Wet-milled GH5 and GH30
阅读说明：本技术 湿磨中的gh5和gh30 (Wet-milled GH5 and GH30 ) 是由 T·P·吉本斯 B·维达尔 J·拉维妮 M·J·阿克曼 沈心妤 曹轶 里伟 张羽 S 于 2018-07-24 设计创作，主要内容包括：本申请提供了在湿磨过程中改善来自玉米籽粒的总淀粉和/或谷蛋白产率的方法,该方法包括将玉米籽粒或玉米籽粒的级分与包含有效量的一种或多种水解酶的酶组合物混合,其中所述水解酶中的至少一种选自由以下组成的组：GH30多肽、GH5多肽或其组合。(The present application provides a method of improving the total starch and/or gluten yield from corn kernel during wet milling, the method comprising mixing corn kernel or a fraction of corn kernel with an enzyme composition comprising an effective amount of one or more hydrolytic enzymes, wherein at least one of the hydrolytic enzymes is selected from the group consisting of: a GH30 polypeptide, a GH5 polypeptide, or a combination thereof.)
1. A method of improving the overall starch yield and/or gluten yield from corn kernel during wet milling, the method comprising mixing corn kernel or a fraction of corn kernel with an enzyme composition comprising an effective amount of one or more hydrolytic enzymes, wherein at least one of the hydrolytic enzymes is selected from the group consisting of: a GH30 polypeptide, a GH5 polypeptide, or a combination thereof.
2. The method according to claim 1, wherein the amount of starch and/or gluten released from the fiber during the wet milling process is increased.
3. The method according to any one of the preceding claims, comprising the steps of:
a) soaking the corn kernels in water to produce soaked kernels;
b) milling the soaked kernels to produce soaked and milled kernels;
c) separating the germ from the soaked and milled kernels to produce a corn kernel material comprising fiber, starch, and gluten; and
d) subjecting the resulting corn kernel material to a fiber washing procedure.
4. The method of any one of the preceding claims, wherein the corn kernel or a fraction of the corn kernel is mixed with the one or more hydrolytic enzymes before, during or after step d) of claim 3.
5. The method of any one of the preceding claims, wherein the corn kernel or a fraction of the corn kernel is mixed with the one or more hydrolytic enzymes during step d) of claim 3.
6. The method of any one of the preceding claims, wherein the corn kernel or a fraction of the corn kernel is allowed to contact the one or more hydrolytic enzymes for at least 15 minutes.
7. The method according to any one of claims 3-6, wherein the fiber washing program comprises using a fiber washing system optimized for introducing one or more hydrolytic enzymes, and wherein the fiber washing system comprises a space configured to provide a total retention time in the fiber washing system of at least 35 minutes and less than 48 hours.
8. The method according to any of the preceding claims, wherein the incubation time in said space in the fibre washing system is configured to be at least 5 minutes and less than 48 hours.
9. The method according to any one of the preceding claims, wherein the incubation temperature is between 25 ℃ and 95 ℃.
10. The method of any one of the preceding claims, wherein the GH5 polypeptide has xylanase activity.
11. The method of any one of the preceding claims, wherein the GH30 polypeptide has xylanase activity.
12. A method according to any one of the preceding claims, wherein the GH5 polypeptide further comprises one or more of endo- β -1, 4-glucanase/cellulase (EC 3.2.1.4) activity and/or endo- β -1, 4-xylanase (EC 3.2.1.8) activity and/or β -glucosidase (EC3.2.1.21) activity and/or β -mannosidase (EC3.2.1.25) activity and/or β -glucosylceramide (EC β) activity and/or glucan β -1, 3-glucosidase (EC β) activity and/or lichenase (EC β) activity and/or exo- β -1, 4-glucanase/dextrinase (EC 3.2.1.74) activity and/or glucan endo-1, 6-glucosidase (EC 72) activity and/or endoglucanase-6-glucosidase (EC 72) activity and/or endoglucanase 1-72-endoglucanase (EC 3.72) activity and/or endoglucanase-72-endoglucanase (EC 1.72) activity and/or endoglucanase (EC 3-72) activity and/or cellobiosidase (EC 3-72) activity and/or a xylanase (EC 3-72) activity.
