Methods for High-Temperature Hydrolysis of Galactose-Containing Oligosaccharides in Complex Mixtures

专利摘要:
Α-galactosidase from an extremely high temperature source is useful for treating animal feed by hydrolyzing galactose oligosaccharides present in animal feed. The α-galactosidase of Thermotoga maritima is useful for hydrolyzing the indigestible oligosaccharides of raffinose, stachiose and verbacose, commonly found in animal feed compositions. The ability to use this enzyme at high temperatures, i.e. temperatures typically used in industrial processes typically associated with animal feed formulations or processing, is advantageous in that it adds nutritional value to the animal feed and provides flexibility in processing. Super high temperature α-galactosidase is also useful as a food additive for human food.
公开号:KR20030036621A
申请号:KR10-2003-7000905
申请日:2001-07-20
公开日:2003-05-09
发明作者:마이클 비. 라나한；에드워드 에스. 주니어 밀러；로버트 엠. 켈리
申请人:신젠타 파티서페이션즈 아게；
IPC主号:

专利说明:

Method for High-Temperature Hydrolysis of Galactose-Containing Oligosaccharides in Complex Mixtures
[1] The present invention relates to a method for processing animal feed and other complex substrates using ultra high temperature enzymes that hydrolyze oligosaccharides.
[2] α-galactosidase (also referred to herein as α-D-galactosid galactose hydrolase, EC 3.2.1.22, α-gal or Gal36) is the non-reducing end of a simple galactose-containing oligosaccharide Is an exo-functional glycosidase that catalyzes the hydrolysis of α-1 → 6 binding α-D-galactosyl residues. Examples of these oligosaccharides include raffinose, starchiose, verbacose and melibiose, as well as more complex polysaccharides.
[3] Intracellular and extracellular α-gal are widely distributed in microorganisms, plants and animals. Genes encoding α-gal have been cloned from a variety of sources including humans, plants, yeast, fibrous fungi and bacteria. Based on similarities in primary structure and hydrophobic cluster analysis, α-gal is divided into three well-conserved groups in the general classification of glycosyl hydrolases. Α-gal derived from bacteria is classified into 4 groups and 36 groups, and α-gal derived from eukaryotic cells is classified into 27 groups.
[4] Isolation of the bacterium Thermotoga maritima is described in Huber et al., Arch. Microbiol. 144, 324-333 (1986). Thermomoto maritima is an oil bacterium that exhibits complete aerobic, rod-like, fermentable and ultra-high temperature, and grows at temperatures between 55 ° C. and 90 ° C. (optimum growth temperature is about 80 ° C.). This eubacteria is isolated from geothermally heated seabeds in Italy and the Azores. Thermotoga neopolitana is another hyperthermic milk bacterium that is associated with Thermomoto Marittima. Enzymes isolated from both Thermomoto Marittima and Thermomoto Neopolitana include β-mannase, β-mannosidase, α-galactosidase and hemicellulase. Of the known α-gal, the hyperthermic bacterium Thermomoto is only active in α-gal ( Tm GalA) of Marittima and the hyperthermic bacterium Thermomoto is neopolytana α-gal ( Tn GalA) at temperatures exceeding 75 ° C. Long term stability was shown.
[5] Animal feed formulations are generally made to have balanced carbohydrate and protein content and are adjusted to suit various stages in the life cycle of a particular animal. In many animal feeds, soy flour makes up a significant amount of the feed. For example, in a feed of bulgogi chicken, soy flour constitutes approximately 20-30% of the protein content. Soybeans have high protein content, especially high amino acid lysine and threonine, but low methionine content. High protein content is the reason why soy is used a lot in animal and human foods (ie, infant formulas). US production of soy flour is $ 6 billion industrially, and about 80% of US soy flour production is used for animal feed each year.
[6] Approximately 15% of soy flour is indigestible by unit animals. This 15% soy flour makes up dietary fiber (as insoluble fiber) in poultry food. Generally, about 3 to 5% of this insoluble fraction is raffino-oligosaccharide. In other foods, such as legumes or wheat based foods, the raffino-oligosaccharide content is much higher by 35% and accounts for most of the anti-nutritive carbohydrates in certain types of food.
[7] The presence of undigested oligosaccharides can lead to undesirable consequences with regard to optimal energy use of animal feed. Enzymatic treatment of animal feed can increase the utilization of digestible soluble carbohydrates. A slight increase in the metabolic apparent energy (AME) content of the feed can result in significant cost savings. AME can be increased by minimizing feed consumption to remove anti-nutritive factors (ie, indigestible oligosaccharides), improving the digestibility of available carbohydrate components, and improving the water solubility of the insoluble fraction.
[8] The general outline of a typical soy flour processing sequence illustrates a typical animal feed processing. During the processing of animal feed, especially animal feed comprising soy flour, the animal feed is treated with boiling hexane to remove the oil present in the soybean material (ie flakes). Next, hexane is recovered by distillation from the oil.
[9] After hexane treatment, the feed was steamed for 1-2 minutes to denature the protein and degrade the protease inhibitors. The main purpose of the heat treatment is to denature the protease inhibitors found in soy flour. This is especially true for soybeans containing excess protease and protease inhibitors. During this stage, the moisture content is raised to about 20%, which is generally the highest moisture content in all animal feed processing steps. Residual urease activity is commonly used as a measure to measure protein denaturation. After steaming, feed is sent to a desolvent / toaster. Here, the feed is heated or "cooked" to remove any remaining hexane and reduce the water content by approximately 14%. Additional protein denaturation occurs at this stage. After the toaster operation, the feed is pelleted (eg by extrusion) at a temperature of approximately 180 ° F. (82 ° C.). The pelletization and extrusion process generally lasts for several tens of seconds. After cooling, the moisture content can be further reduced to a total moisture content of 2% to about 12%.
[10] Current techniques for enzyme treatment of animal feed generally use enzymes derived from mesophilic bacteria to produce animal feed with improved digestibility and nutritional value. These enzymes should generally be used in the final processing stage of feed formulation after pelletization because of the relatively low thermal stability of the enzyme and the high temperatures used for feed processing. Physical pelleting processes typically include heating the feed and extruding the feed through a die. High temperatures are necessary to remove excess moisture that prevents the pellets from remaining in an unseparated form and to "melt" the feed into the pellets. Most pelletizers can handle feeds of approximately 1,000 kg / hr.
[11] Enzymes are added to the newly formed pellets as they exit the pelletizer and air cooler. Typically, the enzyme solution is sprayed from a nozzle perpendicular to the feed pellets coming off. Coating the pellets with enzymes in this way is characterized by: (1) the rate of enzyme use is limited by the water content of the enzyme solution (when the pellets are too wet to be separated from each other, and the high water content in the pellets causes mold to be stored And in the case of promoting fungal growth), and (2) these limitations and high pellet formation rates are inefficient processes in that feed pellets are often incompletely coated with enzymes. When using this technique, it is estimated that only about one of the five pellets is actually coated by the enzyme.
[12] In addition, mesophilic enzymes generally target the intrinsic activity (ie, post-digestion activity) in animals. Because of the presence of pH and proteases in the digestive tract of animals, externally supplied enzymes become significantly less effective.
[13] Therefore, there is a need for the use of enzymes in animal feed which are indigestible oligosaccharides which are decomposed into monomers and stable at high temperatures used in feed processing before the animal is digested.
[14] In addition to reducing the metabolic apparent energy (AME) content in human and animal foods, it is also undesirable for non-digestible oligosaccharides to be present in human and animal foods, which is a gastrointestinal discomfort caused by the presence of such oligosaccharides. (For example, flatulence and other gastrointestinal syndromes). Certain foods that show swelling symptoms include legumes (e.g., peanuts, kidney beans), some mustard plants (e.g., cabbage, brussels sprout), and some fruits (e.g., raisins, Bananas, apricots). The main cause of bloat from the foods mentioned above is that the body is unable to digest certain carbohydrates (ie raffinose, starchiose and verbacose) contained in these foods. The inability of mammals to digest these carbohydrates allows rot bacteria in the large intestine to break down these carbohydrates by fermentation. This forms excessive levels of rectal gas, mainly carbon dioxide, methane and hydrogen. Humans and other unit mammals digest D-galactose by digesting these three oligosaccharides because their digestive systems do not produce α-galactosidase or produce very small amounts of α-galactosidase. it's difficult.
[15] The use of α-galactosidase in vitro to make the above-mentioned oligosaccharides digestible is described in US Pat. Nos. 3,966,555, 4,241,185 and 4,431,737, each of which discloses a variety of microorganisms. Disclosed is a method of culturing to produce and / or stabilize α-galactosidase, suggesting that α-D-galactosidase may be used for food processing and / or added to food products for a period of up to 12 hours. do. In vitro hydrolysis of α-D-galactosid-binding sugars with addition of α-galactosidase is described in R. Cruz, et al., Journal of Food Science 46,1196-1200 (1981). It is described in.
[16] US Pat. No. 5,436,003 to Rohde et al. Describes a method for alleviating gastrointestinal discomfort with a composition containing β-fructofuranosidase, cellulase and hemi-cellulase. Liquid products marketed under the trade name BEANO by AkPharma are enzymes that reduce or eliminate the intestinal gases produced when eating foods such as green beans, broccoli, rice hulls, and other plant and grain sources that are low-fat, high-fiber healthy foods, or It is described as a food additive. The BEANO product contains the enzyme α-galactosidase obtained from Aspergillus niger .
[17] Unfortunately, problems associated with hydrolyzing α-D-galactosid-linked sugars by processing foods in vitro with α-galactosidase as known to reduce symptoms in mammals digesting food. There is. Generally, the enzyme is applied to foods that have already been prepared (ie cooked). Enzymatic treatment of intact (ie undigested or chewed) beans or other vegetables and fruits with enzymes is inefficient and expensive. The solid nature of these foods interferes with efficient, uniform and fully effective enzyme activity in that the enzyme is only in external contact with the substrate. Finally, current methods using α-galactosidase generally involve treating the enzyme immediately before food consumption, so that the activity of the enzyme occurs mainly during the post-consumption digestion process. Currently used enzymes cannot be applied to foods prior to the preparation of foods (ie, before cooking, before heating) because mesophilic α-galactosidase exhibits thermal instability at high temperatures. The ability to use α-galactosidase, which is stable at high temperatures, is flexible to food consumers in terms of (1) the preparation of food containing undesirable oligosaccharides and (2) the ability to hydrolyze unwanted oligosaccharides prior to digestion. It is preferable in that it provides.
