dominant acitivity being lipase. Besides PEPAd, other A. niger aspartic acid proteases of PEPAa, PEPAb and PEPAc (Wang, 2010), porcine elastase, and yeast 30 carboxypeptiase Y all showed certain activity with OTA. This is the first time aspartic proteases other than metallo-protease (i.e., carboxypeptidase A, a Zn 2 ' dependent protease), serine protease elastase and carboxypeptidase Y have been shown to be able to break OTA down. Other proteases, lipases and amidases listed in Table 1 were not able to break OTA down (data not shown).

WO 2012/032472 PCT/IB2011/053901 49 Table 1. Enzymes screened for ochratoxin A degradation Enzyme Organism Ref pH Manufacturer Carboxypeptidase Bovine pancreas C9268 7,5 Sigma A Carboxypeptidase Yeast 12758 7 USB Y Carboxypeptidase Yeast C3888 7 Sigma Y Chymotrypsin 8 Christian Chymostar Hansen Elastase Porcine prancreas E0258 8,5 Sigma Endoproteinase Roche S.Aureus 791156 7,5 Glu-C Diagnostics LG12 Bacillus subtilis 7,5 Danisco Lipase A Amano T M Aspergillus niger 534781 7,5 Amano T M PROACT #3617 Nocardiopsisprasina 7,5/4 Novozymes Proline peptidase 7 proC Bacillus Protease thermoproteolyticus P1512 8 Sigma thermolysinrokko Proteinase K Tritirachium album p0390 8 Sigma Protease P3000 Bacillus subtilis 7,5 Roche Trypsin Bovine pancreas 109819 8 Diagnostics phospholipase 7 Danisco KLM1 lipase 3 5,5 Danisco Lipase Rhizopus arrhizus 62305 8,5 Sigma Lipase Candida cylindracea 62316 6,5 Sigma Lipase Rhizopus niveus 62310 7,5 Sigma GAP3 Aspergillus Niger 6 Danisco PEPAa Aspergillus Niger 6 Danisco WO 2012/032472 PCT/IB2011/053901 50 PEPAb Aspergillus Niger 6 Danisco PEPAc Aspergillus Niger 6 Danisco PEPAd 1 Aspergilus Niger AserilusNier6 Danisco terminal deletion) PEPAd2 (full Aspergillus Niger AserilusNier6 Danisco length) Grindamyl Aspergilius Niger 7,5 Danisco Pseudomonas Amidase A6691 7 Sigma aeruginosa Peptide Amidase Citrus sinensis 17232 7 Fluka Amidase PLY511 7 Profors Penicillin amidase E. coli 76429 7 Sigma Example 2 Purification of ochratoxin A degrading activity from an A. niger fermetation liquid or filtered broth (cell free). 5 Firstly ammonium sulfate precipitation was employed. Several ammonium sulfate concentrations were applied to an A. niger crude protein extract. The OTA degrading activity was determined in both supernatant and pellet. By increasing the ammonium sulfate concentration, the OTA degrading activity was transferred from the supernatant to the pellet. At 60% ammonium sulfate, all the degrading activity was 10 found in the pellet. This demonstrated that the enzyme was able to hydrolyze the mycotoxin precipitates from 40% to 60% ammonium sulfate. At the same salt concentration (60%), a slight lipase activity remained in the pellet (data not shown). The ammonium sulfate fraction from 40% to 60% was employed as the starting 15 material for further ochratoxin degrading enzyme purification. Hydrophobic interaction chromatography (HIC) followed by anion exchange chromatography (AEX) as described above were used to separate lipase activity from OTA degrading activity. The active fraction was suspended in 50 mM tricine-HCI (pH7.0) containing 1M

(NH

4

)

2

SO

4 (Buffer A). 20 Figure 2A shows the percentage of OT-alpha area obtained after 1h reaction of the fractions obtained from Phenyl Sepharose with OTA. This figure shows an increase WO 2012/032472 PCT/IB2011/053901 51 in OTA degrading activity from fraction no. 51 to no. 64, while the highest lipase/esterase activity was found in fraction no. 65-68. This observation indicates that the ochratoxin degrading activity had apparently nothing to with the lipase/esterase activity in the Amano T M lipase product (it has previously been indicated that OTA 5 degrading activity was due to lipase activity, Stander et al., 2000). The protein that degrades OTA was eluted between 90% and 100% buffer B (50 mM tricine-HCI (pH 7.0), indicating that Phenyl Sepharose chromatography medium apparently functioned as an affinity medium. OTA also contains a phenyl group and the OTA degrading enzyme must be able to bind the phenyl group from either OTA or the separation 10 medium used here. Concerning the lipase/esterase activity, which was present in all fractions obtained from this step, it was eluted at 100% buffer B. This is not unexpected because lipases/esterases perform hydrolysis of lipids and triglycerides, which are very hydrophobic substrates and therefore bind tightly with hydrophobic matrices like Phenyl-Sepharose. 15 Figure 2B shows the percentage of OT-alpha area obtained after 30min reaction of the AEX fractions with OTA. The maximum OTA activity was found in fraction no. 26. However, it was also in these fractions that the relative lipase activity was the highest. This indicates that the AEX step was not able to separate lipase/esterase from OTA 20 degrading activity, this was used by earlier investigators as the sole chromatography step in their attempt to purify OTA degrading activity (Abrunhosa et al., 2007). In summary figures 2A and 2B indicate that lipase/esterase is not the enzyme that is responsible for the degradation of OTA (Figure 2A) as it has been assumed earlier 25 (Stander et al. (2000) and the separation of lipase/esterase activity from OTA degrading activity was not achieved by AEX, which was practiced by earlier investigators in an effort to purify the enzyme degrading OTA from Amano T M lipase (Abrunhosa et al., 2007). The earlier investigators did not show that Phenyl Sepharose chromatography medium usually regarded as an hydrophobic interaction 30 chromatography medium separating proteins based on their difference in hydrophobicity could be used here as an affinity separation medium for OTA degrading activity. After the purification by ammonium sulfate fractionation followed by HIC/affinity 35 chromatography on Phenyl Seharose and AEX, the fractions which contained the WO 2012/032472 PCT/IB2011/053901 52 OTA degrading activity still had some lipase/esterase activity. Figure 4 shows the results of analysis of the active fractions on OTA obtained in the AEX purification procedure of Figure 2B by SDS-PAGE. It can be seen that on the gel there are two major bands: A and B. The partial sequencing of those bands by mass spectrometry 5 followed by homology searching in the protein database suggests that Band A comprises at least 3 proteins corresponding respectively to: 1- Predicted acetamidase/formamidase from Aspergillus oryzae 45,012Da (27% coverage) 2- Formamidase from Aspergillus fumigatus 45,277Da (24% coverage) 10 3- Formamidase from Aspergillus nidulans 44,907Da (20% coverage) And that Band B comprises at least 3 proteins corresponding to: 1- Glyoxylate/hydroxypyruvate reductase from Aspergillus oryzae 39,603Da (34% coverage) 2- Acetylesterase from Aspergillus awamori 32,640Da (9% coverage) 15 3- Endo-1,4-beta-xylanase A precursor from Aspergillus niger 35,486Da (17% coverage). From the results indicated in Figure 4, it appeared likely that the enzyme which breaks down OTA was A. niger formamidase. The other identified enzymes have theoretically nothing to do with OTA degradation. Further purification of A. niger formamidase was 20 undertaken. Example 3 Improved Purification of ochratoxin A degrading enzyme Further improvement for the purification by HIC and AEX was not achieved by using 25 different buffers and different gradient programs. Purification using Phenyl Sepharose can be regarded as an affinity step because the OTA molecule has the phenyl group however, the impurities have high hydrophobicity and bind tightly to the phenyl Sepharose medium and therefore elute very late (Figure 4A). When Octyl- and Butyl Sepharose were employed, the OTA degrading activity fraction was found in the 30 unadsorbed flow-through fractions. This indicates that it has low hydrophobicity and WO 2012/032472 PCT/IB2011/053901 53 that its tight biding to Phenyl Sepahrose is of affinity in nature (Figure 4A) whilst the impurities having high hydrophobicity bound tightly and eluted later (Figure 4B) as in the case with Phenyl-Sepharose. 5 The procedure was further improved using ammonium sulfate fractionation (AS), HIC/affinity on Phenyl-Sepharose (HIC1), HIC on Butyl-Sepharose (HIC2) and AEX, the active fraction was concentrated and desalted with a molecular sieve with cutoff of 10kDa. The concentrated and desalted fractions 5 and 6 shown on Figure 4 were analyzed on SDS-PAGE (Figure 5). The protiein bands were isolated from the gel 10 and digested with trypsin. The digest was separated on RP-HPLC and analyzed by MS to identify the identity of the protein bands. The MS results indicate that after 4 steps of purification the OTA degrading activity was not confined to one single band, instead the following enzymes were identified 15 (order of intensity): Formamidase (51% sequence coverage), formamidase degradation product (caused by protease) (30%), unannotated A. niger protein sequence (An14g02080, XP_001400834) having similarity to bacterial amidase (25%), glutamyltranspeptidase (17%), and aldose 1-epimerase (14%). From these results the identified enzyme that most likely breaks down OTA was considered to be 20 Aspergilius niger formamidase as indicated by the results in Figures 3 and 5, followed by an unannotated A. niger protein sequence having similarity to bacterial amidase and glutamyltranspeptidase, all of the three having activities toward C-N bond, which exists in OTA molecule. 25 Example 4 Characterization of the enzymes identified that may have the potential to break OTA down. Based on the results from Figure 3, 4, 5 and 6, further studies were undertaken on the two putative Aspergillus enzymes formamidase and glutamyltranspeptidase, and the 30 hypothetical protein An14g02080 (XP_001400834). The putative formamidase of Aspergillus origin has not previously been characterized with respect to its biochemical properties including substrate specificity though it had been cloned from A. nidulans and expressed as indicated indirectly by the activity of 35 beta-galactosidase activity (Fraser et al., 2001). Formamidase is known to be involved WO 2012/032472 PCT/IB2011/053901 54 in glyoxylate and dicarboxylate metabolism and also nitrogen metabolism. It converts formamide in the presence of water to formic acid (formate) and ammonium (formamide + H 2 0 4 formate + NH 3 ). The presence of formamidase activity was assayed using Megazyme formic acid kit as indicated in the Material and method 5 section above. When this assay was carried out with fractions obtained in the AEX step, formamidase was observed in fractions of 25 to 30 (Figure 6A). This was the first indication that fungi and fungal preparations including the Amano T M lipase product 10 prepared from A. niger had an active formamidase breaking down formamide to formic acid and ammonium. Equally important the formamidase activity was purified using the purification steps designed to purify OTA activity, suggesting that fungal formamidase breaks down OTA. When the same fractions tested for formamidase activity were assayed for degradation of OTA, the conversion increased from fraction 15 26 to 32 (Figure 6B). As can be seen from Figure 6A and Figure 6B the two activities were present in the same fractions. From the data it was further hypothesised that the A. niger formamidase or Aspergillus formamidase that had not been characterized before had OTA degradation activities. 20 Example 5 Analysis of Formamidase and OTA degradation activity in A. niger transformants harboring the A. nidulans formamidase gene A. niger formamidase transformants were analyzed for formamidase activity and OTA degrading activity. As can be seen from Table 2 formamidase activity was detected in 25 12 out of 15 transformants. Surprisingly the formamidase A. niger was apparently not able to break down OTA as can be seen in the transformants that had high formamidase activity but low or no OTA degrading activity. This indicates that fungal formamidase may not have OTA degradation activity but had been co-purified with the OTA activity through the 4 purification steps. 30 Interestingly by integrating the foreign DNA as in the case of A. nidulans formamidase gene into the A. niger genome the inventors have succeeded by unknown mechanism in creating transformants having higher OTA degrading activities compared to the parent strain used for the transformation A. niger see for example transformants 7#, 2#, 8#, 11#, 15#. Note that 16# was wild type (parent strain). Control was the reaction WO 2012/032472 PCT/IB2011/053901 55 without the addition of broth. Fermentation broth was used for all these assays. The reaction mixture contained 10pl broth, 50pl OTA (lpg/ml), 100pl sodium phosphate (67mM, pH7 or pH9). For control, 10p] broth from 5-day old culture in a shake flask fermentation was replaced with 10pl water. At the end of the reaction 160pl 5 acetonitrile was added to stop the reaction, and filtered and 10pl injected for HPLC analysis. Based on the surprising discovery that formamidase of Aspergilli was not able to breakdown OTA the inventors undertook further research to identify if a second 10 candidate for OTA degradation, i.e. the hypothetical A. niger protein (An14g02080, XP_001400834) designated amidase 2 herein had OTA degrading activity. Example 6 Construction of a putative amidase 2 gene expression plasmid The putative amidase 2 gene from Aspergillus niger encodes the amidase protein of 15 480 amino acids (SEQ ID NO: 1). To construct the recombinant expression plasmid for A. niger amidase 2 gene, two primers GGAGATCTATCATGGTCCGCCGAATTG and AATCTAGACTAGTGATGGTGATGGTGATGCAGAAAAGGATTACGTG were used in a Pfu Ultra Il PCR reaction with genomic DNA template obtained from A. niger UVK143 strain (Ward et al., Appl. Microbiol. Biotechnol., 39: 738-743, 1993). The 20 PCR reaction was performed for 30 cycles of 95'C for 30 seconds, 50CC for 30 seconds and 72'C for 1 minute with the two primers. The final extension at 720C was done for 5 minutes and the reaction was chilled to 40C. The nucleotide sequence of the coding region of the resulting PCR amplicon, without the His tag, is listed as SEQ ID NO: 2. The PCR fragment was purified with a Qiagen spin column. It was digested 25 with restriction enzyme Bg/li and Xbal and cloned into the pGAPT plasmid vector (see e.g., US patent no. 6, 426,410) that had been digested with Bg/// and Xbal. The resulting plasmid, pGAPT-amidase 2 (shown in Figure 7) was confirmed by DNA sequencing to have a recombinant gene comprising A. niger amidase 2 inserted between the A. niger glucoamylase promoter and an A. tubingensis glucoamylase 30 terminator. The corresponding encoded protein sequence is shown in SEQ ID No. 4 which is identical to SEQ ID No. 1 except that six histidine residues were added at the c-terminal end of the protein to aid protein purification.

