WO2018005229A1 - In feed assay of microbial proteases using peptide substrates - Google Patents

Abstract

A novel in feed assay for microbial proteases, conducted at a pH below 7, using certain peptide substrates is disclosed. This assay can be designed to use at least one additional exogenous microbial peptidase if colorimetric detection and/or quantification is used. If fluorescence is used, then there is no need to add any additional exogenous peptidase(s).

Description

TITLE

IN FEED ASSAY OF MICROBIAL PROTEASES USING PEPTIDE SUBSTRATES

This application claims the benefit of U.S. Provisional Application No. 62/356,849 filed June 30, 2016, which is incorporated herein by reference in its entirety.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

The sequence listing provided in the file named NB41020USPSP_SequenceListing.txt" with a size of 7.9 KB bytes which was created on June 23, 2016 and which is filed herewith, is incorporated by reference herein in its entirety.

FIELD

The field relates to a novel in-feed assay, performed at a pH below 7, for detecting and/or quantitating microbial proteases microbial aspartic protease, using peptide substrates.

BACKGROUND

Hydrolases are hydrolytic enzymes, specifically, biochemical catalysts that use water to cleave chemical bonds. Examples of common hydrolases include esterases, proteases, glycosidases, nucleosidases and lipases.

Proteases (also called peptidases or proteinases) are enzymes capable of cleaving peptide bonds. Proteases have evolved multiple times, and different classes of protease can perform the same reaction by completely different catalytic mechanisms. Proteases can be found in animals, plants, bacteria, archaea and viruses.

Peptidases play essential roles in protein activation, cell regulation and signaling, as well in the generation of amino acids for protein synthesis or utilization in other metabolic pathways. Depending on their site of cleavage, peptidases can be classified as exopeptidases or

endopeptidases. Exopeptidases prefer to hydrolyze amino acid residues from the terminus of a peptide, whereas, endopeptidases prefer to cleave internal peptide bonds. Exopeptidases can be further divided into amino peptidases and carboxypeptidases depending on whether they hydrolyze residues from the amino or carboxy terminus of a peptide.

Proteases are commonly added to animal feed to increase protein digestibility of the feed. The proteases can be added to the feed in a number of ways such as prior to pelleting or it can be sprayed onto and/or mixed into the feed. In any event, it is desirable to measure the amount of proteases activity in the feed to ensure that the protease was added and added in the correct quantity and that it survived the pelleting and/or mixing process.

Numerous methods are known to detect protease activity in samples. These methods are usually based on using natural protein substrates, synthetic peptide substrates or peptide analog substrates labeled with a chromophore, fluorophore or a radioisotope to detect protease activity.

For example, various assays for detecting and measuring activity of proteases are available for measuring protease activity that are based on the release of acid-soluble peptides from casein or hemoglobin, measured as absorbance at 280 nm or colorimetrically using the Folin method. Other exemplary assays involve the solubilization of chromogenic substrates (See e.g., Ward, "Proteinases," in Fogarty (ed.)., Microbial Enzymes and Biotechnology, Applied Science, London, [1983], pp. 251-317). Other exemplary assays include, but are not limited to succinyl-Ala-Ala-Pro-Phe-para nitroanilide assay (suc-AAPF-pNA) and the 2,4,6-trinitrobenzene sulfonate sodium salt assay (TNBS assay). Numerous additional references known to those in the art provide suitable methods (See e.g., Wells et al., Nucleic Acids Res. 11 :7911-7925 [1983]; Christianson et al., Anal. Biochem. 223 : 119 -129 [1994]; and Hsia et al., Anal Biochem.

242:221-227 [1999]).

US2016-0369320, published Dec. 22, 2016 to Lichtenstein et al., describes methods for measuring activity in feed. The method requires a phosphate-containing buffer and is conducted at a pH in the range from 7-11.

Unfortunately, the detection and/or quantitation of proteases in feed is usually

complicated by the fact that naturally occurring components in the feed, including endogenous protein substrates in the feed, can compete with the labeled substrate being used to detect and/or measure protease activity.

Thus, there remains a need for an in-feed protease assay that can be performed on-site simply without the need for expensive and sophisticated equipment and can be performed under acidic condition below pH 7.

SUMMARY

In one aspect, the disclosure provides an assay for detecting and/or quantitating microbial peptidase activity in feed, said assay comprising:

(a) contacting the feed with (i) a peptidase substrate wherein the substrate has a specific amino acid sequence that is capable of being recognized by a microbial aspartic peptidase in the feed, said substrate having between 2 and 20 amino acid residues and further wherein both N-terminal and C-terminal ends of the peptidase substrate are each modified with the same or with a different detectable moiety and (ii) at least one additional peptidase, and said contacting occurs under acidic conditions below pH 7; and

(b) detecting and/or quantitating the detectable moiety.

In a second aspect, at least one additional microbial peptidase is an exopeptidase.

In a third aspect, the peptidase substrate has the amino acid sequence N-terminus- AAX1X2AAX3- C-terminus (SEQ ID NO: 1) in which both N-terminal and C-terminal ends of the sequence are each modified with the same or with a different detectable group and further wherein Xi is an amino acid selected from the group consisting of Leu, Lys, Arg, Glu, Phe, Asp, Tyr, His, Ala, Thr, Cys, Gly and Tip; X2 is an amino acid selected from the group consisting of Phe, Val, Lys, Ala, Cys, Glu, Leu, Pro, Tyr, His, He, Arg and Sen; X3 can be absent or K.

In a fourth aspect, the peptidase substrate can be Succ-DMFIND-Anb. (SEQ ID NO:2).

In a fifth aspect, the detectable moiety can be a chromophore.

In a sixth aspect, the detectable moiety is detected and/or quantitated by measuring

UV/Vis absorption.

In a seventh aspect,

(a) the N-terminal detectable moiety is selected from the group consisting of acyl groups such as formyl, acetyl, benzoyl, fluorophores of Abz (2-Aminobenzoyl or Anthraniloyl), N-Me- Abz(N-Methyl-anthraniloyl), Dansyl (5-(Dimethylamino)naphthalene-l-sulfonyl), DMACA (7-Dimethylaminocoumarin-4-acetate), EDANS (5-[(2-Aminoethyl)amino]naphthalene-l- sulfonic acid), FITC (Fluorescein isothiocyanate), Lucifer Yellow

(6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino- lH-benz[d,e]isoquinoline-5,8-disulfonic acid), Mca ((7-Methoxycoumarin-4-yl)acetyl), Trp (Tryptophanyl); and

(b) the C-terminal detectable moiety is selected from the group consisting of: pNA(para- Nitroaniline), Anb (5-Amino-2-nitrobenzoic acid), Dnp (2,4-Dinitrophenyl), EDDnp (N-(2,4- Dinitrophenyl)ethylenediamine), 4-Nitro-Phe (4-Nitro-phenylalanine), 3-Nitro-Tyr (3-Nitro- tyrosine), 4-Nitro-Phe(4-Nitro-phenylalanine), DABCYL(4-(4- Dimethylaminophenylazo)benzoyl), NBD (7-Nitro-benzo[2, l,3]oxadiazol-4-yl), Dabsyl (4-(4- Dimethylaminophenylazo)-benzenesulfonyl), 4-Nitro-Z(4-Nitro-benzyloxycarbonyl). In an eighth aspect, disclosed is an assay for detecting and/or quantitating microbial aspartic peptidase activity in feed, said assay comprising:

(a) contacting the feed with a peptidase substrate wherein the substrate has a specific amino acid sequence that is capable of being recognized by the microbial peptidase in the feed, said substrate having between 2 and 20 amino acid residues and further wherein both N-terminal and C-terminal ends of the peptidase substrate are each modified with the same or with a different detectable moiety, and said contacting occurs under acidic conditions below pH 7;; and

(b) detecting and/or quantitating the detectable moiety.

In an ninth aspect, the peptidase substrate has the amino acid sequence N-terminus-

AAKFAAAAX1X2AAX3- C-terminus (SEQ ID NO: 1) in which both N-terminal and C-terminal ends of the sequence are each modified with the same or with a different detectable

whereingroup and further wherein Xi is an amino acid selected from the group consisting of Leu, Lys, Arg, Glu, Phe, Asp, Tyr, His, Ala, Thr, Cys, Gly and Trp; X2 is an amino acid selected from the group consisting of Phe, Val, Lys, Ala, Cys, Glu, Leu, Pro, Tyr, His, He, Arg and Ser; X3 can be absent or K.

