RU2791882C2 - USE OF ALPHA-1,4/1,6-GLYCOSIDE HYDROLASES ACTIVE AT LOW pH VALUE AS FEED ADDITIVE FOR RUMINANTS FOR IMPROVEMENT OF STARCH DIGESTION - Google Patents

Abstract

FIELD: feed technology.

SUBSTANCE: use of a composition based on enzymes for increasing starch digestion and glycose yield in a ruminant is proposed. Moreover, the specified composition contains glucoamylase EC 3.2.1.3 enzyme derived from Trichoderma reesei or Aspergillus fumigatus. The specified glucoamylase has at least 20% activity at pH less than or equal to 3 in the presence of pepsin, compared to glucoamylase activity at pH of 6 in the presence of pepsin. The specified glucoamylase is active in at least two of three digestive chambers of a ruminant, including rumen, read stomach, and small intestine. The specified glucoamylase acts with digestive enzymes present in digestive chambers of a ruminant to provide an increase in starch digestion and glucose yield.

EFFECT: invention is aimed at an increase in starch digestion and glucose yield.

8 cl, 11 dwg, 10 tbl, 19 ex

Description

FIELD OF THE INVENTION

The field of the invention relates to animal nutrition and in particular to the use of low pH alpha-1,4/1,6-glycoside hydrolases (GLCH), such as alpha-amylases, glucoamylases and alpha-glucosidases, as a feed additive for ruminants to improve starch digestion.

BACKGROUND OF THE INVENTION

Ruminants have the unique ability to convert roughage into protein and energy through their microbial/enzymatic digestive systems. Accordingly, ruminants play an important role in the Earth's ecology and in the food chain.

The main difference between ruminants and non-ruminants is that ruminant stomachs have four sections: tripe, reticulum, booklet, and abomasum. In the first two chambers, in the rumen and the mesh, food is mixed with saliva and separated into layers of solid and liquid material. The solids collect together to form a cud or food bolus.

The gum is then regurgitated and chewed to completely mix it with saliva and to reduce the particle size. Fiber, especially cellulose and hemicellulose, is initially broken down in these chambers by microorganisms (mainly bacteria, but also some protozoa, fungi, and yeasts) into the three main volatile fatty acids (VFAs): acetic acid, propionic acid, and butyric acid. Protein and non-structural carbohydrates (pectin, sugars and starches) are also fermented.

Despite the fact that the scar and the mesh have different names, they represent a single functional space, since the digested food can move between them in both directions. Together, these chambers are called the reticulo-scar. The digested food that is now in the lower part of the reticulo-rumen, intended for liquid food, then passes into the next chamber, the book, where water and many inorganic minerals are absorbed into the bloodstream.

After that, the digested food moves into the true stomach, the abomasum. Abomasum is the direct equivalent of a single-chambered stomach and digested food is digested here in much the same way. The digested food finally moves to the small intestine, where digestion and absorption of nutrients takes place. Microorganisms that multiply in the reticulo-rumen are also digested in the small intestine. Fermentation continues in the large intestine in the same way as in the reticulo-rumen.

Enzymes for use as feed additives in ruminants are mainly fibrolytic enzymes such as cellulases, beta-glucanases and hemicellulases (Table 1 in Beauchemin et al., 2004. A rationale for the development of feed enzyme products for ruminants. Can. J. Anim. Sci. 84: 23-36). There are limited reports of starch hydrolases for use in ruminants. Starch hydrolases are grouped into endo- and exoamylases.

Endoamylases, also called alpha-amylases (EC 3.2.1.1), are starch-lytic enzymes that catalyze the hydrolysis of internal alpha-1,4-O-glycosidic bonds to polysaccharides while retaining the alpha-anomeric configuration in products. Most alpha-amylases require calcium ions (Ca ²⁺ ) for their activity, structural integrity and stability.

The alpha-amylase family, ie the GH-H clan of glycoside hydrolases, is the largest family of glycoside hydrolases, transferases and isomerases, comprising almost 30 different enzymatic specificities. A wide variety of enzymes can act on starch. Such enzymes can be divided into four groups: endoamylases, exoamylases, debranching enzymes, and transferases.

Endoamylases cleave internal alpha-1,4 bonds resulting in alpha-anomeric products.

Exoamylases cleave alpha-1,4 or alpha-1,6 bonds of external glucose residues resulting in alpha or beta anomeric products.

Debranching enzymes hydrolyze alpha-1,6 bonds exclusively leaving long linear polysaccharides.

Transferases cleave alpha-1,4-glycosidic bonds of the donor molecule and transfer part of the donor to the acceptor glycoside to form a new glycosidic bond.

Glycoside hydrolases have been divided into classes based on their type of reaction and into families based on their well-defined amino acid sequence similarities. Most amylolytic enzymes belong to the GH-13 family. The GH-13 family can be further classified based on a larger category called clan, which characterizes the three-dimensional structure of the catalytic module. Among the fourteen clans (A-N) defined for glycosidases and transglycosidases, the alpha-amylase family (GH-13) belongs to the eighth clan GH-H.

Animals synthesize and secrete the digestive pancreatic alpha-amylase for starch hydrolysis. This alpha-amylase has an optimum pH of around pH 6-7 and is active in the small intestine.

The addition of microbial amylases to the feed may further increase the digestion of starch by the animals, probably due to the fact that secreted pancreatic amylase is insufficient and/or the passage of feed through the digestive tract of the small intestine is rather rapid, resulting in insufficient time for pancreatic amylase to be thoroughly digested. starch, especially in the case of poultry. (Isaksen, M. F. Cowieson, A. J. Kragh, K. M. (2010). Starch– and protein–degrading enzymes: biochemistry, enzymology and characteristics relevant to animal feed use. In: Enzymes in farm animal nutrition /edited by M. R. Bedford and G. G. Partridge. p. 85–95).

Thus, endoamylases are among the feed enzymes widely used in the feed industry. The most common feed amylases used are from Bacillus subtilis, B. amyloliquefaciens, B. licheniformis and Aspergillus oryzae .

These feed amylases are optimally active in the pH range of pH 4 to pH 8. Most of them show significant pepsin resistance. However, such feed enzymes do not appear to contain a crude starch binding domain (SBD) and may have a limited ability to hydrolyze ungelatinized raw starch.

Planchot et al. (Extensive degradation of native starch granules by alpha-amylase from Aspergillus fumigatus , Journal of Cereal Science, vol. 21, Issue 2, 1995, pp. 163-171) reported low efficiency of alpha-amylase from Bacillus sp . in relation to the breakdown of granular starch compared to pancreatic amylase. EFSA (Scientific Opinion on the safety and efficacy of Ronozyme RumiStar (alpha-amylase) as a feed additive for dairy cows. Additives and Products or Substances used in Animal Feed (FEEDAP). EFSA Journal 2012;10(7):2777) reported on the use of alpha-amylase from B. licheniformis in dairy cows. The effect was in some cases noticeable, and in some cases the effect was negligible. Dettori-Campus et al. (Hydrolysis of starch granules by the amylase from Bacillus stearothermophilus NCA 26. Process Biochemistry, Volume 27, Issue January 1, 1992, pp. 17-21) studied amylases from 16 separate Bacillus strains and demonstrated their preferential activity against soluble starch, rather than crude starch, with the highest ratio of crude starch to soluble starch activity being 0.25 for the B. amylolyticus strain.

Tricarico et al. (Dietary supplementation of ruminant diets with an Aspergillus oryzae alpha-amylase. Animal Feed Science and Technology. Volume 145, Issues 1-4, August 14, 2008, pp. 136-150) reviewed the use of alpha-amylase from Aspergillus oryzae as ruminant dietary supplement and hypothesized that this amylase-based supplement could improve animal performance by modifying ruminal starch digestion without the need to enhance ruminal starch digestion. This effect could well be due to enzymatic impurities, since, by definition, the addition of amylase is expected to increase starch digestion (Beauchemin and Holtshausen, (2010). Developments in enzyme usage in ruminants. In: Developments in enzyme usage in ruminants. In: Enzymes in farm animal nutrition /edited by MR Bedford and GG Partridge, pp. 206–230).

Monteiro de Souza et al. (Application of microbial alpha-amylase in industry - a review. Brazilian Journal of Microbiology (2010) 41: 850-861) reviewed industrial alpha-amylases and almost all of them had an optimal pH above pH 5, with the exception of one from Aspergillus kawachii , which was characterized by optimal pH and stability at 3.0. It was A. kawachii amylase that was used together with glucoamylase for simultaneous liquefaction and fermentation of corn starch to bioethanol due to its ability to break down raw starch or granular starch (Dunn–Coleman et al., 2008, US 7354752 B2).

The digestibility of starch in feed varies greatly and depends on a number of factors, including the physical structure of both the starch and the feed base.

U.S. Patent Application Publication 2005/0037053, published Feb. 17, 2005, discloses the use of an enzyme having amylolytic activity and capable of degrading resistant starch in starch-containing feed for monogastric animals such as poultry and pigs.

WO 2008/06881, published Jan. 17, 2008, discloses the use of bacterial amylases in feed for bovine ruminants to improve milk yield, apparent digestibility of the fed ration, feed dry matter digestibility, weight gain and/or feed conversion rate.

WO 2015/128366, published September 3, 2015, discloses the use of bacterial amylases in combination with one or more proteases in feed for bovine ruminants to improve the digestibility of maize and/or maize silage, in particular to improve milk yield, weight gain and/or feed conversion ratio.

Accordingly, there is still a need to increase starch digestibility, increase glucose yield, increase dry matter digestion and/or gas production for ruminants.

BRIEF DESCRIPTION OF THE INVENTION

In one embodiment, a method is disclosed for increasing starch digestibility and glucose yield in a ruminant, which comprises adding at least one alpha-1,4/1,6-glycoside hydrolase (GLCH) as a feed additive to a ruminant's feed, wherein said hydrolase (a) has at least 20% activity at pH 3 or less in the presence of pepsin compared to hydrolase activity at pH 6 in the presence of pepsin, (b) said hydrolase is active in at least two of the three digestive chambers ruminant, including rumen, abomasum, and small intestine, and (c) the hydrolase acts in conjunction with digestive enzymes present in the ruminant's digestive chambers to increase starch digestibility and glucose output.

In a second embodiment, at least one GLCH enzyme is capable of hydrolyzing raw starch under conditions comparable to those found in rumen or abomasum.

In a third embodiment, the hydrolase is selected from the group consisting of alpha-amylases, glucoamylases, and alpha-glucosidases. Preferably the group consists of

alpha-amylases and glucoamylases.

In a fourth embodiment, the alpha-amylases are members of the GH 13 family or are selected from the group consisting of alpha-amylase (EC 3.2.1.1); pullulanase (EC 3.2.1.41 ); cyclomaltodextrin-glucanotransferase (EC 2.4.1.19 ); cyclomaltodextrinase (EC 3.2.1.54 ); trehalose-6-phosphate hydrolase (EC 3.2.1.93 ); oligo-alpha-glucosidase (EC 3.2.1.10 ); maltogenic amylase (EC 3.2.1.133 ); neopullulanase (EC 3.2.1.135 ); alpha-glucosidase (EC 3.2.1.20 ); maltotetraose-forming alpha-amylase (EC 3.2.1.60 ); isoamylase (EC 3.2.1.68 ); glucodextranase (EC 3.2.1.70 ); maltohexaose-forming alpha-amylase (EC 3.2.1.98 ); maltotriose-forming alpha-amylase (EC 3.2.1.116 ); branching enzyme (EC 2.4.1.18 ); trehalose synthase (EC 5.4.99.16 ); 4-alpha-glucanotransferase (EC 2.4.1.25 ); maltopentaose-forming alpha-amylase (EC 3.2.1.- ) ; amylosucrases (EC 2.4.1.4 ); sucrose phosphorylase (EC 2.4.1.7 ); maltooligosyltrehalose-trehalohydrolase (EC 3.2.1.141 ); isomaltulose synthase (EC 5.4.99.11 ); maltooligosyltrehalose synthase (EC 5.4.99.15 ); amyl-alpha-1,6-glucosidase (EC 3.2.1.33 ) and alpha-1,4-glucan:phosphate-alpha-maltosyltransferase (EC 2.4.99.16 ).

In a fifth embodiment, the glucosidases are members of the GH 13 or GH31 family, or are selected from the group consisting of alpha-glucosidase (EC 3.2.1.20 ); alpha-galactosidase (EC 3.2.1.22 ); alpha-mannosidases (EC 3.2.1.24 ); alpha-1,3-glucosidase (EC 3.2.1.84 ); sucrase-isomaltase (EC 3.2.1.48 ) (EC 3.2.1.10 ); alpha-xylosidases (EC 3.2.1.177 ); alpha-glucanase (EC 4.2.2.13 ); isomaltosyltransferase (EC 2.4.1.– ); oligosaccharide-alpha-1,4-glucosyltransferase (EC 2.4.1.161 ); sulfoquinovosidases (EC 3.2.1.– ).

In a sixth embodiment, the glucoamylases are members of the GH15 family or are selected from the group consisting of glucoamylase (EC 3.2.1.3 ); glucodextranase (EC 3.2.1.70 ); alpha-trehalase (EC 3.2.1.28 ) and dextrandextrinase (EC 2.4.1.2 ).

In a seventh embodiment, a method is disclosed for increasing starch digestibility, increasing glucose yield, increasing dry matter digestion, and increasing gas production during fermentation in a ruminant, comprising adding to the feed an enzyme-based composition containing (i) at least one GLCH enzyme as feed ruminant additives, wherein said enzyme (a) is at least 20% active at pH 3 or less in the presence of pepsin compared to enzyme activity at pH 6 in the presence of pepsin, (b) said enzyme is active in at least two of the ruminant's three digestive chambers, including rumen, abomasum, and small intestine, and (c) the enzyme works in conjunction with pancreatic amylase to increase glucose output, and (ii) at least one protease.

In a ninth embodiment, at least one GLCH enzyme is capable of hydrolyzing raw starch.

In a tenth embodiment, the GLCH enzyme is selected from the group consisting of alpha-amylases, glucoamylases, and alpha-glucosidases. Preferably the enzyme is selected from the group consisting of alpha-amylases and glucoamylases.

In an eleventh embodiment, at least one GLCH enzyme is selected from the group consisting of alpha-amylase (EC 3.2.1.1); pullulanase (EC 3.2.1.41 ); cyclomaltodextrin-glucanotransferase (EC 2.4.1.19 ); cyclomaltodextrinase (EC 3.2.1.54 ); trehalose-6-phosphate hydrolase (EC 3.2.1.93 ); oligo-alpha-glucosidase (EC 3.2.1.10 ); maltogenic amylase (EC 3.2.1.133 ); neopullulanase (EC 3.2.1.135 ); alpha-glucosidase (EC 3.2.1.20 ); maltotetraose-forming alpha-amylase (EC 3.2.1.60 ); isoamylase (EC 3.2.1.68 ); glucodextranase (EC 3.2.1.70 ); maltohexaose-forming alpha-amylase (EC 3.2.1.98 ); maltotriose-forming alpha-amylase (EC 3.2.1.116 ); branching enzyme (EC 2.4.1.18 ); trehalose synthase (EC 5.4.99.16 ); 4-alpha-glucanotransferase (EC 2.4.1.25 ); maltopentaose-forming alpha-amylase (EC 3.2.1.- ) ; amylosucrases (EC 2.4.1.4 ); sucrose phosphorylase (EC 2.4.1.7 ); maltooligosyltrehalose-trehalohydrolases (EC 3.2.1.141 ); isomaltulose synthase (EC 5.4.99.11 ); maltooligosyltrehalose synthase (EC 5.4.99.15 ); amyl-alpha-1,6-glucosidase (EC 3.2.1.33 ) and alpha-1,4-glucan:phosphate-alpha-maltosyltransferase (EC 2.4.99.16 ).

In a twelfth embodiment, the glucosidases are selected from the group consisting of alpha-glucosidase (EC 3.2.1.20 ); alpha-galactosidase (EC 3.2.1.22 ); alpha-mannosidases (EC 3.2.1.24 ); alpha-1,3-glucosidase (EC 3.2.1.84 ); sucrase-isomaltase (EC 3.2.1.48 ) (EC 3.2.1.10 ); alpha-xylosidases (EC 3.2.1.177 ); alpha-glucanase (EC 4.2.2.13 ); isomaltosyltransferase (EC 2.4.1.– ); oligosaccharide-alpha-1,4-glucosyltransferase (EC 2.4.1.161 ); sulfoquinovosidase (EC 3.2.1.– ).

In a thirteenth embodiment, the glucoamylases are selected from the GH15 family of glycosyl hydrolases.

In a fourteenth embodiment, the protease is selected from the group consisting of an acidic protease or a neutral metalloprotease, and preferably the protease is a fungal aspartic protease or a bacterial neutral metalloprotease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the release of glucose and maltose from cornmeal by AkAA alpha-amylase in the presence of pepsin and AFP-protease (example 2).

Figure 2 shows the release of glucose, maltose and maltotriose from cornmeal by AkAA alpha-amylase and pancreatin and the synergy of AkAA and pancreatin to increase glucose release and decrease maltotriose release (Example 4).

Figure 3 shows the activity of AtAA alpha-amylase against cornmeal in the presence of pepsin at pH 2.5 and pancreatin at pH 6.7 and the complete conversion of maltotriose when AtAA and pancreatin are dosed together (Example 5).

Figure 4 shows the stability of AkAA and AcAA alpha-amylases and their mixture with TrGA and CS4 glucoamylases and aspartyl protease when incubated with rumen juice (Example 6).

Figure 5 shows the activity of AkAA and AcAA alpha-amylases in the presence of pepsin, tested at pH 3.2-6.0 (Example 7).

Figure 6 shows the activity of enzyme mixtures containing alpha-amylases AkAA, AcAA, and glucoamylase TrGA, CS4, and aspartyl peptidase AFP, in the presence of pancreatin (example 8).

The figure 7 shows the interaction of glucoamylase with pancreatin in relation to increasing the hydrolysis of corn starch granules (example 10).

The figure 8 shows the interaction of glucoamylase with pancreatin in relation to the increase in the hydrolysis of corn flour (example 10).

Figure 9 shows the release of glucose from cornstarch granules by yeast maltase (alpha-glucosidase) in the presence of a fixed amount of pancreatin (Example 13).

The figure 10 shows the stability of five alpha-glucosidases in rumen juice and the interaction with pancreatin during the release of glucose (example 15).

The figure 11 shows the interaction of five alpha-glucosidases with pancreatin in the release of glucose at pH 6.0 (example 16).

DETAILED DESCRIPTION OF THE INVENTION

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

A number of terms and abbreviations are used in this disclosure. Unless expressly stated otherwise, the following definitions apply.

The singular forms of the element or component are intended to be non-limiting as to the number of examples (ie, instances) of the element or component. Therefore, the singular forms are to be understood as including one or at least one, and the singular form for an element or component also includes the plural, except where the singular is expressly intended.

The term "comprising" means the presence of the stated features, integers, steps, or components as set forth in the claims, but in such a way that it does not exclude the presence or addition of one or more features, integers, steps, components, or groups of them. The term "comprising" is meant to include the embodiments encompassed by the terms "substantially consisting of" and "consisting of". Similarly, the term "substantially consisting of" is intended to include the embodiments encompassed by the term "consisting of".

All ranges, if any, are inclusive and combinable. For example, when referring to the range "1 to 5", said range should be considered to include the ranges "1 to 4", "1 to 3", "1-2", "1-2 and 4-5", "1 -3 and 5", etc.

As used herein in relation to a numerical value, the term "approximately" refers to a range of +/- 0.5 of the numerical value, unless the specified term is otherwise specifically defined in the context. For example, the phrase "pH value of about 6" refers to pH values from 5.5 to 6.5, unless a different pH value is specifically defined.

Each maximum numerical limit disclosed herein is intended to include every lower numerical limit, as if such lower numerical limits would be expressly given herein. Each minimum numerical limit disclosed herein will include each upper numerical limit as if such upper numerical limits would be explicitly given herein. Each numerical range provided herein will include each narrower numerical range that falls within such broader numerical range as if such narrower numerical ranges were expressly given herein.

The terms alpha-1,4/1,6-glycoside hydrolase or alpha-1,4;1,6-glycoside hydrolase are used interchangeably in this document ("GLCH"). They refer to enzymes capable of hydrolyzing alpha-1,4 bonds and/or alpha-1,6 bonds in oligomer or polymer molecules, such glycoside hydrolases (a) being at least 20% active at a pH value of 3 or less in the presence of pepsin compared to enzyme activity at pH 6 in the presence of pepsin, (b) such enzymes are active in at least two of the three digestive chambers of the ruminant, including the rumen, abomasum, and small intestine, and (c) enzymes act in conjunction with pancreatic amylase to increase glucose output. Preferably, such enzymes have one or two carbohydrate-binding modules and are capable of hydrolyzing crude starch. Preferably, such glycoside hydrolase enzymes are selected from the group consisting of alpha-amylases, glucoamylases and/or alpha-glucosidases.

The term "glycoside hydrolase" is used interchangeably with "glycosidases" and "glycosyl hydrolases". Glycoside hydrolases promote the hydrolysis of glycosidic bonds in complex sugars (polysaccharides). Together with glycosyltransferases, glycosidases form the main catalytic apparatus for the synthesis and destruction of glycosidic bonds. Glycoside hydrolases are classified in EC 3.2.1 as enzymes that catalyze the hydrolysis of O- or S-glycosides. Glycoside hydrolases can also be classified according to the stereochemical result of the hydrolysis reaction. Thus, they can be classified as eitherpreserving, orinverting enzymes. Glycoside hydrolases can also be classified as exo- or endo-acting depending on whether they act at the (usually non-reducing) end or in the middle, respectively, of the oligo/polysaccharide chain. Glycoside hydrolases can also be classified in ways based on sequence or structure. They are usually named after the substrate they act on.

The term "glycosyltransferase" refers to an enzyme that catalyzes the formation of a glycosidic bond between monosaccharides.

The terms "glycan" and "polysaccharide" are used interchangeably herein. Glycan refers to a polysaccharide or oligosaccharide, or the carbohydrate moiety of a glycoconjugate such as a glycoprotein, glycolipid, or proteoglycan, even if the carbohydrate is only an oligosaccharide. Glycans can be homo- or heteropolymers of monosaccharide residues. They may be linear or branched molecules. Glycans can be found attached to proteins, as in glycoproteins and proteoglycans. They are usually found on the outer surface of cells. O- and N-linked glycans are very common in eukaryotes but can also be found, albeit less frequently, in prokaryotes.