WO 2012/032472 PCT/IB2011/053901 56 Example 7 Construction of a recombinant amidase 2 production strain The AP4 strain of A. niger (Berka et al, Gene 86: 153-162, 1990) was transformed with the pGAPT-amidase 2 plasmid using a PEG-mediated protoplasts fusion transformation protocol. The transformation protocol utilized a modification of the 5 Campbell method (see, Campbell et at., Curr. Genet. 16:53-56 (1989)) with Lysing Enzyme from Trichoderma harzianum (Sigma, L1412). More than one hundred transformants were obtained and thirty transformants were selected on MM plate. The control AP4 parental strain and fifteen transformants were chosen to be grown in CMA plates first and the mycellium agar plugs containing both mycellium and spores 10 from CMA plates were transferred to the shake flasks containing 30 ml of culture medium. The transformants were grown for 6 days at 28'C. The mycellium pellets from control AP4 strain and all fifteen transformants were washed with water at least twice. The mycellium pellets were suspended in CelLytic Y Cell Lysis Reagent (Sigma, C4482). The intracellular proteins were extracted at room temperature for 2 15 hours. The suspensions were spun down and 15 pl of the supernatants containing extracted intracellular proteins were analyzed using SDS-PAGE gels (Figure 8). The culture broths were spun down in a microfuge for 10 minutes and 15 pl of the supernatant (extracellular proteins) were analyzed using SDS-PAGE gels (Figure 8).

WO 2012/032472 PCT/1B201 1/053901 57 co 00 C-0 (0 Cco N N ICD 0D 0 CC U) Z3 co _D 0) o_ Q) CD C) o ( 0N coN 0 LO Nj UC) I 0D co 4-C CDco C C CD c)oD N CO C) CO c - CO CD N C mB 1) (Y 10 C:) 0z - - -- 0 -. 0) oc 4-0 E) CO -) 1O I 10 a- 10 (D CO ) a) ( < - u 0 CO CO ) (D D a_ D < <-- <__ <_F- t-_ FH CO O 0 C 0 0 L WO 2012/032472 PCT/IB2011/053901 58 Table 3 shows the medium used (per liter) for growing Aspergillus niger and its transformants in 250ml siliconized flasks with 30ml of medium. The A. niger strains were first grown in CMA plates for 3-5 days and 2cm 2 plus were transferred to flask 5 and grown at 30'C for 5 days. For the wild type (control strain or parental strain 620), 0.5 mg/ml of uridine was supplemented. Corn Streep Solids 50g NaH 2

PO

4

.H

2 0 1g MgSO 4 .7H 2 0 1.0238173g 50% (w/v) Staley 7350 Corn Syrup 100mL Sodium Citrate Dihydrate 8g (Na 3

C

6

H

5

O

7 2H 2 O) pH to 5.8 - 6.0 Bring to volume with milliQ H 2 0. Strain can be grown in CMA plate (per liter): Dextrose (Glucose) 20g BD Bacto Malt Extract 20g BD Bacto Peptone 1g BD Bacto Agar 20g Autoclave and poor plates 10 Figure 8. The SDS-PAGE analysis of intracellular proteins of A. niger harboring the Amidase 2 gene. The protein band of amidase 2 was indicated by an arrow bar. M. molecular markers, AP4, the parent strain of A. niger. The transformants were numbered from 1 to 15. 15 Figure 9 shows SDS-PAGE of extracellular proteins of A. niger harboring the Amidase 2 gene. The protein band of amidase 2 is indicated by an arrow bar. The other major bands above the amidase 2 band were a-amylase and glucoamylase secreted by A.

WO 2012/032472 PCT/IB2011/053901 59 niger. The samples loaded to the gels were the fermentation brothes of the different transformants. In order to confirm the supposed amidase 2 band separated on SDS-PAGE was 5 indeed the product of the amidase gene, the band was cut out, digested with trypsin by in-gel digestion. The digests were separated by RP-HPLC and analyzed by mass spectrometry. This revealed 4 amino acid sequences which are found in the putative amidase 2 sequence (SEQ ID NO: 1) (Figure 18A). The four sequences identified were: to 1. EALQNGY; 2. ALPAGEVLGSY; 3. GAGPL; 4. SVGPQAPL. 15 Example 8 Characterization of recombinant amidase 2 with respect to its degradation of OTA and biochemical, catalytical parameters As shown in Figure 10, both the intracellular and extracellular fractions (broth) of the 20 15 transformants of A. niger harboring the putative protein coding gene (amidase 2) plus the parent strain (wt) were assayed for their capability to breakdown OTA. Figure 10 surprisingly shows that the hypothetical protein encoding A. niger gene from Genbank (An14g02080, Acc No. XP_001400834) encodes an active enzyme having OTA degrading activity and this active enzyme was found both intracellularly and 25 extracellularly. One can see the best transformants are transformant 3, 4, 7, 11, 13 and 14 as no detectable OTA was left (i.e. below the detectable level of 2pbb) after the incubation with the intracellular or the extracellular fractions (the broth). Compared to the parent strain (wt) A. niger, transformant 8, 10 and 15 also had much higher OTA degrading activity. 30 The recombinant amidase 2 was active at least between pH 3 to 9 in the degrading OTA. Figure 11 shows that the enzyme activity was the highest at pH 7.0 followed by pH 6 and 8; pH 5 and 9.

WO 2012/032472 PCT/IB2011/053901 60 The recombinant amidase 2 was apparently heat stable as seen from Figure 12 as more than 80% activity remained after 5-30 min pre-incubation at 80 'C compared to control where no enzyme was added. The reaction mixture of 5pl enzyme plus 4 5 pl 67 mM sodium phosphate (pH7.0) was incubated at 80 0C for 0-30 min before adding 5 another 50pi the phosphate buffer and 25 pl 1 pg/ml OTA, and incubated at 30 0C for 60 min. The reaction was stopped by adding 130pl acetonitrile containing 0.2% acetic acid (v/v). The reaction mixture was filtered and injected (10pl) for HPLC analysis. CtrI was control where no amidase was added. 10 Further more the recombinant amidase 2 was quite stable at 70 0C between pH 6 and 9. The stability decreased with the decrease in pH (Figure 13). Activity was assayed at pH 7.0 and 30 0. The pre-incubation mixture contained 5pl amidase 2 mixture (155pg protein/ml), 45pl 67mM phosphate buffer (pH3, 4, 5, 6, 7, 8 and 9). The samples were pre-incubated at 700C for 30min. After cooling to 30'C, 2pl 0.5mg/ml 15 OTA in ethanol and 80pl 0.2M Mops-NaOH (pH 7.0) were added and the reaction incubated at 30 0C for 60 min. The reaction was stopped by adding 130pl acetonitrile containing 0.2% HAC, filtered and injected on RP-HPLC with an injection of 5 pl to monitor the remaining OTA. 20 Example 9 Purification of the recombinant amidase 2 mat from the fermentation broth of A. niger harboring the amidase 2 gene The broth of transformant 13 which had high OTA activity (Figure 10) was used to purify the recombinant amidase 2 mat by ammonium sulfate fractionation, affinity 25 chromatography on Phenyl Sepharose, HIC on Butyl-Sepharose and AEX as described below. 1) (NH 4

)

2

SO

4 fractionation. To 40 ml of the A. niger broth harboring the A. niger amidase 2 gene was added (NH 4 ) 2SO4 to 40% saturation by stirring. After 10 min centrifugation at 3500 rpm, the supernatant was collected and (NH 4 ) 2SO4 added to 30 60% saturation by stirring and the solution centrifuged again. The pellet corresponding to the precipitate between 40 to 60% (NH 4 ) 2

SO

4 saturation was dissolved in 40ml of 50 mM tricine-HCI (pH7.0) containing 1 M (NH 4 ) 2SO4 and filtered with 0.22pm filter.

WO 2012/032472 PCT/IB2011/053901 61 2) Hydrophobic interaction/affinity chromatography. The filtered sample from the above step was loaded a Phenyl Sepharose FF column (2.6xlOcm). Following a wash with tricine-HCI (pH7.0) containing 1 M (NH 4 ) 2

SO

4 , the active fractions were eluted in 10ml per tube at a flow rate of 10ml/min with a linear gradient of 50 mM tricine-HC 5 (pH 7.0) from 1 M to 0 M of (NH 4 ) 2

SO

4 . The purification was achieved on Akta purifier system. 3) Anion exchange chromatography. The active fractions were pooled and desalted on a PD10 column equilibrated in 20 mM Tris/HCI, pH 7.5 (buffer A). The desalted 10 sample was applied to a Source Q15 column equilibrated in buffer A (10 ml/min). The column was washed with buffer A and the bound proteins were eluted with a linear gradient of 0-1 M NaCl in buffer A. During the gradient elution, fractions of 5 ml were collected. The active fractions were analyzed on SDS-PAGE (Figure 14A), which indicates that the amidase 2 mat was purified basically to homogeneity. The activity 15 was related to a protein band having a molecular mass just below 49 kDa on the SDS-PAGE (Figure 14). Mass spectrometry by Maldi-tof measurement indicates that this protein band may have a mass of 47214Da (+2%) (Figure 14B). N-terminal sequencing of fraction 12 indicate that the N-terminal of the Amidase 2 mat started at STDEAKVT (SEQ ID NO. 33). The full length of the 480aa amidase 2 with 6 histidine 20 residues is estimated to have a molecular mass of 51,983 Da while the N-terminal 42aa truncated amidase 2 with 6 histidine residues at its C-terminal is supposed to have a mass of 47,338 Da. These indicate that the amidase 2 secreted into the A. niger broth, i.e., amidase 2mat is the amidase 2 that has been processed by a signal peptidase or other peptidase in the culture broth cleaving between the peptide bond of 25 Ala-Ser in Amidase 2 sig. Example 10 Substrate specificity of amidase 2 Km/nVm determination: Using the fractions obtained in the AEX step of amidase 2 mat the kinetic constants of 30 Km and Vm of the enzyme degrading OTA were 0.29pM/min and 13pM, respectively by using the Lineweaver-Burk calculation method.