13. The method of any one of the preceding claims, wherein the GH30 polypeptide further comprises one or more of endo- β -1, 4-xylanase (EC 3.2.1.8) activity and/or β -glucosidase (EC3.2.1.21) activity and/or β -glucuronidase (EC3.2.1.31) activity and/or β -xylosidase (EC 3.2.1.37) activity and/or β -fucosidase (EC 3.2.1.38) activity and/or glucosylceramidase (EC 3.2.1.45) activity and/or β -1, 6-glucanase (EC 3.2.1.75) activity and/or glucuronoaarabinoxylan- β -1, 4-xylanase (EC3.2.1.136) activity and/or endo- β -1, 6-galactanase (EC:3.2.1.164) activity and/or [ terminal ] reducing enzyme (EC β -xylosidase (EC3.2.1.31) activity.
14. The method of any one of the preceding claims, wherein the GH5 polypeptide is selected from the group consisting of:
i) 1-3, 8, 10 or 12;
ii) a mature polypeptide having at least 60% identity to the mature polypeptide of i)
iii) a subsequence of any one of mature polypeptides in i) and ii).
15. The method of any one of the preceding claims, wherein the GH30 polypeptide is selected from the group consisting of:
i) mature polypeptide of the amino acid sequence shown in SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6 or SEQ ID NO 7;
ii) a mature polypeptide having at least 60% identity to the mature polypeptide of i)
iii) a subsequence of any one of mature polypeptides in i) and ii).
16. The method of any one of the preceding claims, wherein the enzyme composition comprising one or more hydrolases further comprises one or more enzymes selected from the group consisting of cellulases (EC 3.2.1.4), xylanases (EC 3.2.1.8), arabinofuranosidases (EC3.2.1.55 (non-reducing end α -L-arabinofuranosidase), EC 3.2.1.185 (non-reducing end β -L-arabinofuranosidase) cellobiohydrolases I (EC3.2.1.150), cellobiohydrolases II (E.C.3.2.1.91), cellobiosidases (E.C.3.2.1.176), β -glucosidases (E.C.3.2.1.21), β -xylosidases (EC 3.2.1.37) or proteases (E.C XXXXXX).
17. The method according to any one of the preceding claims, wherein the one or more hydrolases are expressed in an organism with a cellulase background, such as trichoderma reesei.
18. The method according to any one of the preceding claims, wherein the one or more hydrolases are purified.
19. The method according to any one of the preceding claims, wherein the one or more hydrolytic enzymes are in a liquid composition.
20. The method according to any one of the preceding claims, wherein the one or more hydrolytic enzymes are in a solid composition.
21. The method according to any one of the preceding claims, wherein the effective amount of the one or more hydrolytic enzymes mixed with the one or more fractions of the corn kernel material is between 0.005-0.5kg enzyme protein per metric ton of corn kernel entering the wet milling process.
22. A composition comprising corn fiber, said composition obtainable by the process of any one of claims 1-21.
Use of a GH30 polypeptide and/or a GH5 polypeptide in a method for improving starch yield and/or total gluten yield from corn kernel during wet milling as defined in any one of claims 1-21.
Technical Field
The present invention relates to a method of improving starch and/or gluten yield from corn kernel during wet milling, preferably during fiber washing, comprising contacting the corn kernel with an enzyme composition comprising a GH30 polypeptide, a GH5 polypeptide, or a combination thereof.
Background
Conventional corn wet milling is a process designed for the recovery and purification of starch and several by-products, including germ, gluten (protein), and fiber. Fiber is the least valuable byproduct, so industry has invested considerable effort to increase the yield of more valuable products (e.g., starch and gluten) while reducing the fiber fraction. High quality starch is valuable because it can be used for various commercial purposes after further processing into products such as dried starch, modified starch, dextrin, sweeteners, and alcohols. Gluten is commonly used in animal feed, such as corn gluten meal (about 60% protein) or corn gluten feed (about 20% protein).
The wet milling process can vary significantly depending on the particular milling equipment used, but the process typically includes: grain cleaning, steeping, milling, germ separation, secondary milling, fiber separation, gluten separation, and starch separation. After cleaning the corn kernel, typically by soaking in water or in diluted SO under controlled time and temperature conditions2Softening them in solution. The kernel is then milled to break down the pericarp, and the germ is separated from the rest of the kernel. The remaining slurry, which consists primarily of fiber, starch and gluten, is finely milled and screened in a fiber washing process to separate the fiber from the starch and gluten, then to separate the gluten and starch, and the starch may be purified in a washing/filtration process.