The term alpha-amylase is used interchangeably with alpha-1,4-D-glucan glucanohydrolase and glycogenase. Alpha-amylases (EC 3.2.1.1) usually, but not always, require calcium to function. Such enzymes catalyze the endohydrolysis of alpha-1,4-glucosidic bonds in oligosaccharides and polysaccharides. Alpha-amylases act randomly on starch, glycogen and related polysaccharides and oligosaccharides, releasing reducing groups in the alpha configuration.

The term "low pH active alpha-amylase" ("LpHAA") refers to an alpha-amylase that is at least 20% active at pH 3.0 or less compared to its activity at pH 6, 0. The term "glucoamylase" (EC 3.2.1.3) is used interchangeably with glucan-1,4-alpha-glucosidase, amyloglucosidase, gamma-amylase, lysosomal alpha-glucosidase, acid maltase, exo-1,4-alpha-glucosidase, glucoamylase, gamma -1,4-glucanglucohydrolase, acid maltase and 1,4-alpha-D-glucanhydrolase. This enzyme cleaves the extreme alpha-1,4-glycosidic bonds at the non-reducing end of amylose and amylopectin to produce glucose. It also cleaves alpha-1,6-glycosidic bonds.

The term alpha-glucosidase (E.C. 3.2.1.20) is used interchangeably with maltase, glucoinvertase, glucosucrose, maltase-glucoamylase, alpha-glucopyranosidase, glucosidoinvertase, alpha-D-glucosidase, alpha-glucoside hydrolase, alpha-1,4-glucosidase, and alpha -D-glucoside hydrolase. This enzyme hydrolyzes terminal non-reducing (1-4)-linked alpha-glucose residues with the release of one molecule of alpha-glucose. Alpha-glucosidase is a carbohydrate hydrolase that releases alpha-glucose rather than beta-glucose.

The term "feed" is used in relation to products that are fed to animals in the rearing of farm animals. The terms "food" and "pet food" are used interchangeably. In a preferred embodiment, the food or feed is suitable for consumption by non-ruminants and ruminants.

As used herein, the term "probiotic"("DFM") is a source of live (viable) naturally occurring microorganisms. DFM categories include Bacillus, lactic acid bacteria and yeast. Bacillus are unique gram-positive rods that form spores. These spores are very stable and can withstand environmental conditions such as heat, moisture and a certain pH range. These spores germinate into active vegetative cells when ingested by animals and can be used in micronized and pelleted diets. Lactic acid bacteria are Gram-positive cocci that produce lactic acid and are pathogen antagonists. Because lactic acid bacteria appear to be heat sensitive to some extent, they are not used in pelleted diets. Lactic acid bacteria include the genus Bifidobacterium , Lactobacillus , and Streptococcus. Yeast are not bacteria. These microorganisms belong to a group of plants called fungi.

As used herein, the term "protease" refers to an enzyme capable of cleaving a peptide bond. The terms "protease", "peptidase" and "proteinase" may be used interchangeably. Proteases can be found in animals, plants, bacteria, archaea, and viruses. Proteolysis can be mediated by enzymes, which are currently divided into six broad groups: aspartic proteases, cysteine proteases, serine proteases, threonine proteases, glutamine proteases, and metalloproteases.

In particular, the term "protease" means the domain of a protein or polypeptide derived from a microorganism, such as a fungus, bacterium, or from a plant or animal, and which is characterized by the ability to catalyze the cleavage of peptide bonds at one or more different positions of the protein backbone (for example, E.C. 3.4) .

The term "acid protease" means a protease characterized by the ability to hydrolyze proteins under acidic conditions. It may encompass any number of protein hydrolysing agents such as endopeptidases or exopeptidases.

The terms "aspartic protease" and "aspartic acid protease" are used interchangeably herein and are a type of acid protease. Aspartic proteases (EC 3.4.23), also known as aspartyl proteases, are characterized by an activated water molecule associated with one or more aspartate catalytic residues to hydrolyze a peptide bond in a polypeptide substrate. Typically, they have two highly conserved aspartates in the active site and are characterized by optimal activity at acidic pH values.

The abbreviation "AFP" refers to fungal aspartic protease, that is, aspartic protease, the source of which is the body of the fungus.

The term "metaloprotease" means any protease in which a metal is involved in the catalytic mechanism. Most metalloproteases require zinc, but some use cobalt. The metal ion is coordinated to the protein through three ligands. The ligands coordinating the metal ion can vary for histidine, glutamate, aspartate, lysine, and arginine. The fourth coordinating position is occupied by the active water molecule.

There are two subgroups of metalloproteinases, including (a) exopeptidases, metalloexopeptidases (EC number: 3.4.17) and (b) metalloendopeptidases (3.4.24).

Well known metalloendopeptidases include ADAM proteins and matrix metalloproteinases.

The term "pepsin" as used herein refers to an enzyme that cleaves proteins into smaller peptides at low pH values of 2.0 to 3.5, i.e. it is a protease belonging to the aspartic peptidase family A1. It is produced and secreted in the stomach and is one of the main digestive enzymes in the digestive systems of humans and animals, where it aids in the digestion of proteins in food or feed.

The term "ruminant" as used herein refers to a mammal that is able to obtain nutrients from food of plant origin by fermenting it in a specialized stomach before digestion, mainly through the action of microorganisms. The process typically requires burping and re-chewing the fermented contents of the rumen (known as cud). The process of re-chewing the cud to further refine plant solids and promote digestion is called cud chewing. Approximately 150 ruminant species include both domesticated and wild species. Ruminants include, without limitation, cattle, cows, goats, sheep, giraffes, yaks, deer, elks, antelopes, buffaloes, and the like.

The term "ruminant digestive chambers" as used herein refers to the rumen, reticulum, booklet, abomasum, and small intestine (McDonald et al., 2011, Animal Nutrition (7th Edition), pp. 156-191). The abomasum is the direct equivalent of a single-chambered stomach.

The term "forage" as used herein refers to a type of animal feed, which is any agricultural food used specifically for feeding domesticated farm animals such as cattle, goats, sheep, horses, chickens and pigs. "Forage" refers specifically to food given to animals (including crushed and fed plants), as opposed to what they obtain on their own (called foraging). Forage is also called livestock feed and includes hay, straw, silage, pressed and pelleted feed, oils, and mixed rations, and sprouted grains and legumes (such as bean sprouts, fresh malt, or malt grains). Most pet foods are plant-based, but some manufacturers add ingredients that are animal-based to processed foods.

The term "feed" is used in relation to products that are fed to animals in the rearing of farm animals. The terms "food" and "pet food" are used interchangeably.

The term "starch" is used interchangeably with "amylum". It is a polymeric carbohydrate consisting of a large number of glucose units linked by glycosidic bonds and is the most common storage carbohydrate in plants. Thus, "starch" can refer to any material composed of plant complex polysaccharide carbohydrates, consisting of amylose and amylopectin, with the formula (C ₆ H ₁₀ O ₅ ) _x where X can be any number. In particular, the term refers to any material of plant origin, including, without limitation, cereals, grasses, tubers and roots, and more specifically wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, potatoes, sweet potatoes. and tapioca.

The terms "starch binding domain (SBD)" or "carbohydrate binding module (CBM)" are used interchangeably herein. SBDs can be divided into nine CBM families. As a source of energy, starch undergoes cleavage by a large number of different amylolytic enzymes. However, only about 10% of them are capable of binding and breaking down raw starch. Such enzymes usually contain a separate sequence, a structural module called the starch-binding domain, which mediates attachment to starch granules. SBD refers to an amino acid sequence that binds preferentially to a starch (polysaccharide) or maltosaccharide, alpha, beta, and gamma cyclodextrin, etc. substrate. 10% microbial amylolytic enzymes.

The term "catalytic domain" refers to a structural region of a polypeptide other than CBM that contains an active site for hydrolysis of a substrate.

The term "granular starch" refers to raw (not cooked) starch, such as granular starch, which has not been gelled.

The terms "granular starch hydrolysing enzyme (GSH)" and "granular starch hydrolytic activity (GSH)" are used interchangeably herein and refer to enzymes that are capable of hydrolyzing starch in granular form under digestive tract conditions comparable to with conditions found in the digestive tract of animals, and in particular ruminants.

The term "gelatinization" refers to the breakdown of the intermolecular bonds of starch molecules in the presence of water and heat, allowing more water to bind at the hydrogen bonding sites. This leads to the irreversible dissolution of the starch granules in water to form a viscous suspension.

The term "degree of polymerization (DP)" refers to the number of monomer units in a macromolecule or molecule of a polymer or oligomer. For example, it may refer to the number (n) of monomer units in the saccharide in question. Examples of DP1 are monosaccharides such as glucose and fructose. Examples of DP2 are disaccharides such as maltose and sucrose. DP>3 denotes polymers with a degree of polymerization greater than 3.

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 without limitation any host cell, enzyme, variant, nucleic acid, protein, peptide, or cofactor, that is at least partially derived from one or more or all of the naturally occurring components with which it is naturally associated; (3) any substance modified by man as compared to that substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance compared to other constituents 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 an RNA or DNA polymer 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 DNA polymer may consist of one or more segments of cDNA, genomic DNA, or synthetic DNA.

The term "purified" in relation to nucleic acids or polypeptides generally means a nucleic acid or polypeptide that is essentially free of other components, as determined by analytical techniques well known in the art (for example, a purified polypeptide or polynucleotide forms a single band in electrophoretic gel, chromatographic eluate and/or media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces virtually one band in an electrophoretic gel is "purified". The purity of the purified nucleic acid or polypeptide is at least about 50%, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 99.5%, approximately 99.6%, approximately 99.7%, about 99.8% or more (eg, weight percent in moles). In other words, a composition is enriched in a molecule if there is a significant increase in the concentration of the molecule following the application of a purification or enrichment procedure. 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 in a relative or absolute concentration that is higher than in the original composition.

The terms "peptides", "proteins" and "polypeptides" are used interchangeably herein and refer to a polymer of amino acids linked together by peptide bonds. A "protein" or "polypeptide" contains a polymeric sequence of amino acid residues. This disclosure uses the one and 3 letter codes for amino acids as defined by the IUPAC-IUB Joint Committee on Biochemical Nomenclature (JCBN). One letter X refers to any of the twenty amino acids. It is also understood that a polypeptide can be encoded by multiple nucleotide sequences due to the degeneracy of the genetic code. Mutations can be named by the one letter code of the original amino acid followed by the position number and then the one letter code of the variant amino acid. For example, a mutation from 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 for any of the listed amino acids. For example, 6(L, I) means that position 6 may be substituted with leucine or isoleucine. The sequence also uses a slash (/) to denote substitutions, for example, F/V indicates that a particular position may be phenylalanine or valine.

Mutations can be named by the one letter code of the original amino acid followed by the position number and then the one letter code of the variant amino acid. For example, a mutation from glycine (G) at position 87 to serine (S) is represented as "G087S" or "G87S".

The term "mature" form of a protein, polypeptide or peptide refers to the functional form of a protein, polypeptide or enzyme without a signal peptide sequence and a propeptide sequence.

The term precursor protein or peptide form refers to the mature form of a protein with a prosequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a "signal" sequence operably linked to the amino terminus of the prosequence. The precursor may also have additional polypeptides that are involved in post-translational activity (eg, polypeptides cleaved from it to leave the mature form of the protein or peptide).

"Prosequence" or "propeptide sequence" refers to the amino acid sequence between the signal peptide sequence and the mature sequence of an enzyme (eg, glycoside hydrolase, as described herein) that is required for proper folding and secretion of the enzyme; they are sometimes called intramolecular chaperones. Cleavage of the prosequence or propeptide sequence results in the formation of the mature active enzyme, which is often expressed as proenzymes.

The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that may be involved in the secretion or direction of transport of the mature form or precursor protein form. The signal sequence is usually located at the N-terminus of the precursor or mature protein sequence. The signal sequence may be endogenous or exogenous. The signal sequence is normally absent in the mature protein. The signal sequence is usually cleaved from the protein by a signal peptidase after the protein has been transported. The gene of interest may be expressed with or without a signal sequence.

The term "wild type" in relation 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 any entities (eg, proteins, amino acids, or nucleic acid sequences) found in nature. Conversely, the term "non-naturally occurring" refers to entities not found in nature (eg, recombinant nucleic acids and laboratory-produced protein sequences or wild-type sequence modifications).

As used herein in relation to amino acid residue positions, the expressions "corresponding to", or "corresponding to", or "corresponds to" refers to an amino acid residue at a numbered position in a protein or peptide, or an amino acid residue that is similar, homologous, or equivalent to a numbered residue in a protein or peptide. As used herein, the term "relevant region" generally refers to a similar position in a related protein or reference protein.

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

The term "amino acid" refers to the basic chemical structural unit of a protein or polypeptide. Thus, the codon for the amino acid alanine, which is a hydrophobic amino acid, can be replaced by a codon that codes for another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes that result in the replacement of one negatively charged residue by another (such as aspartic acid for glutamic acid) or one positively charged residue by another (such as lysine for arginine) can also be expected to give a functionally equivalent product. In many cases, it is expected that nucleotide changes that lead to changes in the N-terminal and C-terminal parts of the protein molecule will also not lead to changes in protein activity. Each of the proposed modifications is within the skill of the ordinary person skilled in the art, as well as determining the retention of the biological activity of the encoded products.

The term "codon-optimized", referring to genes or coding regions of nucleic acid molecules for transformation of different hosts, refers to changing codons in a gene or coding regions of nucleic acid molecules to reflect the normal codon usage of the host organism without changing the polypeptide that encoded by DNA.

The term "gene" refers to a nucleic acid molecule that expresses a particular protein, comprising regulatory sequences preceding (5' non-coding sequences) and following the coding sequence (3' non-coding sequences). The term "native gene" means a gene found in nature, with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene containing regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may contain regulatory sequences and coding sequences that originate from different sources, or regulatory sequences and coding sequences that originate from the same source but are arranged in a different order from that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. Foreign genes may include native genes introduced into a non-native organism or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

The term "coding sequence" refers to a nucleotide sequence that codes for a particular amino acid sequence. "Suitable regulatory sequences" refers to nucleotide sequences located upstream (5'-non-coding sequences), within or downstream (3'-non-coding sequences) of the coding sequence and which affect transcription, processing or stability of the RNA or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leaders, an RNA processing site, effector binding sites, and stem-loop structures.

The term "operably linked" refers to the association of nucleic acid sequences in a single nucleic acid molecule. so that the function of one influences the other. For example, a promoter is operably linked to a coding sequence if it is capable of influencing the expression of that coding sequence, i.e. the coding sequence is under the transcriptional control of a promoter. Coding sequences may be operably linked to regulatory sequences in either sense or antisense orientation.

The terms "regulatory sequence" or "control sequence" are used interchangeably herein and refer to a segment of a nucleotide sequence that is capable of increasing or decreasing the expression of specific genes within an organism. Examples of regulatory sequences include, without limitation, promoters, signal sequences, operators, and the like. As noted above, regulatory sequences can be operably linked in sense or antisense orientation to a coding sequence/gene of interest.

"Promoter" or "promoter sequences" refers to DNA sequences that determine where transcription of a gene begins by RNA polymerase. Promoter sequences are usually located immediately upstream or at the 5'end of the transcription initiation site. Promoters may be derived entirely from a native or naturally occurring sequence, or may be composed of various elements derived from various promoters found in nature, or they may even contain synthetic DNA segments. Those skilled in the art will appreciate that different promoters may direct gene expression in different tissues or cell types, or at different stages of development or in response to different environmental or physiological conditions ("inducible promoters").

The term "3'-non-coding sequences" refers to DNA sequences downstream of a coding sequence and includes sequences encoding regulatory signals capable of influencing mRNA processing or gene expression, such as transcriptional termination.

As used herein, the term "transformation" refers to the transfer or introduction of a nucleic acid molecule into a host organism. The nucleic acid molecule can be administered as a linear or circular form of DNA. The nucleic acid molecule may be a plasmid that replicates autonomously, or it may integrate into the genome of the producing host. Producing hosts containing the transformed nucleic acid are referred to as "transformed" or "recombinant" or "transgenic" organisms or "transformants".

As used herein, the term "recombinant" refers to the artificial combination of two otherwise separated segments of nucleic acid sequences, for example, by chemical synthesis or manipulation of isolated nucleic acid segments using genetic engineering techniques. For example, DNA into which one or more segments or genes have been inserted naturally or through laboratory manipulation from a different molecule, from another part of the same molecule, or from an artificial sequence, resulting in the introduction of a new sequence into a gene and, accordingly, into an organism. The terms "recombinant", "transgenic", "transformed", "engineered", or "modified to express an exogenous gene" are used interchangeably herein.

The terms "recombinant construct", "expression construct", "recombinant expression construct", and "expression cassette" are used interchangeably herein. The recombinant construct contains an artificial combination of nucleic acid fragments, such as regulatory and coding sequences, not all of which occur together in nature. For example, the construct may contain regulatory sequences and coding sequences that come from different sources, or regulatory sequences and coding sequences that come from the same source but are arranged in a different order from that found in nature. Such a construct may be used on its own or may be used in combination with a vector. If a vector is used, then the choice of vector depends on the method that will be used to transform the host cells, as is well known to those skilled in the art. For example, a plasmid vector can be used. One skilled in the art is well aware of the genetic elements that must be present in a vector for successful transformation, selection, and propagation of host cells. One of skill in the art will also appreciate that different independent transformants may exhibit different levels and patterns of expression (Jones et al. , (1985) EMBO J 4:2411-2418; De Almeida et al. , (1989) Mol Gen Genetics 218:78-86), and thus, in order to obtain lines with the desired level and pattern of expression, it is common to screen for multiple transformants. Such screening can be performed using standard molecular biology, biochemical and other assays, including Southern DNA analysis, Northern analysis of mRNA expression, PCR, quantitative real-time PCR (qPCR), reverse transcription PCR (RT-PCR) , protein expression analysis by immunoblotting, enzymatic or activity assays, and/or phenotypic analysis.

The terms "producing host", "host", and "host cell" are used interchangeably herein and refer to any organism or cell thereof, whether human or non-human, into which the recombinant construct for gene expression. This term covers any offspring of a parent cell that is not identical to the parent cell due to mutations that occur during reproduction.

The term "percent identity" is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by sequence comparison. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the number of matching nucleotides or amino acids between strands of such sequences. "Identity" and "similarity" can be easily calculated using known methods, including, without limitation, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987) and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Methods for determining identity and similarity are written in the code of publicly available computer programs.

As used herein, the term "% identity" or "percent identity" or "PID" refers to protein sequence identity. Percent identity can be determined using standard techniques known in the art. Useful algorithms include BLAST algorithms (see, Altschul et al., J Mol Biol, 215:403–410, 1990; and Karlin and Altschul, Proc Natl Acad Sci USA, 90:5873–5787, 1993). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity, but is not recommended for query sequences of less than 20 residues (Altschul et al., Nucleic Acids Res, 25:3389–3402, 1997; and Schaffer et al., Nucleic Acids Res , 29:2994–3005, 2001). Exemplary default BLAST parameters for searching for nucleic acid sequences include: adjacent word length threshold=11; E-value cutoff=10; substitution matrix=NUC.3.1 (match=1, mismatch=-3); gap opening=5 and gap extension=2. Exemplary default BLAST parameters for searching amino acid sequences include: word length=3; cutoff E-values=10; substitution matrix=BLOSUM62; gap opening=11 and gap extension=1. The percent (%) amino acid sequence identity value is determined by the number of identical residues that match, divided by the total number of residues of the "reference" sequence, including any gaps created by the program for optimal/maximum alignment. The BLAST algorithms refer to the "reference" sequence as the "query" sequence.

As used herein, the terms "homologous proteins" or "homologous enzymes" refer to proteins that share a distinct primary, secondary, and/or tertiary structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins align. Homologous searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST with a cutoff (E-value cutoff) at 0.001. (Altschul SF, Madde TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI BLAST a new generation of protein database search programs. Nucleic Acids Res 1997 Set 1;25(17):3389– 402). Using this information, protein sequences can be grouped. It is possible to construct a phylogenetic tree using amino acid sequences.

Sequence alignments and percent identity calculations can be made using the Megalign program from the LASERGENE bioinformatics data calculation software package (DNASTAR Inc., Madison, WI), the AlignX program from Vector NTI, version 7.0 (Informax, Inc., Bethesda, MD), or the EMBOSS open source software package (EMBL–EBI; Riceet al.,Trends in Genetics16, (6):276–277 (2000)). Multiple sequence alignment can be done with a Clustal alignment method (such as CLUSTALW; e.g. version 1.83) (Higgins and Sharp,cabios,5:151–153 (1989); Higginset al.,Nucleic Acids Res. 22:4673–4680 (1994); and Chennaet al.,Nucleic Acids Res 31 (13):3497–500 (2003)), available from the European Molecular Biology Laboratory through the European Bioinformatics Institute) with default parameters. Suitable parameters for protein alignments in CLUSTALW include the following: gap penalty=15, gap extension=0.2, matrix=Gonnet (e.g., Gonnet250), protein ENDGAP=-1, protein GAPDIST=4 and KTUPLE=1. In one embodiment, fast or slow alignment is applied with default settings for slow alignment. Alternatively, the options used in the CLUSTALW method (for example, version 1.83) can be modified to also use KTUPLE=1, gap penalty=10, gap extension=1, matrix=BLOSUM (e.g., BLOSUM64), window=5 and TOP DIAGONALS SAVED=5.

Various amino acid sequences of polypeptides and polynucleotide sequences are disclosed herein as features of certain aspects. In certain embodiments, 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. Alternatively, in certain embodiments, the polypeptide sequence or polynucleotide sequence of a variant may be 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% identical to the sequence disclosed in this document. The amino acid sequence or polynucleotide sequence of the variant has a similar function to 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" in relation to a polypeptide refers to a polypeptide that differs from the specified wild type, parent or reference polypeptide in that it includes one or more naturally occurring or artificial amino acid substitutions, insertions or deletions. Similarly, the term "variant" in relation to a polynucleotide refers to a polynucleotide that differs in nucleotide sequence from said wild-type, parental, or reference polynucleotide. The identity of the wild-type polypeptide or polynucleotide, parent or reference polypeptide or polynucleotide will be apparent from the context.