[18] The inventors have found that certain hyperthermic enzymes can be used as processing additives to improve the quality of animal feed and human food. The present invention relates to α-galactosidase obtained from an ultra-high temperature source for treating animals directly by hydrolysis of galactose-containing oligosaccharides present in animal feed, for example, α-galactosa from Martina DSM3109. Use galactosidase. Enzymatic treatment is achieved by directly adding the hyperthermic α-galactosidase formulation to a substrate composition comprising galactose-containing oligosaccharides (eg, animal feed containing soy flour). One advantage of the present invention is the ability to use the enzymes at high temperatures, ie temperatures typically used in industrial processes typically associated with animal feed formulation or processing.
[19] In addition, because the substrate is more fully accessible to the enzyme at higher temperatures, the enzyme is in complete contact with the substrate. Moisture demand for enzyme activity is generally reduced at elevated temperatures needed for enzyme activity. Enzyme activity on the substrate can also be controlled by adjusting the time the mixture is held at elevated temperature. Thus, one aspect of the present invention is to contact the ultra-high temperature α-galactosidase with a complex substrate containing galactose-containing oligosaccharides (e.g., animal feed) and then heat the mixture to enzyme-mediated hydrolysis. Is a new method of hydrolyzing galactose-containing oligosaccharides by promoting.
[20] Another aspect of the present invention is a composition comprising a mixture of a composite substrate comprising galactose-containing oligosaccharides (eg, soy flour, soy flakes or animal feed) and an ultra high temperature α-galactosidase.
[21] A third aspect of the invention is a composition comprising α-galactosidase obtained from an ultra high temperature source, which can be used as a food additive for reducing gastrointestinal discomfort in humans and animals.
[22] A fourth aspect of the invention is a composition comprising α-galactosidase obtained from an ultra high temperature source, which can be used, for example, as a processing additive in the isolation of vegetable proteins (ie soy protein). Such additives are useful for removing oligosaccharides and galactose monomers from protein products to facilitate preventing or reducing gastrointestinal discomfort in humans and animals.
[23] Because of the high thermal stability of the enzymes disclosed herein and the high temperatures at which these enzymes exhibit activity, the present invention allows enzymatic modification of animal feed to occur during high temperature feed processing before feeding the animal to the animal. Storage problems arising from increased moisture content are reduced or eliminated since the enzyme treatment is no longer necessary after pelletization. As smaller particles are processed (ie compared to the final pelletized product), the enzyme efficiency is increased due to the reduced mass transfer resistance. Finally, hydrolysis of galactose-containing oligosaccharides ultimately increases food value in the sense that the nutritional value of the food is higher (ie, the food is more useful food energy that can be used by animals). .
[24] Accordingly, the present invention is directed to contacting a substrate with an ultra high temperature α-galactosidase, and to hydrolyzing the substrate, galactose-containing oligosaccharides at a temperature at which the high temperature α-galactosidase is active. Provided are methods for hydrolyzing galactose-containing oligosaccharides present in a substrate, comprising heating for a sufficient time. In a preferred embodiment, said oligosaccharide is selected from the group consisting of raffinose, starchiose and verbascose. In another preferred embodiment, the substrate is animal feed, soy flour or human food.
[25] In another preferred embodiment, the hyperthermic α-galactosidase is isolated from the group consisting of Thermomoto maritima, Thermomoto neopolitana, Thermomotoga elfii and Thermomoto species T2. . Preferably, the ultra high temperature α-galactosidase is isolated from thermomoto maritima, more preferably thermomoto maritima DSM3109. In another preferred embodiment, the oligosaccharide is hydrolyzed to galactose monomers. In another preferred embodiment, the method is carried out under 70% moisture conditions or 25% moisture conditions. In another preferred embodiment, the heating occurs at 80 ° C, 85 ° C, 90 ° C or 100 ° C.
[26] In another embodiment, the hyperthermic α-galactosidase is (a) culturing a host cell comprising an expression vector containing the polynucleotide sequence encoding the hyperthermic α-galactosidase; (b) expressing an ultra high temperature α-galactosidase; (c) produced by recovering ultra high temperature α-galactosidase from the host cell culture. Preferably, the polynucleotide has the sequence of SEQ ID NO: 1. In another preferred embodiment, said polynucleotide comprises (a) DNA having the nucleotide sequence of SEQ ID NO: 1; (b) a polynucleotide encoding an ultra high temperature α-galactosidase and hybridizing to the DNA of (a) under stringent conditions; And (c) a super-temperature α-galactosidase and is selected from the group consisting of the DNA of (a) or (b) and other polynucleotides due to the degeneracy of the genetic code. Preferably, the polynucleotide encodes an ultra high temperature α-galactosidase having the amino acid sequence of SEQ ID NO: 2.
[27] The present invention also provides the preparation of an animal feed composition comprising hydrolyzed galactose-containing oligosaccharides, which comprises contacting components of the animal feed composition with an ultra high temperature α-galactosidase during processing of the animal feed. A method is provided wherein said ultra-high temperature α-galactosidase is contacted with animal feed components prior to a heating step during animal feed processing for a time sufficient to hydrolyze the galactose-containing oligosaccharides. In a preferred embodiment, said galactose-containing oligosaccharides are selected from the group consisting of raffinose, starchiose and verbascose. In another preferred embodiment, said animal feed comprises soy flour or soybean play, or is a chicken feed. In another preferred embodiment, the hyperthermic α-galactosidase is isolated from the group consisting of Thermomoto maritima, Thermomoto neopolitana, Thermomoto elpi and Thermomoto species T2. Preferably, the ultra high temperature α-galactosidase is isolated from thermomoto maritima, more preferably thermomoto maritima DSM3109. In another preferred embodiment, the oligosaccharides hydrolyze with galactose monomers. In another preferred embodiment, the contacting of the hyperthermic α-galactosidase with the components of the animal feed composition is performed under 70% moisture conditions, 25% moisture conditions or 45% moisture conditions. In another preferred embodiment, the heating occurs at 80 ° C, 85 ° C, 90 ° C or 100 ° C. In another preferred embodiment, the contact of the hyperthermic α-galactosidase with the components of the animal feed composition occurs before the last pelleting step in animal feed processing.
[28] In another preferred embodiment, the hyperthermic α-galactosidase is (a) culturing a host cell comprising an expression vector containing the polynucleotide sequence encoding the hyperthermic α-galactosidase; (b) expressing an ultra high temperature α-galactosidase; (c) produced by recovering ultra high temperature α-galactosidase from the host cell culture. Preferably, the polynucleotide has the sequence of SEQ ID NO: 1. Preferably, the polynucleotide comprises (a) DNA having the nucleotide sequence of SEQ ID NO: 1; (b) a polynucleotide encoding an ultra high temperature α-galactosidase and hybridizing to the DNA of (a) under stringent conditions; And (c) a super-temperature α-galactosidase and is selected from the group consisting of the DNA of (a) or (b) and other polynucleotides due to the degeneracy of the genetic code. Preferably, the polynucleotide encodes an ultra high temperature α-galactosidase having the amino acid sequence of SEQ ID NO: 2.
[29] In another preferred embodiment, the hyperthermic α-galactosidase is in liquid solution form, dried form, partially purified form or substantially purified form when it comes into contact with the components of the animal feed composition.
[30] The present invention also provides an animal feed produced according to any one of the above methods.
[31] The present invention also provides food additives for reducing gastrointestinal discomfort in mammals, including ultra high temperature α-galactosidase. Preferably, the ultra high temperature α-galactosidase is isolated from the group consisting of thermomoto maritima, thermomoto neopolitana, thermomoto elpi and thermomoto species T2. Preferably, the ultra high temperature α-galactosidase is isolated from thermomoto maritima, more preferably thermomoto maritima DSM3109.
[32] In another preferred embodiment, the hyperthermic α-galactosidase is (a) culturing a host cell comprising an expression vector containing the polynucleotide sequence encoding the hyperthermic α-galactosidase; (b) expressing an ultra high temperature α-galactosidase; (c) produced by recovering ultra high temperature α-galactosidase from the host cell culture. Preferably, the polynucleotide has the sequence of SEQ ID NO: 1. Preferably, the polynucleotide comprises (a) DNA having the nucleotide sequence of SEQ ID NO: 1; (b) a polynucleotide encoding an ultra high temperature α-galactosidase and hybridizing to the DNA of (a) under stringent conditions; And (c) a super-temperature α-galactosidase and is selected from the group consisting of the DNA of (a) or (b) and other polynucleotides due to the degeneracy of the genetic code. Preferably, the polynucleotide encodes an ultra high temperature α-galactosidase having the amino acid sequence of SEQ ID NO: 2.
[33] The present invention comprises the steps of contacting a hyperthermic α-galactosidase with a food comprising one or more oligosaccharides selected from the group consisting of raffinose, starchiose and verbacose; And heating the food for a time sufficient for the ultra high temperature α-galactosidase to hydrolyze the oligosaccharide, thereby providing a method for preventing gastrointestinal discomfort caused by the food in a mammal.
[34] In addition, the present invention provides processing additives for removing galactose-containing oligosaccharides in the process of preparing edible soy protein, including ultra high temperature α-galactosidase.
[35] In addition, the present invention comprises the steps of (a) contacting the soybean substrate with an ultra high temperature α-galactosidase; (b) heating the soybean substrate at a temperature suitable for hydrolysis of the oligosaccharides for a time sufficient to hydrolyze the galactose-containing oligosaccharides; And (c) removing the hydrolyzed galactose-containing oligosaccharides from the soybean substrate prior to final extraction or fractionation of the edible soy protein to remove edible galactose-containing oligosaccharides from the soybean substrate to be processed. Provides a method for producing soy protein. In a preferred embodiment, the heating takes place before the oil is removed from the soybean substrate. In another preferred embodiment, heating occurs after the oil is removed from the soybean substrate. In another preferred embodiment, the soybean substrate is soy flakes.