In a tenth aspect,

(a) the N-terminal detectable moiety is selected from the group consisting of acyl groups such as formyl, acetyl, benzoyl, fluorophores of Abz (2-Aminobenzoyl or Anthraniloyl), N-Me- Abz(N-Methyl-anthraniloyl), Dansyl (5-(Dimethylamino)naphthalene-l-sulfonyl), DMACA (7-Dimethylaminocoumarin-4-acetate), EDANS (5-[(2-Aminoethyl)amino]naphthalene-l- sulfonic acid), FITC (Fluorescein isothiocyanate), Lucifer Yellow

(6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino- lH-benz[d,e]isoquinoline-5,8-disulfonic acid), Mca ((7-Methoxycoumarin-4-yl)acetyl), Trp (Tryptophanyl); and

(b) the C-terminal detectable moiety is selected from the group consisting of: pNA (para- Nitroaniline), Anb (5-Amino-2-nitrobenzoic acid), Aminobenzoyl, Dnp (2,4-Dinitrophenyl), EDDnp (N-(2,4-Dinitrophenyl)ethylenediamine), 4-Nitro-Phe (4-Nitro-phenylalanine), 3-Nitro- Tyr (3-Nitro-tyrosine), 4-Nitro-Phe(4-Nitro-phenylalanine), DABCYL(4-(4- Dimethylaminophenylazo)benzoyl), NBD (7-Nitro-benzo[2, l,3]oxadiazol-4-yl), Dabsyl (4-(4- Dimethylaminophenylazo)-benzenesulfonyl), 4-Nitro-Z(4-Nitro-benzyloxycarbonyl). In an eleventh aspect, the, peptidase substrate has the sequence set forth in SEQ

ID NO:5.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

Figure la depicts progress curves showing AFP activity on Abz-AAFFAA-Anb (SEQ ID NO: 15) substrate with the aid of 3PP in coupled assay Figure lb depicts the reaction rate as a function of AFP concentration ^g/ml) of an assay of AFP using Abz-AAFFAA-Anb (SEQ ID NO: 15) as the peptidase substrate with the aid of 3PP.

Figure 2 depicts the reaction rate of AFP using Succ-DMFIND-Anb (SEQ ID NO:2) as substrate with the aid of 3PP in a coupled assay.

Figure 3 is the absorbance spectrum of SUCC-DMFIND-Anb (SEQ ID NO:2) before and after the hydrolysis by AFP and 3PP proteases in a coupled assay.

Figure 4 depicts the reaction rate of AFP using SUCC-DMFIND-Anb (SEQ ID NO:2) as substrate with the aid of leucine aminopeptidase as the exopeptidase in a coupled assay.

Figure 5 is a coupled in-feed assay of AFP with the aid of 3PP using Abz-AAKFAA-Anb (SEQ ID NO:3) as the substrate. The values plotted were the average 5-6 traces (n=5 or 6).

Figure 6 is an in-feed coupled assay for AFP with the aid 3PP using Abz-AAKFAA-Anb (SEQ ID NO:3) as the substrate. The values plotted were the average 10-11 data points for each reaction condition.

Figure 7 depicts the progress curves for a coupled in-feed assay of AFP with the aid of 3PP using Abz-AAKFAA-Anb (SEQ ID NO:3) as the substrate. The values plotted were the average 3-4 data points for each reaction condition. Each data point was subtracted from the values of control feed extract prepared without AFP enzyme.

Figure 8 is a dose response curve for a coupled assay of AFP in-feed using Abz- AAKFAA-pNA (SEQ ID NO:3) as substrate with the aid of 3PP.

Figure 9a depicts a dose response curve for an in-feed assay of AFP using Abz-

AAKFAA-pNA (SEQ ID NO:3) as substrate at pH 4.0 and Abz-AAKFAA-Anb (SEQ ID NO:3) as substrate at pH 3.0.

Figure 9b depicts dose response curve for an in-feed assay of AFP at low concentrations (1-25 mSAPU/mL) using Abz-AAKFAA-pNA (SEQ ID NO:3) as substrate at pF OFigure 10 depicts an in-feed assay of AFP using Abz-AAKFAA-Anb (SEQ ID NO:3) as substrate. Figure 10 depicts in-feed assay of AFP using Abz-AAKFAA-Anb (SEQ ID NO:3) as substrate.

Figure 11 depicts rate of fluorescence emitted by hydrolysis of Abz-AAFFAA-Anb (SEQ ID NO: 15) for in-feed assay of AFP, after pelleting at elevated temperature (90°C).

Figure 12 depicts fluorescent signal detection for an in-feed assay of AFP using H- Y(N02)DMFINDKPK(Abz)-OH(SEQ ID NO:6) as the substrate after pelleting at elevated temperature (90°C).

The following sequences comply with 37 C.F.R. §§ 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures - the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and the sequence listing requirements of the European Patent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. § 1.822.

Table 1. Summary of Gene and Protein SEQ ID Numbers

AAKFAAK Artificial sequence 16

AAFFAAK Artificial sequence 17

AALFAA Artificial sequence 18

DETAILED DESCRIPTION

All patents, patent applications, and publications cited are incorporated herein by reference in their entirety.

In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.

The articles "a", "an", and "the" preceding an element or component are intended to be nonrestrictive regarding the number of instances (i.e., occurrences) of the element or component.

Therefore "a", "an", and "the" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term "comprising" means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term

"comprising" is intended to include embodiments encompassed by the terms "consisting essentially of and "consisting of . Similarly, the term "consisting essentially of is intended to include embodiments encompassed by the term "consisting of.

Where present, all ranges are inclusive and combinable. For example, when a range of "1 to 5" is recited, the recited range should be construed as including ranges "1 to 4", "1 to 3", "1- 2", "1-2 & 4-5", "1-3 & 5", and the like.

The term "microbial" relates to microbes or microorganisms such as algae, bacteria, yeast, fungi and the like.

The terms "protease", "peptidase" and "proteinase" are used interchangeably herein. A protease means a protein or polypeptide domain of derived from a microorganism, e.g., a fungus, bacterium, or from a plant or animal, and that has the ability to catalyze cleavage of peptide bonds at one or more of various positions of a protein backbone (e.g., E.C. 3.4).

Peptidases can be classified by reaction catalyzed which is a functional classification or by molecular structure and homology which is a MEROPS classification. Table 2. Functional classification:

Table 3. MEROPS classification

The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non- naturally occurring substance, (2) any substance including, but not limited to, any host cell, enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated. The terms "isolated nucleic acid molecule", "isolated polynucleotide", and "isolated nucleic acid fragment" will be used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

The term "purified" as applied to nucleic acids or polypeptides generally denotes a nucleic acid or polypeptide that is essentially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%>, about 65%>, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%) or more pure (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

As used herein, the terms "functional assay" and "assay" are used interchangeably herein and refer to an assay that provides an indication of a protein's activity. In some embodiments, the term refers to assay systems in which a protein is analyzed for its ability to function in its usual capacity. For example, in the case of a protease, a functional assay involves determining the effectiveness of the protease to hydrolyze a proteinaceous substrate.

The terms "peptides", "proteins" and "polypeptides are used interchangeably herein and refer to a polymer of amino acids joined together by peptide bonds. A "protein" or

"polypeptide" comprises a polymeric sequence of amino acid residues. The single and 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure. The single letter X refers to any of the twenty amino acids. 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. Mutations can be named by the one letter code for the parent amino acid, followed by a position number and then the one letter code for the variant amino acid. For example, mutating glycine (G) at position 87 to serine (S) is represented as "G087S" or "G87S". When describing modifications, a position followed by amino acids listed in parentheses indicates a list of substitutions at that position by any of the listed amino acids. For example, 6(L,I) means position 6 can be substituted with a leucine or isoleucine. At times, in a sequence, a slash (/) is used to define substitutions, e.g. F/V, indicates that the particular position may have a phenylalanine or valine at that position.

The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is a native or naturally-occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non- naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

As used herein with regard to amino acid residue positions, "corresponding to" or "corresponds to" or "corresponds" refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide. As used herein, "corresponding region" generally refers to an analogous position in a related protein or a reference protein.

The terms "derived from" and "obtained from" refer to not only a protein produced or producible by a strain of the organism in question, but also a protein encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protein which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protein in question.

The term "amino acid" refers to the basic chemical structural unit of a protein or polypeptide.

The following abbreviations used herein to identify specific amino acids can be found in Table 4. Table 4. One and Three Letter Amino Acid Abbreviations.

Three-Letter One-Letter

Amino Acid Abbreviation Abbreviation

Alanine Ala A

Arginine Arg R

Asparagine Asn N

Aspartic acid Asp D

Cysteine Cys C

Glutamine Gin Q

Glutamic acid Glu E

Glycine Gly G

Histidine His H

Isoleucine He I

Leucine Leu L

Lysine Lys K

Methionine Met M

Phenylalanine Phe F

Proline Pro P

Serine Ser S

Threonine Thr T

Tryptophan Tip W

Tyrosine Tyr Y

Valine Val V

Any amino acid or as defined herein Xaa X

It would be recognized by one of ordinary skill in the art that modifications of amino acid sequences disclosed herein can be made while retaining the function associated with the disclosed amino acid sequences. For example, it is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded protein are common. For example, any particular amino acid in an amino acid sequence disclosed herein may be substituted for another functionally equivalent amino acid. For the purposes of this disclosure, substitutions are defined as exchanges within one of the following five groups:

1. Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic, nonpolar residues: Met, Leu, He, Val (Cys); and

5. Large aromatic residues: Phe, Tyr, and Tip.

Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain aspects. Variants of these sequences that are at least about 70- 85%, 85-90%, or 90%-95% identical to the sequences disclosed herein may be used in certain embodiments. Alternatively, a variant polypeptide sequence or polynucleotide sequence in certain embodiments can have at least 60%, 61%, 62%,63%,64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. The variant amino acid sequence or polynucleotide sequence has the same function of the disclosed sequence, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function of the disclosed sequence.

The term "variant", with respect to a polypeptide, refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally- occurring or man-made substitutions, insertions, or deletions of an amino acid. Similarly, the term "variant," with respect to a polynucleotide, refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.