WO 2012/032472 PCT/IB2011/053901 62 Ochratoxin B degradation: The enzyme solution of Amidase 2 mat obtained after AEX was tested on ochratoxin B at the same concentration under optimum conditions for one hour. The degrading rate with ochratoxin B was 8% compared to OTA. 5 Example 11 The activity of pure amidase 2 mat in breaking down OTA Ochratoxin A (OTA) degrading activity of amidase 2 mat purified from the fermentation broth of A. niger harboring the amidase 2 gene as a function of its concentration is shown in Figure 15. It can be seen from Figure 15 that the 10 degradation of OTA was amidase 2 mat concentration dependent. One can also see that after 30min with an amidase 2 mat concentration of 160 ng/ml the OTA decreased by 83%. From the linear region of Figure 15 the calculated specific activity for amidase 2 mat was 900 nanomole OTA hydrolyzed per min per mg protein at pH7.0 and 400C at an OTA concentration of 1 pg/mI reaction mixture. The reaction for 15 Figure 15 was performed in 0.1 Mops-NaOH (pH 7.0) and 40'C for 30 min before it was stopped by adding equal volume of acetonitrile containing 0.2% acetic acid and analyzed on RP-HPLC on a C18 column as described in the Material and Method section. 20 Example 12 Differences in sensitivity to Ca2+ between the intracellular and extracellular amidase 2 found in A. niger. It was observed that the amidase 2 (i.e., amidase 2 mat) purified from the Amano TM lipase product or from the broth of transgenic A. niger harboring the amidase 2 gene were both inhibited by around 40% of its activity in the hydrolysis of OTA by 8 mM 25 CaCl 2 (Figure 16), while the intracellular fraction of amidase 2 (amidase 2 sig) of transgenic A. niger was not appreciably inhibited by CaCl 2 at 8 mM CaC1 2 (Figure 17). These results indicate that the loss of the N-terminal of amidase 2 resulted in sensitivity to Ca 2 + inhibition. The inhibition by Ca2* was overcome by the addition of divalent chelators including EDTA in an equal molar basis. Also it is known that the 30 OTA degrading activity in the Amano T M lipase product is sensitive to EDTA. This example indicates that use of amidase 2 mat in systems containing divalent ions may be aided by the addition of a chelator such as EDTA. Citric acid and other organic acid such as propionic acid are widely used as feed additives and can chelate the WO 2012/032472 PCT/IB2011/053901 63 divalent ion or trivalent ions, relieving their inhibition of amidase 2 mat. On the other hand extra EDTA also inhibited the recombinant Amidase 2 mat (data not shown). These results suggest that amidase 2 mat can be used for detoxification of OTA but has certain limitations due to its sensitivity to divalent or trivalent metal ions and to 5 chelators like EDTA, while amidase 2 sig was discovered to be much less sensitive to divalent or trivalent metal ions and to chelators like EDTA (see below) indicating the importance of the full length of amidase 2 instead of the secreted or processed or mature amidase 2 (i.e., amidase 2 mat) found in the A. niger broth. 10 Example 13 Degradation of OTA by the amidase 2 extracted from the intracellular fraction (Amidase 2 sig) of A. niger. This example shows the advantage of using the less sensitive form of amidase 2, i.e., amidase 2 sig. As can be seen from Figure 17, 20 mM CaC 2 or 20 mM EDTA had no inhibition to the amidase 2 sig compared to amidase 2 mat (Figure 16). This provides 15 a clear advantage for using amidase 2 sig in additives comprising a chelator in detoxifying OTA. The reaction contained 60pl mops-NaOH (0.2M, pH7.0), 20mM CaC 2 , 20mM EDTA, amidase 2 sig (0.46pg protein) extracted from A. niger cell harboring the amidase gene, 2pl 25pg/ml OTA and water to a final volume of 120pL. The reaction was performed at 370C for 30min and stopped by adding 150pl 20 acetonitrile containing 0.2% acetic acid. OTA and its product OT-alpha (=OTa) were analyzed on RP-HPLC on C18 column with an injection of 5pl. Positive control (PC) contained no additives other than the buffer the amidase and OTA. Blank contained only buffer and OTA. 25 Example 14 Degradation of OTA in corn flour and DDGS (Dried Distillers Grains with Solubles) by the amidase 2 sig extracted from the intracellular fraction (Amidase 2 sig) of A. niger. Corn is the major carrier for OTA and it can be further carried through to and concentrated in DDGS in bioethanol production. Corn and DDGS are widely used as 30 feed ingredients. Mycotoxin level in DDGS is the usual factor that limits the amount of DDGS that can be added to feed. The examples here indicate that ability of amidase 2 sig in degrading OTA in corn flour and DDGS: WO 2012/032472 PCT/IB2011/053901 64 For corn flour, 50mg corn flour was added 210pl water, 8 pl OTA(25pg/ml) and 20pl amidase 2 sig (3.lpg protein), mixed and incubated at 37'C for 22hr. The OTA concentration decreased from 800ppb at the start to 14ppb at the end. OTA analysis by HPLC is described in the materials and methods section above. 5 For DDGS, 1g was suspended in 1Oml water, pH adjusted to 6.7 with NaOH. To 210pl of this slurry was added 8pl OTA (25pg/ml) and 20pl amidase 2 sig (3.lpg protein), mixed and incubated at 370C for 22hr. The OTA concentration decreased from 800ppb at start to 5ppb at the end. OTA analysis by HPLC was described in the 10 materials and methods section above. Example 15 Degradation of OTA in milk by amidase 2 mat. It is known that many food products including milk and cheese can be contaminated with OTA. This example tests OTA degradation in milk using amidase 2 mat. 15 Light milk (1.5% fat, protein 3.4g, carbohydrate 4.7g Calcium 120mg, Phosphorus 95mg in 100ml) from Arla Foods Amba (Aarhus, Denmark) 0.15ml, 3pI OTA (2.5pg/ml), 7 .5pl purified amidase 2 mat (from the broth of A. niger harboring the amidase 2 gene) containing 0.22pg protein were mixed and reacted at 400C for 2.5hr. The OTA decreased from 47ppb at start to undetectable level (<2ppb) after 2.5hr 20 incubation. OTA analysis by HPLC was described in the materials and methods section above. Example 16 Amidase 2 mat in the detoxification of OTA in corn flour The reaction mixture in 1.5 ml eppendorf tubes (C1-3) comprised 60 mg corn flower, 25 200pI Mops-NaOH (0.2 M pH7.0), 4 0pl amidase 2 mixture (which was mixture of an equal volume of the broth of A. niger transformant having amdiase 2 gene 3, 4, 7, 8, 10, 11, 13, 14, and 15), 2 0pl OTA (0.5mg/ml). Total volume of the three components was 260pl; final OTA was 38pg/ml or 38ppm (ppm, parts per million). Control: Tubes of Controls (C-4-6) were the same as C1-3 except that the amidase preparation (the 30 broth) was replaced with water. The reaction was performed at 30"C with skaking for 20 hr before the addition of equal volume of acetonitrile 260pl containing 0.2% acetic acid to stop the reaction and WO 2012/032472 PCT/IB2011/053901 65 to extract the OTA. After centrifugation and filter, the filtrate was analyzed on HPLC. The HPLC results indicate that the remaining OTA was undetectable (less than 2ppb), indicating that amidase 2 mat was able to reduce the OTA in corn flour from 38 ppm to less than 2ppb. 5 Example 17 Amidase 2 mat in the detoxification of OTA in corn-soy based feed. The tests of amidase 2 mat with soy-corn based feed (whose composition is given in Table 3) contaminated with OTA was exactly as the test with corn flour, that is, tubes S1-3 (experimental) were with the broth while for tubes S 5-6 (controls) water 10 replaced the broth. After 20 hr incubation, the OTA level was reduced from 38 ppm to 6.6 ppm, a reduction of 6 times or 82%. Table 3. The composition of corn/soy based feed used Corn Diet Ingredients Percent Corn 60.01% Soybean meal 48 31.52% Soy oil 4.00% Salt 0.40% DL Methionine 0.20% Limestone 1.16% Dicalcium Phos 1.46% VIT/MIN 1.25% Total 100% 15 Example 18 Solving the crystal structure of Amidase 2 15 crystals of amidase 2 were obtained as shown in Table 4. 20 Crystals AM7 and AM15 were x-ray diffracted and the structure of these two crystals of amidase 2 (aa 45-480) was solved at 2.5A resolution.

WO 2012/032472 PCT/IB2011/053901 66 Amidase 2 was shown to have a structure similar to a Tim Barrel structure as shown in Figure 20 and the active site as shown in Figure 21. A summary of the data for the solved crystal structure for the AM7 crystal set is set out below. 5 Dataset: am7 Overall InnerShell OuterShell Low resolution limit 80.00 80.00 2.64 High resolution limit 2.50 7.91 2.50 Rmerge 0.182 0.044 0.611 10 Rmerge in top intensity bin 0.057 - Total number of observations 441395 14486 65195 Total number unique 46056 1566 6711 Mean((I)/sd(l)) 9.4 20.5 3.3 Completeness 96.5 93.3 97.6 15 Multiplicity 9.6 9.3 9.7 The structure was solved using Phaser and analyzing an ensemble of 5 most similar structures. 20 It was found that the active site of amidase 2 (SEQ ID NO:1) includes at least the following residues: His111, His113, His191, Lys246, His287, His289, His307, Asp378 and Val 253. It may accommodate at least two metal ions for catalysis and/structure purposes forming so-called a binuclear metal centre. 25 The coordinates for one monomer of the enzyme which is in the form of a tetramer are shown in Figure 22.

WO 2012/032472 PCT/IB2011/053901 67 Ti [i T I-l 4 I- - -[ - -4 u"- C40 ~ 1% C2 <7. O C CC -Ac -I-I II PA 0 i F IAA o I u J42I c Ni~ hiiiiNOJ 1 CG Ci r~ ~- < <c -- - r, - r - r III -)- - N- WO 2012/032472 PCT/IB2011/053901 68 Table 5. The structural elements of amidase 2 Color coding: white = smaller sandwich domain, Dark gray = catalytic barrel domain, 5 light gray = barrel strands and barrel helices; helix a7 may also be considered a barrel helix 1 48-58 2 65-74 3 76-83 -1 84-86 2 87-94 4 967105 14432-43 51 404-19-4 15 5 2 -04 7a 1_777-181RJ 71b 14-1464 Exm- 196xreso of9midsesfro fuglseisGoee 8 266-280 grmnioaM101 n 2tri9u0ni-3aeA01 3inTihoem 10 303s30. 3 r03 -3 - 44 10 312-2 12 _ _ 32-37 -~~~1 443-4481 2933 127 449-451 15 372-3484 16 443-448 Example 19. Expression of amidases from fungal species Glomerella graminicola MI.001 and Metarhizium anisopliae ARSEF 23 in Trichoderma 10 reeseii.