The terms "plasmid", "vector" and "cassette" refer to an extrachromosomal element, often carrying genes that are not part of the central metabolism of the cell, and usually in the form of double-stranded DNA. Such elements can be autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences, in linear or circular form, single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences are connected or recombined into a unique design. which is capable of introducing the polynucleotide of interest into the cell. "Transformation cassette" refers to a particular vector containing a gene and having elements in addition to the gene that promote transformation of a particular host cell. The terms "expression cassette" and "expression vector" are used interchangeably herein and refer to a particular vector containing a gene and having elements in addition to the gene that allow that gene to be expressed in a host.

As used herein, the term "expression" refers to the production of a functional end product (eg, mRNA or protein) in the form of a precursor protein or in mature form. Expression can also refer to the translation of an mRNA into a polypeptide.

Gene expression involves transcription of the gene and translation of the mRNA into a precursor or mature protein. "Antisense suppression" refers to the production of antisense RNA transcripts capable of providing suppression of the expression of a target protein. "Cosuppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (US Pat. No. 5,231,020). A "mature" protein refers to a post-translationally processed polypeptide; i.e., a polypeptide from which any pre- or pro-peptides present in the primary translation product have been removed. "Precursor protein" refers to the primary translation product of an mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides can be, without limitation, intracellular localization signals. "Stable transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, including the genomes of both the nucleus and organelles, resulting in genetically stable inheritance. In contrast, "transient transformation" refers to the transfer of a nucleic acid fragment into the nucleus or DNA-containing organelle of a host organism, resulting in gene expression without integration or stable inheritance. Host organisms containing transformed nucleic acid fragments are referred to as "transgenic" organisms.

The expression vector may be one of a variety of vectors or cassettes suitable for transforming suitable production hosts known in the art. Typically, the vector or cassette will include sequences that control transcription and translation of the corresponding gene, a selectable marker, and sequences that allow for autonomous replication or integration into the chromosome. Suitable vectors typically include the 5' region of the gene, which carries elements that control transcription initiation, and the 3' region of the DNA fragment, which controls transcription termination. Both control regions can be derived from genes homologous to the genes of the transformed production host and/or genes native to the production host, although such control regions may not be generated in this manner.

As used herein, the terms "homologous proteins" or "homologous enzymes" refer to proteins that share a distinct primary, secondary, and/or tertiary structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins align. Homologous searches for protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST with a cutoff (E-value cutoff) at 0.001. (Altschul SF, Madde TL, Shaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI BLAST a new generation of protein database search programs. Nucleic Acids Res 1997 Set 1; 25(17):3389– 402). Using this information, protein sequences can be grouped. It is possible to construct a phylogenetic tree using amino acid sequences. Amino acid sequences can be entered into a program such as from the Vector NTI Advance package, and a guide tree can be created using neighbor joining (NJ) (Saitou and Nei, Mol Biol Evol, 4:406-425, 1987). The tree construction can be calculated using the Kimura correction for sequence distance and ignoring gapped positions. A program such as AlignX can display the calculated distance values in brackets after the name of the molecule displayed on the phylogenetic tree.

Understanding the homology between molecules can reveal the evolutionary history of molecules as well as information about their function; if the newly sequenced protein is homologous to an already characterized protein, then there is a strong indication of the biochemical function of the new protein. Homology is the most fundamental relationship between two entities; two molecules are said to be homologous if they come from a common ancestor. Homologous molecules or homologs can be divided into two classes: paralogs and orthologs. Paralogs are homologues that are present within the same species. Paralogs often differ in their specific biochemical functions. Orthologs are homologues that are present in different species and have very similar or identical functions. The protein superfamily is the largest group (clade) of proteins for which a common origin can be deduced. Typically, this common origin is based on sequence alignment and mechanism of action similarity. Superfamilies typically contain several families of proteins that are characterized by sequence similarity within the family. The term "protein clan" is commonly used for protease superfamilies based on the MEROPS protease classification system.

The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994). The default parameters for the CLUSTAL W algorithm include the following: gap opening penalty=10.0; gap extension penalty=0.05; protein alignment weight matrix=BLOSUM series; DNA alignment weight matrix=IUB; delay of differing sequences % = 40; distance between gaps=8; weight of transitions in DNA=0.50; list of hydrophilic residues=GPSNDQEKR; use of negative matrix=OFF; toggle remainder-specific penalties=ON; hydrophilicity penalty toggle=ON and end gap toggle penalty=OFF. CLUSTAL algorithms include deletions occurring at either end. For example, a five amino acid deletion variant at either end (or in the polypeptide) of a 500 amino acid polypeptide will have a 99% (495/500 identical residues x 100) percent sequence identity relative to the "reference" polypeptide. Such a variant would be encompassed by a variant having "at least 99% sequence identity" with respect to the polypeptide.

As used herein, the term "functional assay" refers to an assay that provides a determination of enzyme activity. In some embodiments, the term refers to assay systems in which an enzyme is analyzed for its ability to function in the context of its normal functional activity.

Ruminants have the unique ability to convert roughage into protein and energy through their microbial/enzymatic digestive systems. Accordingly, ruminants play an important role in the Earth's ecology and in the food chain.

The main difference between a ruminant and a non-ruminant is that a ruminant has a four-chambered stomach, consisting of a rumen, a net, a book, and an abomasum. Rennet is the direct equivalent of a single chambered stomach (McDonald et al., 2011, Animal Nutrition (7th Edition), pp. 156–191).

The scar is the largest section, with a volume of 150–200 liters (40–50 gallons).

There are billions of microorganisms in the digestive system. They help the cow digest and absorb nutrients from the feed. In order to achieve efficient feed digestion and high milk yields, the bacteria must be in optimal conditions. It is the bacteria that are the ones who break down the food. Feeding a cow is, in fact, feeding the microorganisms in her rumen.

The fermentation process takes place in the rumen and net. The tripe of a cow is like a large fermentation vat. Over 200 different bacteria and 20 types of protozoa help the cow to absorb fibrous feed products and non-protein sources of nitrogen.

During fermentation, microorganisms convert carbohydrates into volatile fatty acids and gases. This process allows the cow to convert cellulose fiber into energy.

Among the gases produced in the rumen during fermentation (500–1500 liters per day) (150–400 gallons), 20–40% are methane and carbon dioxide. The production of fermentation gases represents a significant waste of energy. Certain fermentation modifiers, such as ionophores, improve the energy efficiency of ruminants by reducing such energy losses for gas production.

Fermentation gases are expelled by belching. If burping is not possible or effective, cows may suffer from bloating. When food enters the rumen, it is layered on the scar mat, which is held on the surface of the rumen contents. Through rhythmic contractions of the rumen wall, freshly eaten material accumulates in the back of the mat. The scar mat consists of undigested material with a 15% dry matter content. The bacteria attach to the feed and gradually break down the fermentable material. When chewing the cud, the cow regurgitates portions of the cud from the front layer. Saliva enters the oral cavity, and due to the abrasive action of the teeth, the surfaces exposed to microorganisms increase.

The food particles become smaller as the bacteria attack and the chewing process continues. They gradually absorb the liquid and sink to the bottom of the scar. The content of the scar at the bottom of the reticulo-rumen is characterized by a dry matter content of 5%.

Scar contractions occur once per minute. The contractions ensure the mixing of liquid and solid contents in the rumen to promote fermentation and avoid stagnation. The contractions also serve to release gases trapped either in the mat or in the liquid portion of the scar contents. The fermentation gases are then released through belching. Disruption of this process can lead to bloating. Feed particles of the appropriate size and density are transferred to the liquid in the net by rumen contractions. Subsequent contractions push these particles and some of the liquid content out of the reticulo-scar into the book.

The scar and mesh are essentially the same department, but with different functions. While most of the fermentation action takes place in the rumen, the mesh serves as an intermediate zone for passage into the book or burp.

The ideal pH value in the rumen is between 6 and 7. Ruminal microorganisms are the most viable within this range. If the pH value fluctuates too much, some types of micro-organisms die and this reduces the absorption of the feed. Microorganisms that break down cellulose (hay, silage, etc.) are unable to grow or ferment cellulose at pH values below 6.0. When the pH value in the rumen drops below 6, the rumen is considered acidic. Cicatricial acidosis can be acute with a rapid, severe drop in pH. More common among high performing herds is subclinical acidosis, which is characterized by chronic intermittent periods of low rumen pH.

As noted above, the digestibility of starch in feed is highly variable and depends on a number of factors, including the physical structure of both the starch and the feed base.

It has been found that starch digestibility, especially crude starch hydrolysis, in a ruminant diet can be improved by using at least one alpha-1,4/1,6-glycoside hydrolase (GLCH) as a ruminant feed additive, wherein said hydrolase ( a) has at least 20% activity at pH 3 or less in the presence of pepsin compared to enzyme activity at pH 6 in the presence of pepsin, (b) said hydrolases are active in at least two digestive chambers of the ruminant, including rumen, mesh, book and abomasum; and (c) the hydrolase acts in concert with the digestive enzymes present in the digestive chambers of the ruminant to increase glucose output and increase starch digestibility.

In yet another aspect, a method is described for increasing starch digestibility, increasing glucose yield, increasing dry matter digestion, and increasing gas production during fermentation in a ruminant, comprising adding to a feed an enzyme-based composition comprising (i) at least one GLCH enzyme as feed ruminant additives, wherein said enzyme (a) is at least 20% active at pH 3 or less in the presence of pepsin compared to enzyme activity at pH 6 in the presence of pepsin, (b) said enzyme is active in at least two of the ruminant's three digestive chambers, including rumen, abomasum, and small intestine, and (c) the enzyme works in conjunction with pancreatic amylase to increase glucose output, and (ii) at least one protease.

Some digestive enzymes present in the ruminant's digestive chambers with which the hydrolase can act to increase glucose output include, but are not limited to, ruminal amylolytic enzymes, pepsin, or pancreatin.

Preferably, the hydrolase is capable of hydrolyzing raw starch under conditions comparable to those found in rumen or abomasum.

The hydrolase may be selected from the group consisting of alpha-amylases, glucoamylases and alpha-glucosidases, or the hydrolase may be selected from the group consisting of alpha-amylases and glucoamylases.

Any suitable alpha-amylase (E.C. 3.2.1.1), whether commercially available or not, may be used in the method described herein.

Illustrative alpha-amylases include, without limitation, a wild-type alpha-amylase, a variant or fragment thereof, or a genetically engineered hybrid alpha-amylase that is derived from, for example, a catalytic domain from one microbial source and a starch-binding domain from another microbial source. Non-limiting examples of other alpha-amylases that may be useful for creating such hybrids can be obtained from Bacillus , Aspergillus, Trichoderma, Rhizopus, Fusarium, Penicillium, Neurospora and Humicola .

Mention may also be made of those alpha-amylases which are characterized by hydrolytic activity against granular starch (GSH), or alpha-amylases which have been recombinantly engineered to provide activity against GSH. This GSH activity is advantageous because such enzymes break down more starch, in particular any granular (raw) starch that may be present in any feed containing molasses, etc. Alpha-amylases with GSH activity include without limitation, alpha-amylases derived from Aspergillus kawachi (eg AkAA), Aspergillus niger (eg AnAA), A. clavatus (AcAA), A. terreus (AtAA) and Trichoderma reesei (eg TrAA).

The alpha-amylases AkAA, AcAA, and AtAA have two carbohydrate-binding domains, one of which belongs to the family of carbohydrate-binding modules/domains 20 (CBM20 or CD20), while the other is sometimes referred to as a secondary binding site (SBS). SBS and CBM appear to function by 1) targeting the enzyme to its substrate, 2) targeting the substrate to the active site niche, 3) destabilizing the substrate, 4) increasing processivity, 5) allosteric regulation, 6) donating reaction products, and/or 7) attachment to the cell wall of the parent microorganism.

Many of these putative functions correspond to functions related to non-catalytic binding in CBM. Unlike CBM, SBS are characterized by a fixed position relative to the catalytic site, which makes them more or less suitable for performing specific functions (Cuyvers S., Dornez E., Delcour J. A., Courtin C. M. (2012), Occurrence and functional significance of secondary binding carbohydrate sites in glycoside hydrolases, Crit. Rev. Biotechnol. 32, 93-107).

Some commercially available alpha-amylases that may have GSH activity or enzymes used in carbohydrate hydrolysis processes are commercially available, see e.g. TERMAMYL® 120-L, LC and SC SAN SUPER®, SUPRA® and LIQUEZYME ® SC available from Novo Nordisk A/S, FUELZYME® LF from Verenium and CLARASE® L, SPEZYME® FRED, SPEZYME® XTRA, GC626 and GZYME® G997 available from Danisco, USA, Inc., Genencor Division.

In other embodiments, the alpha-amylase is required to be an acid-stable alpha-amylase having 20% activity at a pH value of 3.0 or less compared to its activity at a pH value of 6.0

Alpha-amylases may be selected from the group consisting of alpha-amylase (EC 3.2.1.1); pullulanase (EC 3.2.1.41 ); cyclomaltodextrin-glucanotransferase (EC 2.4.1.19 ); cyclomaltodextrinase (EC 3.2.1.54 ); trehalose-6-phosphate hydrolase (EC 3.2.1.93 ); oligo-alpha-glucosidase (EC 3.2.1.10 ); maltogenic amylase (EC 3.2.1.133 ); neopullulanase (EC 3.2.1.135 ); alpha-glucosidase (EC 3.2.1.20 ); maltotetraose-forming alpha-amylase (EC 3.2.1.60 ) isoamylase (EC 3.2.1.68 ); glucodextranase (EC 3.2.1.70 ); maltohexaose-forming alpha-amylase (EC 3.2.1.98 ); maltotriose-forming alpha-amylase (EC 3.2.1.116 ); branching enzyme (EC 2.4.1.18 ); trehalose synthase (EC 5.4.99.16 ); 4-alpha-glucanotransferase (EC 2.4.1.25 ); maltopentaose-forming alpha-amylase (EC 3.2.1.- ) ; amylosucrases (EC 2.4.1.4 ); sucrose phosphorylase (EC 2.4.1.7 ); maltooligosyltrehalose-trehalohydrolases (EC 3.2.1.141 ); isomaltulose synthase (EC 5.4.99.11 ); maltooligosyltrehalose synthase (EC 5.4.99.15 ); amyl-alpha-1,6-glucosidase (EC 3.2.1.33 ) and alpha-1,4-glucan:phosphate-alpha-maltosyltransferase (EC 2.4.99.16 ).

Any suitable glucoamylase (GA) (E.C. 3.2.1.3.), whether commercially available or not, can be used in the method described herein.

The suitability of glucoamylase (syn. amyloglucosidase (GA) (EC 3.2.1.3)) will depend on its activity against maltose. Thus, a suitable glucoamylase should have at least 20% maltose activity, on a protein basis, compared to Trichoderma reesei (TrGA) derived glucoamylase when assayed with 2% (w/v) maltose, pH 6 ,0 and 40 ^o C. TrGA is widely used in the production of first generation bioethanol due to its ability to generate glucose from starch (US patent, 2008, US 7413879 B2, Trichoderma reesei Glucoamylase and its homologues). STARGEN™ 002 is a granular starch hydrolyzing enzyme product for ethanol production sold by DuPont.

For animal nutrition, it is important that the rate of hydrolysis of maltose to glucose be high, since it is glucose that is absorbed into the bloodstream in the digestive tract, and not its corresponding dimer, maltose.

Thus, the low activity of Aspergillus niger glucoamylase (AnGA) for maltose renders this glucoamylase unsuitable for use in the methods described herein. Glucoamylase is known to have many subsites for binding near the active site. Subsite theory has been developed to analyze the affinity of glucoamylases for substrates. One of the assumptions of this theory is that the substrate can bind to the enzyme in either an efficient or inefficient manner. It is believed that the low activity of Aspergillus niger glucoamylase (AnGA) may be due to the possibility that maltose binds to AnGA in an inefficient manner. (Swanson et al., 1977, Biotechnol. Bioeng. 19:1715–1718).

Exemplary glucoamylases include, but are not limited to, wild-type glucoamylases, a variant or fragment thereof, or a genetically engineered hybrid glucoamylase that can be derived from, for example, a catalytic domain from one microbial source and a starch-binding domain from another microbial source. Hybrid glucoamylase includes, for example, glucoamylases containing a catalytic domain from a GA from one organism (eg GA Talaromyces ) and a starch binding domain (SBD) from a different organism (eg GA Trichoderma).

Such hybrid glucoamylases can also be made using a peptide to align a catalytic domain from a GA from one organism (eg GA Talaromyces ) and a starch binding domain (SBD) from a different organism (eg GA Trichoderma).

Examples of such hybrid glucoamylases include, but are not limited to, Aspergillus niger glucoamylases G1 and G2 (see, for example, Boel et al. 6352851); glucoamylase Aspergillus awamori (see, for example, WO 84/02921); glucoamylase Aspergillus oryzae (see, for example, Hata et al., (1991) Agric. Biol. Chem. 55:941-949) and Aspergillus shirousami. (See, for example, Chen et al., (1996) Prot. Eng. 9:499–505; Chen et al. (1995) Prot. Eng. 8:575–582; and Chen et al., (1994) Biochem J. 302:275–281).

Some glucoamylases are endogenously expressed by bacteria, plants and/or fungi. For example, some glucoamylases are produced by several strains of filamentous fungi.

Preferably, the glucoamylase will have granular starch (GSH) hydrolytic activity or has been recombinantly engineered to provide GSH activity. This GSH activity is advantageous because such enzymes break down more starch, in particular any granular (raw) starch that may be present in any feed containing molasses, etc.

Glucoamylases that can be used in the method disclosed herein include glucoamylases derived from Talaromyces strains ((e.g. T. emersonii, T. leycettanus, T. duponti and T. thermophilus glucoamylases (see e.g. WO 99/28488 ; US Pat. No. RE: 32153; Pat. US No. 4587215)); strains of Trichoderma, (for example, T. reesei) and glucoamylase, characterized by at least about 80%, about 85%, about 90% and about 95% identity sequences of SEQ ID NO: 4 disclosed in US Pat. e.g., Boel et al., (1984) EMBO J. 3:1097-1102; WO 92/00381; WO 00/04136; Chen et al., (1996) Prot. Eng. 9:499-505; Taylor et al., (1978) Carbohydrate Res. 61:301–308, US Pat. No. 4,514,496, US Pat. No. 4,092,434, US Pat. –223 and SEQ ID NO: 3 WO 2005/052148)). embodiments, the glucoamylase has at least about 85%, about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, and about 99% sequence identity with the amino acid sequence of SEQ ID NO: 3 WO 05/052148. Other glucoamylases useful in the present invention include those derived from Athelia rolfsii and variants thereof (see eg WO 04/111218) and Penicillium spp. (e.g. Penicillium chrysogenum).

Commercially available glucoamylases include, without limitation, Spirizyme Ultra, Spirizyme® Achieve, Spirizyme® Fuel, SPIRIZYME® EXCEL, DISTILLASE®, OPTIDEX® L-400 and G ZYME® G990 4X, GC480, G-ZYME® 480, FERMGEN® 1-400 ( Danisco USA, Inc, Genencor Division), CU.CONC® (Shin Nihon Chemicals, Japan), GLUCZYME® (Amano Pharmaceuticals, Japan (see e.g. Takahashi et al., (1985) J. Biochem. 98:663– 671)). Minor enzymes that find use in the present invention include three forms of glucoamylase (e.g. E.C. 3.2.1.3) produced by Rhizopus sp., namely "Gluc1" (MW 74000), "Gluc2" (MW 58600) and "Gluc3" (MW 61400).

Any suitable alpha-glucosidase (E.C. 3.2.1.20) may be used as described herein.

As noted above, alpha-glucosidase releases glucose from maltose, isomaltose, and maltosaccharides when in its hydrolytic mode. It can also transfer a glucose moiety to acceptor molecules including glucose, maltose, isomaltose and maltosaccharides or any other suitable molecules when in its synthetic mode, especially at higher substrate concentration and high product concentration. In the digestive tract, the synthesis mode or transfer reaction is minimal, since the resulting product, which is glucose, will be removed by absorption.

Proteases (also called peptidases or proteinases) are enzymes capable of cleaving peptide bonds. Proteases have evolved more than once, and different classes of proteases can carry out the same reaction due to completely different catalytic mechanisms. Proteases can be found in animals, plants, bacteria, archaea, and viruses.

Proteolysis can be mediated by enzymes, which are currently divided into six broad groups: aspartic proteases, cysteine proteases, serine proteases, threonine proteases, glutamine proteases, and metalloproteases.

Preferably the protease is an acidic protease, and more preferably it is an acidic fungal protease.

Any acidic or neutral protease can be used in the present invention. For example, acidic fungal proteases include proteases derived from Aspergillus, Trichoderma, Mucor and Rhizopus such as A. niger, A. awamori, A. oryzae, Trichoderma reesei and M. miehei . AFP can be produced by expression of a heterologous or endogenous protein from bacterial, plant, and fungal sources. Preferably AFP is secreted by strains of Trichoderma.

Illustrative acid proteases include, without limitation, a wild-type acid protease, a variant or fragment thereof, or a genetically engineered acid fusion protease that can be derived from, for example, a catalytic domain from one microbial source and a starch-binding domain from another microbial source.

In another aspect, the protease may be a neutral protease such as a metalloprotease (syn. metallopeptidase) and more preferably a bacterial metalloprotease.

In another aspect, any isolated recombinant substantially pure synthetically or non-naturally produced nucleic acid is provided, comprising a nucleotide sequence encoding any polypeptide having any of the glycoside hydrolase activities described herein (including any fusion protein, etc.). ), which was discussed earlier in this document. Also of interest is a vector containing a polynucleotide encoding any of the glycoside hydrolases described herein. One of skill in the art will appreciate that the vector may be any suitable expression vector and that the choice of vector may vary depending on the type of cell into which the vector is to be inserted. Suitable vectors include pGAPT-PG, pRAX1, pGAMD, pGPT-pyrG1, pC194, pJH101, pE194 and pHP13 (see Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons [1990]) . See also Perego, Integrational Vectors for Genetic Manipulations in Bacillus subtilis in Sonenshein et al., [eds.] Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics, American Society for Microbiology, Washington, DC [1993] , pp. 615–624) and p2JM103BBI.

The expression vector may be one of a variety of vectors or cassettes suitable for transforming suitable production hosts known in the art. Typically, the vector or cassette will include sequences that control transcription and translation of the corresponding gene, a selectable marker, and sequences that allow for autonomous replication or integration into the chromosome. Suitable vectors typically include the 5' region of the gene, which carries elements that control the initiation of transcription, and the 3' region of the DNA fragment, which controls the termination of transcription. Both control regions can be derived from genes homologous to genes in the transformed production host and/or genes native to the production host, although such control regions may not be generated in this way.