The terms "animal feed", "feed", "feed sample" and "sample" are used interchangeably herein. Thus, feed means any sample containing a protease activity be detected and/or measured. Exemplary samples include, but are not limited to, feed samples, feed ingredient samples, fermentation broth samples and cell culture medium, food samples, dairy product samples, pet food samples. The term "animal feed" refers to feeds intended exclusively for consumption by animals, including domestic animals (pets, farm animals etc.) or for animals raised to produce food e.g. fish farming.

The term "substrate" means the material or compound upon which an enzyme acts. More specifically, a "peptidase substrate" is a substrate having a specific amino acid sequence capable of being recognized by the peptidase in the feed sample being assayed. It is further modified at the N-terminal and C-terminal ends with either the same or a different detectable moiety as is described herein below. The terms "peptidase substrate" and "peptide substrate" are used interchangeably herein.

The term "pH" means, in chemistry, pH (potential of hydrogen) is a numeric scale used to specify the acidity or basicity of an aqueous solution. It is approximately the negative of the base

10 logarithm of the molar concentration, measured in units of moles per liter, of hydrogen ions.

More precisely it is the negative of the logarithm to base 10 of the activity of the hydrogen ion.

Solutions with a pH less than 7 are acidic and solutions with a pH greater than 7 are basic. Pure water is neutral, at pH 7, being neither an acid nor a base.

The terms "detectable moiety" and "signal producing group" are used interchangeably herein and refer any signal producing moiety such as chromophores and fluorophores that are capable of producing a signal when cleaved by at least one endopeptidases or at least one exopeptidase.

The term "chromophore" refers to the part of a molecule responsible for its color, e.g., a group of atoms in a chemical compound that are responsible for the color of the compound. It can be any chemical group that produces color in a compound. The color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. The chromophore is a region in the molecule where the energy difference between two different molecular orbitals falls within the range of the visible spectrum. Visible light that hits the chromophore can thus be absorbed by exciting an electron from its ground state to an exited stated.

The term "fluorophore" refers to a fluorescent chemical compound that can re-emit light upon excitation. Fluorophores absorb light energy of a specific wavelength and re-emit it at a longer wavelength. The excitation wavelength may be a very narrow or broader band, or it may be all beyond a cutoff level. The emission spectrum is usually sharper than the excitation spectrum, and it is of a longer wavelength and correspondingly lower energy. Excitation energies range from ultraviolet ("UV") through the visible spectrum and emission energies may continue from visible light into the near infrared region.

The quantification of feed additive proteases following supplementation to feed has proved technically challenging. What is described herein is a novel method to allow detection of microbial protease in animal feed which is easy to use and inexpensive to perform. More specifically, the microbial protease is a microbial aspartic protease.

Specifically, what is described is an assay for detecting and/or quantitating microbial peptidase activity in feed, said assay comprising:

(a) contacting the feed with a peptidase substrate with at least one peptidase wherein the substrate has a specific amino acid sequence that is capable of being recognized by the peptidase and said substrate has 2 or more amino acid residues and further wherein both N- terminal and C-terminal ends of the peptidase substrate are each modified with the same or with a different detectable moiety; and

(b) detecting and/or quantitating the detectable moiety.

The peptidase can be an endopeptidase or an exopeptidase. When more than one peptidase is used, then at least one peptidase should an endopeptidase and at least the other one peptidase should be an exopeptidase. When at least one additional peptidase is used, then this can also be referred to as a "coupled assay or coupled enzymic reaction, or cascade enzymic reaction."

The assays described herein, whether coupled or not coupled, can be performed in a pH range of 2 to 6. More preferably, the pH range is 2.5 to 5.5 and most preferably the pH range is from 3 to 4.5

Exemplary microbial endopeptidases include, but are not limited to, microbial aspartic peptidases, bacterial, archaeal, and fungal pepsin including thermopsin, penicillopepsin, rhizopuspepsin, mucorpepsin, candidapepsin, barrierpepsin, endothiapepsin, saccharopepsin, aspergillopepsin, polyporopepsin, phytepsin, plasmepsin, trichodermapepsin, yapsin,

canditropsin, candiparapsin, memapsin, syncephapepsin, podosporapepsin, nothepsin, napsin, and eimepsin, bacterial and fungal trypsin, mesotrypsin, bacterial and fungal chymotrypsin, elastase, thermolysin, glutamyl endopeptidases, neprilysin and the like.

Exemplary microbial exopeptidases include, but are not limited to, aminopeptidases, carboxypeptidases, dipeptidases, tripeptidases and the like. It is important that the peptidase substrate has a specific amino acid sequence capable of being recognized by the microbial peptidase in the feed sample and that the N-terminal and C- terminal ends are blocked with the same or a different detectable moiety.

Preferably, the peptidase substrate has the amino acid sequence N-terminus- AlaAlaXiX2AlaAlaX3-C-terminus (SEQ ID NO: 1) in which both N-terminal and C-terminal ends of the sequence are each modified with the same or with a different detectable group and further wherein Xi is an amino acid selected from the group consisting of Leu, Lys, Arg, Glu, Phe, Asp, Tyr, His, Ala, Thr, Cys, Gly and Tip; X2 is an amino acid selected from the group consisting of Phe, Val, Lys, Ala, Cys, Glu, Leu, Pro, Tyr, His, He, Arg and Ser and

X3 can be absent or K.

Examples of such sequences include, but are not limited to, the following:

Abz-AAKFAA-pNA, (SEQ ID NO:3), Abz-AARFAA-pNA, (SEQ ID NO:7), Abz-AAHFAA- pNA, (SEQ ID NO: 8), Abz-AALFAA-pNA, (SEQ ID NO: 9), Abz-AAYFAA-pNA, (SEQ ID NO: 10), Abz-AAQFAA-pNA, (SEQ ID NO: 11), Abz-AAEFAA-pNA, (SEQ ID NO: 12), Abz- AADFAA-pNA, (SEQ ID NO: 13), Abz-AAAFAA-pNA; (SEQ ID NO: 14);

Abz-AAKFAA-Anb, (SEQ ID NO:3), Abz-AARFAA-Anb, (SEQ ID NO:7), Abz-AAHFAA- Anb, (SEQ ID NO: 8), Abz-AALFAA-Anb, (SEQ ID NO: 9), Abz-AAFFAA-Anb, (SEQ ID NO: 15), Abz-AAYFAA-Anb, (SEQ ID NO: 10), Abz-AAQFAA-Anb, (SEQ ID NO: 11), Abz- AAEFAA-Anb, (SEQ ID NO: 12), Abz-AADFAA-Anb, (SEQ ID NO: 13), Abz-AAAFAA- Anb;(SEQ ID NO: 14);

Abz-AAKFAA-Deed,(SEQ ID NO:3), Abz-AARFAA-Deed,(SEQ ID NO:7), Abz-AAHFAA- Deed,(SEQ ID NO:8), Abz-AALFAA-Ded,(SEQ ID NO:9), Abz-AAYFAA-Deed,(SEQ ID NO: 10), Abz-AAQFAA-Ded,(SEQ ID NO: 11), Abz-AAEFAA-Ded,(SEQ ID NO: 12), Abz- AADFAA-Ded,(SEQ ID NO: 13), Abz-AAAFAA-Ded,(SEQ ID NO: 14), H- Y(N02)DMFINDKPK(Abz)-OH,(SEQ ID NO:6), Cy5Q-AAFFAA-Cy3B,(SEQ ID NO: 15), Cy5Q-AALFAA-Cy3B,(SEQ ID NO:9), Cy5Q-AAKFAAK-Cy3B,(SEQ ID NO: 16), Cy5Q- AAFFAAK-Cy3B,(SEQ ID NO: 17), Cy5Q-AALFAAK-Cy3B,(SEQ ID NO: 18), and Cy5Q- AAKFAA-Cy3B; (SEQ ID NO:3); Succ-AAKFAA-Anb, (SEQ ID NO:3), Succ-AARFAA-Anb, (SEQ ID NO:7), Succ-AAHFAA- Anb,(SEQ ID NO:8), Succ- A ALF A A- Anb , ( SEQ ID N0:9), Succ-AAFFAA-Anb,(SEQ ID NO: 15), Succ- AAYFAA- Anb, (SEQ ID NO: 10), Succ- AAQFAA- Anb, (SEQ ID NO: 11), Succ- AAEFAA-Anb, (SEQ ID NO: 12), Succ-AADFAA-Anb, (SEQ ID NO: 13), Succ-AAAFAA-Anb (SEQ ID NO: 14), Succ-DMFIND-Anb; (SEQ ID NO:2);or

Succ-AAKFAA-pNA, (SEQ ID NO:3), Succ-AARFAA-pNA, (SEQ ID NO:7), Succ-AAHFAA- pNA, (SEQ ID NO: 8), Succ-AALFAA-pNA, (SEQ ID NO: 9), Succ-AAFFAA-pNA, (SEQ ID

NO: 15), Succ-AAYFAA-pNA, (SEQ ID NO: 10), Succ-AAQFAA-pNA, (SEQ ID NO: 1 1), Succ- AAEFAA-pNA, (SEQ ID NO: 12), Succ-AADFAA-pNA, (SEQ ID NO: 13), Succ-AAAFAA- pNA, (SEQ ID NO: 14).

The detectable moiety can be attached to either end of the peptidase substrate by any means, including covalent or non-covalent means.