WO 2012/032472 PCT/IB2011/053901 69 To express amidases from Glomerella graminicola M1.001 and Metarhizium anisopliae ARSEF 23, both genes were synthesized as codon optimized sequences for expression in T. reesei and cloned in pDonor221 vector (Invitrogen, Carlsbad, CA, USA) by Life technologies (Germany). 6xHis codons were added at the 3' coding 5 region of each gene for purification purposes. To enable the expression of the both amidases in Trichoderma reesei, their coding sequences were cloned into the Gateway compatible destination vector pTTT-pyrG13 (described in W02010141779A1 and PCT/US10/57531) via the Gateway@ LR recombination reaction. This vector contains the T. reesei cbhl-derived promoter and terminator 10 regions allowing for a strong inducible expression of a gene of interest, the Aspergillus nidulans amdS and pyrG selective markers conferring growth of transformants on either acetamide as a sole nitrogen source or in the absence of uridine or both, and the T. reesei telomere regions allowing for non-chromosomal plasmid maintenance in a fungal cell. The cbhl promoter and terminator regions are separated by the 15 chloramphenicol resistance gene, CmR, and the lethal E coli gene, ccdB, flanked by the bacteriophage lambda-based specific recombination sites attR1, attR2. Such configuration allows for direct selection of recombinants containing a gene of interest under the control of the cbhl regulatory elements in the correct orientation via the Gateway@ LR recombination reaction. 20 LR recombination reactions between the pEntry clones of each amidase gene and the destination vector pTTT-pyrGl3 were done using the LR clonaseTM 1l enzyme mix according to the protocol from Invitrogen. Recombination products generated were transformed to Ecoli Max Efficiency DH5a, as described by the supplier (Invitrogen), and clones containing the expression constructs pTTT-pyrG13-Amidase G. 25 graminicola and pTTT-pyrG13-Amidase M. anisoplia were selected on 2xYT agar plates (16 g/L Bacto Tryptone (Difco, USA), 10 g/L Bacto Yeast Extract (Difco, USA), 5 g/L NaCl, 16 g/L Bacto Agar (Difco, USA)) with 100pg/ml ampicillin. After growth of bacterial cultures in 2xYT medium with 100pg/ml ampicillin, isolated plasmids were subjected to restriction analysis and clones with correct restriction pattern were used 30 for transformation of the Trichoderma reeei 2830 pyrG negative strain deleted for 4 major cellulases cbhl, cbhll, egli, egll (W02010141779A1). The final expression plasmids pTTT-pyrGl3-Amidase G. graminicola and pTTT-pyrGl3-Amidase M. anisopliae are shown in Figure 23-25. 0.5-2 pg of each expression plasmid was transformed in T. reesei using PEG 35 Protoplast method with slight modifications, as indicated. For protoplasts preparation, WO 2012/032472 PCT/IB2011/053901 70 spores were grown for 16-24 hours at 240C in Trichoderma Minimal Medium MM ( 20g/L glucose, 15g/L KH 2

PO

4 , pH 4.5, Sg/L (NH4) 2

SO

4 , 0.6g/L MgSO 4 x7H 2 0, 0.6 g/L CaCl 2 x2H 2 O, 1 ml of 1000X T. reesei Trace elements solution {5 g/L FeSO 4 x7H 2 0, 1.4 g/L ZnSO 4 x7H 2 O, 1.6 g/L MnSO 4 xH 2 O, 3.7 g/L CoCI 2 x6H 2 O }) with shaking at 150 5 rpm. Germinating spores were harvested by centrifugation and treated with 50mg/ml of Glucanex G200 (Novozymes AG, Switzerland) solution for lyses of the fungal cell walls. Further preparation of protoplasts was performed by a standard method, as described by Penttils et al. [Gene 61(1987) 155-164]. In general, transformation mixtures containing appx 1 pg of DNA and 1-5x 107 10 protoplasts in a total volume of 200 pl were treated with 2 ml of 25% PEG solution, diluted with 2 volumes of 1.2M sorbitol/10mM Tris, pH7.5/ 10mM CaC 2 solution, mixed with 3% selective top agarose MM containing 1 M sorbitol and 20 mM acetamide (the same Minimal medium as mentioned above but (NH4)2SO 4 was substituted with acetamide) and poured onto 2% selective agarose with acetamide. 15 Plates were incubated for 5-7 days at 280C before transformants grew and started sporulation. A spore mixture (106 spores/ml) harvested from a transformation plate was used to inoculate shake flasks with production medium (per 1L): Glycine production medium (4.7 g/L (NH 4

)

2

SO

4 , 33 g/L 1,4-Piperazinebis(propanesulfonic acid), pH 5.5, 6.0 g/L 20 glycine, 5.0 g/L KH 2

PO

4 , 1.0 g/L CaCl 2 x2H 2 0, 1.0 g/L MgSO 4 x7H 2 0, 2.5 ml/L of 400X T. reesei trace elements, 20 g/L Glucose, 6.5 g/L Sophorose). As a control, T. reesei recipient 2830 strain was grown under the same conditions but in the presence of 10 mM uridine. After 5 days of fermentation at 300 C and 200 rpm, cultures were harvested and subjected to enzymatic analysis from both extracellular and 25 intracellular extracts. Example 20. Expression of Aspergillus oryzae amidase in Aspergillus niger GICC#2445 strain To express putative amidase gene from A. oryzae in Aspergillus niger, the amidase 30 encoding sequence was amplified from the Aspergillus oryzae RIB40 genomic DNA (obtained from the Fungal Genetics Stock Collection, FGSC, USA) using the gene specific primers with attB1 and attB2 sites at the 5' and 3' termini for subsequent cloning via a Gateway approach. In addition the reverse primer contains 6 xHis encoding triplets to facilitate purification of the protein expressed.

WO 2012/032472 PCT/IB2011/053901 71 The following set of primers was used: Forward 5' GGGACAAGTTTGTACAAAAAAGCAGGCTCACCATGCACCGGATGCTCACAGACG ACAGAC3' 5 Reverse 5' ACCACTTTGTACAAGAAAGCTGGGTCTAGTGGTGGTGGTGGTGGTGTGATTTAA TCAGCATGCCGCCACGC 3'. All PCR reactions were amplified with a high fidelity Phusion DNA polymerase (Finnzymes OY, Espoo, Finland) under standard conditions recommended by the 10 supplier. After separation of the amplified DNA fragment on a 0.8% agarose gel, it was purified with a Nucleospino Extract PCR clean-up kit (Macherey-Nagel GmbH & co. KG, Duren, Germany) and 150 ng were recombined with the pDonor221 vector (Invitrogen, Carlsbad, CA, USA) according to recommendation of the supplier. E. coli DH5a colonies with pEntry clones containing the A. oryzae amidase were selected on 15 2xYT agar plates with 50pg/ml kanamycin. Plasmids isolated from bacterial cells were analyzed by their restriction digestion pattern for the insert presence and checked by sequence analysis using a ABI3100 sequence analyzer (Applied Biosystems). The resulting pEntry-Amidase A. oryzae plasmid was used for cloning into a destination vector pRAXdest2, as described in patent US07459299, via subsequent Gateway LR 20 reaction according to the protocol from Invitrogen (Carlsbad, CA, USA). Recombination products formed were transformed to E.coli Max Efficiency DH5a, as described by the supplier (Invitrogen), and clones containing the expression construct pRAX-Amidase A. oryzae (Fig.25) were selected on 2xYT agar plates (16 g/L Bacto Tryptone (Difco, USA), 10 g/L Bacto Yeast Extract (Difco, USA), 5 gIL NaCl, 16 g/L 25 Bacto Agar (Difco, USA)) with 100pg/ml ampicillin. After growth of bacterial cultures in 2xYT medium with 100pg/ml ampicillin, isolated plasmids were subjected to restriction analysis followed by sequencing using a ABI3100 sequence analyzer (Applied Biosystems). Besides a bacterial sequence, the final expression plasmid pRAX2-Amidase A. oryzae contains the A. oryzae amidase coding region under the 30 control of the A. niger glucoamylase promoter and terminator, the Aspergillus nidulans pyrG gene as a selective marker and the A. nidulans AMA1 sequence for autonomous replication in fungal cells. 0.5-2ug of this plasmid was transformed in A. niger var awamori GICC#2445 strain developed by Genencor using a common transformation procedure known in the art 35 or described in patent US07459299. This strain is deleted for the endogenous WO 2012/032472 PCT/IB2011/053901 72 glucoamylase glaA gene and carries a mutation in the pyrG gene allowing for selection of transformants for uridine prototrophy. A. niger transformants were grown on MM medium (the same minimal medium as was used for T. reesei transformation but 10 mM NH 4 CI was used instead of acetamide as a Nitrogen source) for 4-5 days 5 at 370 C and a total population of spores (106 spores/ml) from different transformation plates was used to inoculate shake flasks with production medium (per 1L): 12 g Trypton; 8 g Soyton; 15 g (NH 4

)

2

SO

4 ; 12.1 g NaH 2

PO

4 xH 2 0; 2.19 g Na 2

HPO

4 x2H 2 O; 1 g MgSO 4 x7H 2 O; 1 ml Tween 80; 150 g Maltose; pH 5.8. After 5 days of fermentation at 300 C and 200 rpm, cultures were harvested and subjected to enzymatic analysis 10 from both extracellular and intracellular extracts. Example 21. Amidases from three additional fungi for ochratoxin degradation The putative amidase sequences from Glomerella graminicola (GMGM), Metarhizium anisopliae and Aspergilius oryzae all showed activity in degrading ochratoxin A. 15 Table 6 shows the activity of three fungal amidases from Glomerella graminicola, Metarhizium anisopliae and Aspergilius oryzae expressed intracellularly in Trichoderma reesei and Aspergillus niger. The reaction mixture using OTA as substrate consisted of 75 pl phosphate (67mM, pH7), 5 pl OTA (2.4pg/ml) and 20pl 20 intracellular cell-free fraction. The reaction was performed at 23 0C for 17 h and stopped by adding 0.15ml acetonitrile and analyzed on HPLC with an injection volume of 5 pl. Table 6 Strains/and activities Trichoderma Trichoderma Trichoderma Aspergillus reesei parent reesei parent reesei parent niger strain strain used for strain strain transformed the transformed transformed with putative transformation with putative with putative amidase amidase gene amidase gene gene from from from Aspergillus Glomerella Metarhizium oryzae gramicola anisopliae Activity on ochratoxin A 12.26 0.68 0.96 8.57 expressed as ochratoxin A peak area WO 2012/032472 PCT/IB2011/053901 73 The data in the table indicate that the amidase from G. gramicola has the highest activity against ochratoxin, whereas the amidase from A. oryzae has lower activity against this substrate. 5 Example 22 Sequence comparison of putative amidase Sequences Sequence comparisons were done over the full length of the polypeptide sequences using the AlignX VectorNTI program (InvitrogenCorporation) using the default 10 parameters (Gap opening penalty -10, Gap extension penalty 0.1). As can be seen in Table 7 the putative amidases may have low overall homology. However, as can be seen from Figure 26 all of the polypeptides of SEQ ID NOs: 5-11, 13, 14 and 15 have the same motifs as amidase 2 (SEQ ID NO:1 and 3). The sequences are aligned with Multalin version 5.4.1 (F. CORPET, 1988, Nucl. 15 Acids Res., 16: 10881-10890). Parameters used: blosum62, Gap weight: 12, Gap length weight: 2, Consensus levels: high=90% low=50%, Consensus symbols: ! is anyone of IV, $ is anyone of LM, % is anyone of FY, # is anyone of NDQEBZ. Descriptions of the sequences and their web accession number (in parenthsis): 20 Furthermore, as shown in Example 21 (Table 6) SEQ ID NO:13-15 have ochratoxin degrading activity. Table7 Sequence ID NO % identity to SEQ ID NO:1 (full length) 25 SEQ ID NO:5 41.8% SEQ ID NO:6 42.4% SEQ ID NO:7 42.4% SEQ ID NO:8 40.7% SEQ ID NO:9 36.5% SEQ ID NO:10 28.2% SEQ ID NO:11 36.5% SEQ ID NO:13 56.1% 30 SEQ ID NO:14 53.4% SEQ ID NO:15 34.6% The common sequence motifs of the 11 sequences have the Consensus: 1) I/m-P-G-I/m-w/i-D-c/v/s/a-H-x-H-f/y/l-xG, where the two His residues are in the 35 active site; WO 2012/032472 PCT/IB2011/053901 74 2) G-y/f-T; 3) G-t/a/s/v/r-i/f/a-x-G-P; 4) G-H-g/s-D where the His residue is in the active site; 5) D-G-v/e-x-e/d/g-C-x-x-alg/t-v/a-R-x-q/m/a-l/i/v-R-r/h/c-g/n-A-k/r/t/e/d-x-l-K, where 5 the Lys residue is in the active site 6) G-G-V-l/m/v/g-S-x-x-D-x-P, where the Val residue is in the active site; 7) V-a/s/h-A-H-c/v/a-h/q-G-k/r-x-G, where the two His residues are in the active site; 8) H-g/v/a-s/t/i-ylf/e-l/a/i-D, where the His residue is in the active site; 9) G-V-x-l-a/v-l/a-G-T-D, where Asp residue is in the active site. 10 SEQ ID NO:1, is amidase 2 from A. niger, SEQ5, hypothetical protein from A. niger (XP 001400981), SEQ ID NO:6, hypothetical protein from A. flavus (XP_002385313), SEQ ID NO:7, hypothetical protein from Talaromyces stipitatus (XP 002477972), SEQ ID NO:8, hypothetical protein from Neurospora crassa 15 (XP_962238), SEQ ID NO:9, putative amidohydrolase from Streptomyces roseosporus (ZP_04712156), SEQ ID NO: 10, putative amidohydrolase from Thennotoga lettingae (YP 001470371), SEQ ID NO:11, putative amidohydrolase from Salinispora arenicola (YP_001536995), SEQ ID NO:13, putative amidohydrolase from Glomerella grarminicola (EFQ25792), SEQ ID NO:14, putative amidohydrolase from 20 Metarhizium anisopliae(EFZ00058), SEQ ID NO:15, hypothetical protein from Aspergillus oryzae (XP_001826758). Example 23 Sequence comparison of SEQ ID NO:1 to carboxypeptidase having ochratoxin degrading activity 25 Two carboxypeptidases are known which have low ochratoxin degradding activity, namely bovine carboxypeptidase A and yeast carboxypeptidase Y (Luis Abrunhosa, Robert R. M. Paterson and Armando Venancio, Biodegradation of Ochratoxin A for Food and Feed Decontamination, Toxins 2010, 2, 1078-1099 ). 30 The ochratoxin degrading activity of these enzymes has been confirmed. However, as can be seen from Figure 27, these enzymes show no homology to SEQ ID NO:1 and do not contain any of the 9 motifs associated with ochratoxin degrading ability. Furthermore, these enzymes do not have the amidase typeTim Barrel structure.