[36] The present invention also provides an isolated edible soy protein produced according to any one of the above methods.
[37] These and other aspects of the invention are described in detail in the following detailed description of the invention.
[38] Brief description of sequence listing
[39] SEQ ID NO: 1 nucleotide sequence of the Thermomotor Marittima DSM 3109 galA or gal36 gene.
[40] SEQ ID NO: 2 amino acid sequence encoded by SEQ ID NO: 1.
[41] The nucleotide sequence starts at GTG, the translation initiation codon. The upstream ribosomal binding site sequence is omitted. During the cloning of this gene as described herein, the translational start codon GTG was changed to ATG to facilitate the insertion of pET24d + into a single NcoI site immediately following the ribosomal binding site.
[42] FIG. 1 is a graph showing the activity of Thermomoto Maritrima GalA on PNP-galactose as a function of pH. Buffer (50 mM citrate for pH 2.5-3.5; 50 mM Na acetate for pH 4-6; 50 mM Na phosphate for pH 6.5-8) was used.
[43] FIG. 2 is a graph showing Thermomoto's activity against PNP-galactose as a function of temperature. All assays were performed using 50 mM Na acetate buffer, 0.1 M NaCl and 1 mM PNP-galactose.
[44] The invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. However, the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
[45] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise.
[46] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
[47] Unless otherwise specified, cloned genes, expression cassettes, vectors (eg, plasmids), proteins and protein fragments can be produced according to the invention using standard methods. Such techniques are known to those skilled in the art [see, eg, Sambrook et al., Eds., Molecular Cloning: A Laboratory Manual Second Edition (Cold Spring Harbor, NY 1989); FM Ausubel et al, eds., Current Protocols In Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).
[48] The amino acid sequences disclosed herein are described in the carboxy direction from the amino direction from left to right. Amino and carboxy groups are not described in this sequence. Nucleotide sequences are described herein as single strands only in the 5 'to 3' direction from left to right. Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (in the case of amino acids) in three letter codes according to 37 CFR §1.822 and established practices. User Manual, 99-102 (Nov. 1990) and US Patent and Trademark Office.
[49] A. Definition
[50] As used herein, "protein" or "enzyme" refers to two or more amino acids covalently attached, including proteins, polypeptides, oligopeptides and peptides. Proteins can be composed of naturally occurring amino acids and peptide bonds, or synthetic peptide-like structures. Thus, as used herein, "amino acid" or "peptide residue" refers to both naturally occurring and synthetic amino acids. "Amino acid" also includes amino acid residues such as proline and hydroxyproline. The side chain may be of the (R) or (S) structure. If non-naturally occurring side chains are used, non-amino acid substituents can be used, for example, to prevent or delay degradation in vivo. Chemical blocking groups or other chemical substituents may also be added.
[51] As used herein, “amino acid sequence” refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments thereof, and refers to naturally occurring or synthetic molecules. Fragments of α-galactosidase preferably retain the biological activity of α-galactosidase. When “amino acid sequence” as used herein refers to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence and similar terms do not mean that the amino acid sequence is limited to the fully native amino acid sequence associated with the protein molecule.
[52] As used herein, “amplification” refers to the production of additional copies of nucleic acid sequences and is generally performed using polymerase chain reaction (PCR) techniques well known in the art (Dieffenbach, CW and GS Dveksler (1995) PCR). Primer, A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY).
[53] As used herein, the term "nucleic acid derivative" refers to a chemical modification of an α-galactosidase or a nucleic acid or an encoded α-galactosidase that is complementary to an α-galactosidase. Such modifications include, for example, replacing hydrogen with alkyl, acyl or amino groups. Nucleic acid derivatives encode polypeptides that retain the biological or immunological function of natural molecules. An induction polypeptide is a polypeptide that has been modified by glycosylation, PEGylation, or any similar method that retains the biological or immunological function of its parent polypeptide.
[54] The term "homology" as used herein refers to the degree of complementarity. There may be partial homology or complete homology (ie, identity). Partially complementary sequences that at least partially inhibit the same sequence from hybridizing to a target nucleic acid are called using the functional term “substantially homologous”. Inhibition of hybridization of sequences that are completely complementary to the target sequence can be investigated using hybridization assays (such as Southern or Northern blots, solution hybridization, etc.) under conditions of low stringency. Substantially homologous sequences or hybridization probes will compete for and inhibit the binding of sequences that are fully homologous to the target sequence under low stringency conditions. Low stringency conditions allow non-specific binding; It goes without saying that the binding of two sequences requires specific (ie selective) interaction. The absence of non-specific binding can be tested using a second target sequence (eg, a sequence having less than about 30% identity) without even a degree of partial complementarity. In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.
[55] As used herein, "nucleic acid", "oligonucleotide" or grammatically the same term refers to two or more nucleotides joined by covalent bonds. Nucleic acids of the invention, although in some cases, other backbones known in the art (eg, phosphoramide; phosphorothioate; phosphorodithioate; O-methylphosphoroamidite bonds and peptide nucleic acid backbones and Although nucleic acid homologues may be included, they will generally have phosphodiester bonds.
[56] "Nucleic acid sequence" and "polynucleotide" as used interchangeably herein refer to oligonucleotides, nucleotides or polynucleotides and fragments thereof, which may be single or double stranded and represent genomic or synthetic DNA or Refers to RNA.
[57] As used herein, the term “hydrolyzing” refers to the removal of α-D-galactosyl residues from the non-reducing end of an oligosaccharide comprising galactose units via enzymatic activity. In oligosaccharides, hydrolysis of oligosaccharides means that the degree of polymerization (DP) of the oligosaccharides is reduced. The reduction in the degree of polymerization may mean that the oligosaccharides are hydrolyzed to smaller oligosaccharides, preferably that the oligosaccharides are fully hydrolyzed to their monomer galactose units.
[58] The term "substrate" as used herein refers to a compound or mixture comprising oligosaccharides, in particular oligosaccharides starchiose, raffinose and verbacose. Exemplary substrates specifically described herein include oilseed flour (ie, soy flour, canola flour), vegetable protein flakes, any form of animal feed, and human food.
[59] Soy or Glycine max is an exemplary substrate for the present invention, although other substrate sources such as canola, rapeseed, sunflower seed, flaxseed, safflower seed, sesame seed, and cotton seed may be substrate sources according to the present invention. Use as a source. Thus, terms such as "powder", "oil", "flake", "food or feed", "protein" and "product" defined by the term soybean may also apply to other substrate sources. In general, although the present invention may be used with other substrate sources, suitable substrate sources are preferably oilseed seeds.
[60] The term “soy product” as used herein is any edible product or other product for which soybean is its natural source. Thus, a "soy product" includes soy products such as soy flour, soybean oil, soy flakes, soy flour, soy protein and protein concentrates, soy lecithin, pods, soy isolates or concentrates, soy curds, or soy flours. Any animal feed or human food.
[61] In general, “soy flour” is defined as the high-protein residue (typically over 40% protein) remaining after soybean oil is extracted from soybean. Examples of various methods of processing soybeans to produce soy flour are described in US Pat. No. 4,103,034 (Ronai et al.), Which is incorporated herein in its entirety. Soy flour is a common source of protein generally preferred in the manufacture of animal feed, and may be a complete soy flour or peeled soy flour extracted with a solvent or eviction agent, or may be processed by other methods known in the art. .
[62] "Animal feed" generally refers to one or more protein sources, such as oilseed flour (eg, soybean flour), one or more carbohydrate sources, and other ingredients (eg, fillers, swelling agents, added nutritional substances and additions herein) Mixtures of organic materials). Animal feeds are well known in the art and include high quality protein feeds and other feeds consisting of slightly lower quality proteins. Feeds include soybean flour, cottonseed flour, feather flour, blood flour, grasses, meat and bone flour, sunflower seed flour, canola powder, peanut flour, safflower flour, flaxseed flour, sesame flour, early flowering legumes, fish products, distillation By-product protein foodstuffs such as water and beer residues, dairy products, poultry products, hay, corn, wheat, alfalfa, barley, milo, sugar cane and mixtures thereof. Other ingredients that may be included in the animal feed are further described below.
[63] B. Properties of ultra high temperature α-galactosidase
[64] Α-galactosidase isolated from high temperature organisms (also referred to herein as "super high temperature enzymes" or "super high temperature α-galactosidase") is useful in the present invention. Galactosidase can be isolated) including the species of the bacterium thermos (ie, thermos thermophilia) and thermomotoga .. Preferred hyperthermic organisms include thermomoto maritima, thermomoto neopolitana and thermomotoga Particularly preferred are species of the genus Thermotoga, including Elpi and Thermotoga sp. T2, and Thermotoga maritima are particularly preferred, and preferred isolated α-galactosidases include Thermomoto maritima DSM3109 and Thermomoto neopolitana. 5068 and those isolated from mutants or variants thereof (W. Liebel et al., System. Appl. Microbiol. 21, 1-11 (1998) and G. Duffaud et al., Appl. Environmental Microbiol. 63,169 -177 (1997)).
[65] α-galactosidase can be isolated from very high temperature organisms according to the techniques known in the art and described herein. A description of how to isolate enzymes from hyperthermic organisms can also be found in G. Duffaud et al., Appl. Environmental Microbiol. 63, 169-177 (1997). As used herein, α-galactosidase may be natural, synthetic, semi-synthetic or recombinant α-galactosidase. In one preferred embodiment, the hyperthermic a-galactosidase of the invention has the amino acid sequence described herein as SEQ ID NO: 2. The hyperthermic α-galactosidase of the present invention may be encoded by an isolated polynucleotide, a preferred embodiment of the polynucleotide being a cDNA having a nucleotide sequence described herein as SEQ ID NO: 1.