Modification of the N-terminus includes any chemical modifications of the N-terminal amino acid residues, such as acylation, acetylation, adding chromophores and fluorophores etc., or any reagent that can react with free amino groups either enzymatically or non-enzymatically.

Modification of C-terminus includes modification of the C-terminal residues with any chemical reagent capable of reacting with the carboxyl group. The chemical reagent can have chromophores, fluorophores, etc. For example, a typical formula for peptides are: Bl -(Peptide) n-B2 wherein B 1 and B2 are chemical groups attached to the N- and C-terminal residues of the peptide including chromophores or fluorophores that have absorbance or fluorescence emission in the wavelength range of 300 to 700nm when in free form.

Thus, when Bi-(Peptide)n-B2 is hydrolyzed with by any microbial peptidase present in the feed. However, no absorbance will be detected until the detectable moiety attached covalently to the N-terminal or C-terminal is released. The letter "n" is the number of amino acid residues in the peptide to be cleaved by at least one endopeptidase. "n" can range from at least 2 to at least 20 amino acid residues up to about 50 and any integer in between. The amino acid residues may include artificial amino acids.

Addition of the additional at least one exopeptidase or endopeptidase having

exopeptidase activity cleaves either the N-terminal or C-terminal detectable moieties so that they are in a form that can then be detected and/or quantitated as a measure of the microbial peptidase activity present in the feed. For example, when the peptide substrate, Abz-AAFFAA-Anb (SEQ ID NO: 15) is added to a feed sample containing microbial peptidase activity, then any microbial peptidase present will cleave the substrate into Abz-AAF- and -FAA-Anb. The thus generated - FAA-Anb can then be cleaved by an added additional exopeptidase such as the

aminotripeptidase 3PP to release Anb which has increased absorbance at 400nm and can be measured by a spectrophotometer. 3PP is a member of Sedolisin family S53 in the MEROPS database. It was prepared as described in WO2016062857 (Al) published on April 28,

2016.2016-04-28.

In another aspect, if a N- and C-blocked substrate such as Abz-AAKFAA-pNA (SEQ ID NO:3) is first hydrolyzed by an aspartic protease, then if the reaction is stopped, for example, by raising the pH to 7. pNA from the generated FAA-pNA can then be released by exopeptidase like 3PP or by certain endopeptidases that can act on shorter peptide substrates.

Thus, if the coupled reaction involves two steps, then at least one additional peptidase added can be an exopeptidase or it can be any other peptidase that acts on shorter peptides like the generated Abz-AAF, Abz-AAK, FAA-pNA, FAA-Anb as long as the other peptidase can release the detectable moiety. If the couple reaction involves in one step then the reaction mixture contains the aspartic peptidase to be assayed, the both ends blocked peptide substrate, and exopeptidase or any other peptidase that are inactive to the both ends blocked substrate and can only release the detectable moiety after the cleavage of the blocked substrate first by the aspartic peptidase to be assayed.

The following abbreviations used for illustrative detectable moieties can be found in

Mca 7-methoxycoumarin acetyl

Dansyl (5-(Dimethylamino)naphthalene-l-sulfonyl),

DMACA (7-Dimethylaminocoumarin-4-acetate)

EDANS (5-[(2-Aminoethyl)amino]naphthalene-l-sulfonic acid),

FITC (Fluorescein isothiocyanate)

Lucifer Yellow (6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino- lH-benz[d,e]isoquinoline-5,8-disulfonic acid)

Trp (Tryptophanyl)

Dnp (2,4-Dinitrophenyl),

EDDnp (N-(2,4-Dinitrophenyl)ethylenediamine)

4-Nitro-Phe (4-Nitro-phenylalanine),

DABCYL (4-(4-Dimethylaminophenylazo)benzoyl)

NBD (7-Nitro-benzo[2, l,3]oxadiazol-4-yl)

Dabsyl (4-(4-Dimethylaminophenylazo)-benzenesulfonyl)

4-Nitro-Z (4-Nitro-benzyloxycarbonyl)

Examples of N- and C-terminal blocked peptides include, but are not limited to: Abz- AAFFAA-Anb,(SEQ ID NO: 15), Abz-AAKFAA-pNA,(SEQ ID NO:3), Abz-AAFFAA- pNA,(SEQ ID NO: 15), Abz-AAHFAA-pNA(SEQ ID NO:8), Abz-AARFAA-pNA(SEQ ID NO: 7), Abz-AALFAA-pNA(SEQ ID NO: 9), Abz-AAYFAA-pNA(SEQ ID NO: 10), Abz- AAQF AA-pNA(SEQ ID NO : 11 ) , Abz- AAEF AA-pNA(SEQ ID NO : 12) , Abz-AADF AA- pNA(SEQ ID NO: 13), Abz-AAAFAA-pNA(SEQ ID NO: 14), Abz-AAFFAA-Ded(SEQ ID NO: 15), Mca-AAFFAA-Anb(SEQ ID NO: 15), Succ-AAFFAA-Anb(SEQ ID NO: 15), Succ- AAFFAA-pNA,(SEQ ID NO: 15), Succ-DMSIND-Anb(SEQ ID NO:4), Succ-DMSIND- pNA(SEQ ID NO:4), Succ-DMF-Anb, Succ-DMF-pNA.

Such N- and C-terminal blocked peptides have no major absorbance or fluorescence emission in the wavelength of 300-700nm. It is only when the blocked peptide is cleft by endopeptidase or at least one exopeptidase is added after the endopeptidase cleavage that the detectable moieties are released and subsequently detected whether colorimetrically or by fluorescence or by any other means. When peptides such as Succ-DMSIND-pNA (SEQ ID NO:4), Abz-AAKFAA-pNA (SEQ ID NO:3) and Abz-AAFFAA-Anb (SEQ ID NO: 15) are hydrolyzed by endopeptidases, then fluorescence can be measured as an indication of the peptidase activity and absorbance can also be measured for the endopeptidase activity if exopeptidase is added. In addition, absorbance can also be measured for the endopeptidase activity if an exopeptidase is added to the reaction, to release the C-terminal chromophore.

One means by which fluorescence can be detected is Fluorescence Resonance Energy

Transfer ("FRET") which is the non-radiative transfer of energy from an excited fluorophore (or donor) to a suitable quencher (or acceptor) molecule. FRET is used in a variety of applications including measurement of protease activity with substrates, in which the fluorophore is separated from the quencher by a short peptide sequence containing the enzyme cleavage site. Proteolysis of the peptide results in fluorescence as the fluorophore and quencher are separated.

Thus, when peptides such as Abz-AAFFAA-pNA (SEQ ID NO: 15) and Abz-AAFFAA-

Ded (SEQ ID NO: 15) are hydrolyzed by endopeptidases, then fluorescence can be measured as an indication of the mammalian and viral peptidase activity (Filippova et al., 1996, Fluorogenic

Peptide Substrates for Assay of Aspartyl Proteinases, Anal. Biochem. 234: 113-118).

Exemplary N-terminal detectable moieties include, but are not limited to Abz(2-

Aminobenzoyl or Anthraniloyl), N-Me-Abz(N-Methyl-anthraniloyl), Dansyl (5-

(Dimethylamino)naphthalene-l-sulfonyl), DMACA

(7-Dimethylaminocoumarin-4-acetate), EDANS (5-[(2-Aminoethyl)amino]naphthalene-l- sulfonic acid), FITC (Fluorescein isothiocyanate), Lucifer Yellow

(6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino- lH-benz[d,e]isoquinoline-5,8-disulfonic acid), Mca ((7-Methoxycoumarin-4-yl)acetyl), Trp (Tryptophanyl).

Exemplary C-terminal detectable moiety includes, but are not limited to, pNA(para-

Nitroaniline), Anb (5-amino-2-nitrobenzoic acid)), Dnp (2,4-Dinitrophenyl), EDDnp (N-(2,4- Dinitrophenyl)ethylenediamine), 4-Nitro-Phe (4-Nitro-phenylalanine), 3-Nitro-Tyr (3-Nitro- tyrosine), 4-Nitro-Phe(4-Nitro-phenylalanine), DABCYL(4-(4-

Dimethylaminophenylazo)benzoyl), NBD (7-Nitro-benzo[2,l,3]oxadiazol-4-yl), Dabsyl (4-(4- Dimethylaminophenylazo)-benzenesulfonyl), 4-Nitro-Z(4-Nitro-benzyloxycarbonyl). In another aspect, what is described is an assay for detecting and/or quantitating microbial aspartic peptidase activity in feed, said assay comprising:

(a) contacting the feed with a peptidase substrate wherein the substrate has a specific amino acid sequence that is capable of being recognized by the microbial peptidase in the feed, said substrate having 2 or more amino acid residues and further wherein both N- terminal and C-terminal ends of the peptidase substrate are each modified with the same or with a different detectable moiety, and said contacting occurs under acidic conditions below pH 7; and

(b) detecting and/or quantitating the detectable moiety.

The preferred peptidase substrate for this assay has the amino acid sequence N-terminus- AAKFAA-C-terminus in which both N-terminal and C-terminal ends of the sequence are each modified with the same or with a different detectable group selected from the group consisting of Abz, Anb, pNA and the like.