WO 2012/032472 PCT/1B201 1/053901 75 SEQUENCE LISTING SEQ ID NO: 1 5 Amidase 2 sig from A. niger MvVRRIASATP MVQSPMSPLG TTYCVRPNPV SLNLQRRPLV IASTDEAKVT 51 IYAGLLIPG DGEPLRNAAL XTSDKIIAFV GSEAIPKKY LRSTOSTHRV 11PVLMPGLWD~C E-MHFGGDDDY YNDYTSGLAT HPASSGARLA RGCWEAL NG 11YTSYRDLAGY GCEVAKAIND GTIVGPNVYS SGAALSQTAG HGDTFALPAG ,201 EVLGSYGVYSN PRPGYWGAGP LCIADCVEEV KRAVRLQIRRP GAKVIKV\YJAS 21GGVMSRDDNP NFAQFSPEEL KVIVEEAARQ NRIVSAHVG KAGIMAAIKA 01GCKSTIEHVSY ADEEVWELMK EKGILYVATR SVIEIFLASN GEGLVKESWA 31KLQALADSHL KAYQGAIKAG VTIALGTDTA PGGPTALELQ F-7)VERGGMTP 401 LEAIK-AATAN APLSVG3PQP TGQLREGYE ADVILALEENP LEDTKVFQEP K41TAVTHVWKGG KLFKGPGIGP WGEDAPI'PFL SEO ID NO. 2 Sequence type: DNA; DNA name: DNA containing amidase 2 gene, 10 Length: 1443, Organism: Ao-pergillus niger ATGGTCCGCC GAATTGCTTC AGCTACACCT ATGGTGCAAT CGCCCATrGTC GCCATTGGOC ACAACATACT GCGTCCGTCC TAATCCTGTT TCACTGAATC TTCAAAGAAG ACCTCTCGTG ATCGCATCAA CAGACGAGGC CAAGGTCACT ATAATATATG CCOGACTATT AATCCCTGGC 15 GACCGG GAAC CTCTGCGCAA TGCTGCCCTA GTCATCAGCG ATAAOATCA T CGCGTTCGTT GGATCCGZAAG CCGACATCCC TAAGAAATAC CTCCGOTCCA CGCAOTCTAC TCATCGTGTC CCCOTGCTCA TGCCTGGTTT GTGGOATTGC CACATGCATT TTGGCGGGA TGACGATTAT TACAACGATT ATACATCTGG TCTGGCCACT CATCCAOCAT CATCAGGTGC TCGACTAOCC CGTGGTTOCT GGGAAGCATT GCAG-AATGGG TATACATCCT ACCGCGACCT AGCCGGATAC 20 GGGTGCGACG TCGCAAAGGC GATCA-ATGAT GGCACTATCO TTGGTCCAAA CGTGTACTCG TCTGGCGCTG CACTCAGTCA GACAGCTGGA CACGGCGATA TCTTCGCTCT TCCAGCAGGC GAA7 GTACTGG GGAGTTATGG AGTAATGAAC CCACGCCCTG GGTACTOOGG GOCAGGGCCG CTATGTATCG CCGATGGCGT AGAGGAGGTC CGACGAGCAG TGAGGTTGCA CATCCGTCGC GOTGCAAAGG TTATCAAAGT GATGGCCTCT GGGGGTGTCA TGTCGCGAGA CGATAATCCC 25 AACTTTGCAC AGTTCTCTCC AGAAGAACTG IAAGGTGATAG TGGAAGAGGC GGCTCGACAO AACCGGATCG TTTCTGCACA TGTGCATOOC AAGGCGGGGA TTATGGCTGC TATCAAAGCA GOCTGCAAGA GTCTGGAGCA TGTGTCTTAT GCTGACGAGG AGGTCTGGGA GCTCATGAAA GAGAAGGGAA TTTTGTATGT GGCCACACGC TCGGTTATTG AAATCTTTCT GGCTAGTAAT GGAGAGGGGT TGOTGAAAGA GTCGTGGGCC AAGTTGCAGG CCCTTGCCGA TTCGCATTTG 30 AAAGCTTATC AGGGAOCTAT TAAGGCGGGT GTTACCATTO CGTTGGGAAC GOATACCGCC CCCGGTGGTC CTACCGCACT TGAGTTGCAG TTTGCCGTFCG AOAGAGGAGG TATGACGCCG TTGGAGOCCA TCAAAGCCGC AACTGCGAAC GCTCCCCTGT CAGTTGGTCC ACAAGCACCG TTGACGGGTC AC-CTTCGCGA GGGGTATGAG GCAGATGTGA TTGCOTTGGA GGAGAATCCA TTOGAGGACA TCAAAGTCTT TCAGGAGCCG AAOOCAGTTA CCCACGTCTG GAAGGGAGGG 35 -AAACTGTTCA AAGGTCCAGG TATTGGTCCG TGGGGAGAAG ATGCACOTAA TCCTTTTCTG