DNA fragments that direct transcriptional termination can also be obtained from various genes native to the preferred producing host cell. In certain embodiments, the inclusion of a termination control site is optional. In certain embodiments, the expression vector includes a termination control region derived from a preferred host cell.

The expression vector can be incorporated into a production host, in particular into cells of microbial production hosts. Producing host cells can be microbial hosts found in fungal or bacterial families and which grow over a wide range of temperature, pH and solvent tolerance. For example, it is contemplated that any of bacteria, algae, and fungi such as filamentous fungi and yeast can appropriately house the expression vector.

Incorporation of an expression vector into a producing host cell can be used to express a protein of interest such that it can be intracellular, extracellular, or a combination of both inside and outside the cell. Extracellular expression provides an easier recovery of the desired protein from the fermentation product than in the case of methods for extracting the protein obtained by intracellular expression.

The recombinant expression vector can be any vector, such as a plasmid or virus, that can be easily subjected to recombinant DNA procedures and result in the expression of a nucleotide sequence. The choice of vector generally depends on the compatibility of the vector with the producing host into which the vector is to be introduced. Vectors can be linear or closed circular plasmids. The vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into a producing host, integrates into the genome and replicates along with the chromosome(s) into which it has integrated. Some non-limiting examples of such vectors are provided in the Fungal Genetic Stock Center (FGSC, <www.fgsc.net") strain catalog. Additional examples of suitable expression and/or integration vectors are provided in Sambrook et al., (1989)higher,Ausubel (1987)higher,van den Hondel et al.(1991) in Bennett and Lasure (Eds.) MORE GENE MANIPULATIONS IN FUNGI, Academic Press. 396-428 and US Pat. No. 5,874,276. Particularly suitable vectors include pTREX, pFB6, pBR322, PUCI8, pUCI00 and pENTR/D. Plasmids suitable for use in bacterial cells include pBR322 and pUC19, which allow for replication inE. coli, and pE194, for example, which ensures the implementation of replication inbacillus.

Briefly, with regard to production in producing host cells, reference may be made to Sambrook et al., (1989) above, Ausubel (1987) supra, van den Hondel et al. (1991) in Bennett and Lasure (Eds.) MORE GENE MANIPULATIONS IN FUNGI, Academic Press (1991) pp. 70–76 and 396–428; Nunberg et al., (1984)Mol. Cell BioI.4:2306–2315; Boel et al.(1984) 30 EMBO J.3:1581–1585; Finkelstein in BIOTECHNOLOGY OF FILAMENTOUS FUNGI, Finkelstein et al. Eds. Butterworth-Heinemann, Boston, MA (1992), Chap. 6; Kinghorn et al.(1992) APPLIED MOLECULAR GENETICS OF FILAMENTOUS FUNGI, Blackie Academic and Professional, Chapman and Hall, London; Kelley et al. (1985)EMBO J.4:475–479; Pentila et al., (1987)Gene61:155–164; and US Pat. No. 5,874,276. A list of suitable vectors can be found in the Fungal Genetics Stock Center strain catalog (FGSC, www at fgsc.net). Suitable vectors include those obtained from, for example, Invitrogen Life Technologies and Promega. Particular vectors suitable for use in fungal host cells include vectors such as pFB6, pBR322, pUC18, pUC100, pDON^™201,pDONR^™221, pENTR^™, pGEM^®3Z and pGEM^®4Z.

The vector system can be a single vector or plasmid, or two or more vectors or plasmids that together contain the complete DNA to be introduced into the host cell's genome, or transposon.

The vector may also contain one or more selectable markers to allow easy selection of transformed cells. A selectable marker is a gene whose product provides biocidal or viral resistance, and the like. Examples of selectable markers include those that confer antimicrobial resistance. Nutrition-related markers also find use in the present invention, including those markers known in the art as amdS , argB , and pyr4 . Markers suitable for transformation of Trichoderma are known in the art (see, for example, Finkelstein, chapter 6 in Biotechnology of Filamentous Fungi, Finkelstein et al., EDS Butterworth-Heinemann, Boston, MA (1992) and Kinghorn et al., ( 1992) Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London). In some embodiments, expression vectors will also include a replicon, an antibiotic resistance gene to allow selection of bacteria harboring recombinant plasmids, and unique restriction sites in non-essential regions of the plasmid to allow insertion of heterologous sequences. The choice of a specific antibiotic resistance gene is not critical; any of the many resistance genes known in the art is suitable. Prokaryotic sequences are preferably chosen such that they do not interfere with DNA replication or integration in Trichoderma reesei .

The vector may also contain element(s) to enable stable integration of the vector into the genome of the producing host or autonomous replication of the vector in the producing host, regardless of the genome of the cell. For integration into the genome of the host cell, the vector may rely on a nucleotide sequence encoding an aspartic protease, or any other element of the vector for stable integration of the vector into the genome by homologous or non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication allowing the vector to autonomously replicate in a producing host.

More than one copy of the nucleotide sequence encoding the glycoside hydrolase can be inserted into the producing host to increase its production. An increase in the number of copies of the nucleotide sequence can be obtained by integrating at least one additional copy of the sequence into the genome of the producing host or by incorporating an amplifiable selectable marker gene, and additional copies of the nucleotide sequence can be selected by culturing the producing host cells in the presence of a suitable selection agent.

A vector containing a nucleotide sequence encoding any of the glycoside hydrolases is introduced into a producing host such that the vector is maintained as a chromosomal integrated element or as a self-replicating extrachromosomal vector. Generally, integration is seen as an advantage since the nucleotide sequence is more likely to be stably maintained by the producing host. Integration of the vector into the chromosome of the producing host can occur by homologous or non-homologous recombination, as discussed above.

Illustrative vectors include, without limitation, pGXT (similar to the pTTTpyr2 vector described in PCT Published Application WO2015/017256). Mention may also be made of standard bacterial expression vectors, including bacteriophages λ and M13, as well as plasmids such as those based on pBR322, pSKF, pET23D, and fusion protein expression systems such as MBP, GST and LacZ. Also, epitope tags can be added to recombinant proteins to provide convenient isolation methods, such as c-myc.

Examples of suitable expression and/or integration vectors are given in Sambrook et al., (1989) supra, Bennett and Lasure (Eds.) More Gene Manipulations in Fungi, (1991) Academic Press pp. 70–76 and pp. 396–428 and articles cited therein; in US Pat. No. 5,874,276 and the Fungal Genetic Stock Center (FGSC, www.fgsc.net.) strain catalog. Suitable vectors are available from Promega and Invitrogen. Some specific suitable vectors include pBR322, pUC18, pUC100, pDON™201, pENTR™, pGEN®3Z and pGEN®4Z. However, other forms of expression vectors which perform equivalent functions and which are or become known in the art may also be used. Thus, a wide variety of host/expression vector combinations can be used to express the DNA sequences disclosed herein. For example, suitable expression vectors may consist of segments of chromosomal, non-chromosomal, and synthetic DNA sequences such as various known SV40 derivatives and known bacterial plasmids, for example, plasmids from E. coli, including col E1, pCR1, pBR322, pMb9, pUC19, and their derivatives, plasmids for a wider range of hosts, such as RP4, phage DNAs, such as numerous derivatives of the lambda phage, such as NM989, and other DNA-containing phages, such as M13 and filamentous phages containing single-stranded DNA, yeast plasmids, such as plasmid 2 .mu or its derivatives.

The choice of producing host may be any suitable microorganism such as bacteria, fungi and algae. Typically, the choice will depend on the gene encoding the glycoside hydrolase of interest.

Examples of suitable production hosts include, without limitation, bacterial, fungal, plant cells, etc. Preferably, the production host can be selected from E. coli , Streptomyces , Hansenula , Trichoderma (particularly T. reesei ), Bacillus , Lactobacillus , Aspergillus (in in particular A. niger ), plant cell and/or spores of Bacillus , Trichoderma or Aspergillus .

In some embodiments, a recombinant glycoside hydrolase enzyme can be used in the methods and compositions disclosed herein. In a preferred aspect, a food or feed supplement is provided that contains the glycoside hydrolase described herein.

A variety of standard transfection methods can be used to generate cell lines of bacteria and filamentous fungi (eg Aspergillus or Trichoderma ) that express large amounts of the desired glycoside hydrolase. Some of the published methods for introducing DNA constructs into cellulase-producing Trichoderma strains include Lorito, Hayes, DiPietro and Harman, (1993) Curr. Genet. 24:349–356; Goldman, VanMontagu and Herrera-Estrella, (1990) Curr. Genet. 17:169-174; and Penttila, Nevalainen, Ratto, Salminen and Knowles, (1987) Gene 6: 155-164, also see US Pat. No. 6,022,725; U.S. Patent No. 6,268,328 and Nevalainen et al., "The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes" in Molecular Industrial Mycology, Eds, Leong and Berka, Marcel Dekker Inc., NY (1992) pp . 129–148; for Aspergillus include Yelton, Hamer and Timberlake, (1984) Proc. Natl. Acad. sci. USA 81: 1470–1474, for Fusarium include Bajar, Podila and Kolattukudy, (1991) Proc. Natl. Acad. sci. USA 88: 8202–8212, for Streptomyces include Hopwood et al., 1985, Genetic Manipulation of Streptomyces : Laboratory Manual, The John Innes Foundation, Norwich, UK, and Fernandez–Abalos et al., Microbiol 149:1623–1632 (2003 ), and for Bacillus include Brigidi, DeRossi, Bertarini, Riccardi and Matteuzzi, (1990) FEMS Microbiol. Lett. 55:135-138).

However, any of the well known procedures for introducing foreign nucleotide sequences into host cells can be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors, and any other well-known means of introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into the host cell. (see, for example, Sambrook et al. above). The Agrobacterium-mediated transfection method described in US Pat. No. 6,255,115 is also applicable. It is only necessary that the particular genetic engineering procedure employed be able to successfully introduce at least one gene into a host cell capable of expressing that gene.

Depending on the host cell used, post-transcriptional and/or post-translational modifications can be made. One non-limiting example of post-transcriptional and/or post-translational modification is "truncation" or "truncation" of a polypeptide. For example, this may result in the transition of the glycoside hydrolase from an inactive or essentially inactive state to an active state, as in the case of a propeptide undergoing additional post-translational processing to a mature peptide characterized by enzymatic activity. Alternatively, this cut may result in the production of the mature glycoside hydrolase described herein and further removal of the N- or C-terminal amino acids to form truncated forms that retain enzymatic activity.

Other examples of post-transcriptional or post-translational modifications include, without limitation, myristoylation, glycosylation, truncation, lipidation, and tyrosine, serine, or threonine phosphorylation. One of skill in the art will appreciate that the type of post-transcriptional or post-translational modifications a protein may undergo may depend on the host organism in which the protein is expressed.

Methods for transforming Aspergillus and Trichoderma are described, for example, in Yelton et al. (1984) Proc. Natl. Acad. sci. USA 81: 1470–1474; Berka et al., (1991) in Applications of Enzyme Biotechnology, Eds. Kelly and Baldwin, Plenum Press (NY); Cao et al ., (2000) Sc. 9:991–1001; Campbell et al., (1989) Curro Genet. 16:53–56; Pentilla et al., (1987) Gene 61:155–164); de Groot et al., (1998) Nat. Biotechnol. 16:839–842; in US patent No. 6022725; US patent No. 6268328 and European patent No. 238023. Expression of a heterologous protein in Trichoderma is described in US patent No. 6022725; US patent No. 6268328; Harkki et al. (1991); Enzyme Microb. Technol. 13:227–233; Harkki et al ., (1989) Bio Technol. 7:596–603; European Patent No. 244234; EP 215594 and Nevalainen et al., "The Molecular Biology of Trichoderma and its Application to the Expression of Both Homologous and Heterologous Genes", in MOLECULAR INDUSTRIAL MYCOLOGY, Eds. Leong and Berka, Marcel Dekker Inc., NY (1992) pp. 129-148). With regard to the transformation of Fusarium strains, reference is also made to W096100787 and Bajar et al., (1991) Proc. Natl. Acad. sci. USA 88:8202–28212.

After the introduction of the expression vector into the cells, the transfected or transformed cells are cultured under conditions conducive to gene expression under the control of promoter sequences. In some cases, the promoter sequence is the cbh1 promoter. Large batches of transformed cells can be cultured as described in Ilmen et al 1997 ("Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei ." Appl. Envir. Microbiol. 63 :1298-1306).

DNA uptake by the host strain Trichoderma sp. depends on the concentration of calcium ions. Typically, approximately 10–50 mM CaCl2 is used in the absorbed solution. Additional suitable compounds include a buffer system such as TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA) or 10 mM MOPS, pH 6.0, and polyethylene glycol. It is assumed that polyethylene glycol fuses cell membranes, which makes it possible to deliver the contents of the medium into the cytoplasm of a strain from the Trichoderma species. This fusion often ensures the integration of several copies of plasmid DNA into the host chromosome.

Usually during the transformation of Trichoderma sp. use protoplasts or cells that have been subjected to a permeability treatment, typically at a density of 10 ⁵ -10 ⁷ /ml, in particular 2×10 6 /ml. These protoplasts or cells in a volume of 100 µl in an appropriate solution (for example,

1.2 M sorbitol and 50 mM CaCl2) can be mixed with the required DNA. As a rule, a high concentration of PEG is added to the absorbed solution. From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplast suspension; however, it is advisable to add approximately 0.25 volume to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, etc. can also be added to the absorbed solution to promote transformation. Similar techniques are available for other fungal host cells. Cm., for example, US patent No. 6022725.

The cell culture medium may be any conventional medium suitable for growing the host cell and allowing expression of any of the glycoside hydrolases described herein. Suitable media and media components are available from commercial vendors or may be prepared according to published formulations (eg, those described in the catalogs of the American Type Culture Collection).

In some embodiments, the production of a spent whole fermentation broth with a recombinant microorganism can be achieved using any culture method known in the art that results in expression of the enzyme of interest. Thus, fermentation can be understood to include shake flask culture, small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid phase fermentation types) in laboratory or industrial fermenters, performed in a suitable medium and under conditions that allow expression or enzyme isolation. The term "spent whole fermentation broth" is defined herein as the unfractionated content of the fermentation material, which includes the culture medium, extracellular proteins (e.g., enzymes) and cell biomass. It is understood that the term "spent whole fermentation broth" also encompasses cell biomass that has been lysed or permeabilized using methods well known in the art.

Host cells can be cultured under suitable conditions that allow expression of any of the glycoside hydrolases described herein. The expression of enzymes can be constitutive, such that they are produced continuously, or inducible, requiring stimulation to initiate expression. In the case of inducible expression, protein production can be initiated when needed by, for example, adding a stimulant substance, eg dexamethasone, or IPTG, or sophorose, to the culture medium.

Polypeptides can also be produced recombinantly in a cell-free systemin vitrosuch as the TNT™ rabbit reticulocyte system (Promega). The expressing host can also be cultured in an appropriate environment for the host under aerobic conditions. Shaking or a combination of agitation and aeration may be provided, with production occurring at the appropriate temperature for that host, e.g. from about 25°C to about 75°C (for example, 30°C to 45°C), depending on the needs of the host and the production of the required alpha-glucosidase. Cultivation can occur for from about 12 to about 100 hours or more (and any value for time in that span, e.g. 24–72 hours). Typically, the pH of the culture broth is from about 4.0 to about 8.0, also depending on the culture conditions required by the host, respectively, to produce the enzyme of interest, such as the glycoside hydrolase described herein. Because of this, producing hosts and transformed cells can be cultured in conventional nutrient media. The culture medium for transformed host cells can be modified as appropriate to activate promoters and select transformed cells. The specific culture conditions, such as temperature, pH, and the like, may be those used for the host cell chosen for expression and will be apparent to those skilled in the art. In addition, preferred culture conditions can be found in scientific literature such as Sambrook, (1982) higher; Kieser, T, MJ. Bibb, MJ. Buttner, KF Chater and D.A. Hopwood (2000) Practical Streptomyces Genetics. John Innes Foundation, Norwich UK; Harwood, et al., (1990) Molecular Biological Methods forbacillus, John Wiley and/or from the American Type Culture Collection (ATCC; www.atcc.org).

Any of the fermentation methods well known in the art can be suitably used to ferment the transformed or derived fungal strain described above.

Classical batch fermentation is a closed system in which the composition of the medium is set at the beginning of the fermentation and this composition is not changed during the fermentation. At the start of fermentation, the medium is inoculated with the desired organism(s). In other words, the entire fermentation process takes place without adding any components to the fermentation system during the entire period.

Alternatively, batch fermentation is defined as "batch" with respect to the addition of a carbon source. In addition, attempts are often made to control factors such as pH and oxygen concentration during the fermentation process. Typically, the compositions of metabolites and biomass of the periodic system are constantly changing until the end of fermentation. In batch cultures, cells go through a static lag phase to an exponential growth phase and finally to a stationary phase where the growth rate slows down or stops. Untreated cells in stationary phase eventually die. Typically, cells in the exponential phase are responsible for the bulk of product production. A suitable variation of the standard batch system is the "fed-batch" system. In this variant of the typical batch system, the substrate is added gradually as the fermentation proceeds. Fed-batch systems are useful where catabolite repression is known to inhibit cell metabolism and/or where it is desirable to have a limited amount of substrates in the fermentation medium. Measuring the actual substrate concentration in fed-batch systems is difficult and is therefore estimated based on changes in measurable factors such as pH, dissolved oxygen and off-gas partial pressure such as CO2. Batch and fed-batch types of fermentation are well known in the art.

Continuous fermentation is another known fermentation method. It is an open system in which a certain fermentation medium is continuously added to the bioreactor and an equal amount of the conditioned medium is simultaneously removed for processing. In continuous fermentation cultures are generally maintained at a constant density where the cells are predominantly in an exponential growth phase. Continuous fermentation allows modulation of one or more factors affecting cell growth and/or product concentration. For example, a limiting nutrient such as a carbon source or a nitrogen source may be kept at a fixed level while all other parameters are allowed to decrease. In other systems, a number of growth influencing factors may vary continuously while the cell concentration, as measured by the turbidity of the medium, is kept constant. Continuous systems strive to maintain stable growth conditions. Thus, cell loss due to media recovery should be proportional to the rate of cell growth during fermentation. Methods for modulating nutrients and growth factors for continuous fermentation processes, as well as techniques for maximizing the rate of product formation, are well known in the field of industrial microbiology.

Separation and concentration techniques are known in the art, and conventional methods can be used to prepare a concentrated solution or broth containing the alpha-glucosidase polypeptide of the present invention.

After fermentation, a fermentation broth is obtained, microbial cells and various suspended solids, including residual fermentation raw materials, are removed using conventional separation techniques to obtain a solution containing the enzyme. Generally, filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction or chromatography, or the like are used.

Sometimes it may be desirable to concentrate the solution or broth containing the polypeptide of interest in order to optimize recovery. The use of non-strength solutions or broth usually extends the incubation time to collect the enriched or purified enzyme precipitate.

The solution containing the enzyme can be concentrated using conventional concentration techniques until the desired level of enzyme is reached. The concentration of the solution containing the enzyme can be achieved by any of the methods discussed in this document. Examples of enrichment and purification methods include, without limitation, rotary vacuum filtration and/or ultrafiltration.

The glycoside hydrolase described herein can be tested for activity using a number of tests known in the art. For example, activity can be tested by combining the enzyme with a glycoprotein or oligosaccharide and water, if necessary. Activity can be measured by analysis of reaction products that can be isolated and visualized, for example, by thin layer chromatography or by HPLC using columns to analyze monosaccharides and oligosaccharides, coupled enzyme analysis using glucose oxidase-peroxidase to quantify glucose, and a reagent representing is 3,5-dinitrosalicylic acid (DNS), for a general analysis of reducing sugar (see examples below).

In addition, the glycoside hydrolase described herein, whether encapsulated or not, may be in the form of beads.

It is contemplated that the glycoside hydrolase described herein may be used in combination with one or more additional enzymes. In some embodiments, one or more additional enzymes, esterases, formamidases, α-galactosidases, e.g. invertase, isomerases, laccases, phenol oxidases, lipases, lyases, mannosidases, oxidases, oxidoreductases, pectinases, pectate lyases, pectin acetylesterases, pectin depolymerases, peptidases, pectin methyl esterases, pectinolytic enzymes, peroxidases, phenol oxidases, phytases, polygalacturonases, rhamnogalacturonases, ribonucleases, transferases, transporters proteins, transglutaminases, xylanases, hexose oxidases (D-hexoses: (3/4-oxidoreductase, EC 1.1.3.5), acid phosphatases and/or others or combinations thereof. These include enzymes which, for example, modulate the viscosity of the formulation or feed.

Any of the GLCH enzymes discussed herein, preferably alpha-amylase, glucoamylase and alpha-glucosidase, can be used in combination with probiotics. DFM categories include Bacillus, lactic acid bacteria and yeast. Bacilli are unique Gram-positive rods that form spores. These spores are very stable and can withstand environmental conditions such as heat, moisture and a certain pH range. These spores germinate into active vegetative cells when ingested by animals and can be used in finely ground and pelleted diets. Lactic acid bacteria are Gram-positive cocci that produce lactic acid and are pathogen antagonists. Because lactic acid bacteria appear to be heat sensitive to some extent, they are not used in pelleted diets. Lactic acid bacteria include the genus Bifidobacterium , Lactobacillus , and Streptococcus. Yeast are not bacteria. These microorganisms belong to a group of plants called fungi.

Any of the GLCH enzymes discussed herein, preferably alpha-amylase, glucoamylase and alpha-glucosidase, can be used in combination with suitable veterinary drugs to reduce the degree of acidosis or to reduce protozoa in the rumen.

Such veterinary medicinal products may include ionophores such as monensin and its derivatives.

Toxin binders are designed to bind toxins in feed and protect animals from their harmful effects. Accordingly, any of the GLCH enzymes discussed herein may also be used in combination with toxin binders or toxin adsorbents. An example of a toxin binder is a mycotoxin binder, including, but not limited to, various hydrated sodium calcium aluminosilicates (HSCAS), bentonite, zeolite, clays, yeast cell walls, and toxin degrading enzymes.

In another aspect, any of the GLCH enzymes discussed herein can also be used in combination with herbs, essential oils, and anti-caking agents.