It has been suggested to use enzymes in several steps of the wet milling process, for example to use enzymes for the impregnation step of the wet milling process. It has been shown that commercial enzyme products
More recently, "enzymatic milling" has been developed, which is a modified wet milling process that uses proteases to significantly reduce the overall processing time during corn wet milling and eliminates the need for sulfur dioxide as a processing agent. Johnston et al, Cereal Chem [ Cereal chemistry ],81, page 626-632 (2004).
US 6,566,125 discloses a method for obtaining starch from maize involving steeping maize kernels in water to produce steeped maize kernels, milling the steeped maize kernels to produce a milled maize slurry and incubating the milled maize slurry with an enzyme (e.g., a protease).
US 5,066,218 discloses a method of grinding grain, particularly corn, which comprises cleaning the grain, steeping the grain in water to soften it, and then grinding the grain with cellulase enzymes.
WO 2002/000731 discloses a process for treating crop kernels which comprises soaking the kernels in water for 1-12 hours, wet milling the soaked kernels and treating the kernels with one or more enzymes, including acid protease.
WO 2002/000911 discloses a process for isolating starch gluten, which process comprises subjecting milled starch to an acid protease.
WO 2002/002644 discloses a process for washing a starch slurry obtained from the starch gluten separation step of a milling process, said process comprising washing the starch slurry with an aqueous solution comprising an effective amount of an acidic protease.
WO 2014/082566 and WO 2014/082564 disclose cellulolytic compositions for use in wet milling.
Although the art has investigated the effects of using enzymes in corn wet milling, there remains a need for improved enzyme technology that can reduce the energy expenditure and cost associated with corn wet milling and provide increased starch and gluten yields during steeping/steeping of corn kernels, during milling of corn kernels and during starch gluten separation.
Disclosure of Invention
In a first aspect, the present invention relates to a method for improving the overall starch yield and/or gluten yield that can be obtained from corn kernel during wet milling, the method comprising: mixing corn kernel or a fraction of corn kernel with an enzyme composition comprising an effective amount of one or more hydrolytic enzymes, wherein at least one of the hydrolytic enzymes is selected from the group consisting of: GH30 polypeptides, GH5 polypeptides, and combinations thereof.
In a second aspect, the present invention relates to an enzyme composition comprising an isolated GH30 polypeptide, an isolated GH5 polypeptide, or both, and the use of such an enzyme composition to improve the overall starch yield and/or gluten yield that can be obtained from corn kernel during wet milling.
In a third aspect, the present invention relates to a composition comprising corn starch, corn gluten or corn fiber, said composition being obtained by the process described in the first aspect and examples of the present invention.
In other aspects, the invention relates to polypeptides having xylanase activity and polynucleotides encoding the polypeptides.
In a fourth aspect, the present invention relates to an isolated polypeptide having xylanase activity, selected from the group consisting of:
(a) a polypeptide having at least 80% sequence identity to the mature polypeptide of
(b) a polypeptide encoded by a polynucleotide having at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO 9 or SEQ ID NO 11;
(c) 10 or 12, which variant comprises a substitution, selection and/or insertion at one or more positions; and
(d) a fragment of the polypeptide of (a), (b), or (c), which fragment has xylanase activity.
In a fifth aspect, the present invention relates to a composition comprising a polypeptide according to the fourth aspect of the invention.
The invention also relates to a nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide of the fourth aspect of the invention operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.
Drawings
The present invention, and in particular preferred embodiments according to the present invention, will be described in more detail with reference to the accompanying drawings. The drawings illustrate ways of implementing the invention and should not be construed as limiting other possible embodiments falling within the scope of the appended set of claims.
Figure 1 schematically shows a first embodiment of a counter current fibre washing system according to the invention,
fig. 2 schematically shows another embodiment of the system according to the invention.
FIG. 3 schematically illustrates a screen unit with a built-in incubator
Figure 4 shows schematically a screen unit in the form of a hydrocyclone
Detailed Description
It is an object of the present invention to provide a method for improving the starch and/or gluten yield obtainable from corn kernel by treating the corn kernel with a hydrolytic enzyme composition during hydrolysis, preferably in a fiber washing procedure. The present inventors have surprisingly found that treatment of corn kernel with a GH5 polypeptide or a GH30 polypeptide or a combination thereof enzyme improves the release of bound starch and gluten from the fiber and thus improves the yield of starch and/or gluten obtainable.