[66] Enzymes of the invention may be naturally purified products, products prepared by chemical synthetic methods, or prokaryotic or eukaryotic hosts (eg, bacteria, yeasts, higher plants, insects in culture and Mammalian cells). Depending on the host used in the recombinant production method, the enzymes of the present invention may or may not be glycosylated. The enzymes of the invention may or may not comprise starting methionine amino acid residues.
[67] The optimal temperature at which an enzyme of the invention will be active will depend on each enzyme and each organism (the enzyme of the invention is first isolated from this organism). In general, the enzymes of the present invention exhibit activity at temperatures higher than about 75 ° C, more preferably higher than about 80 ° C, and most preferably higher than about 85 ° C. The enzymes of the present invention may exhibit activity at temperatures as high as 90 ° C. or even at 100 ° C. In the most preferred embodiment, the enzyme of the present invention exhibits little or no activity at normal ambient temperature or room temperature (ie, about 25 ° C.). In general, the enzymes of the present invention will generally have a maximum half-life at their optimum temperature of about 80 ° C. to 98 ° C., more preferably about 85 ° C. to 98 ° C. This enzyme will generally exhibit activity at about 100 ° C., although its half life is generally shorter at 100 ° C.
[68] The ultra high temperature α-galactosidase of the present invention has various moisture contents and shows activity under a wide environment. For example, the ultra high temperature α-galactosidase of the present invention exhibits activity at about 70% water content, about 45% water content, about 25% water content, and even lower water content.
[69] Those skilled in the art will appreciate, for example, "induced evolution, such as those described in US Pat. No. 5,837,458 (Minshull et al.), US Pat. No. 5,837,500 (Ladner et al.) And US Pat. No. 5,811,238 (Stemmer et al.). Or alternatively, it will be appreciated that metabolic engineering techniques can be used to design useful variants of the enzymes of the invention to have optimal activity in the use of a particular substrate or under certain conditions, the patents of which are incorporated herein in their entirety. .
[70] C. Production of ultra high temperature α-galactosidase
[71] In one embodiment, Cryogenic α-galactosidase can be isolated and optionally purified from their natural hyperthermic organisms according to techniques known in the art. Exemplary descriptions of how naturally-occurring hyperthermic α-galactosidase can be isolated from their naturally occurring hyperthermic organisms and suitable conditions and reagents thereof can be found in G. Duffaud et al., Appl. Environmental Microbiol. 63, 169-177 (1997).
[72] In another embodiment, polynucleotides encoding the hyperthermic α-galactosidase (preferably, DNA) may be cloned and expressed (or overexpressed) to produce enzymes useful in the present invention. The expressed protein is then isolated and used in the methods and compounds of the invention. The hyperthermic enzymes prepared in this way can then be optionally purified, but these enzymes can be used in the present invention in unpurified or partially purified form.
[73] The polynucleotide sequence used to express α-galactosidase may be genomic polynucleotides, cDNA polynucleotides, synthesized polynucleotides or any combination thereof. Said polynucleotide sequence is cloned into a suitable vector a cDNA library derived from a strain producing any ultra high temperature α-galactosidase; Transforming a suitable host cell with said vector; culturing the host cell under conditions suitable for expressing the enzyme encoded by the clone in the cDNA library; Screening positive clones by measuring any hyperthermic α-galactosidase activity of the enzymes produced by the clones; And cloning the enzyme-encoding DNA from this clone.
[74] Polynucleotides used to express α-galactosidase are well known using synthetic oligonucleotide probes prepared based on the DNA sequence described as SEQ ID NO: 1, any suitable subsequence thereof or the amino acid sequence described as SEQ ID NO: 2. Hybridization can be conveniently cloned from any ultra high temperature α-galactosidase-producing organism by hybridization according to the methods described. Alternatively, the DNA sequences can be cloned using PCR primers prepared based on the DNA sequences disclosed herein.
[75] As mentioned above, the present invention uses isolated and optionally purified ultra high temperature α-galactosidase. Such proteins may be isolated from host cells expressing them according to known techniques, or may be prepared synthetically. Nucleic acids of the invention, constructs containing them, and host cells that express the encoded protein are useful for preparing the enzymes of the invention.
[76] Certain initiation signals may be used to more efficiently translate sequences encoding ultra high temperature α-galactosidase. Such signals include initiation codons and contiguous sequences. If the sequence encoding the hyperthermic α-galactosidase, its start codon and upstream sequence are inserted into a suitable expression vector, no additional transcriptional or translational control signal may be required. However, when only the coding sequence or fragment thereof is inserted, an exogenous translational control signal comprising an initiation codon must be provided. In addition, the start codon must be in the correct reading frame so that the entire insert is translated. Exogenous translation elements and initiation codons can be from a variety of sources and can be both naturally occurring and synthesized. Expression efficiency can be enhanced by including an enhancer suitable for the particular cellular system used, such as those described in D. Scharf et al., Results Probl. Cell Differ. 20, 125-162 (1994). have.
[77] Polynucleotides encoding the hyperthermic α-galactosidase of the present invention include proteins homologous to the proteins disclosed herein, and polynucleotides encoding proteins having biological properties that are essentially the same as the proteins disclosed herein. Included herein are DNAs disclosed as SEQ ID NO: 1 and DNA encoding the ultra high temperature α-galactosidase described herein as SEQ ID NO: 2. This definition includes its natural allelic sequence. Thus, polynucleotides that hybridize to the DNA disclosed herein as SEQ ID NO: 1 (or fragments or derivatives thereof used as hybridization probes discussed below) and which encode the protein of the invention (eg, the protein of SEQ ID NO: 2) upon expression Also useful in the practice of the present invention.
[78] Conditions for causing other polynucleotides encoding the proteins of the invention to hybridize to the DNA of SEQ ID NO: 1 disclosed herein upon expression can be determined according to known techniques. For example, in standard hybridization assays, hybridization of the polynucleotide sequence with the DNA of SEQ ID NO: 1 disclosed herein can be performed under reduced stringency, moderate stringency, or even stringent conditions (eg, 37 ° C.). Indicated by the washing stringency of 35-40% formaldehyde containing 5x Denhardt's solution, 0.5% SDS and 1x SSPE at; containing 5x Denhartz solution, 0.5% SDS and 1x SSPE at 42 ° C. Conditions indicated by washing stringency of 40-45% formaldehyde, and wash stringency of 50% formaldehyde containing 5x Denharz's solution, 0.5% SDS and 1x SSPE at 42 ° C. have. In general, a sequence encoding a protein of the invention and hybridizing to the DNA of SEQ ID NO: 1 disclosed herein will have at least 75% homology, at least 85% homology, and even at least 95% homology with the DNA of SEQ ID NO: 1. In addition, polynucleotides encoding proteins of the invention, or polynucleotides that hybridize to DNA of SEQ ID NO: 1 but are different in SEQ ID NO: 1 and codon sequences due to the degeneracy of the genetic code, are also useful in the practice of the present invention. The degeneracy of the genetic code to allow different nucleic acid sequences to encode the same protein or the same peptide is well known in the table (Table 1 of US Pat. No. 4,757,006 (Toole et al.)).
[79] Although it is desirable that the nucleotide sequence encoding the hyperthermic α-galactosidase and variants thereof be capable of hybridizing with the nucleotide sequence of a naturally occurring hyperthermic α-galactosidase under stringent conditions appropriately selected, the hyperthermic α-galactosidase It may be advantageous to make variants thereof using galactosidase or substantially other codons. Codons can be chosen to increase the rate at which expression of a peptide occurs in a particular prokaryotic or eukaryotic host depending on the frequency with which the particular codon is used by the host. Nucleotide sequences encoding hyperthermic a-galactosidase, and other reasons for substantially altering derivatives thereof without altering the encoded amino acid sequence, have higher desirable properties than transcripts produced from naturally occurring sequences, for example. Production of RNA transcripts with half-life.
[80] The present invention also includes the preparation of DNA sequences or fragments thereof encoding ultra high temperature α-galactosidase and derivatives thereof according to synthetic chemistry. After preparation, the synthetic sequences can be inserted into any of many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry can be used to introduce mutations into sequences encoding ultra high temperature α-galactosidase or any fragment thereof.
[81] The nucleotide sequences set forth in SEQ ID NO: 1 herein can be used to make hybridization probes that specifically bind to polynucleotides (ie, cDNAs) or mRNAs of the invention to determine the presence of amplification or overexpression of a protein of the invention. .
[82] It is well known to produce cloned genes, recombinant DNA, vectors, transformed host cells, proteins and protein fragments by genetic engineering techniques. US Pat. No. 4,761,371 to Bell et al. At Col. 6 line 3 to Col. 9 line 65); US Pat. No. 4,877,729 to Clark et al. At Col. 4 line 38 to Col. 7 line 6; US Patent No. 4,912,038 to Schilling at Col. 3 line 26 to Col. 14 line 12; And US Patent No. 4,879,224 (Wallner at Col. 6 line 8 to Col. 8 line 59) (Applicant specifically refers to all patent references cited herein, the entire contents of which are incorporated herein by reference. ).
[83] Vectors are replicable nucleic acid (preferably DNA) constructs. Vectors can be used in the present invention to amplify DNA encoding a protein of the invention, or to express a protein of the invention. Expression vectors are replicable nucleic acid constructs in which a nucleic acid sequence encoding an enzyme of the invention is operably linked to a suitable regulatory sequence that can affect the expression of the enzyme of the invention in a suitable host. The need for such regulatory sequences will depend on the host selected and the method of transformation chosen. Typically, regulatory sequences include transcriptional promoters, optionally operator sequences that control transcription, sequences encoding appropriate mRNA ribosomal binding sites, and sequences encoding end of transcription and translation. Amplification vectors do not require expression control domains. All that is required is a selection gene to facilitate recognition of the ability to replicate in the host, and transformants, typically conferred by the origin of replication.