Surprisingly and unexpectedly, it has been found that when detectable moiety-AAKFAA- detectable moiety (SEQ ID NO:3) is used in lieu of detectable moiety-AAFFAA-detectable moiety (SEQ ID NO: 15), that assay sensitivity is improved at least 2-10 times when K replaces F in the peptidase substrate sequence. Additionally, the substrate detectable moiety-AAKFAA- detectable moiety (SEQ ID NO:3) was shown to have increased water solubility over the substrate detectable moiety-AAFFAA-detectable moiety.

Non-limiting examples of compositions and methods disclosed herein include:

1. An assay for detecting and/or quantitating microbial aspartic peptidase activity in feed, said assay comprising:

(a) contacting the feed with (i) a peptidase substrate wherein the substrate has a specific amino acid sequence that is capable of being recognized by a microbial aspartic peptidase in the feed, said substrate having between 2 and 20 amino acid residues and further wherein both N-terminal and C-terminal ends of the peptidase substrate are each modified with the same or with a different detectable moiety and (ii) at least one additional peptidase , and said contacting occurs under acidic conditions below pH 7; and

(b) detecting and/or quantitating the detectable moiety.

2. The assay of claim 1 wherein the at least one additional microbial peptidase is an exopeptidase. 3. The assay of claim 1 wherein the peptidase substrate has the amino acid sequence N- terminus-AAXiX2AAX3- C-terminus (SEQ ID NO: l) in which both N-terminal and C-terminal ends of the sequence are each modified with the same or with a different detectable group and further wherein Xi is an amino acid selected from the group consisting of Leu, Lys, Arg, Glu, Phe, Asp, Tyr, and His; X2 is an amino acid selected from the group consisting of Phe, Val, Lys, Ala, Cys, Glu, Leu, Pro, Tyr, His, He, Arg and Ser; X3 can be absent or K.

4. The assay of claim 1 wherein the peptidase substrate is Succ-DMFIND-Anb (SEQ ID

NO:2).

5. The assay of claim 1 wherein the detectable moiety is a chromophore.

6. The assay of claim 1 wherein the detectable moiety is detected and/or quantitated by measuring UV/Vis absorption.

7. The assay of claim 1 or 3 wherein

(a) the N-terminal detectable moiety is selected from the group consisting of acyl groups such as formyl, acetyl, benzoyl, fluorophores of Abz (2-Aminobenzoyl or Anthraniloyl), N-Me- Abz(N-Methyl-anthraniloyl), Dansyl (5-(Dimethylamino)naphthalene-l-sulfonyl), DMACA (7-Dimethylaminocoumarin-4-acetate), EDANS (5-[(2-Aminoethyl)amino]naphthalene-l- sulfonic acid), FITC (Fluorescein isothiocyanate), Lucifer Yellow

(6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino- lH-benz[d,e]isoquinoline-5,8-disulfonic acid), Mca ((7-Methoxycoumarin-4-yl)acetyl), Trp (Tryptophanyl); and

(b) the C-terminal detectable moiety is selected from the group consisting of: pNA(para- Nitroaniline), Anb (5-Amino-2-nitrobenzoic acid), Dnp (2,4-Dinitrophenyl), EDDnp (N-(2,4- Dinitrophenyl)ethylenediamine), 4-Nitro-Phe (4-Nitro-phenylalanine), 3-Nitro-Tyr (3-Nitro- tyrosine), 4-Nitro-Phe(4-Nitro-phenylalanine), DABCYL(4-(4- Dimethylaminophenylazo)benzoyl), NBD (7-Nitro-benzo[2,l,3]oxadiazol-4-yl), Dabsyl (4-(4- Dimethylaminophenylazo)-benzenesulfonyl), 4-Nitro-Z(4-Nitro-benzyloxycarbonyl).

8. An assay for detecting and/or quantitating microbial aspartic peptidase activity in feed, said assay comprising:

(a) contacting the feed with a peptidase substrate wherein the substrate has a specific amino acid sequence that is capable of being recognized by the microbial peptidase in the feed, said substrate having between 2 and 20 amino acid residues and further wherein both N-terminal and C-terminal ends of the peptidase substrate are each modified with the same or with a different detectable moiety, and said contacting occurs under acidic conditions below pH 7; and

(b) detecting and/or quantitating the detectable moiety.

9. The assay of claim 8 wherein the peptidase substrate has the amino acid sequence N- terminus-AAXiX2AAX3- C-terminus (SEQ ID NO: l) in which both N-terminal and C-terminal ends of the sequence are each modified with the same or with a different detectable group and further wherein Xi is an amino acid selected from the group consisting of Leu, Lys, Arg, Glu, Phe, Asp, Tyr, His, Ala, Thr, Cys, Gly and Tip; X₂ is an amino acid selected from the group consisting of Phe, Val, Lys, Ala, Cys, Glu, Leu, Pro, Tyr, His, He, Arg and Ser; X₃ can be absent or K.

10. The assay of claim 8 or 9 wherein

(a) the N-terminal detectable moiety is selected from the group consisting of acyl groups such as formyl, acetyl, benzoyl, fluorophores of Abz (2-Aminobenzoyl or Anthraniloyl), N-Me-

Abz(N-Methyl-anthraniloyl), Dansyl (5-(Dimethylamino)naphthalene-l-sulfonyl), DMACA (7-Dimethylaminocoumarin-4-acetate), EDANS (5-[(2-Aminoethyl)amino]naphthalene-l- sulfonic acid), FITC (Fluorescein isothiocyanate), Lucifer Yellow

(6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino- lH-benz[d,e]isoquinoline-5,8-disulfonic acid), Mca ((7-Methoxycoumarin-4-yl)acetyl), Trp (Tryptophanyl); and

(b) the C-terminal detectable moiety is selected from the group consisting of: pNA(para- Nitroaniline), Anb (5-Amino-2-nitrobenzoic acid), Aminobenzoyl, Dnp (2,4-Dinitrophenyl), EDDnp (N-(2,4-Dinitrophenyl)ethylenediamine), 4-Nitro-Phe (4-Nitro-phenylalanine), 3-Nitro- Tyr (3-Nitro-tyrosine), 4-Nitro-Phe(4-Nitro-phenylalanine), DABCYL(4-(4-

Dimethylaminophenylazo)benzoyl), NBD (7-Nitro-benzo[2,l,3]oxadiazol-4-yl), Dabsyl (4-(4- Dimethylaminophenylazo)-benzenesulfonyl), 4-Nitro-Z(4-Nitro-benzyloxycarbonyl).

11. The assay of claim 8 wherein the peptidase substrate has the sequence set forth in SEQ ID NO:5. EXAMPLES

Unless defined otherwise herein, all technical and scientific terms used herein have the 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, 2D ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE

HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with a general dictionary of many of the terms used with this disclosure.

The disclosure is further defined in the following Examples. It should be understood that the Examples, while indicating certain embodiments, is given by way of illustration only. From the above discussion and the Examples, one skilled in the art can ascertain essential

characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt to various uses and conditions.

The meaning of abbreviations is as follows: "sec" or "s" means second(s), "ms" mean milliseconds, "min" means minute(s), "h" or "hr" means hour(s), "μί" means microliter(s), "mL" means milliliter(s), "L" means liter(s); "mL/min" is milliliters per minute; '^g/mL" is microgram(s) per milliliter(s); "LB" is Luria broth; "μτη" is micrometers, "nm" is nanometers; "OD" is optical density; "IPTG" is isopropyl-P-D-thio-galactoside; "g" is gravitational force; "mM" is millimolar; "SDS-PAGE" is sodium dodecyl sulfate polyacrylamide; "mg/mL" is milligrams per milliliters; "N" is normal; "w/v" is weight for volume; "DTT" is dithiothreitol; "BCA" is bicinchoninic acid; "DMAc" is N, N'- dimethyl acetamide; "LiCl" is Lithium chloride' "NMR" is nuclear magnetic resonance; "DMSO" is dimethylsulfoxide; "SEC" is size exclusion chromatography; "GI" or "gi" means Genlnfo Identifier, a system used by

GENBANK^® and other sequence databases to uniquely identify polynucleotide and/or polypeptide sequences within the respective databases; "DPx" means glucan degree of polymerization having "x" units in length; "ATCC" means American Type Culture Collection (Manassas, VA), "DSMZ" and "DSM" refer to Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, (Braunschweig, Germany); "EELA" is the Finish Food Safety Authority (Helsinki, Finland;) "CCUG" refer to the Culture Collection, University of Goteborg, Sweden; "Sue." means sucrose; "Glue." means glucose; "Fruc." means fructose; "Leuc." means leucrose; and "Rxn" means reaction. General Methods

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described by Sambrook, J. and Russell, D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, NY (1984); and by Ausubel, F. M. et. al., Short Protocols in Molecular Biology, 5^th Ed. Current Protocols and John Wiley and Sons, Inc., N.Y., 2002.

Materials and methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for use in the following Examples may be found in Manual of Methods for General Bacteriology, Phillipp Gerhardt, R. G. E. Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds., (American Society for Microbiology Press, Washington, DC (1994)), Biotechnology: A

Textbook of Industrial Microbiology by Wulf Crueger and Anneliese Crueger (authors), Second Edition, (Sinauer Associates, Inc., Sunderland, MA (1990)), and Manual of Industrial

Microbiology and Biotechnology, Third Edition, Richard H. Baltz, Arnold L. Demain, and Julian E. Davis (Editors), (American Society of Microbiology Press, Washington, DC (2010).