TAG

WO 2012/032472 PCT/IB2011/053901 76 SEQ ID NO. 3 Sequence type: Protein; Protein name: amidase 2 mat, Length: 438, Organism: Aspergillus niger 5 SerThrAspGluAlaLysValThrIleIleTyrAlaGlyLeuLeuIleProGlyAspGly GluProLeuArgAsnAlaAlaLeuValIleSerAspLysIleIleAlaPheValGlySer GluAlaAspIleProLysLysTyrLeuArgSerThrGlnSerThrHlisArgValProVal LeuMetProGlyLeuTrpAspCysHisMetHisPheGlyGlyAspAspAspTyrTyrAsnl AspTyrThrSerGlyLeuAlaThrHisPrOAlaSerSerGlyAlaArgLeuAlaArgGly 10 CysTrpGluAlaLeuGlnAsnGlyTyrThrSerTyrArgAspLeuAlaGlyTyrGlyCys GluValAlaLysAlaIleAsnAspGlyThrIleValGlyProAsnValTyrSerSerGly AlaAlaLeuSerGlnThrAlaGlyHisGlyAspIlePheAlaLeuProAlaGlyGluVal LeuGlySerTyrGlyValMetAsnProArgProGlyTyrTrpGlyAlaGlyProLeuCys IleAlaAspGlyValGluGluValArgArgAlaValArgLeuGlnIleArgArgGlyAla 15 LysValIleLysValMetAlaSerGlyGlyValMetSerArgAspAspAsnProAsnPhe AlaGlnPheSerProGluGluLeuLysValIleValGluGluAlaAlaArgGlnAsnArg IleValSerAlaHisValHisGlyLysAlaGlyIleMetAlaAlaIleLysAlaGlyCys LysSerLeuGluHisValSerTyrAlaAspGluGluValTrpGluLeuMetLysGluLys GlyIleLeuTyrValAlaThrArgSerValIleGluIlePheLeuAlaSerAsnGlyGlu 20 GlyLeuValLysGluSerTrpAlaLysLeuGlnAlaLeuiAlaAspSerHisLeuLysAla TyrGlnGlyAlaIleLysAlaGlyValThrIleAlaLeuGlyThrASpThrAlaProGly GlyProThrAlaLeulGluLeuGlnPheAlaValGluArgGlyGlyMetThrProLeuGlu AlaIleLysAlaAlaThrAlaAsnAlaProLeuSerValGlyProGlnAlaProLeuThr GlyGlnLeuArgGluGlyTyrGluAlaAspValIleAlaLeuGluGluAsnProLeuGlu 25 AspIleLysValPheGlnGluProLysAlaValThrHisValTrpLysGlyGlyLysLeu PheLysGlyProGiyIleGlyProTrpGlyGluAspAlaArgAsnProPheLeu WO 2012/032472 PCT/IB2011/053901 77 SEQ ID NO. 4 Amidase 2 A. niger +c terminal GH MVRRIASATPMVQSPMSPLGTTYCVRPNPVSLNLORRPLVIASTDEAKVTIIYAGLLIPGDGE PLRNAALVISDKIIAFVGSEADIPKKYLRSTQSTHRVPVLMPGLWDCHMHFGGDDDYYNDYTS GLATHPASSGARLARGCWEALQNGYTSYRDLAGYGCEVAKA.INDGTIVGPNVYSSGAALSOTA GHGDIFALPAGEVLGSYGVMNPRPGYWGAGPLCIADGVEEVRRAVRLQIRRGAKVIKVIASGG VMSRDDNPNFAQFSPEELKVIVEEAARQNRIVSAHVHGKAGIMAAIKAGCKSLEHVSYADEEV WELMKEKGILYVATRSVIEIFLASNGEGLVKESWAKLQALADSHLKAYQGAIIKAGVTIALGTD TAPGGPTALELQFAVERGGMTPLEAIKAATANAPLSVGPQAPLTGOLREGYEADVIALEENPL EDIKVFOEPKAVTHVWKGGKLFKGPGTGPWGEDARNPFLHHHHHH 5 SEQ ID NO. 5 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 420, Organism: Aspergillus niger MetHisArgProLeuThrAspSerThrPheTyrArgIleAsnAlaAspIleLeuIlePro 10 GlyArgGlyAlaProIleAlaArgGlyAlaValValTrpLysSerLysThrIleLeuTyr SerGlyProGlnHisGluValProValGluTyrGlnAspAlaValThrThrHisValPro ValAlaMetProGlyMetTrpAspCysHisIleHisPheLeuGlyAlaThrAlaAlaThr MetAspSerIleValAsnThrProGlnAlaLeuAlaGlyAl aArgSerValProTyrLeu TyrAlaThrIleIleAlaGlyPheThrSerValArgGluValGlyGlyTyrGlyCysGlu 15 LeuAlaLysValIleAspGluGlyArgIleProGlyProThrIleTyrGlyAlaHisSer AlaIleSerMetThrAlaGlyHisGlyAspValHisGlyValAsnProGluGlyLeuArg AspLeuCysThrHisGlyLeuProLeuThrIleAlaAspGlyValProGluCysLeuGln AlaValArgLysGlnLeuArgHisGlyAlaArgIleIleLysValCysAlaSerGlyGiy ValValSerAlaTleAspAspProGlnHisGlnGluPheSerPheGluGluLeuLysAla 20 IleValAspGluAlaAlaArgAlaArgArgValValAlaAlaHisCysHisGlyLysAla GlyIleMetAsnAlaLeuArgAlaGlyCysArgThrIleGluHisGiySerTyrLeuAsp GluGluAlaIleAspLeuMetLeuGluLysGlyAlaMetLeuValAlaThrArgSerVal IleGluSerGlyLeuAlaMetArgAspLeuPheThrProGlySerTyrGlnLysLeuLeu GluValAlaAspThrHisLysArgAlaTyrGluLeuAlaValArgArgGlyValProIle 25 AlaLeuGlyThrAspGlnPheIleSerSerAspAsnProAlaLeuGlyTyrGlyArgAsn GlyLysGluLeuValTyrAlaValAlaAlaGlyMetThrProLeuAlaAlaIleGluAla AlaThrAlaAsnGlyProLeuThrLeuGlyAspGlnAlaProLysSerGlyGlnLeuArg GluGlyPheAspAlaAspIleIleAlaLeuThrAlaAsnProLeuGluAsnIleIleVal WO 2012/032472 PCT/IB2011/053901 78 ValSerAspProLysAsnValThrHisValTrpArgTyrGlyLysLeuValLysSerAsn SEQ ID NO. 6 Sequence type: Protein; Protein name: putative amidase 2 5 homologue, Length: 419, Organism: Aspergillus flavus MetHisArgMetLeuThrAspAspArgLeuTyrArgValAspAlaAspLeuLeuIlePro GlyLysGlyAspProIleProHi sGlyAlaValValTrpGlnCysLysThrIleArgTyr AlaGlyProArgSerGluValProAlaGluPheGlnGlyAlaThrThrThrHisValPro 10 ValValMetProGlyMetTrpAspCysHisIleHisPheLeuGlyAlaThrAlaAlaThr MetAsnAlaIleValAspThrProGlnAlaLeuAlaGlyAlaArgSerValProAspLeu HisAlaThrValMetAlaGlyPheThrSerValArgGluValGlyGlyTyrGlyCysAsp LeuAlaLysAlaValGlyGluGlyArgIleProGlyProAsnIleTyrSerSerHisSer AlalleSerMetThrAlaGlyHisGlyAspValHisGlyValHisArgAspSerLeuLeu 15 AspLeuCysAlaHisGlyLeuProLeuThrIleAlaAspGlyValProGluCysLeuLeu AlaValArgLysGlnLeuArgArgGlyAlaThrValIleLysValCysAlaSerGlyGly ValValSerAlaIleAspAspProGlnHisGlnGluPheSerPheGluGluLeuLysAla IleValAspGluAlaAlaArgAlaArgArgValValAlaAlaHisCysHisGlyLysAla GlyIleMetAsnAlaLeuArgAlaGlyCysArgThrIleGluHisGlySerPheLeuAsp 20 GluGluAlaValGluLeuMetLysGluLysGlyAlaIleLeuValAlaThrArgSerVal IleGluSerGlyLeuAlaMetLysAspLeuPheThrProSerSerTyrGlnLysLeuLeu GluValAlaAspAlaHisArgLysAlaTyrGlnLeuAlaIleSerArgGlyValThrIle AlaLeuGlyThrAspGlnPheIleSerSerAspAsnProMetIleGlyTyrGlyArgAsn GlyHisGluValArgTyrAlaValAspAlaGlyLeuThrProLeuAlaAlaIleGluAla 25 AlaThrAlaAsnGlyProLeuThrLeuGlyTyrGlnAlaProGlnSerGlyGnLeuLys GluGlyTyrAspAlaAspIleIleAlaValArgGluAsnProLeuGluAsnValAlaVal LeuSerAsnSerLysAsnValThrHisValTrpArgGlyGlyMetLeuIlleLysSer SEQ ID NO. 7 30 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 405, Organism: Talaromyces stipitatus MetArgValArgLeuLysAlaSerIleLeuIleProGlyArgGlyGluProIleGluAsn GlyAlaLeuIleIleAspGlyProLysIleAlaTrpValGlyGlnGlnSerAlaIlePro 35 ThrLysTyrGlnAspValAspPheGluTyrLeuProValLeuMetProGlyLeuTrpAsp WO 2012/032472 PCT/IB2011/053901 79 CysHisThrHisPheMetGlyLeuSerAspGluSerAspThrMetGlnAlaValPheGly SerAlaAlaLeuAlaGlyAlalleAlaAlaLysGluLeuGluThrAlaLeuMetAlaGly PheThrThrIleArgGluValGlyGlyValAlaGlyGluIleTyrProAlalleLysAsn GlyThrIleValGlyProAsnValTyrSerSerIleGlyValLeuGlyIleThrGlyGly 5 HisSerAspValHisAsnValProIleGluAlaValIleAlaLysArgAsnGluGlyThr PheValValCysAspGlyValSerAspCysTleLysThrValArgMetMetValArgArg GlyAlaThrLeuIleLysIleCysAlaThrGlyGlyValGliySerLeuLeuAspAspPro GluAspAlaGlnPheSerProGluGluIleLysAlaIleValAspGluAlaAlaArgSer LysArgIleValAlaAlaHisCysHisGlyLysGluGlyIleMetAsnAlaLeuHisAla 10 GlyVali-isThrIleGluHisGlySerTyrLeuAspGluGluValAlaAlaLeuMetLys GluLysLysAlaLeuPheValSerThrArgLeuI leIleGluGluGlyLeuLysAsnPro LysLeuTrpProProSerSerTyrArgLysLeuThrLysIleSerGluAlaHisLysLys AlaTyrAlaLeuAlaValLysSerGlyValLysIleValLeuGlyThrAspTrpThrAla GlyGluAsnGlyLysGluLeuAlaTyrAlaValGluAlaGlyMetSerProLeuGluAla 15 IleGluAlaSerThrAlaArgCysProGluThrLeuGlySerHisPheAlaProLeuSer GlyGlnLeuLysGluGlyTyrGlyAlaAspValIleAlaValAlaSerAsnProLeuAsp AspIleLysValLeuGlyGluProLysAsnIleThrHisValTrpLysGlyGlyLysLeu TyrLysGlyThrLeu 20 SEQ ID NO. 8 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 440, Organism: Neurospora crassa MetGlnIleLysValThrLeuProAsnAspThrIleAsnArgAspSerValAspAspArg 25 AlaSerTyrHisGlyIleLeuAlaAspValLeuIleProGlyArgGlyGluProLeuLys AsnGlyAlaLeuValValLysAspSerValIleGluTrpValGlyProSerAspGluI le ProSerGluTyrSerSerIleArgValSerArgValProValLeuMetProGlyMetTrp AspValHisThrHisTyrGluGlyValGlyValAlaGlnGlyIleArgGluSerMetLys ProPheLeuProGlyThrAlaThrLeuIleGlyAlaValIleValAspAspMetArgArg 30 ThrLeuMetAlaGlyPheThrSerTleArgGluLeuGlyGlyTyrAlaGlyAspValAla ProAlaIleAspMetGlyAlaIleValGlyProHisValTyrAlaAlaMetSerLeuLeu SerIleThrGlyGlyHisGlyAspLeuHisAspValProLeuArgThrValLeuAspAla CysAlaAsnGlySerSerSerCysPleLeuCysAspGlyValAspGlyCysIleAsnAla ValArgGlnGlnIleArgArgGlyAlaLysValIleLysValCysSerThrGlyGlyVal 35 LeuSerLeuAsnAspGlnProGluAspThrGlnPheSerAlaGluGluLeuArgAlalle WO 2012/032472 PCT/IB2011/053901 80 ValGflGluAlaLysArgSerSerArgValValAlaAlaHisAlaHisGlyLysProGly IleMetAlaAlaLeuAspAlaGlyValLysSerIlieGluHisGlySerPheLeuAspGlu GluValAlaAlaLysMetLysGluLysAspAlaIleLeuValProThrArgHisValVal GluGlyMetAlaAlaAsnAsnAspAspLeu.AspProArgGinArgAlaLysLeuGluArg 5 ThrMetGlnLeuSerArgAspSerIleLysLeuAlaHisArgMetGlyValLysIleAla LeuGlyThrAspThrPheArgSerAspLysAsnHisAlaValAlaHisGlyLysAsnAla MetGluLeuArgTyrAlaIleGluAlaGlyMetThrProLeuGlnAlaIleGluMetAla ThrAlaThrProProGluThrLeuGlyPro(lnAlaArgLysSerGlyGlnLeuLysAla GlyTyrAspAlaAspLeuleAlaIleSerSerAsnProLeuGluAspIleGluIleLeu 10 IleAspProAspAsnIleThrHisValTrpLysGlyGlyValLeuPheLysCysProGln SEQ ID NO. 9 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 413, Organism: Streptomyces roseosporus 15 MetGluHisArgIleAspAlaAspLeuLeulIleProGlyAlaGlyGluProThrValAsn GlySerValValHisAlaAspGlyArgIleArgPheAlaGlyProThrAlaGluLeuPro ArgGluHisArgAlaLeuGluProThrArgValAlaThrLeuLeuProGlyLeuTrpAsp CysHisValHisPheAlaGlyIleArgGlyArgValSerThrGluGluLeuMetLeuThr 20 ProGluThrLeuAlaValValArgSerValLysAspAlaGluThrAlaLeuArgAlaGly PheThrSerValArgAspMetGlyGlyHisGlyCysValLeuAlaGluAlaValArgGlu GlyThrPheThrGlyProAsnIleTyrSerAlaAsnGlnValIleGlyGlnThrGlyGly HisSerAspAlaHisArgLeuProTyrArgTrpValThrAspProCysArgSerGlyGly ThrLeuArgIleAlaAspGlyValAspGluCysValArgAlaValArgLeuGlnLeuArg 25 AlaGlyAlaGluLeuIleLysIleCysThrSerGlyGlyValLeuSerGlu2ValAspAsn ProValHisGlnGlnTyrArgSerGluGluLeuAsnAlaIleValThrGluAlaAlaArg AlaAspArgValValAlaAlaHisCysHisGlyArgAlaGlyIleLeuAlaAlaIleAsp AlaGlyCysHisThrValGluHisGlyThrGluIleAspGluArgThrAlaAspLeuMet AlaGluArgGlyMetThrLeuValProThrArgThrIleTyrGluAlaPheArgGlnAsp 30 ValAlaAlaLeuProProAlaTrpArgAspArgPheAlaLeuMetAlaGluArgHisLeu ThrAlaIleGlyIleAlaHisArgAlaGlyValThrIleAaLeuGlyThrAspLeuGly ThrSerAspArgGlyGlyProLeuSerTrpGlyGlylisGlySerGluPheAlaHisLeu ValSerAlaGlyLeuSerProLeuGluAlaIleLysAlaAlaThrAlaHisGlyProGly ThrLeuGlyProArgAlaProArgSerGlyArgLeuGluAlaGlyTyrAspAlaAspLeu 35 LeuAlaValAspGlyAsnProLeuAlaAspIleThrValLeuAlaAspProAspArgIle WO 2012/032472 PCT/IB2011/053901 81 ThrArgValTrpLysSerGlyGluProValProSerGly SEQ ID NO. 10 Sequence type: Protein.; Protein name: putative