Wet grinding process:
corn kernel is wet milled to open the kernel and separate the kernel into its four major components: starch, germ, fiber, and gluten.
The wet milling process can vary significantly between mills, but conventional wet milling typically includes the following steps:
1. the germ is impregnated and separated, and then,
2. fiber washing program
3. Starch/gluten separation, and
4. and (4) washing the starch.
1. Macerating, milling and separating the germ
The corn kernel is softened by soaking in water at a temperature of about 50 ℃ (e.g., between about 45 ℃ to 60 ℃) for between about 30 minutes to about 48 hours (preferably 30 minutes to about 15 hours, e.g., about 1 hour to about 6 hours). During the steeping period, the kernel absorbs water, increasing its moisture content from 15% to 45% and doubling the size. Optionally adding, for example, 0.1% sulfur dioxide (SO) to the water2) And/or NaHSO3To prevent bacterial growth in warm environments. As the corn swells and softens, the mild acidity of the steep water begins to loosen gluten bonds within the corn and release starch. After the corn kernels are steeped, they are cracked to release the germ. The germ comprises corn oil. The germ is separated from the heavier density mixture of starch, gluten and fiber essentially by "floating" the germ segment free of other substances under closely controlled conditions. This method is used to eliminate any adverse effects of trace amounts of corn oil in later processing steps.
2. Fiber washing program
In order to obtain maximum starch and gluten recovery while keeping any fiber in the final product to an absolute minimum, free starch and gluten must be washed out of the fiber during processing. The fiber is collected and slurried and screened to recover any residual starch or gluten.
3. Isolation of starch gluten
The starch-gluten suspension from the fibre washing step (called ground starch) is separated into starch and gluten. Gluten has a lower density than starch. Gluten is easily spun out by passing the ground starch through a centrifuge.
4. Washing starch
The starch slurry from the starch separation step contains some insoluble protein and many solubles. Before top quality starch (high purity starch) can be produced, it must be removed. In a hydrocyclone, the starch, which is only 1% or 2% protein left, is diluted, washed 8 to 14 times, re-diluted and washed again to remove the last traces of protein and produce high quality starch, typically with a purity greater than 99.5%.
Product of wet milling:wet milling can be used to produce, but is not limited to, corn steep liquor, corn gluten feed, germ, corn oil, corn gluten meal, corn starch, modified corn starch, syrups (e.g., corn syrup), and corn ethanol.
Definition of the enzyme:
cellulolytic enzymes or cellulases/polypeptides having cellulase activity or cellulolytic activity the terms "cellulolytic enzyme", "cellulase" and polypeptide having cellulase activity or cellulolytic activity are used interchangeably herein and refer to one or more (e.g. several) enzymes which hydrolyse a cellulosic material, said material comprising any material comprising cellulose (e.g. fiber), cellulolytic enzymes including one or more endoglucanases (e.g. c3.2.1.4), one or more cellobiohydrolases (E.C.2.1.91 and E.C 3.2.1.150), one or more β -glucosidases (e.c.3.2.1.21), or combinations thereof two basic methods for measuring cellulolytic enzyme activity include (2006) measuring total cellulolytic enzyme activity and (2) measuring individual cellulolytic enzyme activity (endoglucanase, cellobiohydrolase, and β -glucosidase), as described in chehang et al, biochemy [ technologies for [ 24 ] evolution technologies ] and as a Pure cellulase activity determination by filter paper chemistry, analytical cellulose degradation [ 1, wo ] analytical chemistry, wo 10, wo, 10, usa, koshikagaku koku no.
Hydrolysis of cellulosic material by one or more cellulolytic enzymes under the following conditions may also be measuredDuring this time, the increase in sugar production/release determines the cellulolytic enzyme activity: 1-50mg cellulolytic enzyme protein per g of cellulose in Pretreated Corn Stover (PCS) (or other pretreated cellulosic material), for 3-7 days at a suitable temperature (e.g., 40 ℃ -80 ℃, e.g., 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, or 80 ℃) and at a suitable pH (e.g., 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0), compared to a control hydrolysis without cellulolytic enzyme protein added. Typical conditions are: 1ml of reacted, washed or unwashed PCS, 5% insoluble solids (dry weight), 50mM sodium acetate 5(pH 5), 1mM MnSO4, 50 ℃, 55 ℃, or 60 ℃, 72 hours by
Hydrolases having hydrolase activity (hydrolytical enzymes) or hydrolases (hydrolases)/polypeptides "hydrolase" and polypeptides having hydrolase activity are used interchangeably herein and refer to any catalytic protein that uses a water-splitting substrate the hydrolases comprise cellulase (EC 3.2.1.4), xylanase (EC 3.2.1.8), arabinofuranosidase (EC3.2.1.55 (non-reducing end α -L-arabinofuranosidase), EC 3.2.1.185 (non-reducing end β -L-arabinofuranosidase) cellobiohydrolase I (EC3.2.1.150), cellobiohydrolase II (E.C.3.2.1.91), cellobiosidase (E.C.3.2.1.176), β -glucosidase (E.C.3.2.1.21), β -xylosidase (EC 3.2.1.37).