[84] Vectors include, but are not limited to, plasmids, viruses (eg, adenoviruses, cytomegaloviruses), phages, retroviruses, and insertable DNA fragments (ie, fragments that can be inserted into the host genome by recombination). . Vectors can replicate and function independently of the host genome, or in some cases can be inserted into the host genome itself. The expression vector preferably comprises a promoter and an RNA binding site that is operably linked to the gene to be expressed and can be operated in the host organism.
[85] Nucleic acid regions are operably linked or operably linked when they are functionally related to each other. For example, a promoter is operably linked to a coding sequence when he controls the transcription of the sequence, and a ribosomal binding site is operably linked to the coding sequence when he is positioned to allow translation. In general, operably linked means contiguous, and in the case of leader sequences, contiguous in the reading frame. The transformed host cell is transfected or transfected with a vector containing a polynucleotide encoding the hyperthermic α-galactosidase of the present invention and preferably, but not necessarily, the hyperthermic α-galactosidase. It is a cell that expresses. Suitable host cells include prokaryotic cells, yeast cells or higher eukaryotic organism cells.
[86] Prokaryotic host cells include gram negative or gram positive organisms such as E. coli or Bacillus, and E. coli . E. Coli is preferred. this. E. coli is typically transformed using a plasmid originally derived from pBR322 or a vector derived from this plasmid (Bolivar et al., Gene 2,95 (1977)).
[87] The expression vector preferably contains a promoter recognized by the host organism. This generally, but not necessarily, means a promoter obtained from a desired host. The promoter and Shine-Dalgarno sequence (for prokaryotic expression) are operably linked to the DNA of the invention. That is, the sequence is located to promote transcription of mRNA from the DNA. In the present invention, preferred promoters include known λ _pL , T ₇ and P _m promoters. Other promoters commonly used in recombinant microbial expression vectors include beta-lactamase (penicillinase) and lactose promoter systems (Chang et al., Nature 275, 615 (1978); and Goeddel et al., Nature 281, 544 (1979). )); Tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980) and EPO App. Publ. No. 36,776); tac promoter (H. De Boer et al., Proc. Natl. Acad. Sci. USA 80, 21 (1983)). While these are commonly used, other microbial promoters are also suitable. Details regarding many nucleotide sequences are disclosed to enable those skilled in the art to operably ligate the sequences to protein coding DNA in plasmids or viral vectors (Siebenlist et al., Cell 20, 269 (1980)).
[88] Eukaryotic microorganisms, such as yeast cultures, can also be transformed with suitable hyperthermic α-galactosidase coding vectors (see, eg, US Pat. No. 4,745,057). Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although many other strains may be commonly used. Yeast vectors may include origins of replication, promoters, DNAs encoding desired proteins, sequences for polyadenylation and termination of transcription, and selection genes derived from 2 micron yeast plasmids or autologous replication sequences (ARS). Exemplary plasmids are YRp7 (Stinchcomb et al., Nature 282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al., Gene 10, 157 (1980)). This plasmid contains the trp1 gene, which provides a selection marker for selecting yeast mutant strains that do not have the ability to grow in tryptophan medium, for example ATCC 44076 or PEP4-1 (Jones, Genetics 85, 12 (1977). The presence of the trp1 region in the yeast host cell genome then provides an environment for detecting transformation when grown in the absence of tryptophan.
[89] Suitable promoter sequences in yeast vectors include metallothionein, 3-phospho-glycerate kinase (Hitzeman et al., J. Biol. Chem. 255, 2073 (1980)), or other glycolytic enzymes (Hess). et al., J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, Hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosphosphate isomerase, phosphoglucose isomerase And promoters for glucokinase. Vectors and promoters suitable for use in yeast expression are further described in R. Hitzeman et al., EPO Publn. No. 73,657.
[90] Cultures of cells derived from multicellular organisms can also be used for recombinant protein synthesis. In principle, any higher eukaryotic cell culture can be used, whether derived from vertebrate culture or from invertebrate culture, including insect cells. Proliferation of these cells in cell culture is a common process (Tissue Culture (Academic Press, Kruse and Patterson, eds.) (1973)). Expression vectors for these cells are usually the origin of replication (if needed); A promoter, located along with the ribosomal binding site, upstream from the gene to be expressed; RNA splicing site (if intron containing genomic DNA is used); Polyadenylation sites; And transcription termination sequences.
[91] Expression vectors, such as host cells and baculovirus expression vectors, such as insect cells (eg, cultured Spodoptera frugiperda cells), are also described in US Pat. Nos. 4,745,051 and 4,879,236 (Smith et al. As described in.), It can be used to make proteins useful for carrying out the invention. In general, baculovirus expression vectors contain genes to be expressed that are inserted into the polyhedrin gene at a position within the range from polyhedrin transcription initiation signal to ATG initiation signal under the transcriptional control of the baculovirus polyhedrin promoter. The baculovirus genome.
[92] In addition, host cell strains may be selected for their ability to modulate the expression of the inserted sequence or to process the expressed protein in a desired manner. Modifications of such polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processes that cleave the “bulb” form of the protein can also be used to facilitate accurate insertion, folding and / or action. Various host cells (eg, CHO, HeLa, MDCK, HEK293 and WI38) with specific cellular means and characteristic mechanisms for post-translational activity can be purchased from the American Type Culture Collection (ATCC; Bethesda, Md.), It can be chosen to ensure that the correct modification and processing of the foreign protein is achieved.
[93] Stable expression is desirable for long term high yield production of recombinant proteins. For example, cell lines stably expressing hyperthermia a-galactosidase can be expressed using expression vectors that may contain viral origins of origin and / or endogenous expression elements and selectable marker genes on the same or separate vectors. Can be transformed. After introduction of the vector, cells can be grown in proliferation medium for 1-2 days and then transferred to selection medium. The purpose of the selection marker is to confer resistance to selection, the presence of which allows for the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.
[94] Host cells transformed with the nucleotide sequence encoding the hyperthermic α-galactosidase can be cultured under conditions suitable for expressing the protein and recovering the protein from the cell culture. The enzyme produced by the transformed cell may be secreted or contained within the cell, depending on the sequence and / or vector used. As will be appreciated by those skilled in the art, an expression vector containing a polynucleotide encoding an ultra high temperature α-galactosidase contains a signal sequence that allows the super high temperature α-galactosidase to be secreted directly through the prokaryotic or eukaryotic membrane. Can be designed to Other constructions can be used to link the sequences encoding the hyperthermic α-galactosidase with the nucleotide sequences encoding the polypeptide domains that will facilitate purification of soluble proteins.
[95] Enzymes are recombinant cells by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lecithin chromatography. Can be recovered and purified from the culture. If necessary, protein refolding steps can be used to complete the conformation of the mature protein. Finally, high performance liquid chromatography (HPLC) can be used for the final purification step.
[96] In general, those skilled in the art will recognize that small deletions or substitutions can occur in the amino acid sequence of the peptide without adversely affecting the activity of the peptide of the invention. Thus, peptides having such deletions or substitutions are also other aspects of the present invention. In peptides comprising substitution or replacement of amino acids, one or more amino acids of the peptide sequence may be replaced by one or more other amino acids, which replacement does not affect the function of the peptide sequence. These changes are governed by known similarities between amino acids in physical features such as charge density, hydrophobicity / hydrophilicity, size and structure such that the amino acids are replaced with other amino acids that function essentially the same. For example, Ala can be changed to Val or Ser, Val can be changed to Ala, Leu, Met or Ile, preferably Ala or Leu, and Leu is Ala, Val or Ile, preferably Val or Ile. Gly can be changed to Pro or Cys, preferably Pro, Pro can be changed to Gly, Cys, Ser or Met, preferably Gly, Cys or Ser, and Cys can be Gly, Pro, Ser or Met, preferably Pro or Met, Met can be converted to Pro or Cys, preferably Cys, His can be changed to Phe or Gln, preferably Phe, Phe is His, Tyr or Trp , Preferably, His or Tyr, and Tyr may be changed to His, Phe or Trp, preferably Phe or Trp; Trp can be changed to Phe or Tyr, preferably Tyr, Asn to Gln or Ser, preferably Gln, and Gln to His, Lys, Glu, Asn or Ser, preferably Asn or Ser Ser can be changed to Gln, Thr, Pro, Cys or Ala, Thr can be changed to Gln or Ser, preferably Ser, Lys can be changed to Gln or Arg, Arg can be Lys, Asp or Glu, preferably Lys or Asp, Asp can be changed to Lys, Arg or Glu, preferably Arg or Glu, and Glu can be changed to Arg or Asp, preferably Asp. Once the change is made, the change can be routinely screened to determine the effect of the change on function using an enzyme. In addition to recombinant production, fragments of ultra high temperature α-galactosidase can be produced by direct peptide synthesis using solid phase techniques (J. Merrifield, J. Am. Chem. Soc. 85, 2149-2154 (1963)). Protein synthesis can be performed using manual techniques or through automation. Automated synthesis can be accomplished using, for example, an Applied Biosystem 431A Peptide Synthesizer (Perkin Elmer). Full-length molecules can be obtained by chemically synthesizing the various fragments of the hyperthermic α-galactosidase separately and linking using chemical methods.
[97] D. Methods and Compositions Using Ultra-High Temperature α-Galactosidase
[98] Isolated α-galactosidase is useful for hydrolysis of galactose-containing oligosaccharides and compounds, substrates and complex mixtures comprising the same. Oligosaccharides hydrolyzed by the α-galactosidase of the present invention include, but are not limited to, raffinose, starchiose, verbacose and PNP-galactose.
[99] In a preferred embodiment, the α-galactosidase of the invention is useful for the preparation of animal feed. Animals include mammals, birds, fish and reptiles, with mammals and birds being particularly preferred. If the animal is a mammal, livestock, including cattle, pigs, horses and goats, are preferred. If the animal is a bird, preferred animals are chickens and turkeys. If the animal is fish, the preferred animal is maggie.