All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from BD Diagnostic Systems (Sparks, MD), Invitrogen/Life Technologies Corp. (Carlsbad, CA), Life Technologies (Rockville, MD), QIAGEN (Valencia, CA), Sigma-Aldrich Chemical Company (St. Louis, MO) or Pierce Chemical Co. (A division of Thermo Fisher Scientific Inc., Rockford, IL) unless otherwise specified. IPTG, (cat#I6758) and triphenyltetrazolium chloride were obtained from the Sigma Co., (St. Louis, MO). Bellco spin flask was from the Bellco Co., (Vineland, NJ). LB medium was from Becton, Dickinson and Company (Franklin Lakes, New Jersey). BCA protein assay was from Sigma-Aldrich (St Louis, MO).

Example 1.

Assay of AFP using Abz-AAFFAA-Anb (SEQ ID NO: 15) as the peptidase substrate with the aid of the exopeptidase 3PP

The substrate Abz-AAFFAA-Anb (SEQ ID NO: 15) was designed by the inventors and contract synthesized by Schafer-N (Lerso Parkalle 42, 2100 Copenhagen, Denmark) with purity greater than 90% according to the supplier. It was dissolved in DMSO at a concentration of lOmM. AFP (US 7,429,476 B2) is an acidic fungal protease product from DuPont with a protein concentration of 27.7 μg/ml. The term "3PP" refers to a proline tolerant tripeptidyl

aminopeptidase. 3PP is an exopeptidase which is a member of Sedolisin family S53 in the MEROPS database. It was prepared as described earlier (ref: WO2016062857 (Al)— 2016-04- 28) with a concentration of 450 μg/ml. The three components were added to a half bottom area of 96 well microplate (Costar 3695, Corning Inc. NY, USA): 1 μΐ the substrate Abz-AAFFAA- Anb (SEQ ID NO: 15), 1-10 μΐ AFP, 2 μΐ 3PP and 90-100 μΐ 0.1M HAC (pH4.0) having tween 80 (0.1% w/v) to a final volume of 103 μΐ. In controls either AFP or 3PP was omitted. The reaction was started by adding AFP and was followed at 410 nm at 30°C for up to 60 min with reading intervals of 1 min.

Figure la shows the increased absorbance at 410 nm as a function of reaction time and AFP concentrations in the presence of 3PP enzyme. That is, the absorbance increase was in proportional to the amount of AFP present, which can be used to quantify the AFP present in a feed product sample. For example, at 0.277 μg AFP/ml reaction mixture, the linearity was between 2.5 and 7.5 min. In control without AFP, no increase in absorbance was observed during the reaction period. In control without 3PP, the absorbance first decreased and then levelled off (Figure la).

Figure lb shows the reaction rate (mOD/min at 410nm) increases as the AFP

concentration is increased, in a coupled reaction with the aid of 3PP. It is evident that the linear response range for AFP on the peptidase substrate Abz-AAFFAA-Anb (SEQ ID NO: 15) lies between 0.28-1.0 μg/ml (Figure lb).

Example 2

Assay of AFP using Succ-DMFIND-Anb (SEQ ID NO:2) as peptide substrate with the aid of 3PP

The N- and C-terminus blocked peptide substrate, Succ-DMFIND-Anb (SEQ ID NO:2), was designed by the inventors and contract synthesized by Schafer-N with purity greater than 95% according to the supplier. The stock solution of AFP was 27.7 μg/ml, 3PP, 450 μg/ml and Succ-DMFIND-Anb in DMSO, 5 mg/ml. To a half bottom area of 96 well microplate (Costar 3695, Corning Inc. NY, USA) were added 5 μΐ Succ-DMFIND-Anb (SEQ ID NO:2), 1-45 μΐ AFP, 2 μΐ 3PP and 20 mM acetate (pH 4.0) containing 0.05% (w/v) Tween 80 to a final volume of 50 μΐ. The reaction was followed at 410 nm at 30°C for 60 min with reading intervals of 1 min.

Figure 2 shows the increase of reaction rate at 410nm as a function of AFP concentration, in a coupled reaction with the aid of 3PP. It is seen that there was linear response for AFP at concentrations roughly in the range of 0.5 to 10 μg/ml reaction mixture.

Example 3

Absorbance spectrum of Succ-DMFIND-Anb (SEQ ID NO:2) before and after the hydrolysis by AFP and 3PP

The reaction mixture contained 50 μΐ Buffer A (0.1M sodium acetate containing 0.05% w/v Tween 80 and 0.5M NaCl, pH4.0), 20 μΐ AFP (27.7 μ^πιΐ), 2 μΐ 3PP (450 μg/ml) to a volume of 95 μΐ. AFP was omitted from the control. The reaction was started with the addition of 5 μΐ Succ-DMFIND-Anb (SEQ ID NO:2) purchased from Schafer-N in DMSO at 5 mM and was carried out at 30°C for 103min. The reaction mixture was scanned between 300 and 500nm at 5 nm intervals at the end of the reaction.

Figure 3 shows the scanning spectra of the reaction mixture with and without AFP, in a coupled reaction with the aid of 3PP. It can be seen the largest difference between absorbance at 390nm and 410nm, that is, even though the reaction could be monitored between 360 and 440nm, the peak absorbance is 390-410nm, in line with the detection of a released Anb moiety.

Example 4

Coupled assay of AFP using Succ-DMFIND-Anb (SEQ ID NO:2) with the aid of leucine aminopeptidase as the exopeptidase

In this example 3PP was replaced with the leucine aminopeptidase (Corolase^® Lap) from Aspergillus soiae and expressed in A. oyzae (AB Enzymes, Darmstadt, Germany) (Schuster et al., 2001, US 6,228,632B1), The reaction mixture contained 50 μΐ Buffer A, 1 μΐ AFP (10 μg/ml), 0.5 μΐ leucine aminopeptidase Corolase Lap (38.5 μg/ml) and 5 μΐ of the peptide substrate, Succ-DMFIND-Anb (SEQ ID NO:2) (5 mg/ml in DMSO). AFP was omitted from the control. The reaction was started by the addition of the peptide substrate 5 μΐ Succ-DMFIND- Anb at 30°C and followed at 390 nm every 1 min for 120 min.

Figure 4 shows that AFP can be assayed using the N- and C-terminal blocked substrate Succ-DMFIND-Anb (SEQ ID NO:2) with the aid of the exopeptidase leucine aminopeptidase, when the reaction is monitored at 390 nm. The linear range of the reaction was roughly between 10-70 min.

Example 5

Coupled assay of AFP in corn soy feed using Abz-AAKFAA-Anb (SEQ ID NO:3) with the aid of 3PP

Corn soy mash feed (described in: S. Yu, A. Cowieson, C. Gilbert, P. Plumstead, and S. Dalsgaard. 2012. Interactions of phytate and myo-inositol phosphate esters (IPl-5) including IP5 isomers with dietary protein and iron and inhibition of pepsin. Online supplement. J. Anim. Sci. 90: 1824-1832) was ground and passed through a 2 mm sieve using Laboratory Mill 3100 from Perten Instruments (Hagersten, Sweden). The reaction mixture contained 10 μΐ 3PP, 15 μΐ corn soy feed extract having AFP at 0.2g/kg feed (AFP in-feed extract) extracted in Buffer A, 70 μΐ Buffer A in 96 well half area plate. The reaction was started with the addition of 5 μΐ Abz- AAKFAA-Anb (SEQ ID NO:3) designed by the inventors and contract synthesized by Schafer-N (10 mM in DMSO) and was followed after mixing every one min at 400 nm and 45°C for 107 min (Figure 5). For controls, Buffer A was used instead of 3PP (AFP in-feed - 3PP") or the feed extract had no AFP added ("Feed extract having no AFP"). AFP in the final assay mixture in Figure 5 was 3 μg/ml. If lg feed is extracted in 10 ml buffer and 15 μΐ extract is used in an assay mixture of 0.1 ml, the feed should contain 30 g AFP/ton or 30 ppm.

For making the AFP in-feed extract used in Figure 5, 2g AFP TPT (which was coated with multiple layers by Thermal Platform Technology as described in EP 1940241 B l) having 0.2 g pure AFP was mixed with 1kg corn soy feed, ground. To 1 g of the ground feed was added 10ml Buffer A, mixed and extracted at 22°C for 20min. After that it was then filtered through glass fiber filter. The filtrate 15 μΐ was used for the assay. For feed having no AFP, there was no AFP added.

Example 6

Coupled assay of AFP in corn soy feed using Abz-AAKFAA-Anb (SEQ ID NO:3) as substrate with the aid of 3PP

The reaction mixture contained the following: 10 μΐ 3PP (10 μg/ml) and 85 μΐ corn soy feed extract which was made by suspending 1 g corn soy feed having AFP 0.2 mg in 10 ml Buffer A and further diluted 20 times with corn soy feed extract having no AFP made in 0.1 M sodium phosphate (pH3.0) so that the final AFP concentration in the assay mixture was 0.85 μg/ml ("AFP in-feed extract+3PP"). For control, the 85 μΐ corn soy feed extract having no AFP ("Feed having no AFP+3PP") was used. The reaction was started with the addition of 5 μΐ Abz- AAKFAA-Anb (SEQ ID NO:3) (lOmM in DMSO). The reaction was followed after mixing every one min at 400 nm and 45°C for 120min (Figure 6).