amidase 2 5 homologue, Length: 407, Organism: Thermotoga lettingae GluSerAlaValPheIleGluGlyAlaArgIleLeuAlaValGluLysIleLysArgSer GlnIleProSerGlyPheThrGlnIleAspLeuGinGlyArgTyrLeuMetProGlyLeu IleAspAlaHisLeuHisLeuAlaGlyMetArgSerGly spMetValLysGluHisLeu 10 LeuThrProTyrGluThrLeuValAlaArgThrValThrAspLeuLysSerLeuIleGlu AlaGlyPheThrThrValValAspAlaGlyGlySerIleAlaIleAsnLeuLysLysAla IleGlnGluGlyThrIleAlaGlyProArgIleValAlaAlaGlyHisSerLeuSerGln ThrPheGlyHisGlyAspGluHisPheLeuProIleAspTyrValAspProArgThrSer LysPheLysGlyGlyPheGlySerLeuIleCysAspGlyValAlaGluCysIleLysAla 15 AlaArgTyrAlaLeuArgCysGlyAlaAspPheIleLysTleMetAlaThrGlyGlyVal LeuSerGluArgAspArgProGluTyrThrGlnPheThrValGluGluIleLysAlaIle ValGluGluAlaAsnHisAlaArgLysPheValHisAlaHisAlaGlnOlyLysAspGly IleMetAsnAlaLeuLeuGlyGlyValLysValIleAlaHlisAlaIleTyrIleAspAsp GluSerCysLysLeuAlaLysGluLysAsnAlaIleIleValProThrLeuSerIleVal 20 GluHisLeuIleIleHisGlyLysGlnIleGlyAlaProGluTrpGlyLeuArgLysSer GluGluValTyrLysIleHisValGluAsnIleLysLysAlaTyrGluHisGlyValLys IleAlaAlaGlyThrAspPheIleGlyGlyThrLysAlaPheLysHisGlyGluAsnAla LeuGlul ieLeuLeuLeuValAspLysIleGlyMetLysProiUGllnAlaLeuLeuSer AlaThrLysValAlaAlaGluAlaAlaGlyLeuSerGlnLeuValGlySerIleAspLys 25 GlyLysLeuAlaAspLeuLeuleValGluAspAsnProI~euSerAsnValLysIleLeuI MetAspHisSerLysIleSerAlaValPheLysGluGlyIleLeuPheLysAspLysIle GlyLeuGluLysTyrPheAsn SEQ ID NO. 11 30 Sequence type: Protein; Protein name: putative amidase 2 homologue, Length: 408, Organism: Salinispora arenicola MetIleGluCysIleGluAlaAspGlnLeuIleProGlyArgGlyGluProValAlaAsn AlaValValValLeuGluAspAlaThrIleArgTyrAlaGlyProAlaGluHisAlaPro 35 LysValAlaGluAlaArgArgSerArgAlaHisThrValLeuProGlyLeuTrpAspSer WO 2012/032472 PCT/IB2011/053901 82 HisValHisPheMetGlyLeuArgSerAlaAspValGlyIleLeuProGlnGluProVal AlaLeuArgAlaAlaArgThrValAlaAspLeuArgAlaAlaLeiAspAlaGlyPheThr SerValArgGluValGlyGlyLeuGlyLeuAspLeuAlaArgAlaValGluGluGlyThr AlaValGlyProSerValTyrAlaAlaGlyCysAlaLeuSerThrThrGlyGlyHisGly 5 AspLeuHisSerTyrProLeuAlaTrpMetGluGluPheAlaArgHisGlyGlyGluLeu ArgLeuAlaAspGlyGluAlaGluCysValArgAlaValArgGluGlnLeuArgArgAsn AlaLysValIleLysValTyrAlaSerGlyGlyValLeuSerGluValAspHisProIle HisArgGlnPheThrAspArgGluLeuArgAlaIleValGluValAlaGlyLeuAlaAsp ArgValValAlaAlaHisCysHisGlyLysProGlyMetMetAlaAlaIleGluAlaGly 10 ValArgThrIleGluHisGlyThrTyrLeuAspGluGluValAlaAlaAlaMetArgGlu ThrGlyAlaIleLeuValThrThrArgThrIleMetGlnGluLeuIleAspSerArgAla LeuProProTyrAlaLeuArgLysLeuGluSerIleValAspArgHisAlaGluAlaIle ValIleAlaArgGluSerGlyValArgIleAlaAlaGlyThrAspValAlaLeuThrGly AlaGluLeuProAspSerTrpGlyArgAsnGlyArgGluLeuProLeuLeuAlaGluIle 15 GlyPheSerProLeuGluValIleGluAlaAlaThrAlaAlaAlaProAlaThrLeuGly ProGlnAlaProArgSerGlyGlnLeuValGluGlyTyrAspAlaAspValIleThrLeu AspAlaAspProLeuAlaAspIleGlyValLeuAlaLysProAlaHisIleThrGlyVal TrpLysAlaGlyCysArgValAla 20 SEQ ID NO. 12. Sequence type: Protein; Protein name: pepAd2 (protease), Length: 480, Organism: Aspergillus niger MetHisLeuProGlnArgLeuValThrAlaAlaCysLeuCysAlaSerAlaThrAlaPhe 25 IleProTyrThrIleLysLeuAspThrSerAspAspIleSerAlaArgAspSerLeu1Ala ArgArgPheLeuProValProAsnProSerAspAlaLeuAlaAspAspSerThrSerSer AlaSerAspGluSerLeuSerLeuAsnIleLysArgIleProValArgArgAspAsnAsp PheLysIleValValAlaGluThrProSerTrpSerAsnThrAlaAlaLeuAspGlnAsp GlySerAspIleSerTyrIleSerValValAsnIleGlySerAspGluLysSerMetTyr 30 MetLeuLeuAspThrGlyGlySerAspThrTrpValPheGlySerAsnCysThrSerThr ProCysThrMetHisAsnThrPheGlySerAspAspSerSerThrLeuGluMetThrSer luGluTrpSerValGlyTyrGlyThrGlySerValSerGlyLeuLeuGlyLysAspLys LeuThrIleAlaAsnValThrValArgMetThrPheGlyLeuAlaSerAsnAlaSerAsp AsnPheGluSerTyrProMetAspGlyIleLeuGlyLeuGlyArgThrAsnAspSerSer 35 TyrAspA.snProThrPheMetAspAlaValAlaGluSerAsnValPheLysSerAsnIle WO 2012/032472 PCT/IB2011/053901 83 ValGlyPheAlaLeuSerArgSerProAlaLysAspGlyThrValSerPheGlyThrThr AspLysAspLysTyrThrGlyAspIleFhrTyrThrAspThrValGlySerAspSerTyr TrpArgIleProValAspAspValTyrValGlyGlyThrSerCysAspPheSerAsnLys SerAlaIleIleAspThrGlyThrSerTyrAlaMetLeuProSerSerAspSerLysThr 5 LeuHisSerLeuIleProGlyAlaLysSerSerGlySerTyrHisIleIleProCysAsn ThrThrThrLysLeuGflnValAlaPheSerGlyValAsnTyrThrIleSerProLysAsp TyrValGlyAlaThrSerGlySerGlyCysValSerAsnIleIleSerTyrAspLeuPhe GlyAspAspIleTrpLeuLeuGlyAspThrPheLeuLysAsnValTyrAlaValPheAsp TyrAspGluLeuArgValGlyPheAlaGluArgSerSerAsnThrThrSerAlaSerAsn 10 SerThrSerSerGlyThrSerSerThrSerGlySerThrThrThrGlySerSerThrThr ThrThrSerSerAlaSerSerSerSerSerSerAspAlaGluSerGlySerSerMetThr IleProAlaProGlnTyrPhePheSerAlaLeuAlaIleAlaSerPheMetLeuTrpLeu SEQ ID NO:13 15 Organism Glomerella graminicola M1.001 (fungus), 472aa 1 mggsfrpnya dgpeppfspt kkttlviikt sllipgdgep lkdgalviss kviawvgpqs 61 slpseyadsp hrsytvpylm pglwdchahf ggespnddgg ndpyqvfite hpaasgarlt 121 rgcwlalqrg ytslrdvagl gcevsraied gsivgpnvys sgsglsqlag hgdifslpag 20 181 dvllnlgvsq itpghfgtha tmivdgvdec rravrlqirr gakcikvmas ggvmsrddnp 241 nyaqfsaael etiveeatrq nrvvaahvhg kagilaaina gvttlehasf adrecidlik 301 ekgivyiatr ivvhlllstg gkglpptvwe kaklvaknhm taykmaiesg vqialgtdtg 361 pgynmatele caveagmsnl eaikaatang plsvggqapk tgqlkvgyea dvigllqnpv 421 edvkvlqkvd nvgwvwkggk lfkgpgvgpw geepgvwedi tgvsrlrctt gy 25 SEQ ID NO:14 Organism Metarhizium anisopliae ARSEF 23. 485aa, 30 1 mtrrviphse saesvddaqh vsgriystgf giavrtgqpq sqdddaetgv kkvfytivmt 61 kliipgdgep ikdaalvvkn kiidwvgrqa dlpneytekp hklhnvpylm pglwdchvhf 121 agsngereae egstglsfla dhpttagarl argcwdaiqr gytsmrdlag fgceiskaie 181 dgviigpniy sagaclsqla ghgdvfalpa gdallnlgla svkagqfgag msclvdgvde 241 crrgvrlqir rgakcikvma sggvlsrddn plyaqfsree ldtivseakr mertvaahvh 35 301 gkpgilqave agvtsvehvs fadqecidli kdrgtifvgt rtivnlllds kgegmpkkmw 361 ekaklvgths legykkaika gctialgtdt epgfnmaiel qyaveagmss leaikaatan 421 gpltvagqap ltgqlkagye admigvcdnp vedvkvlqkk snigwvwkgg klfkgpgigp 481 wgeel 40 SEQ ID NO:15 A. oryzae amidohydrolase Mhrmltddrlyrvdadllipgkgdpiphgavvwqcktiryagprsgvptefqgattthvpvv\fmpgmwdc hihflgataatmnaivdtpqalagarsvpdlhatvmagftsvrevggygcdlakavgegripgpniyss WO 2012/032472 PCT/IB2011/053901 84 hsaismtaghgdvhgvhrdslldlcahglpltiadgvpecllavrkqlrrgakvikvcasggvvsaidd pqhqefsfeelkaivdeaararrvvaahchgkagimnalragcrtiehgsfldeeavglmkekgailva trsviesglamkdlftpssyqkllavadahrkayqlaisrgvtialgtdqfissdnpmigygrnghevr yavdagltplaaieaatangpltlgyqapqsgqlkegydadiiavrenplenvavlsnsknvthvwrgg 5 mliks SEQ ID NO:16 Bovine carboxypeptidase A (419 aa). Accession: NP_777175. Sigma product no. C0261. 10 1 mqgililsvl lgaalgkedt vghqvlrita adeaevqtvk eledlehlql dfwrgpgqpg 61 spidvrvpfp slqavkvfle ahgiryrimi edvqslldee qeqmfasqsr arstntfnya 121 tyhtldeiyd fmdllvaehp qlvsklqigr syegrpiyvl kfstggsnrp aiwidlgihs 181 rewitqatgv wfakkftedy gqdpsftail dsmdifleiv tnpdgfafth sqnrlwrktr 241 svtsssicvg vdanrnwdag fgkagasssp csetyhgkya nsevevksiv dfvkdhgnfk 15 301 aflsihsysq lllypygyt t qsipdkteln qvaksaveal ksiygtsyky gsiittiyqa 361 sggsidwsyn qgikysftfe lrdtgrygfl lpasgiipta qetwlgvlti mehtlnnly SEQ ID NO:17 20 Carboxypeptidase Y from baker's yeast (Saccharomyces cerevisiae), 532 aa, Accession: EDV11788. Sigma product no. C3888. 1 mkaftsllcg lglsttlaka islqrplgld kdvilqaaek fglnldidhl lkeldsnvld 25 61 awaqiehlyp nqvmsietst kpkfpeaikt kkdwdfvvkn daienygirv nkikdpkilg 121 idpnvtqytg yldvededkh fffwtfesrn dpakdpvilw lnggpgcssl tglffelgps 181 sigpdlkpig npyswnsnat vifldqpvv gfsysgssgv sntvaagkdv ynflelffdq 241 fpeyvnkgqd fhiagesyag hyipvfasei ishkdrnfnl tsvlignglt dpltgynyye 301 pmacgeggep svlpseecsa medslerclg liescydsqs vwscvpatiy cnnaqlapyq 30 361 rtgrnvydir kdceggnlcy ptlqdiddyl nqdyvkeavg aevdhyescn fdinrnflfa 421 gdwmkpyhta vtdllngdlp ilvyagdkdf icnwlgnkaw tdvlpwkyde efasqkvrnw 481 tasitdevag evksykhfty Irvfngghmv pfdvpenals mvnewihggf sl WO 2012/032472 PCT/IB2011/053901 85 Reference list 1 Abrunhosa, L. and Venancio, A. (2007). Isolation and purification of an 5 enzyme hydrolyzing ochratoxin A from Aspergillus niger. Biotechnol.Lett. 29: 1909 1914 2 Abrunhosa, L., Paterson, R. R. M. and Venancio, A. (2010). Biodegradation of Ochratoxin A for Food and Feed Decontamination. Toxins, 2: 1078-1099 3 Fraser, J.A., Davis, M.A., and Hynes, M. J. (2001). The Formamidase Gene 10 of Aspergillus nidulans: Regulation byNitrogen Metabolite Repression and Transcriptional Interference by an Overlapping Upstream Gene. Genetics 157: 19 131 4 Jelinek, C. F., Pohland, A. E., and Wood, G. E. (1989). Worldwide Occurrence of Mycotoxins in Foods and Feeds - An Update. Journal of the Association of Official 15 Analytical Chemists 72: 223-230 5 Li, S., Marquardt, R. R., Frohlich, A. A., Vitti, T. G., and Crow, G. (1997). Pharmacokinetics of ochratoxin A and its metabolites in rats. Toxicol.Appl.Pharmacol. 145: 82-90 6 Pitout, M. J. (1969). The hydrolysis of ochratoxin A by some proteolytic 20 enzymes. Biochem.Pharmacol. 18: 485-491 7 Stander, M. A., Bornscheuer, U. T., Henke, E., and Steyn, P. S. (2000). Screening of commercial hydrolases for the degradation of ochratoxin A. J.Agric.Food Chem. 48: 5736-5739 8 Van der Merwe, K. J., Steyn, P. S., and Fourie, L. (1965). Mycotoxins. II. The 25 constitution of ochratoxins A, B, and C, metabolites of Aspergillus ochraceus Wilh. J.Chem.Soc.[Perkin 1] 7083-7088 9 WHO/FAO (1998). Food irradiation. A Technique for Preserving and Improving the Safety of Food. World Health Organization in collaboration with the Food and Agricultural Organization, Geneva WO 2012/032472 PCT/IB2011/053901 86 10 Wang, H (2010). Increased production of aspartic proteases in filamentous fungal cells. WO 2010014574 (A2)