Xylanase/polypeptide having xylanase Activity the terms "xylanase" and polypeptide having xylanase activity are used interchangeably herein and refer to 1,4- β -D-xylan-xylanase (E.C.3.2.1.8) which catalyzes the endo-hydrolysis of 1,4- β -D-xylosidic bonds in xylan xylanase activity may be 0.01% at 37 ℃
Other definitions:
in this context, terms are used in a manner that is common to the skilled person. Some of these terms are set forth below:
contact time: for one or more enzymes to react with a substrate, the one or more enzymes must be in contact with the substrate. By "contact time" is meant the period of time that an effective amount of one or more enzymes is in contact with at least a portion of the substrate material. The enzyme may not be in contact with all of the substrate material during the contact time, however mixing one or more enzymes with the substrate material allows the potential for the enzyme to catalyze hydrolysis of a portion of the substrate material during the contact time.
Corn kernel: a variety of corn kernels are known, including, for example, dent corn, hard corn, palea corn, striped corn, sweet corn, waxy corn, and the like.
Some corn kernels have an outer covering called the "Pericarp" (Pericarp), protecting the germ in the kernel. It is water and water vapor resistant and undesirable for insects and microorganisms. The only area of the kernel not covered by the "pericarp" is the "top Cap" (Tip Cap), which is the attachment point of the kernel to the cob.
Corn kernel or fractions of corn kernel: this term is used to describe corn kernel in the wet milling process. When the term is used when the corn kernel is broken down and processed, all fractionated parts of the corn kernel should be considered as included. The term includes, for example: soaked kernel, milled kernel, corn kernel material, a first fraction, a second fraction, one or more fractions of corn kernel material, and the like.
Corn kernel material: preferably for reference to a material comprising fibre, gluten and starch, preferably by steaming and grinding the crop kernel and separating the material comprising fibre, gluten and starch from the germ. As the corn kernel material moves through the fiber wash, it is separated into several fractions, including a first fraction(s) and a second fraction (f). Thus, "fraction of corn kernel material" and "one or more fractions of corn kernel material" refer to, inter alia, these first fraction(s) and second fraction (f).
cDNA: the term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor of mRNA that is processed through a series of steps, including splicing, and then rendered into mature spliced mRNA.
A coding sequence: the term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.
And (3) control sequence: the term "control sequence" means a nucleic acid sequence necessary for expression of a polynucleotide encoding a mature polypeptide of the invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or foreign with respect to one another. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
Expressing: the term "expression" includes any step involved in the production of a polypeptide, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to control sequences that provide for its expression.
Fragment (b): the term "fragment" means a polypeptide lacking one or more (e.g., several) amino acids at the amino and/or carboxy terminus of the mature polypeptide; wherein the fragment has pectin lyase activity.
Embryo: the "germ" is the only viable portion of the corn kernel. It contains the genetic information, enzymes, vitamins and minerals necessary for grain growth into a maize plant. In yellow dent corn, about 25% of the germ is corn oil. The endosperm covered or surrounded by the germ contains about 82% of the dry weight of the kernel and is the energy (starch) and protein source for seed germination. There are two types of endosperm, soft endosperm and hard endosperm. In the hard endosperm, the starch is tightly packed together. In soft endosperm, starch is loose.
GH5 polypeptide: refers to a polypeptide having enzymatic activity which is classified as a member of glycoside hydrolase family 5 (http:// www.cazy.org /) in a database of carbohydrate active enzymes (CAZymes).
GH30 polypeptide: refers to a polypeptide having enzymatic activity that is classified as a member of glycoside hydrolase family 30 (http:// www.cazy.org /) in a database of carbohydrate active enzymes (CAZyme).