Essential oil is a concentrated hydrophobic liquid containing volatile aromatic compounds from plants. Essential oils are also known as volatile oils, essential oils, essential oils, or simply as the oil of the plant from which they were extracted, such as clove oil. An "essential" oil is in the sense that it contains the "essence" of a plant aroma - the characteristic aroma of the plant from which it is derived. The term "essential" as used herein does not mean indispensable, as is the case with the terms "essential amino acid" or "essential fatty acid", which are so named because their consumption is necessary for a particular living organism. Essential oils are usually extracted by distillation, often using steam. Other methods include squeezing, solvent extraction, oil-absolute extraction, resin pumping, and cold pressing. They are used in perfumes, cosmetics, soaps and other products, to flavor food and drinks, and to flavor incense and household cleaners.

Animal feeds may include plant material such as corn, wheat, sorghum, soybean, canola, sunflower, or mixtures of any of such plant materials, or sources of plant protein for ruminants. It is expected that animal performance indicators such as growth, feed intake and feed efficiency will be improved, as well as feed uniformity will be improved, ammonia concentration in the animal house will be reduced and therefore the quality of life and health of the animals will be improved. More specifically, as used herein, the expression "animal performance" may be determined by feed efficiency and/or animal body weight gain and/or feed conversion rate and/or feed nutrient digestibility (e.g., amino acid digestibility) and/or digestible energy or metabolic energy in the feed, and/or nitrogen retention, and/or the animal's ability to avoid the adverse effects of necrotic enteritis, and/or the subject's immune response.

The terms "animal feed", "feed", "feed" and "forage" are used interchangeably and may include one or more feed materials selected from the group consisting of a) cereals such as small grains (e.g. wheat, barley, rye, oats and combinations thereof) and/or coarse grains such as maize or sorghum; b) grain processing wastes such as corn gluten meal, dried vinasse with solubles (DDGS) (in particular dried vinasse with solubles based on corn (cDDGS)), wheat bran, wheat semolina, wheat fine bran, rice bran , rice husk, oat husk, coconut kernel and citrus pulp; c) protein derived from sources such as soybeans, sunflowers, peanuts, lupins, peas, fava beans, cotton, canola, fishmeal, dry blood plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats derived from vegetable and animal sources and/or e) minerals and vitamins.

In the case of use as a feed, such as a functional feed, or in its preparation, the enzyme-based composition or feed additive composition of the present invention can be used in combination with one or more of a food-acceptable carrier, a food-acceptance diluent, an acceptable a food-grade excipient, a food-acceptable excipient, a food-active ingredient. For example, at least one component selected from the group consisting of protein, peptide, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride can be mentioned. , potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulphite, methylparaben and propylparaben.

In a preferred embodiment, the enzyme based composition or feed additive composition of the present invention is admixed with a feed component to form a feed product. As used herein, the term "feed component" means all or part of a food product. A food portion may mean one food constituent or more than one food constituent, for example 2 or 3 or 4 or more. In one embodiment, the term "feed component" encompasses the premix or premix constituents. Preferably, the feed may be forage or a premix thereof, compound feed or a premix thereof.

Compound feed, compound feed component or compound feed premix or forage, forage component or forage premix may be admixed to the feed additive composition according to the present invention.

Any feed described herein may include one or more feed materials selected from the group consisting of a) cereals such as small grains (e.g. wheat, barley, rye, oats, triticale and combinations thereof) and/or coarse grains, such as maize or sorghum; b) grain processing wastes such as corn gluten meal, wet vinasse (in particular corn wet vinasse), dried vinasse (DDG) (in particular corn dried vinasse (cDDG)), dried vinasse with solubles (DDGS) (in particular dry corn vinasse with solubles (cDDGS)), wheat bran, wheat semolina, wheat fine bran, rice bran, rice husk, oat husk, coconut kernel and citrus pulp; c) protein derived from sources such as soybeans, sunflowers, peanuts, lupins, pea species, fava beans, cotton, canola, fishmeal, dry plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats derived from vegetable and animal sources; e) minerals and vitamins.

The term "forage" as used herein means any food that is given to an animal (as opposed to food that the animal obtains on its own). Forage covers plants that have been cut. Furthermore, forage includes silage, pressed and pelleted feeds, oils and mixed rations, as well as sprouted grains and legumes.

The forage may be obtained from one or more plants selected from: corn (maize), alfalfa (alfalfa), barley, lollipop, representatives of the genus cabbage, cabbage variety Chau moellier, fodder cabbage, rapeseed (canola) seed, turnip ( swede), turnip, clover, hybrid clover, red clover, underground clover, white clover, fescue, brome, millet, oats, sorghum, soybeans, trees (cut sprouts for tree hay), wheat and legumes.

The term "compound feed" means industrial feed in the form of flour, granules, nuts, cake or crumbs. Compound feed can be mixed with various raw materials and additives. These mixtures are formulated according to the specific requirements of the target animal.

Compound feeds can be complete feeds that provide all the daily required nutrients, concentrates that provide part of the diet (protein, energy), or supplements that provide only additional micronutrients such as minerals and vitamins.

The main ingredients used in compound feed are feed grains, which include corn, wheat, canola meal, rapeseed meal, lupins, soybeans, sorghum, oats and barley.

Accordingly, a premix, as referred to herein, may be a composition consisting of micro-ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other necessary ingredients. Premixes are typically compositions suitable for mixing in industrial rations.

In one embodiment, the food product contains or consists of corn, DDGS (such as cDDGS), wheat, wheat bran, or any combination thereof.

In one embodiment, the feed component may be corn, DDGS (eg, cDDGS), wheat, wheat bran, or a combination thereof. In one embodiment, the food product contains or consists of corn, DDGS (such as cDDGS), or a combination thereof.

The food product described herein may contain at least 30%, at least 40%, at least 50%, or at least 60% by weight of corn and soy flour or corn and full fat soy or wheat flour, or fodder meal from cake of sunflower seeds.

For example, the food product may contain from about 5% to about 40% corn DDGS. For poultry, the feed product may contain, on average, from about 7% to about 15% corn DDGS. For domestic pigs (pigs), the feed product may contain an average of 5–40% corn DDGS. It may also contain whole grain corn, in which case the feed product may contain from about 35% to about 80% corn.

In feed products containing a grain mixture, such as those containing corn and wheat, for example, the feed product may contain at least 10% corn.

Additionally or alternatively, the food product may also comprise at least one high fiber food material and/or at least one high fiber food material processed to produce a high fiber food product. Examples of high fiber feed materials include wheat, barley, rye, oats, cereal by-products such as corn gluten meal, corn gluten feed, wet vinasse, dried vinasse (DDG), dried vinasse with solubles (DDGS), wheat bran, wheat grits, wheat fine bran, rice bran, rice husk, oat husk, coconut kernel and citrus pulp. Some sources of protein can also be considered high-fiber sources: protein derived from sources such as sunflowers, lupins, fava beans, and cottonseed. In one aspect, a food product described herein comprises at least one high fiber food material and/or at least one processed product of at least one high fiber food material selected from the group consisting of, for example, dry vinasse with solubles (DDGS), in particular cDDGS, wet vinasse, dried vinasse (DDG), in particular cDDG, wheat bran and wheat. In one embodiment of the invention, the food product of the present invention comprises at least one high fiber material and/or at least one processed product of at least one high fiber food material selected from the group consisting of, for example, dried solubles vinasses (DDGS), in particular cDDGS, wheat bran and wheat.

The feed may be one or more of the following: compound feed and premix, including pellets, nuts or cake (for cattle); crop or crop residues: corn, soybeans, sorghum, oats, barley, copra, straw, chaff, sugar beet waste; fish flour; meat and bone meal; molasses; oil cake and pressed cake; oligosaccharides; preserved fodder plants: silage; seaweed; seeds and grains, whole or prepared by crushing, grinding, etc.; sprouted grains and legumes; yeast extract.

As used herein, the term "food" encompasses, in some embodiments, pet food. Pet food is a material of plant or animal origin intended for consumption by pets, such as dog food or cat food. Pet food, such as dog and cat food, may be either in a dry form, such as dog food pellets, or in a wet, canned form. Cat food may contain the amino acid taurine.

Animal food may also include fish food. Fish food normally contains the macronutrients, micronutrients and vitamins needed to maintain the health of captive fish. The fish food may be in the form of a flake, granule or tablet. Granular forms, some of which sink quickly in water, are often used for larger or bottom-feeding fish. Some fish foods also contain additives such as beta-carotene or sex hormones to artificially enhance the color and pattern of the fish.

In yet another aspect, pet food encompasses bird food. Bird food includes food that is used in both bird feeders and pet birds. Bird food usually contains various seeds, but may also include lard (beef or mutton fat).

As used herein, the term "contacted" refers to the indirect or direct use of the glycoside hydrolase described herein.

(or to a composition containing glycoside hydrolase), in relation to the product (for example, feed). Examples of uses that can be used include, without limitation, treating the product in a material containing the feed additive composition, direct application by mixing the feed additive composition with the product, spraying the feed additive composition onto the surface of the product, or dipping the product into a feed additive formulation formulation. In one embodiment, the feed additive composition of the present invention is preferably admixed to a product (eg, a feed product). Alternatively, the feed additive composition may be included in the emulsion or raw ingredients of the feed product. This improves the performance of the composition.

The term "thermally stable" means that at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 98% an enzyme that was present/active in the supplement prior to heating to the indicated temperature is still present/active after cooling to room temperature. Preferably, at least about 80% of the enzyme present and active in the supplement prior to heating to the indicated temperature is still present and active after cooling to room temperature.

It is also possible that the glycoside hydrolases (or an enzyme-based composition containing such glycoside hydrolases described herein) described herein can be homogenized to form a powder.

In an alternative preferred embodiment, the glycoside hydrolase described herein (or an enzyme-based composition containing the glycoside hydrolase described herein) can be formulated into beads as described in WO 2007/044968 (referred to as TPT beads) or WO 1997/ 016076, or WO 1992/012645, incorporated herein by reference. "TPT" stands for Thermal Protection Technology.

In another aspect, when the feed additive composition is formulated as granules, the granules contain a hydrated salt that acts as a barrier that coats the protein core. The advantage of this salt coating is improved thermal stability, improved storage stability and protection from other feed additives that adversely affect the enzyme in any way. Preferably, the salt used for salt coating has a water activity of more than 0.25 or a constant moisture content of more than 60% at 20°C. In some embodiments, the implementation of the salt coating contains Na ₂ SO ₄ .

The method for preparing the glycoside hydrolase described herein (or the enzyme-based composition containing the glycoside hydrolase described herein) may also include an additional powder granulation step. The powder can be mixed with other components known in the art. The powder or mixture containing the powder can be forced through a mold and the resulting filaments cut into suitable granules of various lengths.

Optionally, the granulation step may include steaming or a conditioning step prior to forming the granules. The mixture containing the powder can be placed in a conditioning device such as a steam injection mixer. The mixture is heated in a conditioning unit to a predetermined temperature, eg 60-100ºC, typical temperatures would be 70ºC, 80ºC, 85ºC, 90ºC or 95ºC. The residence time can vary from seconds to minutes and even hours. For example, 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour. It will be understood that the glycoside hydrolase described herein (or an enzyme-based composition containing a glycoside hydrolase described herein) is suitable for addition to any suitable feed material.

One skilled in the art will appreciate that different animals require different foods, and even the same animal may require different foods depending on the purpose for which the animal is raised.

Optionally, the food product may also contain additional minerals such as, for example, calcium and/or additional vitamins. In some embodiments, the food product is a mixture of corn and soy flour.

The feed product is usually obtained in feed mills, in which the raw material is first crushed to a suitable particle size and then mixed with the appropriate additives. The feed product can then be made into a slurry or granules; the latter generally involves a method by which the temperature is raised to a predetermined level and then the feed is passed through a mold to obtain pellets of a specific size. The granules are allowed to cool. Subsequently, liquid additives such as fat and enzyme can be added. The preparation of the feed product may also include an additional step which, prior to granulation, includes extrusion or swelling, in particular by suitable techniques, which may include at least the use of steam.

The feed product may be a feed product for monogastric animals such as poultry (e.g. broiler, laying hen, broiler-type layers, turkey, duck, geese, waterfowl) and domestic pig (all age categories), ruminants such as as cattle (e.g. cows or bulls (including calves)), horses, sheep, domestic animals (e.g. dogs, cats) or fish (e.g. fish without a stomach, fish with a stomach, freshwater fish such as salmon , cod, trout, and carp such as koi, saltwater fish such as sea bass, and crustaceans such as small shrimp, mussels, and scallops). Preferably, the feed product is for pigs.

The feed additive composition and/or feed product containing it may be used in any suitable form. The feed additive composition may be administered in the form of solid or liquid formulations, or alternatives thereof. Examples of solid preparations include powders, pastes, boluses, capsules, dragees, tablets, powders and granules, which can be wetted, spray dried or lyophilized. Examples of liquid preparations include, without limitation, aqueous, organic or aqueous-organic solutions, suspensions, and emulsions.

In some applications, feed additive compositions may be mixed with feed or introduced into drinking water.

A feed additive composition comprising admixing a glycoside hydrolase as described herein with a feed-acceptable carrier, diluent or excipient and (optionally) packaging.

The feed product and/or feed additive composition can be combined with at least one mineral and/or at least one vitamin. The compositions thus obtained may be referred to herein as a premix. The feed product may contain at least 0.0001% by weight of the feed additive. Accordingly, the feed product may contain 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.

Preferably, the food or feed additive composition may further comprise at least one physiologically acceptable carrier. The physiologically acceptable carrier is preferably selected from at least one of maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat component, sucrose, starch, Na ₂ SO ₄ , talc, PVA and mixtures thereof. In a further embodiment, the food or feed additive may further comprise a metal ion chelator. The metal ion chelator can be selected from EDTA or citric acid.

In some embodiments, the food or feed additive composition contains the glycoside hydrolase described herein at a level of at least 0.0001 g/kg, 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 least 25.0 g/kg. In some embodiments, the food or feed additive contains at such a level that when added to the food or feed material, the feed material contains the glycoside hydrolase described herein in the range of 1-500 mg/kg, 1-100 mg/kg, 2- 50 mg/kg or 2–10 mg/kg. In some embodiments of the present invention, the food or feed material contains at least 100, 1000, 2000, 3000, 4000, 5000, 10000, 20000, 30000, 50000, 100000, 500000, 1000000 or 2000000 units of glycoside hydrolase per kilogram of feed or food material.

The ranges may include, without limitation, any combination of the lower and upper limits of the ranges discussed above.

Compositions containing any of the glycoside hydrolases described herein and the compositions described herein can be prepared by any suitable method, with the assurance that the composition will contain active enzymes. Such formulations may be in the form of a liquid, a dry powder, or a granule. Preferably the feed additive composition is in solid form suitable for addition to or in a feed pellet.

Dry powder or granules can be obtained by methods known to those skilled in the art such as high shear granulation, drum granulation, extrusion, spheronization, fluid bed agglomeration, fluid bed spray drying.

The glycoside hydrolases and compositions described herein may be coated, eg encapsulated. In one embodiment, the coating protects the enzymes from heat and may be considered a thermal protectant. In one embodiment, the coating protects the enzyme from exposure to low pH. Eudragit® is one example of a coating material that can be used.

The feed additive composition described herein may be formulated as a dry powder or granules as described in WO2007/044968 (referred to as TPT granules) or WO1997/016076 or WO1992/012645 (each of which is incorporated herein by reference).

In one embodiment, the pet food may be formulated as pellets for feed compositions containing: a core; an active agent and at least one coating, wherein the active agent of the granule retains at least 50% activity, at least 60% activity, at least 70% activity, at least 80% activity after exposure to conditions selected from one or several of: a) a feed pelleting process, b) a steam heated feed pretreatment method, c) storage, d) storage as an ingredient in a non-granulated mixture, and e) storage as an ingredient in an initial feed mixture or feed premix containing at least at least one compound selected from trace elements, organic acids, reducing sugars, vitamins, choline chloride, and compounds that provide acidity or basicity to the original feed mixture or feed premix.

With respect to the granule, at least one coating may comprise a moisturizing hydrating material that makes up at least 55% w/w of the granule and/or at least one coating may comprise two coatings. The two coatings may be a moisturizing hydrating coating and a moisture-retaining coating. In some embodiments, the moisturizing hydrating coating may comprise 25% to 60% w/w of the granule and the humectant coating may comprise 2% to 15% w/w of the granule. The moisturizing hydrating coating may be selected from inorganic salts, sucrose, starch and maltodextrin, and the moisture retaining coating may be selected from polymers, resins, whey and starch.

The pellet can be obtained by a feed pelleting process, and the feed pretreatment process can be carried out at 70°C-95°C for several minutes, for example at 85°C-95°C.

The feed additive composition may be formulated as animal feed pellets containing: a core; an active agent, wherein the active agent of the granule retains at least 80% activity after storage and after the steam heated granulation process, where the granule is an ingredient; a moisture-retaining coating; and a moisturizing hydrating coating that makes up at least 25% w/w of the granule, wherein the granule has a water activity of less than 0.5 prior to the steam heated granulation process.

The granule may have a moisture-retaining coating selected from polymers and resins, and the moisturizing hydrating material may be an inorganic salt. The moisturizing hydrating coating may comprise from 25% to 45% w/w of the granule and the humectant coating may comprise from 2% to 10% w/w of the granule.

The granule can be produced by a steam heated granulation process, which can be carried out at 85°C-95°C for several minutes.

Alternatively, the composition is in the form of a liquid formulation suitable for consumption, preferably such consumable liquid contains one or more of the following: buffer, salt, sorbitol and/or glycerin.

In addition, the composition of the feed additive can be formulated by applying, for example spraying, the enzyme(s) onto a carrier substrate, such as, for example, milled wheat.

In one embodiment, the feed additive composition may be formulated as a premix. By way of example only, the premix may contain one or more feed components such as one or more minerals and/or one or more vitamins.

In some embodiments, the glycoside hydrolase will be in a physiologically acceptable carrier. Suitable carriers can be large slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive viral particles. Pharmaceutically acceptable salts may be used, for example mineral acid salts such as hydrochlorides, hydrobromides, phosphates and sulfates, or organic acid salts such as acetates, propionates, malonates and benzoates. Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, adjuvants such as wetting or emulsifying agents or pH buffering agents may be present in such compositions. Such carriers make it possible to formulate pharmaceutical compositions in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries and suspensions for ingestion by the patient. Once formulated, the composition of the present invention may be administered directly to a ruminant.

Non-limiting examples of compositions and methods disclosed herein include the following.

1. A method of increasing starch digestibility and glucose yield in a ruminant, which comprises adding at least one alpha-1,4/1,6-glycoside hydrolase (GLCH) as a feed additive to a ruminant feed, wherein said hydrolase (a) characterized by at least 20% activity at a pH value of 3 or less in the presence of pepsin compared to the activity of a hydrolase at a pH value of 6 in the presence of pepsin, (b) said hydrolase is active in at least two of the three digestive chambers of a ruminant animal, including rumen, abomasum and small intestine, and (c) the hydrolase acts in concert with the digestive enzymes present in the digestive chambers of the ruminant to increase starch digestibility and glucose output.

2. A hydrolase according to embodiment 1 wherein at least one GLCH enzyme is capable of hydrolyzing crude starch under conditions comparable to those found in rumen or abomasum.

3. A hydrolase according to embodiment 1, wherein said hydrolase is selected from the group consisting of alpha-amylases, glucoamylases, and alpha-glucosidases.

4. The hydrolase according to embodiment 2, wherein said hydrolase is selected from the group consisting of alpha-amylases and glucoamylases.

5. A hydrolase according to embodiment 3 or 4, wherein the alpha-amylases are members of the GH 13 family or are selected from the group consisting of alpha-amylase (EC 3.2.1.1); pullulanase (EC 3.2.1.41 ); cyclomaltodextrin-glucanotransferase (EC 2.4.1.19 ); cyclomaltodextrinase (EC 3.2.1.54 ); trehalose-6-phosphate hydrolase (EC 3.2.1.93 ); oligo-alpha-glucosidase (EC 3.2.1.10 ); maltogenic amylase (EC 3.2.1.133 ); neopullulanase (EC 3.2.1.135 ); alpha-glucosidase (EC 3.2.1.20 ); maltotetraose-forming alpha-amylase (EC 3.2.1.60 ); isoamylase (EC 3.2.1.68 ); glucodextranase (EC 3.2.1.70 ); maltohexaose-forming alpha-amylase (EC 3.2.1.98 ); maltotriose-forming alpha-amylase (EC 3.2.1.116 ); branching enzyme (EC 2.4.1.18 ); trehalose synthase (EC 5.4.99.16 ); 4-alpha-glucanotransferase (EC 2.4.1.25 ); maltopentaose-forming alpha-amylase (EC 3.2.1.- ) ; amylosucrases (EC 2.4.1.4 ); sucrose phosphorylase (EC 2.4.1.7 ); maltooligosyltrehalose-trehalohydrolases (EC 3.2.1.141 ); isomaltulose synthase (EC 5.4.99.11 ); maltooligosyltrehalose synthase (EC 5.4.99.15 ); amyl-alpha-1,6-glucosidase (EC 3.2.1.33 ) and alpha-1,4-glucan:phosphate-alpha-maltosyltransferase (EC 2.4.99.16 ).

6. A hydrolase according to embodiment 3 or 4, wherein the glucosidases are members of the GH 13 or GH31 family or are selected from the group consisting of alpha-glucosidase (EC 3.2.1.20 ); alpha-galactosidase (EC 3.2.1.22 ); alpha-mannosidases (EC 3.2.1.24 ); alpha-1,3-glucosidase (EC 3.2.1.84 ); sucrase-isomaltase (EC 3.2.1.48 ) (EC 3.2.1.10 ); alpha-xylosidases (EC 3.2.1.177 ); alpha-glucanase (EC 4.2.2.13 ); isomaltosyltransferase (EC 2.4.1.– ); oligosaccharide-alpha-1,4-glucosyltransferase (EC 2.4.1.161 ); sulfoquinovosidases (EC 3.2.1.– ).

7. Hydrolase according to embodiment 3 or 4, wherein the glucoamylases are members of the GH15 family or are selected from the group consisting of glucoamylase (EC 3.2.1.3 ); glucodextranase (EC 3.2.1.70 ); alpha-trehalase (EC 3.2.1.28 ) and dextrandextrinase (EC 2.4.1.2 ).