[100] In general, animal feed (e.g., chicken and other poultry feed, livestock feed, pet animal feed) is essential for providing the feed in the desired form with a variety of ingredients or components found to be necessary (i.e., "active ingredients"). Prepared by mixing with a carrier material. The feed or feed ingredient may preferably be any ingredient desired, including protein and carbohydrate sources. The choice of active ingredient may depend on the particular value or nutritional value that can be obtained by its activity. Enzymes or proteins, amino acids, pigments, vitamins, antioxidants, antibiotics, colorants and carotenoids may also be added to the feed. In particular, these components can be added simultaneously or sequentially.
[101] The protein component of the animal feed is preferably in the form of several kinds of protein flour (ie soy flour). Suitable forms of protein flour have been described in detail above. Other exemplary sources of protein include single cell proteins or hydrolysates of proteins such as yeast, algae or bacteria; Isolated animal proteins, peptides or hydrolysates of proteins such as hemoglobin, myosin, plasma or other serum proteins, collagen, casein, albumin or keratin; Complex protein sources or hydrolysates of proteins such as milk, blood, whey, blood flour, meat flour, feather flour, fish meal, meat and bone flour, poultry excretion, poultry byproduct flour, hatchery by-products, egg excretion, egg whites, Egg yolks, and shellless eggs; Plant proteins or hydrolysates of proteins such as isolated soy protein, wheat protein, wheat germ, distilled grains and gluten. In a preferred embodiment of the invention, the protein source of the animal feed is a vegetable protein source, and in a more preferred embodiment the animal feed is soybean in any of the soy forms available including soy flour, soy flakes, coarse soy flour, and the like. .
[102] Carbohydrates included in animal feed provide a nutritional source for the animal and may also help in the formation of a solid feed. Useful carbohydrates include corn starch, potato starch, wheat starch, rice starch, cellulose, pectin, agarose and gum; Bioavailable sugars such as glucose, fructose and sucrose; Chemically modified starches such as modified corn starch, methylcellulose, carboxymethylcellulose and dextrins; Wetting agents, such as glycerol or propylene glycol; Telephone party; And corn, rice, oats, in whole form, crushed form, broken form, milled form, rolled form, extruded form, pellet form, degreased form, dehydrated form, solvent extracted form or other processed form. Ground carbohydrates include ground carbohydrates such as barley, wheat, sugar cane, rye, sorghum, cassava, rye wheat and tapioca.
[103] Animal feed may include and preferably contain water (ie, water) with the ingredients. In one embodiment, the animal feed may be formed from a colloidal solution containing gum dissolved in water. The gums that can be used for this purpose can produce gels in suitable solvents and generally have colloidal properties, high molecular weight molecules of plant or animal origin, such as agar, algin and carrageenan derived from seaweed; Vegetable exudate such as gum arabic, ghatti and tragacanth; Vegetable extracts such as pectin; Plant seeds such as guar, locust beans; And animal exudates such as plasma, serum albumin, albuminine, chitin and gelatin. Other rubbers include amylose, amylopectin, and rubber from bacteria.
[104] Animal feed is preferably stabilized against microbial growth. That is, if properly treated, the animal feed will not show signs of microbial growth when sealed and stored for a long time at least about 8 weeks at room temperature. The feed can be stabilized, for example, by sterilization, by adding a microbial growth inhibitor such as methyl paraben or sorbate thereof, or by adjusting the pH of the mixture from which the feed is formed.
[105] In some cases, such as in the case of long term feeding, the feed preferably comprises an amino acid source such as protein (s), amino acids, precursors or homologues of amino acids, and mixtures thereof. Exemplary amino acids are essential amino acids such as methionine, tryptophan, threonine, arginine and lysine. Exemplary amino acid precursors are 2-hydroxy-4- (methylthio) butanoic acid, and calcium and sodium salts sold under the trade name Aliment® by Novus International (St. Louis, Mo.). Salts of 2-hydroxy-4- (methylthio) butanoic acid.
[106] Fats or lipids may also be included in the feed in relatively small proportions, although in certain cases undesirable. Suitable fats include fatty acids such as linoleic acid; Isolated vegetable oils such as sunflower, safflower, soybean, peanut, canola, corn, rapeseed, olive, flaxseed and palm; Fat flour such as cottonseed, peanut, rapeseed, palm flour and nut flour; And fats from animals such as egg yolk, lard, butter, poultry fat, resins and fish oils.
[107] Animal feeds may also contain vitamins and minerals. Vitamin additives can be selected from, for example, vitamins A, B ₁₂ , biotin, choline, polarine, niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, C, D, 25-hydroxy D, E and K. Mineral additives can be selected from, for example, calcium, phosphorus, selenium, chlorine, magnesium, potassium, sodium, copper, iodine, iron, manganese and chromium picolinate.
[108] Animal feed may also include other non-α-galactosidase enzymes such as hydrolases that target other classes of compounds such as proteins, non-starch polysaccharides, lipids, and the like. A more complete list of enzymes, hormones, antibiotics, colorants, stabilizers, amino acid sources and enzymes that can be used in the present invention is described in US Pat. No. 5,985,336 (Ivey et al.), Which is incorporated by reference in its entirety. It shall be included in this specification.
[109] Processing of the components of the animal feed (eg, soy flour) and the animal feed itself may involve several steps performed at high temperatures (ie, temperatures above 60 ° C., 70 ° C. or 80 ° C.). In particular, the components of the animal feed can be exposed to steam treatment during processing, for example, to obtain specific nutritional properties by removing solvents or "cooking" the flour. Generally, the processing of animal feed ends with an extrusion process or a molding process in which the feed is molded into pellets or other forms suitable for animal ingestion. Said suitable form may be a powder, pellet, solution or suspension. Preferred forms will depend on the conditions of use, the composition, and the method of delivery to the end user's destination.
[110] In the present invention, the ultra-high temperature α-galactosidase described herein can be used in shells, hulls or shells such as soybeans, other beans, legumes, corn, wheat, oats, sugar beets, canola, rice or other cereals. Or after removal from the protein source, it may be added to the animal feed at any point in the feed or flour processing below the pelleting or extrusion process of the animal feed. For example, super high temperature α-galactosidase may be added when formulating various components of the feed. The only restriction on the timing at which super high temperature α-galactosidase can be added in the feed processing process is that the enzyme is run under high temperature (ie, temperatures above about 60 ° C, 70 ° C, 75 ° C or 80 ° C) Must be added before the processing step is done. Preferably, the hyperthermic α-galactosidase is added before the ingredient of the animal feed or any steam treatment the animal feed may receive during processing.
[111] The hyperthermic α-galactosidase may be added to the animal feed or to the components of the animal feed in any suitable form, including in liquid form (ie, in the form of enzymes in solution or culture) or in dry powder form. When added in dried form, the ultra high temperature α-galactosidase may be spray dried, lyophilized, freeze dried or dried by any other suitable method known in the art. The hyperthermic α-galactosidase may be added in crude form, partially purified form, substantially purified form or in purified form.
[112] The addition of ultra high temperature α-galactosidase during the processing of feed yields advantages that are not available in the prior art. The ultra high temperature α-galactosidase is active at high temperatures used in animal feed processing, thus eliminating the need for enzymes after pelletization or extrusion processes. After treatment with the hyperthermic α-galactosidase, the animal feed contains galactose and sucrose monomers as a usable and digestible energy source for the animal. Since anti-nutritive factors (ie, indigestible oligosaccharides) are removed by the enzymes, energy values are increased due to increased galactose and sucrose utilization, and protein utilization is also increased. Finally, since the hyperthermic α-galactosidase exhibits activity before digestion of the powder by the animal, it is possible to reliably obtain any benefit of nutrition provided by the degradation of the oligosaccharides by the animal.
[113] In another embodiment of the invention, the ultra high temperature α-galactosidase is used as a food additive for human food. The advantage of using the super high temperature α-galactosidase of the present invention lies in the high temperature activity of the enzyme. That is, the enzyme can be added to the food prior to the preparation process such as cooking, because the increased temperature processed in the food by cooking or heat treatment activates the enzyme. In this embodiment, the hyperthermic α-galactosidase is incorporated into the food prior to the packaging of the food (eg, for soymilk to be heated) or on the food prior to cooking. Thus, the activity of the hyperthermic α-galactosidase that degrades indigestible oligosaccharides acts to reduce gastrointestinal discomfort as described above.
[114] When used as a food additive, ultra high temperature α-galactosidase can be used in any of several forms, including liquids or powders. The enzyme in powder form can be packaged or stored in a “salt-shaker” or other type of powder dispenser, and the powder can be sprayed onto food before cooking. The ultra high temperature α-galactosidase in powder form may be combined with one or more excipients which may be in powder or dry form. Representative examples of dry ingredients that may be combined with food grade α-galactosidase include, but are not limited to, dextrose, dicalcium phosphate, microcrystalline cellulose, modified cellulose and modified starch. These excipients can be purchased from known commercial vendors. In addition to the function of these excipients as non-digestible non-toxic carriers of α-galactosidase, the criteria for selecting such excipients are their taste and ease of flow.
[115] Super high temperature α-galactosidase in liquid form may be added to food from bottles, cans or other containers. Concentrated (highly pure) liquid α-galactosidase may be formed by dissolving the enzyme in dry or powder form in a solvent such as water or mixing with the solvent. The enzyme in liquid form may be diluted with other suitable diluents or excipients. Dilution will depend on the intended use. Representative examples of liquid excipients include, but are not limited to, water, glycerol and sorbitol. Criteria for selecting a suitable liquid excipient include miscibility, stabilization quality and taste.
[116] In another embodiment of the present invention, processing that is useful for the production of edible vegetable protein products (also referred to herein as edible protein isolates or edible protein concentrates), such as edible soy protein products. It can be used as an additive. Specifically, the hyperthermic α-galactosidase of the present invention may assist in the process of removing unwanted oligosaccharides and galactose monomers from protein products, so that the galactose or oligosaccharide component is partially, substantially or It is possible to prepare completely free plant protein products.