Claims (26)

1. An isolated amidase enzyme that degrades ochratoxin. 5

2. The amidase according to claim 1, wherein the ochratoxin is ochratoxin A.

3. The amidase according to claim 1 or claim 2, wherein the amidase comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or nine of the amino acid sequence motifs: 10 1) x-P-G-x-x-D-x-H-x-H-x-xG,; 2) G-x-T; 3) G-x-x-x-G-P;

4) G-H-x-D; 5) D-G-x-x-x-C-x-x-x-x-R-x-x-x-R-x-x-A-x-x-l-K; 15 6) G-G-V-x-S-x-x-D-x-P; 7) V-x-A-H-x-x-G-x-x-G; 8) H-x-x-x-x-D; 9) G-V-x-1-x-x-G-T-D. 20 4. The amidase according to any one of claims 1 to 3, wherein the amidase has a Tim barrel structure including an active site comprising 6 histidine residues, 1 lysine residue and 1 aspartic acid residue, wherein the amino acid residues in the active site correspond to positions H1 11, H1 13, H191, K246, H287, H289, H307 and D378 of SEQ ID NO:1 when the tertiary structure of the amidase and SEQ ID NO:1 are 25 compared.

5. The amidase according to any preceding claim wherein the amidase enzyme comprises a polypeptide sequence having the sequence of any one of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NOs:5-11, 13, 14 or 15, or a sequence having at least 30 70%, 75%, 80%, 90%, 95%, 98%, 99% identity to any one of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NOs:5-11, 13, 14 or 15, or a polypeptide which differs from any one of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NOs:5-11, 13, 14 or 15 by one or several amino acid additions, deletions and/or substitutions, or a polypeptide which is produced by expression of a polynucleotide comprising the sequence of SEQ ID NO: 35 2 or a sequence having at least 70%, 75%, 80%, 90%, 95%, 98%, 99% identity thereto, or a polynucleotide which differs from SEQ ID NO: 2 due to degeneracy of the genetic code; or a polynucleotide which differs from SEQ ID NO: 2 by one or 88 several nucleotide additions, deletions and/or substitutions, or a sequence which hybridises under stringent conditions to the complement of SEQ ID NO: 2 or a sequence which has at least 70%, 75%, 80%, 90%, 95%, 98%, 99% identity thereto. 5

6. The amidase according to any preceding claim, wherein the amidase hydrolyzes about at least 100, at least 200, at least 300, at least 400 to about 900 nanomoles ochratoxin A per min per mg protein at pH7.0 and 402C.

7. A feed or food additive comprising an amidase enzyme capable of degrading 10 ochratoxin according to any one of claims 1 to 6.

8. The feed or food additive according to claim 7, further comprising at least one physiologically acceptable carrier, selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat, wheat bran or a wheat 15 component, rice or rice bran, sucrose, starch, Na 2 SO 4 , Talc, and PVA, and mixtures thereof.

9. The feed or food additive according to claim 7 or claim 8, wherein the feed additive comprises one or more further feed enzyme(s) selected from the group 20 consisting of those involved in protein degradation including carboxypeptidases preferably carboxypeptidase A, carboxypeptidase Y, A. niger aspartic acid proteases of PEPAa, PEPAb, PEPAc and PEPAd, elastase, amino peptidases, pepsin or pepsin-like, trypsin or trypsin -like proteases and bacterial proteases including subtilisin and its variants, and of those involved in starch metabolism, fibre 25 degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism, amylases, arabinases, arabinofuranosidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, -galactosidases, glucanases, glucan lysases, endo-glucanases, glucoamylases, glucose oxidases, glucosidases, including P glucosidase, glucuronidases, hemicellulases, hexose 30 oxidases, hydrolases, invertases, isomerases, lipolytic enzymes, laccases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, 35 xylanases, hexose oxidase (D-hexose: 0 2 -oxidoreductase, EC 1.1.3.5), acid phosphatases and/or others or combinations thereof. 89

10. A feed or food material comprising the feed additive of any one of claims 7 to 9. 5

11. A feedstuff or foodstuff comprising the food or feed material according to claim 10.

12. The feedstuff according to claim 11, comprising one or more feed materials selected from the group comprising a) cereals, such as small grains (e.g., wheat, 10 barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) byproducts from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) silage; d) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, 15 dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; e) oils and fats obtained from vegetable and animal sources; f) minerals and vitamins.

13. The foodstuff according to claim 11, wherein said foodstuff comprises a cereal, for example corn, wheat, barley, sorghum, a cereal product, for example, 20 porridges, noodles, bread or cakes, coffee, cocoa, wine, beer , pulses, spices, dried fruit, grape juice, milk, milk products, for example cheese, meat or meat products.

14. A method of making a foodstuff or feedstuff according to any one of claims 11 to 13 comprising adding to a food or feed material a food or feed additive according 25 to any one of claims 7 to 9.

15. A method of making a food or feed additive according to any one of claims 7 to 9 comprising admixing the amidase with at least one physiologically acceptable carrier. 30

16. A composition comprising an ochratoxin contaminated material and an ochratoxin degrading amidase enzyme according to any one of claims 1 to 6.

17. The composition according to claim 16, comprising one or more further 35 enzyme(s selected from the group consisting of those involved in protein degradation including carboxypeptidases preferably carboxypeptidase A, carboxypeptidase Y, A. niger aspartic acid proteases, preferably PEPAa, PEPAb, PEPAc and PEPAd, 90 elastase, amino peptidases, pepsin or pepsin-like, trypsin or trypsin - like proteases and bacterial proteases including subtilisin and its variants, and of those involved in starch metabolism, fibre degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism, amylases, arabinases, arabinofuranosidases, catalases, 5 cellulases, chitinases, chymosin, cutinase, deoxyribonucleases, epimerases, esterases, -galactosidases, glucanases, glucan lysases, endo-glucanases, glucoamylases, glucose oxidases, -glucosidases, including P glucosidase, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, lipolytic enzymes, laccases, lyases, mannosidases, oxidases, 10 oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: 0 2 -oxidoreductase, EC 1.1.3.5) acid phosphatases and/or others 15 or combinations thereof, and optionally further comprising at least one of a detoxifiying enzyme selected from the group consisting of a mycotoxin degrading enzyme, for example, aflatoxin detoxizyme, zearalenone esterases, zearalenone lactonases, fumonisin carboxylesterases, fumonisin aminotransferases, aminopolyol amine oxidases, deoxynivalenol expoxide hydrolases, a mycotoxin degrading 20 microorganism, for example, Bacillus subtilis, or an absorbent (i.e., myctoxin binders) including at least one polymer, for example, microbial cell walls or an inorganic material such as bentonite.

18. The composition according to claim 16 or claim 17, wherein said composition 25 is a feed composition, a fermentation broth or waste water.

19. A method of reducing ochratoxin contamination of a material comprising adding to said material an ochratoxin degrading amidase enzyme according to any one of claims 1 to 6. 30

20. The method according claim 19, wherein the amidase hydrolyzes about at least 100, at least 200, at least 300, at least 400 to about 900 nanomoles ochratoxin A per min per mg protein when incubated at pH7.0 and 402C. 35

21. The method according to claim 19 or claim 20, further comprising adding one or more further enzyme(s) selected from the group consisting of those involved in 91 protein degradation including carboxypeptidases preferably carboxypeptidase A, carboxypeptidase Y, A. niger aspartic acid proteases of PEPAa, PEPAb, PEPAc and PEPAd, elastase, amino peptidases, pepsin or pepsin-like, trypsin or trypsin -like proteases and bacterial proteases including subtilisin and its variants, and of those 5 involved in, starch metabolism, fibre degradation, lipid metabolism, proteins or enzymes involved in glycogen metabolism, acetyl esterases, aminopeptidases, amylases, arabinases, arabinofuranosidases, catalases, cellulases, chitinases, chymosin, cutinase, deoxyribon ucleases, epimerases, esterases, -galactosidases, glucanases, glucan lysases, endo-glucanases, glucoamylases, glucose oxidases, 10 glucosidases, including P glucosidase, glucuronidases, hemicellulases, hexose oxidases, hydrolases, invertases, isomerases, lipolytic enzymes, laccases, lyases, mannosidases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin depolymerases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenoloxidases, phytases, polygalacturonases, proteases, rhamno-galacturonases, 15 ribonucleases, thaumatin, transferases, transport proteins, transglutaminases, xylanases, hexose oxidase (D-hexose: 0 2 -oxidoreductase, EC 1.1.3.5), acid phosphatases and/or others or combinations thereof, optionally further comprising adding at least one of a detoxifiying enzyme selected from the group consisting of a mycotoxin degrading enzyme, for example, aflatoxin detoxizyme, zearalenone 20 esterases, zearalenone lactonases, fumonisin carboxylesterases, fumonisin aminotransf erases, aminopolyol amine oxidases, deoxynivalenol expoxide hydrolases, a mycotoxin degrading microorganism, for example, Bacillus subtilis, or an absorbent (i.e., myctoxin binders) including at least one polymer, for example, microbial cell walls or an inorganic material such as bentonite. 25

22. A feed material obtained by the method according to any one of claims 19 to 21.

23. A recombinant cell or spore encoding an amidase enzyme which degrades 30 ochratoxin according to any one of claims 1 to 6.

24. The amidse according to claim 3, wherein the motifs comprise: 1) xl-P-G-x 2 -x 3 -D-x 4 -H-x 5 -H-X 6 -x 7 -G,; 2) G-x 8 -T; 35 3) G-x 9 -x 10 -x 1 -G-P; 4) G-H-x 12 -D; 92 5) D-G-x"-x 14 -x 5 -C-x 1 6 x 17 -x 1 x 1 9 -Rx 2 0 x 2 1 -x 2 2 -R-x 23 -x 24 -A-x 25 -x 26 - I-K, 6) G-G-V-x 27 -S-x 28 _X 29 -D-x 30 -P,; 7) V-x 31 -A-H-x 32 -x 33 -G-x 34 -x 35 -G,; 8) H-x 36 -x 37 -x 38 -x 39 -0; 5 9) G-V-x 40 -I-x 4 1 -x 42 -G-T-D; wherein: x 1 = I/m; x 2 = I/m; x 3 = w/i; x 4 = c/v/s/a; x 5 = any amino acid; x 6 = f/y/I; x 7 = any amino acid; x 8 = y/f; x 9 = t/a/s/v/r; x 1 0 = i/f/a; xll=- any amino acid; x 1 2 = g/s; x 13 = v/e; x 1 4 = any amino acid; x 15 = e/d/g; x 16 = any amino acid; x 17 = any amino acid; x' 8 = a/g/t; x 19 = v/a; x 2 0 = 10 any amino acid; x 21 = q/m/a; x 22 = l/i/v; x 23 = r/h/c; x 24 = g/n; x 25 = k/r/t/e/d; x 26 = any amino acid; x 27 =/m/v/g; x 28 = any amino acid; x 29 = any amino acid; x 30 = any amino acid; X31 = a/s/h; x 32 = c/v/a; x 33 =h/q; x 34 =k/r; x 35 = any amino acid; x 36 = g/v/a; x 37 = s/t/i; x 38 = y/f/e; x 39 = I/a/i; X 40 = any amino acid; x 4 1 = a/v; x 42 =-I/a. 15 25. The amidase according to claim 24, wherein the motifs comprise: 1) I/m-P-G-1/m-w-D-c-H-x-H-f-x-G; 2) G-y/f-T; 3) G-t-i-x-G-P; 4) G-H-g-D; 20 5) D-G-v-x-e-C-x-x-a-v-R-x-q-1-R-r-g-A-k-x-I-K; 6) G-G-V-1/-S-x-x-D-x-P; 7) V-a-A-H-c-h-G-k-x-G; 8) H-g-s-y-l-D; 9) G-V-x-l-a-l-G-T-D.

25

26. A feed or food additive comprising an amidase, or a feedstuff or foodstuff comprising a feed additive, or a method of making a feedstuff or foodstuff substantially as herein described with reference to the Examples.