8. A method of increasing starch digestibility, increasing glucose yield, increasing dry matter digestion, and increasing gas production during fermentation in a ruminant, comprising adding to the feed an enzyme-based composition containing (i) at least one GLCH enzyme as a feed additive for the ruminant animal, where the specified enzyme (a) is characterized by at least 20% activity at a pH value of 3 or less in the presence of pepsin compared with the activity of enzymes at a pH value of 6 in the presence of pepsin, (b) the specified enzyme is active at least two of the three digestive chambers of a ruminant, including rumen, abomasum, and small intestine, and (c) the enzyme works in conjunction with pancreatic amylase to increase glucose output, and (ii) at least one protease.

9. An enzyme-based composition according to embodiment 8, wherein at least one GLCH enzyme is capable of hydrolyzing crude starch.

10. An enzyme-based composition according to embodiment 8, wherein said GLCH enzyme is selected from the group consisting of alpha-amylases, glucoamylases, and alpha-glucosidases.

11. An enzyme-based composition according to embodiment 9, wherein said enzyme is selected from the group consisting of alpha-amylases and glucoamylases.

12. An enzyme-based composition according to embodiments 10 and 11, wherein at least one GLCH enzyme is selected from the group consisting of alpha-amylase (EC 3.2.1.1); pullulanase (EC 3.2.1.41 ); cyclomaltodextrin-glucanotransferase (EC 2.4.1.19 ); cyclomaltodextrinase (EC 3.2.1.54 ); trehalose-6-phosphate hydrolase (EC 3.2.1.93 ); oligo-alpha-glucosidase (EC 3.2.1.10 ); maltogenic amylase (EC 3.2.1.133 ); neopullulanase (EC 3.2.1.135 ); alpha-glucosidase (EC 3.2.1.20 ); maltotetraose-forming alpha-amylase (EC 3.2.1.60 ); isoamylase (EC 3.2.1.68 ); glucodextranase (EC 3.2.1.70 ); maltohexaose-forming alpha-amylase (EC 3.2.1.98 ); maltotriose-forming alpha-amylase (EC 3.2.1.116 ); branching enzyme (EC 2.4.1.18 ); trehalose synthase (EC 5.4.99.16 ); 4-alpha-glucanotransferase (EC 2.4.1.25 ); maltopentaose-forming alpha-amylase (EC 3.2.1.- ); amylosucrases (EC 2.4.1.4 ); sucrose phosphorylase (EC 2.4.1.7 ); maltooligosyltrehalose-trehalohydrolases (EC 3.2.1.141 ); isomaltulose synthase (EC 5.4.99.11 ); maltooligosyltrehalose synthase (EC 5.4.99.15 ); amyl-alpha-1,6-glucosidase (EC 3.2.1.33 ) and alpha-1,4-glucan:phosphate-alpha-maltosyltransferase (EC 2.4.99.16 ).

13. An enzyme-based composition according to claim 10 or claim 11, where the glucosidases are selected from the group consisting of alpha-glucosidase (EC 3.2.1.20 ); alpha-galactosidase (EC 3.2.1.22 ); alpha-mannosidases (EC 3.2.1.24 ); alpha-1,3-glucosidase (EC 3.2.1.84 ); sucrase-isomaltase (EC 3.2.1.48 ) (EC 3.2.1.10 ); alpha-xylosidases (EC 3.2.1.177 ); alpha-glucanase (EC 4.2.2.13 ); isomaltosyltransferase (EC 2.4.1.– ); oligosaccharide-alpha-1,4-glucosyltransferase (EC 2.4.1.161 ); sulfoquinovosidases (EC 3.2.1.– ).

14. An enzyme-based composition according to embodiment 10 or 11, wherein the glucoamylases are selected from the GH15 family of glycosyl hydrolases.

15. An enzyme-based composition according to embodiment 8 wherein the protease is selected from the group consisting of an acidic protease or a neutral metalloprotease.

16. An enzyme-based composition according to embodiment 15, wherein the protease is a fungal aspartic protease or a bacterial neutral metalloprotease.

EXAMPLES

Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as generally understood by a person skilled in the art to which the present invention 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, NY (1991) provide a general a dictionary for many of the terms used in this disclosure.

The present invention is further defined in the following examples. It should be understood that the examples, while showing certain embodiments, are provided for purposes of illustration only. From the above discussion and examples, one skilled in the art will be able to ascertain the essential characteristics of the present invention, and without deviating from its spirit and scope, will be able to make various changes and modifications to adapt it to various applications and conditions.

EXAMPLE 1

Materials used in the following examples

The protein samples listed in Table 1 were used in the following examples. Table 1 shows the type of enzyme, the source organism (if known), and the domestic or commercial source of the samples, and patent references for the sequences.

Table 1 . List of evaluated enzyme components

protein name Enzyme type Source organism Link AkAA alpha-amylase Recombinant, source of Aspergillus kawachii Patent US 7354752 ACAA alpha-amylase Recombinant, source of Aspergillus clavatus Patent US 8945889 AtAA alpha-amylase Recombinant, source of Aspergillus terreus Patent US 20150376668 TrGA glucoamylase Recombinant, T. reesei source Patent US 7413879 CS4 glucoamylase Recombinant, variant Patent US 8058033 Brew1 glucoamylase Recombinant, variant Patent US 8809023 FvGA glucoamylase Recombinant, source of Fusarium verticillioides Patent WO 2016100871 AfuGA glucoamylase Recombinant, source of Aspergillus fumigatus Patent US 20160115509 AFP protease Recombinant, T. reesei source Patent US 8288517 Yeast maltase alpha-glucosidase Megazyme E-MALTS catalog no. TG L–2000 alpha-glucosidase Recombinant, source of Aspergillus niger US 2015/0240279 Aclglu1 alpha-glucosidase Recombinant, A. clavatus source US 20150240279 Nfiglu1 alpha-glucosidase Recombinant, source of Neosartorya fischeri US 20150240279 TauSec098 alpha-glucosidase Rasamsonia composticola US 20150240279 TauSec099 alpha-glucosidase Rasamsonia composticola US 20150240279 pork pepsin protease Sigma Catalog No. P7000 Pork pancreatin Many enzymes Sigma Catalog No. P7545 P14L neutral metalloendopeptidase (thermolysin) Geobacillus caldoproteolyticus Patent US 8114656 P7L neutral metalloendopeptidase (bacillolysin) Bacillus amyloliquefaciens Patent US 8574884 AnGA glucoamylase Aspergillus niger , Megazyme Catalog No. E–AMGDF

EXAMPLE 2

Hydrolysis of maize flour by AkAA amylase in the presence of porcine pepsin acid aspartic protease and AFP Trichoderma reesei

The reaction mixture contained 1 ml slurry of cornmeal (20%, w/w, pH 3.2), 20 µl AkAA (209 mg/ml), 50 µl porcine pepsin (Sigma, P7000, 10,000 U/ml in water), 5 µl AFP from Trichoderma reesei (5 mg/ml) and 50 µl and water to a final volume of 1.07 ml. The reaction was carried out at 40 ^° C. for 5 hours with shaking at 200 rpm. At the end of the reaction, 30 μl of the supernatant of the reaction mixture was mixed with 0.23 ml of water and filtered through a 0.22 μm membrane filter. The filtrate (40 µl) was subjected to HPLC analysis using an Aminex HPX-87N HPLC column (Bio-Rad) at a flow rate of 0.6 ml/min. at 78 ^o C for 15 minutes. using water as eluent. Glucose and maltose peaks were determined using an inline RI (Refractive Index) detector and peak areas were integrated using Chromeleon software (Dionex) according to the manufacturer's instructions.

Pepsin should not interfere with the activity of ruminant feed enzymes if they are to function in the abomasum and small intestine.

Figure 1 shows that neither pepsin nor AFP had a negative effect on the release of glucose (G1) and maltose (G2) from cornmeal at pH 3.2. pH 3.2 is one of the pH values that can occur in abomasum, especially after feeding (Constable et al., 2005. Effect of suckling cow's milk or milk replacer on abomasal luminal pH in dairy calves, J. Vet. Intern. Med. 19:97-102). In fact, the addition of pepsin and AFP to the reaction mixture containing AkAA increased the release of G1 by 4% and 22%, respectively. Pepsin alone caused little or no release of G1 and G2 from cornmeal, while AFP itself caused some release of G1 but not G2. AFP added to the reaction mixture containing AkAA not only increased the release of G1, but also increased the amount of G1 compared to G2, indicating that AFP is more conducive to the release of glucose, a monomer (G1). It is G1 that can be directly absorbed into the bloodstream by animals.

It is believed that, under conditions comparable to those found in ruminant stomach and abomasum, two aspartic proteases likely made the cornstarch granules more accessible to AkAA by hydrolyzing proteins surrounding the granules and proteins found within the granule (Mu-Forster et al. al., 1996. Physical association of starch biosynthetic enzymes with starch granules of maize endosperm granule–associated forms of starch synthase I and starch branching enzyme II, Plant Physiol 111: 821–829 Mu–Forster et al., 1998. Surface localization of zein storage proteins in starch granules from maize endosperm, proteolytic removal by thermolysin and in vitro cross-linking of granule-associated polypeptides Plant Physiol 116: 1563-1571). Pepsin is the most important proteolytic enzyme produced in the stomach and abomasum. It digests proteins by cleaving internal peptide bonds with an optimal pH of 2-4. AFP is an extracellular endopeptidase from T. reesei with an optimal pH in the range of 3–4.5 (Table 1).

The corn used in Example 2 and other examples, unless otherwise noted, consisted of 88.2% dry matter (DM), 9.3% crude protein (CP), 2.0% acid detergent fiber (ADF), 6 .6% Neutral Detergent Amylase Treated Fiber (aNDF), 78.5% Non-Fibrous Carbohydrates (NFC), 89.0% Total Digestible Nutrients (TDN).

EXAMPLE 3

Stability of AkAA amylase in the presence of rumen juice

In order for an enzyme supplement in animal feed to be effective in ruminants, it is important that the enzyme exhibit significant stability in rumen juice and rumen-specific conditions. The data in Table 2 shows that AkAA is stable in the presence of cell-free rumen sap when incubated at 40 ^° C. for 4.5 hours, since the release of glucose and maltose was not significantly affected by increasing amounts of rumen sap.

The AkAA used had a total protein concentration of 209 mg/mL, rice starch granules (Sigma S7260) were prepared in water as an 8% (w/w) slurry with pH adjusted to 3.2. The pH value became pH 5.3 after mixing with the pre-incubation mix. Cage-free rumen juice having a pH value of 6.1 was collected from a dairy cow fed a mixed diet of grass (41.66%), whole grain barley (14.79%), maize (16.78%), dehydrated beet pulp sugar beet and cake of sugar beet residues (3.02%), ground barley (6.66%), rapeseed flour (4.88%), soy flour (4.46%), Komix (mixture of vitamins and minerals, 0.2%), Roed Suplex Caps (vitamin blend 0.05%), Suplex E-5000 (vitamin E and selenium, 0.03%), chalk (0.07%), salt (0.06%) and water (7.33%). The tripe juice was provided by the Department of Animal Science, Aarhus University (Blichers Alley 20, DK-8830 Tjele, Denmark). The sugar released during the reaction was analyzed by HPLC using an Aminex HPX-87N HPLC column from Bio-Rad as described in Example 2.

Table 2. Stability of AkAA in rumen juice at 40 ^° C for 4.5 hours.

Treatment No. (n=3) 1 2 3 4 5 6 7 8 9 10 Rumen juice, µl 0 0 90 90 140 140 190 190 240 240 AkAA, µl 0 10 0 10 0 10 0 10 0 10 Thermally treated cicatricial juice, µl 240 240 150 150 100 100 50 50 0 0 Pre-incubation at 40 ^o C for 4.5 hours with shaking at 200 rpm, and then adding additional AkAA and rice starch substrate to initiate the catalytic reaction AkAA, µl 10 0 10 0 10 0 10 0 10 0 Suspension of rice starch, ml 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Enzymatic reaction at 40 ^° C. for 3 hours with shaking at 200 rpm. The supernatant obtained at the end of the reaction was diluted 4 times with water and 40 μl was injected for HPLC analysis of released glucose (G1) and maltose (G2) G1 peak area 5.78 6.25 5.75 5.48 6.86 7.15 7.43 6.62 6.70 6.28 G2 peak area 4.33 4.08 4.18 4.07 3.96 3.89 3.89 3.74 4.06 3.83 Peak area G1+G2 10.11 10.32 9.93 9.56 10.82 11.04 11.32 10.36 10.76 10.11 Percentage of Processing #1 100 102 98 95 107 109 112 102 106 100

EXAMPLE 4

Stability of AkAA in the presence of pancreatin and the effect of AkAA and pancreatin on increasing glucose yield and decreasing maltotriose yield

The reaction mixture contained 0, 5, 10 and 20 µl of porcine pancreatin (Sigma P7545, 25 mg/ml prepared in 0.1% NaCl), 20 µl of AkAA (209 mg/ml), 60 µl of 0.1 M Mes (pH 6.7) containing 5 mM CaCl ₂ , Tween 80 (0.01% w/v). A 0.1% NaCl solution was added to a final reaction volume of 0.12 ml. The reaction mixture was incubated at 40 ^° C. for 3 hours and then a substrate based on 1.5 ml of cornmeal slurry (10% w/w) was added and further incubated at 40 ^° C. for 13 hours. 8.33-fold dilution, and 40 µl were analyzed by HPLC for glucose, maltose and maltotriose using an RI detector as described in example 2.

Figure 2 shows that AkAA was active under conditions simulating those of the ruminant small intestine. Both additive and complementary effects were shown for the 3 doses of porcine pancreatin (Pan) tested. Combining AkAA and Pan increased the production of glucose and maltose and reduced the formation/accumulation of maltotriose, which is nutritionally advantageous because glucose can be absorbed directly, while maltose can be hydrolyzed by intestinal mucosal alpha-glucosidase (maltase) (Nichols et al., 1998. Human small intestinal maltase–glucoamylase cDNA cloning, homology to sucrase–isomaltase, J Biol Chem 273:3076–3081). Pancreatin is an essential digestive enzyme blend for animals, especially monogastric and ruminant animals. It contains enzymatic components such as trypsin, amylase and lipase, ribonuclease and protease produced by exocrine cells of the porcine pancreas. Thus, pancreatin is a combination of enzymes that provides the hydrolysis of proteins, starch and fats.

EXAMPLE 5

AtAA amylase activity against cornmeal in the presence of pepsin and pancreatin

To carry out the reaction with pepsin, the reaction mixture contained 0.1 ml of 10% suspension of corn starch (w/w) (Sigma S4126) prepared in 60 mM glycine–HCl (pH 2.5), 10 µl of pepsin (Sigma P7000, 10000 U ./ml in water) and 0.6 µl (16.76 mg/ml) AtAA amylase. The reaction was carried out at 40 ^o C for 3 hours the Samples were centrifuged. The supernatant was diluted, filtered and analyzed by HPLC using an RI detector for sugar analysis as described in Example 2. The pancreatin reaction was the same as the pepsin reaction, except that a slurry of cornstarch was obtained at 0.1 M Mes (pH 6.7) and 5 μl of 25 mg/ml porcine pancreatin (Pan) (Sigma P7545, dissolved in 0.1% NaCl) was added.

Figure 3 shows that Aspergillus terreus alpha-amylase AtAA (AtAmy1) was active when tested in the presence of both pepsin at pH 2.5 and pancreatin at pH 6.7. In addition, AtAA and pancreatin showed a complementary effect in converting all maltotriose, in fact, into glucose, which can be directly absorbed by animals.

EXAMPLE 6

Hydrolysis of corn starch by AkAA and AcAA amylases in the presence of rumen juice

240 µl of cell-free rumen juice was mixed with 10 µl of enzyme samples: AkAA (209 mg/ml), AcAA (172 mg/ml), AkAA with Trichoderma reesei GA (TrGA) and Trichoderma reesei AFP (223 mg/ml) or AcAA with variant TrGA CS4 and AFP (125 mg/ml) (Table 1) and incubated at 40°C for 4 hours followed by the addition of 250 μl of a suspension of corn starch (Sigma S4126) dissolved in 0.1 M acetate containing 5 mM CaCl ₂ (pH 4.5) and further incubated for 30 minutes. At the end of the reaction, reaction samples were filtered and supernatants were analyzed for total reducing sugars using the alkaline 3,5-dinitrosalicylic acid (DNS) reagent according to Yu et al., (Yu et al, 1997. Methods for the assay of 1,5-anhydro-D-fructose and alpha-1,4-glucan lyase. Carbohydr. Res. 305:73-82) with minor modification. Namely, 10 µl diluted reaction samples were transferred to a 96-well PCR plate containing 120 µl of the DNS reagent. The plate was incubated at 95°C for 5 min. followed by 5 min. cooling to 20°C, and then 100 μl of these mixtures were transferred into a new 96-well plate, which was used to measure the absorbance at 550 nm in a spectrophotometer.

Figure 4 shows a very slight decrease in corn hydrolysis activity for all 4 enzyme samples tested after incubation in rumen juice. The presence of Trichoderma reesei GA and its variant (CS4) and Trichoderma reesei AFP had a positive effect on the total substrate hydrolysis catalyzed by both AkAA and AcAA amylases. Both amylase/glucoamylase/protease enzyme mixtures were prepared with a total protein ratio of 29:70:1. As shown in Figure 4, AkAA, AcAA amylases and their mixtures with TrGA and CS4 glucoamylases and AFP aspartic protease lost less than 13% of their total activity after 4 hours of pre-incubation in rumen juice at 40°C followed by 30 minutes. incubation with corn starch at 40°C at pH 4.5 (compared to untreated mixtures).

These results, obtained under simulated rumen conditions, indicate that these two amylases and their mixtures with glucoamylases and aspartic peptidase can persist in the ruminant's digestive tract.

EXAMPLE 7

Activity of AkAA and AcAA amylases in the presence of pepsin under different pH conditions

The reaction mixture contained 1.0 ml of a suspension of cornmeal (20% w/w) prepared in 0.1 M glycine-HCl (pH 3.2), 0.1 M acetate (pH 4.1, 4.7) or 0.1 M Mes (pH 6.0), each containing 5 mM CaCl ₂ , 25 μl AkAA (209 mg/ml) or AcAA (172 mg/ml), 25 μl pepsin (Sigma P7000, 10,000 units/ml) ml in water). The reaction was carried out at 40 ^° C. for 2 hours. At the end of the reaction, the supernatant obtained by centrifugation was filtered through a 0.45 μm filter and diluted 20 times. The diluted samples were analyzed for total reducing sugar as described in Example 6.

Figure 5 shows hydrolysis of cornmeal with AkAA and AcAA samples incubated at pH 3.2, 4.1, 4.7 and 6.0 in the absence or presence of pepsin. Amylolytic activity, measured as total reducing sugar release by AkAA and AcAA, was not significantly affected by the presence of pepsin compared to the absence of pepsin. This further indicates that these two amylases, which are 70% identical, are active under conditions comparable to those found in the ruminant digestive tract: part of the small intestine (pH 4.1–4.7) and conditions comparable to those found in the small intestine and rumen (both at or near pH 6.0). Thus, the AkAA and AcAA amylases appear to be more active at rumen pH than other amylases currently in use as feed additives for monogastric animals, such as the alpha-amylases from Bacillus amyloliquefaciens, B licheniformis, B. subtilis, Aspergillus niger and A. oryzae (Monteiro de Souza et al., Application of microbial alpha-amylase in industry - a review. Brazilian Journal of Microbiology (2010) 41: 850-861).

EXAMPLE 8

Activity of mixtures of endo-/exoglucan hydrolases and endoprotease in the presence of pancreatin

The reaction mixture contained 1.0 ml slurry of cornmeal (20% w/w in 0.1 M Mes, 5 Mm CaCl ₂ pH 6.0), 25 µl AkAA+TrGA+AFP enzyme mixture (223 mg/ml) or AcAA+CS4+AFP (125 mg/ml), 25 µl porcine pancreatin (Sigma P7545, 25 mg/ml prepared in 0.1% NaCl). The reaction was carried out at 40° C. for 2 hours. At the end of the reaction, the supernatant obtained by centrifugation was filtered through a 0.45 μm filter and diluted 20 times. The diluted samples were analyzed for total reducing sugar as described in Example 6. Figure 6 shows that after 2 hours incubation with cornmeal (20% w/w) at 40° C. at pH 6.0 in the presence of pancreatin the mixture of enzymes AkAA+TrGA+AFP and the mixture of enzymes AcAA+CS4+AFP increased the production of reducing sugar by 4.2- and 3.8-fold, respectively. This study demonstrates that mixtures of AkAA+TrGA+AFP and AcAA+CS4+AFP, preserved during passage through the rumen and abomasum, can act synergistically with pancreatic enzymes to promote sugar release.

Found that like AkAA (example 4), AcAA also acts synergistically with pancreatin. Table 3 shows that when dosing AcAA together with pancreatin, the reduction power generation (DNS-value) is increased by 1.6 times the sum of the reducing powers generated separately by the two enzymes at pH 6.0, and the corresponding value at pH 6 .7 increases by 1.25 times.

The reaction mixture contained 1 ml of 20% w/v. suspension of cornstarch in 0.1 M Mes (pH 6.0 and 6.7) containing 5 mM CaCl ₂ with and without 25 µl of porcine pancreatin (2.5% w/v) and 25 µl of AcAA (17 .2 mg/ml) at 40°C for 2 hours. The resulting reducing sugar was analyzed as described in example 6.

Table 3 . Synergistic effect of AcAA and pancreatin at pH 6.0 and 6.7

Treatment pH 6.0 pH 6.7 Control (no amylase or pancreatin) 1.16 1.00 Pancreatin only minus control 1.96 1.80 AcAA only minus control 1.24 1.06 AcAA dosed with pancreatin minus control 5.12 3.57

EXAMPLE 9

Reducing sugar releasing activity of mixtures of endo-, exoglucan hydrolases and endoprotease with AFP in the presence of pepsin at pH 2.5 relative to pH 6.0

Table 4 shows the activity of a mixture of AkAA, AcAA, TrGA, CS4 and AFP in the absence and presence of pepsin at pH 2.5 relative to pH 6.0. It can be seen that the activity at pH 2.5 is 20% higher than the activity at pH 6.0 for both enzyme mixtures under experimental conditions. It appears to be advantageous that the enzymes in enzyme mixtures are both stable and active under conditions comparable to those found in the stomach or abomasum of a ruminant, where lower pH values predominate.