Gluten: gluten is a protein, consisting of two smaller proteins, glutenin and gliadin. By "gluten" herein is meant the majority of proteins found in corn kernel. The major products of gluten from corn wet milling are corn gluten meal (about 60% protein) and corn gluten feed (about 20% protein).
Milling (grind or grinding): the term "milling" refers to the breaking down of corn kernel into smaller components.
Host cell: the term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Separating: the term "isolated" means a substance in a form or environment not found in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or more or all of the naturally occurring components associated with its property; (3) any substance that is modified by man relative to substances found in nature; or (4) any substance that is modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinantly produced in a host cell; multiple copies of a gene encoding the substance; and using a promoter that is stronger than the promoter with which the gene encoding the substance is naturally associated).
Incubation time: one or more fractions of the corn kernel material are contacted with a hydrolase during fiber washing without a screening period.
In many preferred embodiments, the method according to the invention makes use of a system comprising a space (V) or "incubator" in which the material is "left affected" by the enzyme, and in this case the incubation time can be determined by:
alternatively, if the inflow to the incubator is expressed in volume per time unit:
mature polypeptide: the term "mature polypeptide" means a polypeptide that is in its final form following translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like. In one aspect, the mature polypeptide is amino acids 21 to 678 of SEQ ID NO:2, based on the computer program SignalP (Nielsen et al, 1997, protein engineering 10:1-6) which predicts that amino acids 1 to 20 of SEQ ID NO:2 are signal peptides. It is known in the art that host cells can produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and/or N-terminal amino acids) expressed from the same polynucleotide. It is also known in the art that different host cells process polypeptides in different ways, and thus one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) when compared to another host cell expressing the same polynucleotide.
Mature polypeptide coding sequence: the term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide.
Grinding equipment: "grinding apparatus" means all apparatus used on a mill. The wet milling process will vary depending on the milling equipment available. Examples of milling equipment may be impregnation tanks, evaporators, screw presses, spin dryers, dewatering screens, centrifuges, hydrocyclones, etc. The size and number of each grinding device/wire can vary on different mills, which will affect the grinding process. For example, the number of fiber wash screen units may vary, as may the size of the centrifuge.
Nucleic acid construct: the term "nucleic acid construct" means a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain segments of nucleic acids in a manner not otherwise found in nature, or that is synthetic, that contains one or more control sequences.
Operatively connected to: the term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
Retention time: allowing time for the one or more hydrolytic enzymes to react with the corn kernel or a fraction of the corn kernel in a fiber washing procedure.
In some embodiments, the retention time is the period of time that the corn kernel material and one or more fractions thereof received in the first screen unit (S1) are contacted with an effective amount of one or more hydrolytic enzymes before exiting the fiber washing system again. During the retention time, one or more fractions of the corn kernel material are incubated with one or more hydrolytic enzymes in space (V) and then exit the fiber washing system as part of a first fraction (S1) from the most upstream screen unit (S1) or as part of a second fraction (f4) from the most downstream screen unit (S4).
The retention time may preferably be estimated as the average duration of solids spent in a fiber washing system as defined in the present invention. This can be estimated by the following relationship:
alternatively, if the inflow to the system is expressed in volume per time unit:
the volume of the system is typically set equal to the sum of the volumes of all the voids in the system; however, since the piping in the system is typically made small, it may be preferable to ignore the volume of the piping.
Screening: the term "screened" or "screening" refers to the process of separating corn kernel material into a first fraction s and a second fraction f and moving the fractions from one screen unit to another. The non-screening phase is a non-separation phase provided for incubating the corn kernel material or fraction thereof with an enzyme.
Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
For The purposes of The present invention, The degree of sequence identity between two amino acid sequences is determined using The Needman-Wunsch algorithm (Needleman and Wunsch,1970, J.Mol.biol. [ J.Mol.Biol. [ J.Mol ]48: 443-. Version 6.1.0 was used.
Optional parameters used are gap opening penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The output of the Needle labeled "longest identity" (obtained using-non-simplified options) is used as the percent identity and is calculated as follows: (same residue x 100)/(alignment length-total number of gaps in alignment).
Starch: the term "starch" means a complex polysaccharide from plants
Composed of glucose units in the form of granules which occur widely in plant tissue, composed of amylose and amylopectin and denoted by (C)6H10O5) n (where n is any number).
Dipping or soaking: the term "impregnation" means the use of water and optionally SO2And (4) soaking the crop seeds.