For quantification of AFP in feed, an AFP standard curve shall be made by dissolving AFP in feed extract to a final AFP concentration 0.1-30 μg/ml. Take this extract 85 μΐ and do the assay as above. Figure 6 shows that this method is also applicable for detecting AFP in-feed at AFP level of 8.5 g pure protein/ton treated feed (i.e., at 8.5 ppm) when 1 g feed containing AFP 8.5 μg was extracted in 10 ml buffer and 85 μΐ extract was used in an assay mixture with a final volume of 0.1 ml. The reaction had a wide linear range from 4 to 40 min. The reaction rate for AFP in-feed+3PP was 13.51+/-2.30 (n=10) mOD/min; for the control (feed having no AFP+3PP) it was 1.29+/0.07 (n=l 1), thus, the signal-to-noise noise ratio was 10.

It is known that animal feed may contain high concentration of minerals, vitamins and other secondary metabolites of plants. These materials may interfere with such in feed assays. For example, a high salts concentration may quench fluorescence if a fluorescence assay method is used for detection and/or quantification.

The background absorbance values at reaction time zero increased from 0.12 in Figure 5 to 0.3 in Figure 6 when volume of the feed extract increased from 15 to 85% but the reaction linearity was not affected in either of the figures. Thus, it shows that extraneous materials present in the commercial corn soy feed did not interfere with the assay adopting the AFP and 3PP coupled assay using Abz-AAKFAA-Anb (SEQ ID NO:3) as substrate and monitoring 400 nm absorbance change in a microplate format.

Example 7

Coupled assay of AFP in corn soy feed using Abz-AAKFAA-Anb (SEQ ID NO:3) with the aid of 3PP

The reaction mixture contained: 10 μΐ 3PP (10 μ /ηι1), 85 μΐ corn soy feed extract having AFP at 0.2 g/kg feed extracted in Buffer A and diluted 20, 25, 33, 50 and 100 times with feed extract having no AFP made in Buffer A. For control, 85 μΐ corn soy feed extract having no AFP extracted in Buffer A was used instead. The reaction was started with the addition of 5 μΐ Abz- AAKFAA-Anb (SEQ ID NO:3) (10 mM in DMSO). The reaction was followed after mixing every one min at 400 nm and 45°C for 40 min (Figure 7).

The final AFP concentration per ml reaction mixture in Figure 7 was 0.2, 0.4, 0.52, 0.68 and 0.85 μg/ml. If 1 g feed is extracted with 10 ml buffer A and 8 5μ1 of the extract is used in an assay format given in Figure 7, the assayable AFP level in feed is thus between 1.7 and 8.5 ppm AFP in feed (i.e., 1.7-8.5g AFP per ton treated feed) as shown in Figure 7.

Example 8

Coupled assay of AFP in-feed using Abz-AAKFAA-pNA (SEQ ID NO:3) with the aid of

3PP

Five grams of ground mash feed was added to 50 ml Buffer G (lOOmM Glycine-HCl,

50mM NaCl, 0.05% (w/v) Tween 80, pH 3.0) and various amount of AFP in Buffer G. This feed AFP mixture was extracted at 22°C for 20 min under stirring with a magnetic bar at 600 rpm in a plastic beaker and was then filtered through a GA55 glass fiber filter from Advantec. The filtrate was used directly for the AFP activity assay which was monitored using a spectrophotomer set at 410 nm, 30°C for a 100 min period, with reading intervals of lmin. Five μΐ of the filtrate was added to a Costar™ 96-Well Half-Area Plate with 4.5mm bottom diameter) having 2 μΐ 3PP (0.6 mg/ml) and 90 μΐ Buffer A. The reaction was started by adding 5μ1 Abz-AAKFAA-pNA designed in-house and contract synthesized by GL Biochem (Shanghai, China) substrate using a pre-prepared stock solution of SEQ ID NO:3 10 mM in DMSO. For controls, the filtrate containing AFP and 3PP were replaced with Buffer A. The reaction rate (mOD/min at 410 nm) was calculated as the average of 6 independent readings for different AFP concentrations, subtracting the control having no AFP. Figure 8 shows that AFP could be measured in the presence corn soy feed matrix using the peptidase substrate Abz-AAKFAA-pNA (SEQ ID NO:3) and with the aid of 3PP that released pNA (p-nitroaniline) from FAA-pNA produced by the action of AFP on Abz-AAKFAA-pNA (SEQ ID NO:3). The linear assay range for AFP in the presence of corn soy feed matrix by this couple enzymic assay was from 4 tol2 mSAPU/ml reaction mixture (0.11-0.33 μg/ml, lmSAPU=27.7 ng AFP, 1 SAPU=1000 mSAPU)) (Figure 8). In other word, the assay range is between 1.1 to 3.3g AFP per ton feed when 1 g feed is extracted with 10 ml buffer and 5 μΐ of the extracted is assayed in a final reaction volume 0.1 ml.

One Spectrophotometric Acid Protease Unit (SAPU) is the amount of AFP needed which liberates 1 micromole of tyrosine equivalents per minute under defined assay conditions: solubilized casein peptides from a 30 min proteolytic hydrolysis of a purified high nitrogen casein substrate at pH 3.0 and 37°C. Unhydrolyzed substrate is precipitated with trichloroacetic acid and removed by filtration. Solubilized casein is then measured spectrophotometrically with tyrosine as standard in the UV range.

Example 9

Use of Abz-AAKFAA-pNA (SEQ ID NO:3) and Abz-AAKFAA-Anb (SEQ ID NO:3) as substrates for in-feed assay of AFP

Buffer G 50 ml and various amounts of AFP in this volume of Buffer G were added to 5 g of ground mash corn soy feed. This feed AFP mixture was mixed at 22°C for 20 min under stirring with a magnetic bar in plastic beaker and was then filtered through GA55 glass fiber filter. The filtrate was directly used for the AFP activity assay which was monitored using Spectramax M5 using bottom reading mode at excitation of 320 nm and emission of 420 nm. The filtrate 5 μΐ having AFP and 90 μΐ Buffer A (pH4.0) was added to a 96 well microplate with transparent bottom (Thermo scientific Nunc 265301). After incubation at 37°C for 5 min, 5 μΐ of the peptidase substrate, Abz-AAKFAA-pNA (SEQ ID NO:3) (10 mM in DMSO) was added to the microplate to start the reaction.

When the peptidase substrate, Abz-AAKFAA-Anb (SEQ ID NO:3) was used, (purchased from Schaefer-N), 5 μΐ of a stock solution (10 mM in DMSO) was added to the pre-incubation mixture containing 5 μΐ filtrate and 90 μΐ Buffer G (pH3.0). The reaction was followed by recording the fluorescence at 37°C at every 0.5min. The slopes were obtained by linear regression and used to draw the curve against AFP concentration.

Figure 9a shows that AFP could be assayed in the presence corn soy feed matrix at pFB.O in glycine buffer and pH4.0 in acetate buffer. The rate at pH4.0 with Abz-AAKFAA-pNA was 3.7 faster than at pFB.O with Abz-AAKFAA-Anb. The assay range for AFP was from 0.02 to 0.1 SAPU/ml.

When AFP mash feed extract from Figure 9a was further diluted 5 times with corn soy mash feed extract having no AFP, it was still detectable in a range of 4 to 20 mSAPU/ml reaction mixture corresponding to l . l-5.5g AFP per ton feed when this feed 1 g was extracted in 10 ml buffer and 5 μΐ of the extract was used in the assay format given above (Figure 9b).

Example 10

In-feed assay of AFP using Abz-AAKFAA-Anb (SEQ ID NO:3) substrate Buffer G 50 ml were used to suspend 5g corn soy feed having AFP at 3.8 to 25.2 μg/g (3.8-25.2g AFP per ton feed). This mixture was stirred with a magnetic bar at 22 °C for 20 min. The slurry was filtered through a glass fiber filter and the filtrate obtained was used for the assay of AFP activity.

The reaction mixture contained 20 μΐ the feed filtrate, 75 μΐ Buffer G, pre-incubated at

37°C for 5 min, and 5 μΐ substrate Abz-AAKFAA-Anb (SEQ ID NO:3) prepared in DMSO at 10 mM. The final AFP concentration per ml reaction mixture was 76-504 ng AFP or 2.7-18.2 mSPAU. The reaction was started by adding the substrate to a 96 well clear bottom microplate. Bottom reading mode was adopted. The reaction mixture was mixed 3 seconds before each fluorescence reading with intervals of 0.5 min for a total reading time of 10 min at 37°C. The reaction was monitored continually with an excitation wavelength of 320 nm, emission wavelength of 420 nm and 6 readings per well. The slope (the reaction rate, RFU/min) was obtained for each reaction containing various amount of AFP.

Figure 10 shows that the slope of fluorescence increased linearly versus the amount of AFP dosed in corn soy feed from 3.8 to 25.2 ppm, or 3.8 to 25.2 g AFP per ton feed.

Example 11

Use of Abz-AAFFAA-Anb (SEQ ID NO: 15) as substrate for in-feed assay of in high temperature pelleted feed.