The reaction mixture contained 1.0 ml of a suspension of cornmeal (10%, w/w) in 0.1 M glycine–HCl (pH 2.5) containing 5 mM CaCl ₂ or in 0.1 M Mes–NaOH (pH 6 ,0) containing 5 mM CaCl ₂ , 25 µl of the enzyme mixture AkAA+TrGA+AFP (223 mg/ml) or AcAA+CS4+AFP (125 mg/ml) and 25 µl of pepsin (10000 units/ml). The reaction was carried out at 40 ^° C. for 2 hours with shaking at 300 rpm. At the end of the reaction, the substrate was separated from the enzyme and products by centrifugation at 3500 rpm. within 5 min. using a 96-well filter plate with a 0.45 µm filter. The filtrate was diluted 2-fold and analyzed for the reducing sugar value as described in Example 6.

Table 4. Reducing sugar releasing activity of a mixture of endo-, exoglucan hydrolases and endoprotease including AkAA, AcAA, TrGA, CS4 and AFP in the presence and absence of pepsin at pH 2.5 vs. pH 6.0. Activity at pH 6.0 is considered as 100%

pH reactions Control Control
+pepsin AkAA+TrGA
+AFP AkAA+TrGA
+AFP+pepsin AcAA+CS4
+AFP AcAA+CS4
+AFP+pepsin pH 2.5 0.375 0.368 0.569 0.610 0.424 0.450 pH 6.0 0.390 0.395 0.547 0.587 0.624 0.612 pH 2.5 relative to pH 6.0, % 123 126 21 38

EXAMPLE 10

Hydrolysis of corn starch by glucoamylase under digestion conditions in the small intestine in the presence of pancreatin

The reaction mixture contained 0.5 ml slurry of corn starch (Sigma S4126) or slurry of corn flour (10% w/v) in 0.1 M Mes (pH 6.0), 50 μl of pancreatin (final concentration in the reaction mixture 0, 25%) and glucoamylase at a concentration of 400 ppm in a final volume of 0.56 ml. The reaction was carried out at 40 ^° C. for 1 hour with shaking at 300 rpm. At the end of the reaction, the supernatant was obtained by centrifugation separation from the starch granules using a benchtop centrifuge and analyzed for total reducing sugar by the DNS reagent method as described in Example 6, that is, 10 μl of the supernatant or diluted supernatant was mixed with 100 µl of DNS reagent in a 96-well PCR plate. The PCR plate was then incubated at 95°C for 5 min. followed by 5 min. cooling down to 20°C. 90 µl of the reaction mixture was transferred to a 96-well reading plate and the absorbance at 550 nm was measured as a DNS value.

Figure 7 with cornstarch as substrate and Figure 8 with cornmeal as substrate demonstrate that all five CS4, TrGA, Brew1, AfuGA and FvGA glucoamylases (see Table 1 for details) act synergistically with pancreatin to produce reducing agents. sugars, as indicated by the DNS values. Glucoamylases (GA) (1,4-alpha-D-glucanglucohydrolase (EC 3.2.1.3)) catalyze the hydrolysis of alpha-1,4- and alpha-1,6-glucosidic bonds to release beta-D-glucose from the non-reducing ends of starch and related poly- and oligosaccharides (Sauer et al. 2000. Glucoamylase: structure/function relationships, and protein engineering, Biochim. Biophys. Acta, 1543:275–293). Thus, unlike certain types of alpha-glucosidases, GA is also capable of hydrolyzing crude starch and the alpha-1,4- and alpha-1,6-glucosidic bonds found in alpha-glucans and their oligomers.

Figures 7 and 8 show that porcine pancreatin and all five GAs had a certain ability to hydrolyze cornstarch granules with the highest activity observed for FvGA at pH 6.0 and 40 ^° C. A significant synergistic effect was observed when the reaction the mixture contained both GA and porcine pancreatin. It is believed that this synergy may be due not only to the pancreatic alpha-amylase present in the pancreatin-based product purchased from Sigma, but also likely due to proteases also present in this pancreatin-based product, which can interact with any protein that can be present on the surface of the cornstarch granules as well as inside the granules.

This example shows that glucoamylases can act synergistically with pancreatic digestive enzymes to generate glucose from corn chemistries shown in Example 2. All five GAs used in this experiment and the three alpha-amylases AkAA, AcAA and AtAA used in examples 2–9 above have carbohydrate binding modules belonging to the family 20 (CBM20) (Christiansen et al., 2009. The carbohydrate–binding module family 20 – diversity, structure, and function, FEBS Journal, 276: 5006–5029; Janeceka et al., 2011. Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals (Enzyme and Microbial Technology, 49: 429–440). Such enzymes may have an additional carbohydrate-binding site near the carbohydrate-binding module (Sorimachi et al., 1997. Solution structure of the granular starch binding domain of Aspergillus niger glucoamylase bound to beta-cyclodextrin, Structure, 5:647-661). These carbohydrate-binding modules and carbohydrate-binding sites are likely to be necessary to provide activity against starch granules and maltosaccharides and thus contribute to the synergistic effect when combined with pancreatin.

EXAMPLE 11

Hydrolysis of cornstarch by glucoamylase under rumen-specific conditions

Table 5 shows that five glucoamylases have at least 80% residual activity after 4 hours of incubation in rumen sap in the absence of starch substrate, with the exception of AfuGA which has 65% residual activity. As far as feed enzyme for ruminants is concerned, it needs to be highly stable in rumen juice at 40 ^° C for 4 hours.

Thus, such enzymes show utility as ruminant feed enzymes as they are stable and active in rumen juice at 40 ^° C for 4 hours.

The pre-incubation mixture contained 0.25 ml cell-free rumen juice, 5 μl each of 5 glucoamylases at a concentration of 400 ppm. Pre-incubation was carried out at 40 ^o C for 4 hours with shaking at 300 rpm./min. in a 48-well plate. For controls, the preincubation time was zero. After pre-incubation, 0.25 ml of a slurry of corn starch (Sigma S4126) (10% w/v) in 0.1 M Mes (pH 6.0) was added and the reaction was carried out for 30 minutes. at 40 ^° C. with shaking. The supernatant was obtained by centrifugation separation from the starch granules using a benchtop centrifuge and analyzed for total reducing sugar by the DNS method as described in Example 10.

Table 5. Stability of five glucoamylases in the presence of rumen juice at 40 ^o C for 4 hours.

Glucoamylase Residual activity against corn starch (%) CS4 81 TrGA 98 Brew1 86 AfuGA 65 FvGA 80

EXAMPLE 12

Activity of five glucoamylases at pH 2.5 relative to pH 6.0 in the presence of pepsin

It appears to be advantageous for the enzymes to be both stable and active under conditions simulating those found in the stomach or abomasum of a ruminant, where lower pH values predominate. Activity at pH 2.5 should be quite high, enough to have a significant effect on digestibility in this low pH digestive chamber relative to activity in the rumen and small intestine at pH around 6 when using the diet based on starch. Table 6 shows that all five glucoamylases were more than 20% active at pH 2.5 compared to their activity at pH 6.0. Thus, a balanced activity in the rumen, abomasum and small intestine will increase the ability to digest starch nutrients.

The reaction mixture in a 48-well microplate contained 0.5 ml of a suspension of cornmeal (10% w/v) obtained in glycine-HCl (60 mM, pH 2.5) or in Mes-NaOH (100 mM, pH 6, 0), 50 µl porcine pepsin (final concentration 1000 U/ml) and 5 µl glucoamylase (final concentration 400 ppm). The reaction was carried out at 40 ^° C. for 60 minutes. The released reducing sugar was analyzed using the DNS reagent as described in Example 10. The activity at pH 6.0 was considered as 100%.

Table 6 . Activity coefficient of five glucoamylases at pH 2.5 and pH 6.0. Activity at pH 6.0 for each enzyme is considered as 100%

Glucoamylase pH 2.5 pH 6.0 pH 2.5 relative to pH 6.0, % CS4 0.497 0.793 63 TrGA 0.498 0.778 64 Brew1 0.491 0.578 85 AfuGA 0.663 0.715 93 FvGA 0.220 0.692 32

EXAMPLE 13

Release of glucose from corn starch by yeast alpha-glucosidase in the presence of pancreatin under conditions simulating conditions in the small intestine

The reaction mixture contained 0.5 ml slurry of corn starch (Sigma S4126) (10% w/v) suspended in 0.1 M Mes (pH 6.0) containing 5 mM CaCl ₂ , 0, 10, 20 or 35 µl yeast alpha-glucosidase (which can also be referred to as maltase, purchased from Megazyme, E-MALTS, 1000 U/ml), 0–25 µl porcine pancreatin (2.5% w/v in 1% NaCl, purchased from Sigma cat. no. P7545) and 0-60 µl of water for a total volume of 0.56 ml. The reaction mixtures were incubated in a shaking incubator at 40°C for 60 min. in a 48-well plate. The plate was centrifuged at 3500 rpm. within 10 min. using a tabletop centrifuge to separate undigested starch granules. The resulting supernatant was transferred to a 96-well glucose assay plate using a glucose oxidase/peroxidase glucose assay kit (GOPOD).

For the GOPOD glucose assay, 8 µl of the supernatant was transferred to a 96-well readout plate containing 240 µl of the GOPOD reagent from Megazyme (K-GLUC 09/14). The plate was incubated at 50°C for 20 min. and absorbance at 510 nm was read according to instructions from Megazyme.

It is well known that the pancreatin-based product (Sigma P7545) contains pancreatic alpha-amylase, lipase, ribonuclease and several proteases, including trypsin, chymotrypsin, elastase. The hydrolysis products of food starch by porcine and bovine alpha-amylases are mainly maltose (Banks et al., 1976. The action pattern of bovine pancreatic alpha-amylase. Intl J. Biochem., 7: 107-110). Animals, including monogastric and ruminant animals, are known to hydrolyze maltose via a small intestinal membrane-bound enzyme (Nichols et al., 1998. Human Small Intestinal Maltase–glucoamylase cDNA Cloning, homology to sucrase–isomaltase, J. Biol. Chem. 273:3076-3081). A free-moving enzyme is characterized by a higher catalytic efficiency than a membrane-bound (immobilized) enzyme.

Figure 9 shows that, without pancreatin, yeast alpha-glucosidase, dosed from 0, 10, 20 to 35 µl, did not have the ability to increase the release of glucose from cornstarch granules.

However, in the presence of 2.5 µl of pancreatin, glucose was released and the addition of 10 µl of alpha-glucosidase doubled the amount of glucose released. The output of glucose increased further with increasing dose of alpha-glucosidase. This indicates that yeast alpha-glucosidase acted with enzymes present in pancreatin in a synergistic manner to release glucose from cornstarch. It is believed that the proteases present in pancreatin may contribute to some extent to this synergy, since the protease can hydrolyze proteins present on the surface of the starch granules as well as proteins present within the granule. Such proteins, found on the surface and within starch granules, can adversely affect starch digestion (McAllister et al., 1993. Effect of the protein matrix on the digestion of cereal grains by ruminal microorganisms. J. Anim. sci. 71:205-212).

The alpha-glucosidase used in Example 13 is a highly purified form from Saccharomyces cerevisiae purchased from Megazyme International Ireland (EC 3.2.1.20, CAZy family: GH13, protein database positions: P53341, P38158, CAS: 9001–42– 7). This example demonstrates that the free form of microbial alpha-glucosidase has significant synergy with pancreatic digestive enzymes. However, this yeast alpha-glucosidase has been found to be unstable and inactive under conditions comparable to those found in the stomach and abomasum of a ruminant. It is believed that additional manipulations, such as coating of this yeast alpha-glucosidase or protein engineering, may be required to render it stable at such low pH values in the presence of pepsin to be able to function in the ruminant small intestine.

EXAMPLE 14

Release of glucose from cornstarch by fungal alpha-glucosidase in the presence of pancreatin under conditions simulating those of the small intestine

The three fungal alpha-glucosidases TG L-2000, Aclglu1 and Nfiglu1 (Table 1) were tested for stability and activity in the presence of pepsin, pancreatin and rumen juice (Tables 7.1, 7.2 and 7.3 below). Tables 7.1-7.3 show that all three alpha-glucosidases acted synergistically with pancreatin to increase glucose release from corn starch.

The study of the synergistic effect with pancreatin was carried out as follows: 0.5 ml suspension of corn starch (Sigma S4126) (10% w/v) in 0.1 M Mes (pH 6.0) containing 5 mm CaCl ₂ was incubated with 0 or 2.5 µl alpha-glucosidase TG L-2000 (3.1 mg/ml) and 0 or 2.5 µl porcine pancreatin (Sigma P7545, 2.5% w/v in 1% NaCl). The reaction was carried out at 40 ^° C. for 1 hour. The reaction mixture was then filtered through a 0.22 μm filter. The released glucose in the filtrate was analyzed using the Glucose Assay Kit from Megazyme (GOPOD) (K-GLUC 09/14) (as described in Example 13 above) and analyzed as follows: 8 µl of the filtrate was mixed with 0.24 ml of the GOPOD reagent in a 96-well plate and read at 510 nm after incubation at 50 ^° C for 20 minutes. Units of OD, measured at 510 nm, were used as a measure of glucose release.

A synergistic study with pepsin and pancreatin (Pan) was performed as follows: the pre-incubation mixture contained 0.1 ml glycine-HCl (pH 3.0), 5 mM CaCl2, 25 µl porcine pepsin (Sigma P7000, 5000 U/ml ) and 2.5 μl TG L-2000. This mixture was incubated at 4 ^° C. for 2 hours. 2.5%) was added and further incubated at 40 ^° C. for 1 hour. Released glucose assay was performed as described above in accordance with the pancreatin assay using the Megazyme Glucose Assay Kit.

The study of stability and interaction with ruminal starch hydrolase was carried out as follows: 2.5 μl of alpha-glucosidase TG L-2000 with 0.24 ml of cell-free rumen juice and incubation at 40 ^o C for 4 hours. At the end of incubation, 0.25 ml of cornstarch (10% w/v) in 0.1M acetate (pH 4.5) and further incubated at 40 ^° C for 30 minutes. Released glucose was analyzed using the Megazyme Glucose Assay Kit as described above for the pancreatin assay.

Alpha-glucosidase and porcine pancreatin, used alone, provided a limited release of glucose from cornstarch (Table 7.1). The combination of alpha-glucosidase TG L-2000 and pancreatin provided glucose release in an amount 5.2 times higher than the sum of the individual glucose release rates for each of the two enzyme preparations (table 7.1). Similarly, when Aclglu1 and Nfiglu1 were combined with pancreatin under the same conditions, the increase in glucose release was 8.3-fold (Table 7.2) and 7.8-fold (Table 7.3) compared to the sum of the individual release rates for Aclglu1 and pancreatin or for Nfiglu1 and pancreatin, respectively.

The pepsin stability of the three alpha-glucosidases was tested at pH 3.0 in the presence of pepsin (Table 7.1). It seems clear that the combination of 2.5 µl alpha-glucosidase TG L-2000 and pancreatin increased the release of glucose 7.3-fold even after pre-incubation with pepsin in the absence of substrate at pH 3.0 and 40 ^o C for 2 hours. (table 7.1). For alpha-glucosidases Aclglu1 and Nfiglu1 tested under the same conditions, the increase in glucose release by the combination was 4.1-fold (Table 7.2) and 6.9-fold (Table 7.3). All three alpha-glucosidases showed pepsin stability because, despite preincubation with pepsin, each still showed a synergistic effect when combined with pancreatin in the assay mixture.

Similarly, the synergistic effect and stability of the three alpha-glucosidases in rumen juice were evaluated (Tables 7.1–7.3). Table 7.1 shows that for alpha-glucosidase TG L-2000 added to rumen juice, glucose release increased 2.0-fold compared to the total amount of glucose released separately by alpha-glucosidase TG L-2000 and rumen juice and with pre-incubation in rumen juice in the presence of TG L-2000, glucose release also increased 2.0-fold compared to the release value in the absence of alpha-glucosidase (Table 7.1), meaning that this alpha-glucosidase TG L-2000 is stable in rumen juice . As regards the alpha-glucosidases Aclglu1 and Nfiglu1, their presence resulted in a 2.0- and 2.8-fold increase in glucose release, respectively, for the "treatments without pre-incubation and with pre-incubation" (table 7.2-7.3) with rumen juice.

Table 7.1 . Synergistic effect of alpha-glucosidase TG L-2000 from Aspergillus niger with pancreatin (Pan), amylolytic activity in rumen juice and stability in the presence of pepsin in the release of glucose (G1) from corn starch. The number of repetitions (n) was 2–4

Without Pan With Pan With Pan Pepsin+Pan with incubation at 40 ^o C within 2 hours. Pepsin +
Pan with incubation at 40 ^o C within 2 hours. No pre-incubation with rumen juice No pre-incubation with rumen juice With pre-incubation with rumen juice at 40 ^o C within 4 hours. With pre-incubation with rumen juice at 40 ^o C within 4 hours. TG L–2000 + - + - + - + - + OD510 nm 0.16 0.086 1.29 1.175 8.544 0.494 0.885 0.982 1.822 Standard deviation 0.005 0.001 0.014 0.145 0.276 0.199 0.05 0.117 0.11 The fold increase in G1 release due to the addition of TG L-2000 5.2 7.3 2.0 2.0 n 2 4 2 4 2 4 2 4 2

Table 7.2 . Synergistic effect of alpha-glucosidase from Aspergillus clavatus (Aclglu1) with pancreatin (Pan), amylolytic activity in rumen juice and stability in the presence of pepsin in the release of glucose (G1) from corn starch.

The reaction and assay conditions were the same as for Table 7.1, except that 0 or 50 μg Aclglu1 alpha-glucosidase (Table 1) from A. clavatus was used. The number of repetitions (n) was 2–4

Without Pan With Pan With Pan Pepsin+Pan with incubation at 40 ^o C within 2 hours. Pepsin+Pan with incubation at 40 ^o C within 2 hours. No pre-incubation with rumen juice No pre-incubation with rumen juice With pre-incubation with rumen juice at 40 ^o C within 4 hours. With pre-incubation with rumen juice at 40 ^o C within 4 hours. Aclglu1 + - + - + - + - + OD510 nm 0.06 0.086 1.22 1.175 4.845 0.494 0.75 0.982 2.05 Standard deviation 0.003 0.001 0.013 0.145 0.52 0.199 0.03 0.117 0.08 The fold increase in G1 release due to the addition of Aclglu1 8.3 4.1 2.0 2.8 n 2 4 2 4 2 4 2 4 2

Table 7.3 . Synergistic effect of alpha-glucosidase from Neosartorya fischeri (Nfiglu1) with pancreatin (Pan), amylolytic activity in rumen juice and stability in the presence of pepsin in the release of glucose (G1) from corn starch.

The reaction and assay conditions were the same as described above in relation to Table 7.1, except that 0 or 50 μg Nfiglu1 alpha-glucosidase from N. fischeri was used. The number of repetitions (n) was 2–4

Without Pan With Pan With Pan Pepsin+Pan with incubation at 40 ^o C within 2 hours. Pepsin+Pan with incubation at 40 ^o C within 2 hours. No pre-incubation with rumen juice No pre-incubation with rumen juice With pre-incubation with rumen juice at 40 ^o C within 4 hours. With pre-incubation with rumen juice at 40 ^o C within 4 hours. Nfiglu1 + - + - + - + - + OD510 nm 0.08 0.086 1.29 1.175 8.12 0.494 0.73 0.982 2.06 Standard deviation 0.002 0.001 0.077 0.145 0.13 0.199 0.01 0.117 0.00 The fold increase in G1 release due to the addition of Nfiglu1 7.8 6.9 2.0 2.8 n 2 4 2 4 2 4 2 4 2

EXAMPLE 15

Stability of fungal alpha-glucosidase in rumen juice and interaction with pancreatin during glucose release

The pre-incubation mixture contained 50 µg each of fungal alpha-glucosidases TG L-2000, Aclglu1, Nfiglu1, TauSec098 and TauSec099 (Table 1) dissolved in 2.5 µl and 240 µl cell-free rumen juice mixed in wells of a 24-well plate. . Alpha-glucosidase was not added to the control sample. The mixtures were incubated at ₄₀ °C for 4 h. .5 μl of pancreatin (Sigma P7545 in 0.1% NaCl, 2.5% w/v) and the reaction was carried out at 40° C. for 30 min. (n=4). The resulting supernatant after centrifugation was diluted 10-fold and the released glucose was quantified as described in Example 13 using the Megazyme Glucose Assay Kit.

Figure 10 shows that pre-incubation of five alpha-glucosidases in rumen juice at 40° C. for 4 hours did not cause loss of activity compared to "no pre-incubation" treatment. It appears that more glucose was released in pre-incubated samples than was released in non-pre-incubated samples (Figure 10). This likely indicates that these alpha-glucosidases interacted with starch hydrolases present in rumen sap that contained a residual level of starch substrate.

Example 16

Synergistic effect of five alpha-glucosidases with pancreatin in the release of glucose from corn starch at pH 6.0

The reaction mixture contained 0.5 ml slurry of corn starch (Sigma S4126) (10% w/v) in 0.1 M Mes (pH 6.0) containing 5 mM CaCl ₂ , 50 µg alpha-glucosidase in 2.5 µl and 0 or 2.5 µl pancreatin solution (Sigma P7545, 2.5% w/v in 0.1% NaCl). Alpha-glucosidases were not added to the control sample. The reaction was carried out at 40°C for 60 min. (n=4) and released glucose was quantified using the glucose assay kit described in Example 13.

Figure 11 shows that the production of glucose for control using pancreatin alone and without additional enzyme was limited (0.67 mg/ml in the reaction mixture). With alpha-glucosidase alone, glucose release was low, except for TG L-2000, which showed a glucose release of 0.80 mg/mL in the reaction mixture. With the combined use of alpha-glucosidase and pancreatin, glucose release increased in the range of 11-20-fold increase, depending on the amount of alpha-glucosidase used. This illustrates the synergistic effect on glucose release from corn starch that can be obtained with any of the five alpha-glucosidases and pancreatin.

Example 17

Activity of alpha-glucosidases treated with pepsin at pH 2.5 and pH 6.0

The reaction mixture contained 50 μg of five alpha-glucosidases TG L-2000, Aclglu1, Nfiglu1, TauSec098 and TauSec099 (Table 1) in 2.5 μl, 75 μl of pepsin (Sigma P7000, stock solution 5000 units/ml) and 1.5 ml maltose (21.5 mg/ml) in 0.1 M glycine-HCl containing 5 mM CaCl ₂ pH 2.5 or 0.1 M Mes-NaOH containing 5 mM CaCl ₂ pH 6, 0. The reaction was carried out at 40°C for 30 min. At the end of the reaction, the reaction mixture was then diluted 10-fold and analyzed for glucose release using the Megazyme Glucose Assay Kit as described in Example 13 above.