[117] Methods for obtaining isolated soy protein or other vegetable proteins are known (see, eg, US Pat. No. 5,936,069 (Johnson et al.) And website (www.centralsoya.com)). Removing oligosaccharides and carbohydrates from isolated protein products is often desirable for nutritional reasons. Current methods of removing oligosaccharides from protein isolates are often time consuming, expensive and difficult.
[118] As an example, in the process of the present invention using the processing of isolated soy protein, ultra-high α-galactosidase is added to the soy substrate (eg, soy flake mixture) during the processing of edible soy protein. Subsequently, this mixture containing soybean substrate and ultra high temperature a-galactosidase is subjected to the temperature at which the super high temperature a-galactosidase is active for a time sufficient to cause the oligosaccharides in the soy mixture to be hydrolyzed as described above. Heat to The hyperthermic α-galactosidase may be added before or after removing the oil from the soybean substrate, but preferably in its isolated form prior to extraction or fractionation of the soy protein. Once hydrolyzed, oligosaccharides can be removed from soy protein isolated by methods known in the art, such as washing the protein with water or aqueous alcohol, or isoelectric leaching. Thus, it is possible to produce edible protein products derived from soybean processing, which are partly or completely absent in galactose-containing oligosaccharides. In this way, it is possible to reduce or prevent gastrointestinal discomfort in the consumer of isolated soy protein in that undesirable oligosaccharides are removed from the isolated soy protein as described above.
[119] The following examples are intended to illustrate the invention and should not be construed as limiting the invention.
[120] Example 1
[121] Tm gal Cloning and Expression of A
[122] Tm gal A was cloned via PCR from genomic samples of Tm total DNA. After 35 cycles, a single PCR product of approximately 1.65 kb in length was obtained. Restriction mapping with BamHI, XhoI, NdeI, KpnI and HindIII produced DNA bands of the correct band pattern and the correct size when compared to restriction maps obtained from published DNA sequences. SEQ ID NO: 1 is the Tm gal A nucleotide sequence published in Genebank Database (Accession No. 2660640). This sequence was used to make PCR primers used for cloning this gene.
[123] Tm gal A was transformed into E. coli using pET24d + as the expression vector. It was expressed in E. coli BL21 (λDE3) (Novagen, Madison, Wi; Stratagene, La Jolla, Calif.). After heat treatment (80 ° C., 30 minutes) of French press cell extracts, approximately 330 units of soluble Tm GalA activity were recovered from 4 L of culture (1 unit of enzyme activity was determined by 1 μmol of PNP from PNP-galactose per minute). Defined as the amount of enzyme required to liberate). A single protein band of approximately 64 kDa was clearly identified on the 12% SDS-PAGE gel. This band corresponds to a single monomer of Tm GalA as already described in W. Liebl et al., System. Appl. Microbiol. 21,1-11 (1998).
[124] Example 2
[125] Tm Activity of GalA
[126] Temperature and pH optimum values were determined using PNP-galactose as the substrate in endpoint analysis measuring release of free PNP at 405 nm after 10 minutes. In summary, 1 mL of assay solution contained enzymes diluted appropriately in 1 mM PNP-galactose, and 50 mM Na acetate buffer containing 1 mM NaCl. After 10 minutes, the reaction was stopped by adding 100 μl of 1 M Na ₂ CO ₃ and placing the reaction mixture on ice. 1 and 2 show the activity (%) of the enzyme as a function of pH and temperature, respectively. From these figures, the optimum reaction appears to occur near a pH of 4.5 and a temperature of 85 ° C.
[127] As shown in Table 1, Tm GalA activity decreases with increasing degree of polymerization (DP) of the substrate. Maximum Tm GalA activity is achieved using PNP-galactose as substrate, the maximum activity is reduced by approximately 2.5-fold with Raffinose (DP3) and approximately 20 with Stakiose (DP4) and Verbacose (DP5). Decreases fold. It is expected that a further decrease in specific activity will be observed when the substrate is changed from Stachios to Verbascose. However, these observations are not visible within the error of the analytical technique used (Consumer-Nelson technique). Sobigy-Nelson technology analyzes the production of total sugars that can be reduced. Thus, it is not possible to distinguish galactose freed from Verbacose or galactose freed from Starchis and the product obtained after removing galactose from Verbacose.
[128] Tm GalA specific activity temperamentSpecific activity (μmol min ^-1 , mg protein ^-1 ) PNP-galactose32.5 ± 1.6 Raffinos12.79 ± 1.31 Stachios0.55 ± 0.19 Verbascos0.44 ± 0.16
[129] Example 3
[130] Of chicken feed Tm GalA decomposition
[131] The definite effect of Tm GalA degradation on soluble chicken feed composition was seen both in the direct enzymatic treatment of the feed and in the treatment of the redissolved ethanol extract components. Ethanol extraction provides a means of more precisely performing Tm GalA degradation of chicken feed components by extracting the water soluble carbohydrate fraction from the feed matrix. Carbohydrates extracted by this technique are typically limited to DP <8. 25 g of the feed were extracted with 250 mL of boiling 80% ethanol for 2 hours under complete reflux. Upon evaporation of ethanol, an orange residue remained. This residue could be mixed for 5 minutes, heated at 85 ° C. for 30 minutes and then partially dissolved in 10 volumes of water (w / v). HPLC analysis of this soluble fraction showed three distinct peaks at approximately 37, 42 and 46 minutes. Peaks appearing at approximately 37 minutes and 42 minutes could be identified as starchiose and sucrose, respectively, based on retention time compared to known standards. After treating the soluble fraction with 15 units of Tm GalA for 1 hour, it can be observed that the peak of stachyose disappeared completely. In this particular experiment the initial starchiose concentration was estimated to be approximately 5.6 mM. In addition to the disappearance of starchios, not only the appearance of galactose peaks at approximately 47 minutes, but also an increase in the sucrose peaks from an area count of 4.77 x 10 ^{7 to} an area count of 5.44 x 10 ⁷ at t = 0 after 1 hour can be observed simultaneously. Can be.
[132] When feed (100 mg / ml) was directly treated with 50 units of Tm GalA, similar results were obtained when digesting the soluble fraction extracted. In this experiment, feed was preheated at 98 ° C. for 2 hours before the addition of enzyme. As in the previous study, it can be observed that within 1 hour, the starchiose peak disappears completely and the galactose peak appears in the HPLC chromatogram.
[133] Example 4
[134] Soybean Powder and Soy Flakes Tm Effect of Temperature and Moisture Content on GalA Decomposition
[135] The effect of temperature on direct Tm GalA degradation of soy flour and soy flakes is listed in Table 2. Experiments were performed using 4 g soy flour or soy flakes, 500 U of flour or 50 U of α-Gal and 70% moisture content per flake. Soy flour / bean flake-α-Gal mixtures were incubated for a total of 45 minutes at the temperatures listed in Table 2. At 5 minute intervals during the course of the experiment, samples of the mixture were placed on ice and immediately extracted with 80% ethanol and further treated as described in Example 3. The resuspended fractions were then analyzed by HPLC. Peaks appearing at approximately 35 minutes, 39 minutes and 42 minutes were confirmed to be Stachiose, Raffinose and Sucrose, respectively. Maltohexaose or maltopentaose were used as internal standard. Residual stachios and rapitose concentrations were linearly regression analyzed over time to obtain the rates shown in Table 2. From this data, the rate at which starchiose and raffinose are removed from soy flour and soy flakes decreases as the temperature decreases. From this one can predict the given temperature / activity profile of the enzyme.
[136] Rate of oligosaccharide removal as a function of temperature ^{a, b} Soybean PowderBean flakes TemperatureStachiosRaffinosStachiosRaffinos 90 ℃1.50 (0.951) ^c 5.70 (0.967)1.25 (0.829)4.50 (0.999) 80 ℃0.76 (0.903)2.69 (0.996)0.84 (0.936)3.28 (0.975) 70 ℃Can be ignored1.71 (0.926)Can be ignored1.56 (0.675) a Experiment was performed using 50 U of α-Gal at an excess moisture content of 70%. b Rate is expressed as% oligosaccharides removed per minute, per g of substrate, per U of α-Gal. The number in the horizontal column represents the R ² value for a given experiment.
[137] The effect of moisture content on direct Tm GalA degradation of soy flour and soy flakes is listed in Table 3. This experiment was performed under the same conditions as described above except that the temperature was fixed at 90 ° C. and the moisture content varied as described in Table 3. Prior to incubation at 90 ° C., the soy flour / soy flake-α-Gal mixture was incubated at 45 ° C. under vacuum until the proper moisture content was obtained to vary the water content. After this treatment, the experiment was performed as described above. It can be clearly seen from the data in Table 3 that the water content does not significantly affect the rate of α-Gal degradation of soy flour and soy flakes until perhaps some critical water content is reached.
[138] Removal rate of oligosaccharides as a function of excess moisture content ^{a, b} Soy flourBean flakes Moisture contentStachiosRaffinosStachiosRaffinos 70%1.50 (0.951) cc5.70 (0.967)1.25 (0.829)4.50 (0.999) 45%1.70 (0.9541)2.91 (0.769)1.74 (0.966)Not measured 25%1.26 (0.915)5.20 (0.943)1.26 (0.918)Not measured 10%---- a Experiment was performed using 50 U of α-Gal at 90 ° C. b Rate is expressed as% oligosaccharides removed per minute, per g of substrate, per 1 U of α-Gal. Represents the R ² value of a given experiment.
[139] Example 5
[140] Summary of results
[141] Thermomoto successfully cloned the α-galactosidase (GalA) of Marittima (TM) DSM3109 and first characterized it. This enzyme has an optimal pH of about 4.5-5.0 and an optimal temperature of about 85-90 ° C. This enzyme shows activity against PNP-galactose, raffinose (DP3), stachiose (DP4) and verbacose (DP5). The specific activity of Tm GalA with various substrates is listed in Table 1. In addition, the enzyme was found to have a half-life of 70 minutes at pH 7 and 90 ° C., suggesting the ability to survive in the steaming step during feed processing. In addition, Tm GalA showed only 3% of its maximum activity against PNP-galactose at 25 ° C. (pH 4.5), indicating that Tm GalA activity at room temperature for the higher degree of polymerization of raffino-oligosaccharides may be minimal. It means that there is.
[142] Early Tm GalA digests of chicken feeds with high protein and carbohydrate content showed good results. Tm GalA degradation of dissolved ethanol extracted chicken feed components has been shown to be effective in removing what Tm GalA has tentatively identified as starchiose from the feed by the inventors. It has also been observed that soluble stachyose is removed from raw and unprocessed chicken feed.
[143] The foregoing is intended to illustrate the invention but not to limit the invention. The invention is defined by the following claims, together with the same scope as the following claims, which are included herein.