Polymer coated AFP (AFP TPT granule) 60 g (EP 1940241 B l) was mixed with 120 kg corn soy feed. The final AFP protein was 50 mg per kilo feed. The feed-AFP mixture was steam heated at 90°C for 60 seconds. After cooling it was ground and extracted for AFP activity analysis. Two grams of the ground feed with and without added AFP were extracted in duplicate with 20 ml Buffer A at 22°C for 20 min stirred with a magnetic bar. The expected final AFP concentration in the extract was 5 μg/ml or 180 mSPAU/ml. The extract was filtered through a glass filter. The reaction mixture contained 10 μΐ of the feed extract filtrate with or without AFP, not steam heated, or steam heated, 85 μΐ Buffer A, pre-incubated at 40°C for 5min, 5 μΐ substrate Abz-AAFFAA-Anb (SEQ ID NO: 15) was added to start the reaction in a clear bottom 96 well microplate. The expected final AFP concentration in the assay reaction mixture was

18mSAPU/ml or 0.5 μg AFP/ml. For blank, Buffer A was used instead of 10 μΐ filtrate. Bottom reading was adopted with excitation wavelength of 320 nm, emission wavelength of 420 nm and 6 readings per well. The reaction mixture was mixed 3 seconds before each reading at 40°C using a Spectramax M5 fluorescence spectrophotometer.

Figure 11 shows that the peptidase substrate, Abz-AAFFAA-Anb (SEQ ID NO: 15), could be used for the in-feed assay of polymer coated (granulated) AFP after high temperature pelletingat 90°C by detecting release of fluorescence signal (excitation 320 nm/emission 420 nm).

Example 12.

Use of the fluorescence substrate H-Y(N02)DMFINDKPK(Abz)-OH (SEQ ID NO:6) as substrate for in-feed assay of AFP.

Preparation of mash feed (polymer coated AFP granule added corn soy feed): 60 g AFP granule containing 6 g pure AFP was mixed with 120 kg corn soy feed. For mash feed blank, no AFP granule was added. Part of the mash feed having AFP granule was steam heated at 90°C with a holding time of 60 seconds. After cooling, this feed was ground to a size of standard sieve of 2 mm. To each 1 g of the ground feed with AFP granule heated at 90°C, and mash feed having no AFP granule was added 10 ml Buffer A. The expected final AFP concentration in the extract was 5 μg/ml or 180 mSPAU/ml. AFP was extracted from the feed at 22°C for 20 min stirred with a magnetic bar in a beaker. The extract obtained was centrifuged at 4000 rpm for lOmin in a bench top centrifuge. The supernatant was used for the assay of AFP activity. The AFP activity assay mixture contained 10 μΐ of the substrate H-Y(N02)-DMFINDKPK(Abz)-OH (SEQ ID NO: 6) at 10 mM in DMSO, designed by the inventors and contract made by Schaefer-N and the AFP feed extract filtrate 190 μΐ in a 96 well microplate with transparent bottom. Fluorescence was recorded in bottom reading mode at 22°C every 40 seconds for 24 min with excitation at 315 nm and emission at 445 nm. Each data point in Figure 12 is the average readings of two wells.

Figure 12 shows that polymer coated AFP granule in the presence of corn soy feed matrix could be assayed using H-Y(N02)DMFINDKPK(Abz)-OH (SEQ ID NO: 6) as fluorescence substrate by measuring fluorescence release (excitation 315 nm/emission 445nm). The results also show that heat treatment at 90°C had an impact on AFP in feed activity compared to the unheated control.

Claims

CLAIMS What is claimed is:

1. An assay for detecting and/or quantitating microbial aspartic peptidase activity in feed, said assay comprising:

(a) contacting the feed with (i) a peptidase substrate wherein the substrate has a specific amino acid sequence that is capable of being recognized by a microbial aspartic peptidase in the feed, said substrate having between 2 and 20 amino acid residues and further wherein both N-terminal and C-terminal ends of the peptidase substrate are each modified with the same or with a different detectable moiety and (ii) at least one additional peptidase , and said contacting occurs under acidic conditions below pH 7; and

(b) detecting and/or quantitating the detectable moiety.

2. The assay of claim 1 wherein the at least one additional microbial peptidase is an exopeptidase.

3. The assay of claim 1 wherein the peptidase substrate has the amino acid sequence N- terminus-AAXiX2AAX3- C-terminus (SEQ ID NO: l) in which both N-terminal and C-terminal ends of the sequence are each modified with the same or with a different detectable group and further wherein Xi is an amino acid selected from the group consisting of Leu, Lys, Arg, Glu, Phe, Asp, Tyr, and His; X2 is an amino acid selected from the group consisting of Phe, Val, Lys, Ala, Cys, Glu, Leu, Pro, Tyr, His, He, Arg and Ser; X3 can be absent or K.

4. The assay of claim 1 wherein the peptidase substrate is Succ-DMFIND-Anb (SEQ ID

NO:2).

5. The assay of claim 1 wherein the detectable moiety is a chromophore.

6. The assay of claim 1 wherein the detectable moiety is detected and/or quantitated by measuring UV/Vis absorption.

7. The assay of claim 1 or 3 wherein

(a) the N-terminal detectable moiety is selected from the group consisting of acyl groups such as formyl, acetyl, benzoyl, fluorophores of Abz (2-Aminobenzoyl or Anthraniloyl), N-Me- Abz(N-Methyl-anthraniloyl), Dansyl (5-(Dimethylamino)naphthalene-l-sulfonyl), DMACA (7-Dimethylaminocoumarin-4-acetate), EDANS (5-[(2-Aminoethyl)amino]naphthalene-l- sulfonic acid), FITC (Fluorescein isothiocyanate), Lucifer Yellow

(6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino- lH-benz[d,e]isoquinoline-5,8-disulfonic acid), Mca ((7-Methoxycoumarin-4-yl)acetyl), Trp (Tryptophanyl); and

(b) the C-terminal detectable moiety is selected from the group consisting of: pNA(para- Nitroaniline), Anb (5-Amino-2-nitrobenzoic acid), Dnp (2,4-Dinitrophenyl), EDDnp (N-(2,4- Dinitrophenyl)ethylenediamine), 4-Nitro-Phe (4-Nitro-phenylalanine), 3-Nitro-Tyr (3-Nitro- tyrosine), 4-Nitro-Phe(4-Nitro-phenylalanine), DABCYL(4-(4- Dimethylaminophenylazo)benzoyl), BD (7-Nitro-benzo[2,l,3]oxadiazol-4-yl), Dabsyl (4-(4- Dimethylaminophenylazo)-benzenesulfonyl), 4-Nitro-Z(4-Nitro-benzyloxycarbonyl).

8. An assay for detecting and/or quantitating microbial aspartic peptidase activity in feed, said assay comprising:

(a) contacting the feed with a peptidase substrate wherein the substrate has a specific amino acid sequence that is capable of being recognized by the microbial peptidase in the feed, said substrate having between 2 and 20 amino acid residues and further wherein both N-terminal and C-terminal ends of the peptidase substrate are each modified with the same or with a different detectable moiety, and said contacting occurs under acidic conditions below pH 7; and

(b) detecting and/or quantitating the detectable moiety.

9. The assay of claim 8 wherein the peptidase substrate has the amino acid sequence N- terminus-AAXiX2AAX3- C-terminus (SEQ ID NO: l) in which both N-terminal and C-terminal ends of the sequence are each modified with the same or with a different detectable group and further wherein Xi is an amino acid selected from the group consisting of Leu, Lys, Arg, Glu, Phe, Asp, Tyr, His, Ala, Thr, Cys, Gly and Trp; X₂ is an amino acid selected from the group consisting of Phe, Val, Lys, Ala, Cys, Glu, Leu, Pro, Tyr, His, He, Arg and Ser; X₃ can be absent or K.

10. The assay of claim 8 or 9 wherein

(a) the N-terminal detectable moiety is selected from the group consisting of acyl groups such as formyl, acetyl, benzoyl, fluorophores of Abz (2-Aminobenzoyl or Anthraniloyl), N-Me- Abz(N-Methyl-anthraniloyl), Dansyl (5-(Dimethylamino)naphthalene-l-sulfonyl), DMACA (7-Dimethylaminocoumarin-4-acetate), EDANS (5-[(2-Aminoethyl)amino]naphthalene-l- sulfonic acid), FITC (Fluorescein isothiocyanate), Lucifer Yellow

(6-Amino-2,3-dihydro-l,3-dioxo-2-hydrazinocarbonylamino- lH-benz[d,e]isoquinoline-5,8-disulfonic acid), Mca ((7-Methoxycoumarin-4-yl)acetyl), Trp (Tryptophanyl); and

(b) the C-terminal detectable moiety is selected from the group consisting of: pNA(para- Nitroaniline), Anb (5-Amino-2-nitrobenzoic acid), Aminobenzoyl, Dnp (2,4-Dinitrophenyl), EDDnp (N-(2,4-Dinitrophenyl)ethylenediamine), 4-Nitro-Phe (4-Nitro-phenylalanine), 3-Nitro- Tyr (3-Nitro-tyrosine), 4-Nitro-Phe(4-Nitro-phenylalanine), DABCYL(4-(4- Dimethylaminophenylazo)benzoyl), BD (7-Nitro-benzo[2,l,3]oxadiazol-4-yl), Dabsyl (4-(4- Dimethylaminophenylazo)-benzenesulfonyl), 4-Nitro-Z(4-Nitro-benzyloxycarbonyl).

11. The assay of claim 8 wherein the peptidase substrate has the sequence set forth in SEQ ID NO:5.