Table 8. Ratio of activity at pH 2.5 and pH 6.0 for five alpha-glucosidases. Activity at pH 6.0 is considered as 100%

Glucosidases Activity at pH 2.5 vs. pH 6.0 (%) TG L–2000 138 Aclglu1 107 Nfiglu1 84 TauSec098 120 TauSec099 167

Table 8 shows that all four glucosidases were more active at pH 2.5 than at pH 6.0 in the presence of pepsin, except for Nfiglu1, which was 84% active at pH 2.5 compared to pH 6.0. Thus, such enzymes show utility as ruminant feed enzymes as they are stable and active in rumen juice at 40 ^° C for 4 hours.

Example 18

The use of glucoamylases in combination with P14L and P7L metallopeptidases to increase dry matter digestion and gas formation during rumen corn fermentation

Effects of 4 glucoamylases (CS4, TrGA, AfuGA, FvGA) and two metallopeptidases/proteases (P14L, P7L) and their combinations on dry matter digestion (DMD) and gas production at 0, 3, 7, 9, 12 and 24 h incubation /fermentations were evaluated as described below. Both glucoamylases and proteases were used at a concentration of 0.25 mg/g.

The effects of 4 glucoamylases (CS4, TrGA, AfuGA, FvGA) and two metallopeptidases/proteases (P14L, P7L) and combinations thereof were evaluated on rumen microbial fermentation using dent corn as a substrate for in vitro batch culture of rumen sap. The experiments were based on a protocol established and published previously (Adesogan, AT, Krueger, NA, and Kim, SC 2005. Anim. Feed Sci. Technol. 123: 211-223). The dent corn used in this in vitro batch fermentation system contained 88.8% dry matter (DM), 9.3% crude protein (CP), 3.2% acid detergent fiber (ADF), 8.3% neutral -Amylase treated detergent fiber (aNDF), 76.9% non-fibrous carbohydrates (NFC), 88.0% total digestible nutrients (TDN). It was crushed to 4 mm. All enzymes were applied at 0.25 mg enzyme protein per gram dented corn in three separate runs with 4 replicates per treatment and fermentation or incubation continued for 0, 3, 7, 9, 12 and 24 hours at 39 ^° C Crushed dent corn was weighed 6 replicates per series in F57 filter bags (ANKOM Technology, Macedon, NY). Enzymes were diluted in 0.1 M citrate-phosphate buffer (pH 6.0) before being placed into vials and added to 0.5 g ground dent corn in F57 bags (Krueger, NA, and AT Adesogan. 2008. Anim. Feed Sci. Tech. 145: 84–94 Goering, HK and PJ Van Soest 1970 Agric Handbook No 379 ARS USDA Washington DC pp 20 The F57 filter bags were sealed using a Uline table top plastic bag sealer (Impulse® type AIE-200), then immediately placed into the fermentation vessels, which were 160 ml rubber stoppered vials. Also provided were empty vials containing only the filter bag, as well as controls containing only ground dent corn without enzyme. Rumen juice was representatively sampled from three lactating Holstein dairy cows with rumen cannulation 2–3 hours after complete ration (TMR) consumption. The ingredient composition of TMR fed to fistula dairy cows was based on a dry matter content of 38.2% corn silage, 27.3% ground corn, 14.5% soy flour with 44% crude protein, 9.1 % citrus pulp, 4.5% open feed premix, 4.0% mid-bloom alfalfa hay, 1.8% energy booster (MS Specialty Nutrition, Dundee, IL), 0.5% Novasil ( BASF, Germany), adding up to 100%.

The collected rumen juice was filtered through four layers of cheesecloth before being mixed with pre-warmed artificial saliva (39 ^° C.). The composition of the artificial saliva included a micromineral solution of CaCl ₂ ×2H ₂ O, MnCl ₂ ×4H ₂ O, CoCl ₂ ×6H ₂ O, FeCl ₂ ×6H ₂ O; macromineral solution of Na ₂ HPO ₄ × 12H ₂ O, KH ₂ PO ₄ , MgSO ₄ × 7H ₂ O; buffer solution (NH ₄ ) ₂ HCO ₃ and NaHCO ₃ ; trypticase-peptone solution (tryptone, Sigma-Aldrich, St. Louis, MO, USA); redox indicator resazurin and reducing solution containing cysteine-HCl, 1 M NaOH, Na ₂ S×9H ₂ O, and distilled water. The volume ratio between cicatricial juice and artificial saliva was 1:2. Buffered rumen juice (52 ml) was added to 160 ml rubber stoppered vials containing filter bags, and the vials were closed with rubber stoppers and sealed with aluminum seals. The flasks were incubated for 0, 3, 7, 9, 12 and 24 hours at 39 ^° C. in a forced ventilation incubator. At the end of the incubation, the filter bags containing the residues were dried in an oven at 60 ^° C. for 48 hours and weighed to quantify the DMD. The gas pressure inside the flasks was measured using a pressure transducer and converted a posteriori to gas volume at 0, 3, 7, 9, 12, and 24 hours of incubation. The following formula was used to convert gas pressure to gas volume:

gas volume (ml) = (gas pressure (psi) * 4.8843) + 3.1296.

The equation was derived from experimental conditions using 160 ml rubber stoppered vials and applied to all experiments in Tables 9.1-9.2.

Statistical analysis. For all experiments in Example 18, collected data was analyzed using the GLIMMIX SAS procedure (version 9.1; SAS Institute, Cary, NC). The experiments were designed as a completely randomized block design, where each series was considered as a block. The dose was used as a fixed effect in the model when enzymes were tested at various doses. The response variable included DMD and in-vitro gassing. The series was treated as a random factor. Model fixed effects included sampling time in relation to fermentation parameters measured at different hours after incubation. The UNIVARIATE SAS procedure was used to test the residuals for outliers and normality before final analyses. Treatment effects were considered statistically significant at P < 0.05, while any trends were determined at 0.05 ≤ P ≤ 0.10.

Results of evaluation of four glucoamylases (CS4, TrGA, AfuGA, FvGA) and two metallopeptidases/proteases (P14L, P7L) and their combinations in terms of dry matter digestion (DMD) and gas production at 0, 3, 7, 9, 12 and 24 hours. incubation/fermentation can be found in tables 9.1.1 to 9.1.4. Both glucoamylases and proteases were used at a concentration of 0.25 mg/g.

CS4+/–P14L processing. No significant effects were observed on DMD in response to the addition of CS4 at 0, 3 and 7 hours of incubation or fermentation (Table 9.1.1). However, DMD improved by 56, 36, and 25% at 9, 12, and 24 hours of incubation, respectively ( P < 0.05). Similarly, P14L protease improved DMD by 79, 59, and 32% at 9, 12, and 24 hours of incubation, respectively ( P < 0.05). The combination of CS4 and P14L improved DMD by 109, 86, and 45% at 9, 12, and 24 hours of incubation, respectively ( P < 0.05).

TrGA+/–P14L DMD treatment increased by 62% and 79% with TrGA and P14L at 9 h incubation, respectively (Table 9.1.2). The combination of both enzymes at 9 hours of incubation increased DMD to 124% compared to control ( P < 0.01).

Treatment with AfuGA+/–P14L The effects of the combination of AfuGA and P14L on rumen fermentation parameters are presented in Table 9.1.3. DMD increased at 7, 9, 12 and 24 hours of incubation with AfuGA, P14L and the combination ( P < 0.05). The effects of both enzymes in combination are evident as it increased DMD by 119, 146, 108 and 60% at 7, 9, 12 and 24 hours of incubation, respectively, and the magnitude of the effects was greater than that provided by the enzymes alone ( P < 0.05).

Treatment FvGA+/–P14L The effects of the combination of FvGA and P14L on rumen fermentation parameters are presented in Table 9.1.4. No effects were observed on DMD in response to the addition of FvGA at 0, 3 and 7 hours of incubation; however, DMD improved by 42, 79 and 93% at 9 h incubation (P < 0.01) with FvGA, P14L and their combination, respectively.

All four glucoamylases, metallopeptidase P14L, and combinations thereof were found to increase fermentation gas production significantly at at least 12 and 24 hours of incubation ( P < 0.05) (Tables 9.1.1–9.14).

Treatment CS4, TrGA, AfuGA, FvGA +/– P7L The effects of the combination of 4 glucoamylases (CS4, TrGA, AfuGA, FvGA) with metallopeptidase/protease P7L on DMD at 0, 3, 7, 9, 12 and 24 h incubation/fermentation are shown in tables 9.2.1–9.2.4. Similar to Tables 9.1.1-9.1.4, both glucoamylase and protease were used at a concentration of 0.25 mg/g.

Based on the observed results, synergistic or additive effects of 4 glucoamylases in combination with P7L are observed regardless of incubation time. Additional effects with the combination of CS4 and P7L provided 49%, 112%, 72%, 60%, 55%, and 19% improvement in DMD at 0, 3, 7, 9, 12, and 24 h of incubation, respectively (Table 9.2.1; P < 0.01 ). DMD also increased by 41, 109, 73, 64, 60, and 25% at 0, 3, 7, 9, 12, and 24 hours, respectively, with the combination of TrGA and P7L (Table 9.2.2; P < 0.01).

The combination of AfuGA (Table 9.2.3) and P7L improved DMD by 23, 116, 85, 66, 58, and 21% at 0, 3, 7, 9, 12, and 24 hours, respectively ( P < 0.01). Despite the efficacy of FvGA in combination with P7L in improving DMD at 3, 7, 9 and 12 hours of incubation, no effects were observed at 0 and 24 hours compared to controls (Table 9.2.4). Improvement in DMD was further evidenced by the higher total gas volume observed at most of the incubation time points for all 4 glucoamylases, P7L protease, and combinations thereof ( P < 0.05) (Tables 9.2.1–9.2.4).

Table 9.1. 1. Effects of CS4 and protease (P14L) and their combinations on dry matter digestibility and gas production in vitro. ^a–c Row means with different superscripts differ ( P < 0.05)

Time, h. Control CS4 P14L Combination SE P-value Digestibility of dry matter, % 0 7.2 8.64 9.97 9.5 2.16 0.81 3 11.95 15.7 22.21 23.49 4.42 0.28 7 13.48 18.46 19.35 24.16 2.76 0.13 9 16.07 ^{s 25.15b 28.81ab 33.71a} 1.60 <0.01 12 ^{23.63b 32.27ab 37.47ab 44.04a} 4.32 0.05 24 ^{42b 52.59ab 55.49a 61.04a} 4.04 0.05 Gas volume, ml 0 2.79 3.0 2.84 2.95 0.59 0.99 3 9.72 9.92 10.81 10.69 0.63 0.55 7 19.5 19.69 21.82 22.85 1.67 0.45 9 30.75 33.69 32.04 38.93 2.42 0.16 12 ^{47.87b 53.87ab 50.29b 62.54a} 3.13 0.04 24 75.49 ^{s 91.12ab 79.4bc 99.7a} 4.69 0.02

Table 9.1. 2. Effects of TrGA and protease (P14L) and their combinations on dry matter digestibility and gas production in vitro. ^a–c Row means with different superscripts differ (P < 0.05)

Time, h. Control TrGA P14L Combination SE P-value Digestibility of dry matter, % 0 7.2 8.23 9.97 10.22 1.71 0.57 3 11.95 16.65 22.20 25.81 8.02 0.17 7 13.48 20.65 19.35 26.11 3.28 0.13 9 16.07 ^{s 26.11b 28.81b 36.01a} 1.54 <0.01 12 ^{23.62b 34.11ab 37.47a 44.05a} 4.14 0.05 24 ^{42b 56.75a 55.49ab 63.63a} 4.17 0.04 Gas volume, ml 0 2.79 3.09 2.84 3.01 0.51 0.97 3 9.72 10.08 10.82 12.31 0.87 0.23 7 19.5 20.7 21.82 23.56 1.98 0.54 9 30.75 35.57 32.05 41.69 2.70 0.08 12 ^{47.87b 56.28ab 20.29b 65.99a} 3.42 0.02 24 ^{75.49b 97.24a 79.41b 106.26a} 5.09 <0.01

Table 9.1. 3. Effects of AfuGA and protease (P14L) and their combinations on dry matter digestibility and gas production in vitro. ^a–c Row means with different superscripts differ (P < 0.05)

Time, h. Control AfuGA P14L Combination SE P-value Digestibility of dry matter, % 0 7.2 10.19 9.97 11.94 2.02 0.46 3 11.95 18.48 22.20 26.42 4.65 0.23 7 ^{13.48b 23.26ab} 19.35 ^a.b. 3.17 0.04 9 16.07 ^{s 30.73b 28.81b} 1.56 <0.01 12 ^{23.62b 37.18ab 37.47ab} 5.04 0.04 24 ^42b 3.64 <0.01 Gas volume, ml 0 2.78 2.83 2.84 2.96 0.50 0.99 3 9.72 10.47 10.83 13.19 0.80 0.07 7 19.5 21.12 21.83 28.76 1.52 0.01 9 ^{30.74b 37.62b 32.06b 48.37a} 2.40 <0.01 12 ^{47.87s 59.98b} 50.30 ^{s 75.11a} 2.60 <0.01 24 75.49 ^{s 102.68b} 79.45 ^{s 121.87a} 3.37 <0.01

Table 9.1. 4. Effects of FvGA and protease (P14L) and their combinations on dry matter digestibility and gas production in vitro. ^a–c Row means with different superscripts differ (P < 0.05)

Time, h. Control FvGA P14L Combination SE P-value Digestibility of dry matter, % 0 7.2 7.09 9.97 9.10 2.54 0.81 3 11.95 15.17 22.22 24.28 4.13 0.20 7 13.48 16.47 19.35 21.36 2.55 0.22 9 16.07 ^{s 22.74b 28.81ab} 1.99 <0.01 12 23.63 29.42 37.47 37.42 5.0 0.17 24 42 44.89 55.49 56.47 3.74 0.06 Gas volume, ml 0 2.79 2.99 2.84 3.03 0.46 0.98 3 9.72 10.54 10.82 11.93 0.64 0.19 7 ^{19.5b 21.77ab 21.82ab 25.42a} 1.22 0.05 9 ^{30.75b 35.77ab 32.05b 41.47a} 2.09 0.03 12 ^{47.87b 54.39b 50.30b 62.26a} 2.21 <0.01 24 ^{75.49b 85.68b 79.45b 96.57a} 3.19 <0.01

Table 9.2. 1 Effects of CS4 and protease (P7L) and their combinations on dry matter digestibility and gas production in vitro. ^a–c Row means with different superscripts differ (P < 0.05)

Time, h. Control CS4 P7L Combination SE P-value Digestibility of dry matter, % 0 ^9.21b 0.77 <0.01 3 ^14.7d 17.8 ^{s 24.2b} 1.01 <0.01 7 25.0 ^{s 29.7b} 1.43 <0.01 9 ^{30.3d 36.0s 44.7b} 1.37 <0.01 12 31.9 ^{s 40.2b 43.8b} 1.86 <0.01 24 ^{52.5b 56.8b 57.7ab} 1.83 <0.01 Gas volume, ml 0 2.98 3.64 3.24 3.39 0.23 0.23 3 ^{6.50b 6.45b} 0.54 <0.01 7 ^11.27d 13.31 ^{s 14.50b} 0.34 <0.01 9 14.46 ^{s 20.82b 19.67b} 0.98 <0.01 12 13.59 ^{s 20.12b 21.02b} 0.52 <0.01 24 ^{31.88b 31.42b} 1.25 <0.01

Table 9.2. 2. Effects of TrGA and protease (P7L) and their combinations on dry matter digestibility and gas production in vitro. ^a–c Row means with different superscripts differ (P < 0.05)

Time, h. Control TrGA P7L Combination SE P-value Digestibility of dry matter, % 0 9.21 ^{s 10.8b 11.1b} 0.45 <0.01 3 ^{14.7d 18.0s 24.2b} 1.04 <0.01 7 25.0 ^{s 28.9b} 1.33 <0.01 9 30.3 ^s 36.5 ^{s 44.7b} 1.06 <0.01 12 31.9 ^{s 41.0b 43.8b} 1.66 <0.01 24 52.5 ^{s 58.8b 57.7b} 1.76 <0.01 Gas volume, ml 0 2.98 3.53 3.24 3.53 0.22 0.24 3 ^{6.50b 6.82b} 0.47 <0.01 7 ^11.27d 13.03 ^{s 14.26b} 0.47 <0.01 9 14.46 ^{s 22.03ab 19.67b} 1.13 <0.01 12 13.59 ^{s 20.33b 21.02b} 0.60 <0.01 24 31.88 ^{s 41.60b} 31.42 ^s 1.45 <0.01

Table 9.2. 3. Effects of AfuGA and protease (P7L) and their combinations on dry matter digestibility and gas production in vitro. ^a–c Row means with different superscripts differ (P < 0.05)

Time, h. Control AfuGA P7L Combination SE P-value Digestibility of dry matter, % 0 ^9.21b 0.55 <0.01 3 ^14.7d 20.1 ^{s 24.2b} 0.92 <0.01 7 ^25.0d 33.5 ^{s 40.2b} 1.45 <0.01 9 ^30.2d 38.5 ^{s 44.7b} 1.00 <0.01 12 31.9 ^{s 42.0b 43.8b} 1.81 <0.01 24 52.5 ^{s 60.4ab 57.7bc} 1.90 <0.01 Gas volume, ml 0 2.98 3.85 3.24 3.24 0.25 0.12 3 6.50 ^{s 8.35b 8.84b} 0.53 <0.01 7 11.27 ^{s 15.05b 14.50b} 0.35 <0.01 9 ^14.46b 1.18 0.02 12 13.59 ^{s 22.55b 21.02b} 0.65 <0.01 24 ^{31.88b 31.42b} 1.29 <0.01

Table 9.2. 4. Effects of FvGA and protease (P7L) and their combinations on dry matter digestibility and gas production in vitro. ^a–c Row means with different superscripts differ (P < 0.05)

Time, h. Control FvGA P7L Combination SE P-value Digestibility of dry matter, % 0 9.20 9.85 11.1 10.7 0.51 0.05 3 14.7 ^{s 18.0b} 1.09 <0.01 7 ^{25.1b 28.6b} 1.48 <0.01 9 30.3 ^{s 33.7b 44.7b 45.9b} 1.10 <0.01 12 ^{31.9b 34.9b} 1.99 <0.01 24 ^{52.5ab 50.2b} 1.93 0.03 Gas volume, ml 0 2.98 3.64 3.24 3.8 0.35 0.35 3 6.50 ^{s 8.34b 8.84ab} 0.49 <0.01 7 11.27 ^{s 13.46b 14.50b} 0.39 <0.01 9 ^14.46b 0.94 0.16 12 13.59 ^{s 19.23b} 0.60 <0.01 24 ^{31.88b 33.94ab 31.42b} 1.31 0.041

Example 19

Hydrolysis of maltose by glucoamylases

As a rule, it is glucose that is absorbed in animals. Maltose is a disaccharide formed from two glucose units. Thus, maltose needs to be hydrolyzed to glucose monomers in order to be absorbed. For glucoamylase, it is advantageous to have high catalytic activity with respect to the maltose substrate, in addition to other preferred properties for use as an enzyme feed additive. Preferably, the glucoamylase should have at least 20% activity against the maltose substrate compared to TrGA activity under the test conditions described herein.

The activity of glucoamylase against maltose was determined as follows. A reaction mixture was obtained that contained 2% (w/v) or 8% (w/v) maltose (M-5885, Sigma) and 10 μg of glucoamylase in 50 mM Mes-NaOH (pH 6.0) containing 5 mM CaCl ₂ , with a final volume of 0.05 ml in a 96-well microplate. Each reaction of glucoamylase with maltose was repeated three times.

The reaction was carried out at 40 ^o C for 20 minutes, which was in a linear range with respect to the release of glucose. The reaction was carried out in a PCR machine at 95 ^o C for 5 min. After cooling to room temperature (23 ^o C), 10 µl of the reaction mixture, which was diluted to a concentration in the range from 0.5 to 0.9 mg glucose per ml, was mixed with 0.24 ml of the GOPOD reagent (D-glucose assay kit GOPOD Format from Megazyme) in a microplate. The mixture was then incubated at 50 ^° C. for 20 minutes. and the absorbance was read at 510 nm. The manufacturer's glucose standard was used to generate a standard curve for calibration.

The data in Table 10 show that all of the glucoamylases tested had maltose activity under conditions similar or close to those found in vivo , such as near neutral pH of 6.0.

It was noted that AnGA was an exception to the maltose substrate under these test conditions. Thus, AnGA is not a suitable glucoamylase for use as described herein.

Table 10 . Hydrolysis of maltose by four glucoamylases at two maltose concentrations and pH 6.0

Treatment 2% maltose (w/v) 8% maltose (w/v) TrGA* 100 100 CS4 44 44 AfuGA 41 33 AnGA 15 12 Control (minus enzyme) 0.2 2

*TrGA activity against maltose was considered as 100%.

Claims (8)

1. The use of an enzyme-based composition to increase starch digestibility and glucose yield in a ruminant, said composition comprising the enzyme glucoamylase EC 3.2.1.3 derived from Trichoderma reesei or Aspergillus fumigatus, wherein (a) said glucoamylase has at least 20% activity at a pH less than or equal to 3 in the presence of pepsin compared to the activity of glucoamylase at pH 6 in the presence of pepsin, (b) said glucoamylase is active in at least two of the three digestive chambers of the ruminant, including the rumen, abomasum, and small intestine, and (c) glucoamylase acts with digestive enzymes present in the ruminant's digestive chambers to increase starch digestibility and glucose output. 2. Use according to claim 1, wherein at least one glucoamylase is a Trichoderma reesei glucoamylase. 3. Use according to claim 1 or 2, wherein said composition further comprises at least one alpha-amylase. 4. Use according to claim 3, wherein the alpha-amylase is a member of the GH 13 family or is selected from the group consisting of alpha-amylases from EC 3.2.1.1. 5. Use according to claim 4, wherein at least one alpha-amylase is Aspergillus kawachii alpha-amylase, Aspergillus clavatus alpha-amylase, or Aspergillus terreus alpha-amylase, or Trichoderma reesei alpha-amylase. 6. Application according to any one of paragraphs. 1-5, wherein the enzyme glucoamylase acts with pancreatic amylase to increase glucose output, and the composition further comprises at least one protease. 7. Use according to claim 6 wherein the protease is selected from the group consisting of an acidic protease or a neutral metalloprotease. 8. Use according to claim 7 wherein the protease is a fungal aspartic protease or a bacterial neutral metalloprotease.

Images