权利要求:
Claims (45)
[1" claim-type="Currently amended] Contacting the substrate with an ultra high temperature α-galactosidase; And
Heating the substrate to a temperature at which the hyperthermic α-galactosidase is active for a time sufficient to hydrolyze the galactose-containing oligosaccharides
A method of hydrolyzing galactose-containing oligosaccharides present in a substrate comprising a.
[2" claim-type="Currently amended] The method of claim 1, wherein said oligosaccharide is selected from the group consisting of raffinose, starchiose and verbacose.
[3" claim-type="Currently amended] The method of claim 1, wherein the substrate is selected from the group consisting of animal feed, soy flour, and human food.
[4" claim-type="Currently amended] According to claim 1, in a high temperature property α- galactosidase is I moto MOTO is Thermotoga maritima (Thermotoga maritima), writing write Neo poly appear (Thenmotoga neopolitana), write a moto elpiyi (Thermotoga elfii) and write morpho species T2 Isolated from the group consisting of.
[5" claim-type="Currently amended] The method of claim 1, wherein the ultra high temperature α-galactosidase is isolated from the marimotima.
[6" claim-type="Currently amended] The method of claim 1, wherein the ultra high temperature α-galactosidase is isolated from Marimotima DSM3109.
[7" claim-type="Currently amended] The method of claim 1, wherein the oligosaccharide is hydrolyzed to galactose monomers.
[8" claim-type="Currently amended] The process of claim 1 which is carried out under 70% moisture conditions.
[9" claim-type="Currently amended] The method of claim 1 wherein the process is performed under 25% moisture conditions.
[10" claim-type="Currently amended] The method of claim 1, wherein heating occurs at 80 ° C., 85 ° C., 90 ° C., or 100 ° C. 7.
[11" claim-type="Currently amended] The method of claim 1, wherein the ultra high temperature α-galactosidase
(a) culturing a host cell comprising an expression vector containing a polynucleotide sequence encoding an ultra high temperature α-galactosidase;
(b) expressing an ultra high temperature α-galactosidase;
(c) recovering the ultra high temperature α-galactosidase from the host cell culture.
That is produced.
[12" claim-type="Currently amended] The method of claim 11, wherein said polynucleotide has the sequence of SEQ ID NO: 1.
[13" claim-type="Currently amended] The method of claim 11, wherein the polynucleotide is
(a) DNA having a nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide encoding an ultra high temperature α-galactosidase and hybridizing to the DNA of (a) under stringent conditions; And
(c) polynucleotides encoding ultra high temperature α-galactosidase and differing from the DNA of (a) or (b) due to the degeneracy of the genetic code
And selected from the group consisting of:
[14" claim-type="Currently amended] The method of claim 11, wherein said polynucleotide encodes an ultra high temperature α-galactosidase having the amino acid sequence of SEQ ID NO: 2.
[15" claim-type="Currently amended] Contacting the components of the animal feed composition with the hyperthermic α-galactosidase for a time sufficient for the hyperthermic α-galactosidase to hydrolyze the galactose-containing oligosaccharides prior to the heating step in the processing of the animal feed. A method of producing an animal feed composition comprising a hydrolyzed galactose-containing oligosaccharide comprising a.
[16" claim-type="Currently amended] The method of claim 15, wherein said galactose-containing oligosaccharides are selected from the group consisting of raffinose, starchiose and verbacose.
[17" claim-type="Currently amended] The method of claim 15, wherein said animal feed is a feed or chicken feed comprising soy flour or soy flakes.
[18" claim-type="Currently amended] The method of claim 15, wherein the hyperthermic α-galactosidase is isolated from the group consisting of thermomoto maritima, thermomoto neopolitana, thermomoto elpi and thermomoto species T2.
[19" claim-type="Currently amended] The method of claim 15, wherein the hyperthermic α-galactosidase is isolated from the marimoto from the marimoto.
[20" claim-type="Currently amended] The method of claim 15, wherein the ultra high temperature α-galactosidase is isolated from Marimotima DSM3109.
[21" claim-type="Currently amended] The method of claim 15, wherein said oligosaccharide is hydrolyzed to galactose monomers.
[22" claim-type="Currently amended] The method of claim 15, wherein the contact of the hyperthermic α-galactosidase with the components of the animal feed composition is performed under 70% moisture, 25% moisture, or 45% moisture conditions.
[23" claim-type="Currently amended] The method of claim 15, wherein the heating occurs at 80 ° C., 85 ° C., 90 ° C., or 100 ° C. 16.
[24" claim-type="Currently amended] The contact of the components of the animal feed composition with the hyperthermic α-galactosidase occurs before the final pelletization step in animal feed processing.
[25" claim-type="Currently amended] The method according to claim 15, wherein the ultra high temperature α-galactosidase
(a) culturing a host cell comprising an expression vector containing a polynucleotide sequence encoding an ultra high temperature α-galactosidase;
(b) expressing an ultra high temperature α-galactosidase;
(c) recovering the ultra high temperature α-galactosidase from the host cell culture.
That is produced.
[26" claim-type="Currently amended] The method of claim 25, wherein the polynucleotide has the sequence of SEQ ID NO: 1.
[27" claim-type="Currently amended] The method of claim 25, wherein said polynucleotide is
(a) DNA having a nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide encoding an ultra high temperature α-galactosidase and hybridizing to the DNA of (a) under stringent conditions; And
(c) polynucleotides encoding ultra high temperature α-galactosidase and differing from the DNA of (a) or (b) due to the degeneracy of the genetic code
And selected from the group consisting of:
[28" claim-type="Currently amended] The method of claim 25, wherein said polynucleotide encodes an ultra high temperature α-galactosidase having the amino acid sequence of SEQ ID NO: 2.
[29" claim-type="Currently amended] The method of claim 15, wherein the hyperthermic α-galactosidase is in liquid solution form, dried form, partially purified form or substantially purified form when contacted with the components of the animal feed composition.
[30" claim-type="Currently amended] Animal feed produced according to the method of claim 15.
[31" claim-type="Currently amended] A food additive for reducing gastrointestinal discomfort in a mammal, comprising ultra high temperature α-galactosidase.
[32" claim-type="Currently amended] 32. The food additive of claim 31, wherein the hyperthermic α-galactosidase is isolated from the group consisting of thermomoto maritima, thermomoto neopolitana, thermomoto elpi and thermomoto species T2.
[33" claim-type="Currently amended] 32. The food additive of claim 31, wherein the hyperthermic α-galactosidase is isolated from the marimoto from the thermomoto.
[34" claim-type="Currently amended] 32. The food additive of claim 31, wherein the ultra high temperature α-galactosidase is isolated from marimotoma DSM3109.
[35" claim-type="Currently amended] 32. The method of claim 31, wherein the hyperthermic α-galactosidase is
(a) culturing a host cell comprising an expression vector containing a polynucleotide sequence encoding an ultra high temperature α-galactosidase;
(b) expressing an ultra high temperature α-galactosidase;
(c) recovering the ultra high temperature α-galactosidase from the host cell culture.
Food additives that are produced.
[36" claim-type="Currently amended] The food additive of claim 35 wherein the polynucleotide has the sequence of SEQ ID NO: 1.
[37" claim-type="Currently amended] 36. The method of claim 35, wherein said polynucleotide is
(a) DNA having a nucleotide sequence of SEQ ID NO: 1;
(b) a polynucleotide encoding an ultra high temperature α-galactosidase and hybridizing to the DNA of (a) under stringent conditions; And
(c) polynucleotides encoding ultra high temperature α-galactosidase and differing from the DNA of (a) or (b) due to the degeneracy of the genetic code
Food additives selected from the group consisting of.
[38" claim-type="Currently amended] 36. The food additive of claim 35 wherein the polynucleotide encodes an ultra high temperature α-galactosidase having the amino acid sequence of SEQ ID NO: 2.
[39" claim-type="Currently amended] Contacting a food containing at least one oligosaccharide selected from the group consisting of raffinose, starchiose and verbacose with an ultra high temperature α-galactosidase; And heating said food for a time sufficient for said ultra high temperature a-galactosidase to hydrolyze said oligosaccharide. 2. The method of preventing gastrointestinal discomfort caused by said food in a mammal.
[40" claim-type="Currently amended] A processing additive for removing galactose-containing oligosaccharides in the manufacturing process of edible soy protein, comprising ultra high temperature α-galactosidase.
[41" claim-type="Currently amended] (a) contacting the soybean substrate with an ultra high temperature α-galactosidase;
(b) heating the soybean substrate at a temperature suitable for hydrolysis of the oligosaccharides for a time sufficient to hydrolyze the galactose-containing oligosaccharides; And
(c) removing the hydrolyzed galactose-containing oligosaccharides from the soybean substrate prior to final extraction or fractionation of the edible soy protein.
A method of producing edible soy protein by removing galactose-containing oligosaccharides from soybean substrates to be processed.
[42" claim-type="Currently amended] 42. The method of claim 41, wherein heating occurs before removing oil from the soybean substrate.
[43" claim-type="Currently amended] 42. The method of claim 41 wherein the heating occurs after removing oil from the soybean substrate.
[44" claim-type="Currently amended] 42. The method of claim 41 wherein the soybean substrate is soybean flakes.
[45" claim-type="Currently amended] An isolated edible soy protein produced by the method of claim 41.

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同族专利:

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公开号 | 公开日

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JP2004504043A|2004-02-12|

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PL365191A1|2004-12-27|

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AU8584901A|2002-02-05|

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MXPA03000601A|2004-09-09|

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ZA200300473B|2004-04-20|

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US20020102329A1|2002-08-01|

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IL153080D0|2003-06-24|

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WO2002007529A3|2002-09-19|

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WO2002007529A2|2002-01-31|

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CA2409415A1|2002-01-31|

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EP1305433A2|2003-05-02|

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HU0302045A2|2003-09-29|

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CN1610746A|2005-04-27|

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BR0112651A|2003-06-24|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-07-22|Priority to US22021100P

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2000-07-22|Priority to US60/220,211

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2001-07-20|Application filed by 신젠타 파티서페이션즈 아게

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2001-07-20|Priority to PCT/EP2001/008420

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2003-05-09|Publication of KR20030036621A

优先权:

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申请号 | 申请日 | 专利标题

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US22021100P| true| 2000-07-22|2000-07-22|

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US60/220,211|2000-07-22|

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PCT/EP2001/008420|WO2002007529A2|2000-07-22|2001-07-20|Methods for high-temperature hydrolysis of galactose-containing oligosaccharides in complex mixtures|