US20190010470A1

US20190010470A1 - Processes for producing fermentation products

Info

Publication number: US20190010470A1
Application number: US16/064,627
Authority: US
Inventors: Nathaniel Edward Kreel
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2015-12-22
Filing date: 2016-12-16
Publication date: 2019-01-10
Also published as: MX2018007712A; WO2017112539A1; US10597645B2; US20230303986A1; CA3006775A1; CN108699570A; US10689630B2; EP3394275A1; US11781123B2; US20180371505A1; CA3007103A1; CA3006992A1; US20180371439A1; EP3394276A1; US20200165586A1; US20210269781A1; CN108699571A; CN108699572A; CA3006991A1; WO2017112533A1

Abstract

The present disclosure relates to processes for producing fermentation products from starch-containing material, wherein a thermostable phospholipase C is present and/or added during liquefaction to increase fermentation product yield, such as ethanol yield. The disclosure also relates to the use of a thermostable phospholipase C in processes of the disclosure, for example, to increase fermentation product yield, such as ethanol yield.

Description

FIELD OF THE INVENTION

The present disclosure relates to processes for producing fermentation products, especially ethanol, from starch-containing material. The disclosure also relates to use of a thermostable phospholipase C during liquefaction in a fermentation product production process of the disclosure to increase fermentation product yield, especially ethanol.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Production of fermentation products, such as ethanol, from starch-containing material is well-known in the art. Industrially two different kinds of processes are used today. The most commonly used process, often referred to as a “conventional process”, includes liquefying gelatinized starch at high temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation carried out in the presence of a glucoamylase and a fermentation organism. Another well-known process, often referred to as a “raw starch hydrolysis”-process (RSH process), includes simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of at least a glucoamylase.
Despite significant improvement of fermentation product production processes over the past decade a significant amount of residual starch material is not converted into the desired fermentation product, such as ethanol.
Therefore, there is still a desire and need for providing processes for producing fermentation products, such as ethanol, from starch-containing material that can provide a higher fermentation product yield, or other advantages, compared to conventional processes.

SUMMARY OF THE INVENTION

The object of the present disclosure is to provide processes for producing fermentation products, such as ethanol, from starch-containing material that can provide a higher fermentation product yield, or other advantages, compared to conventional processes.
In the first aspect the present disclosure relates to a method of increasing fermentation product yield during fermentation product production process, wherein a phospholipase C is present and/or added during a liquefaction step of the fermentation product production process.
In another aspect the disclosure relates to processes of producing fermentation products, comprising:
(a) liquefying a starch-containing material using an alpha-amylase in the presence of a phospholipase C;
(b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme to form fermentable sugars; and
(c) fermenting the fermentable sugars using a fermenting organism to product the fermentation product.
In preferred embodiments the fermentation production product is ethanol and the ethanol yield is increased compared to performance of the method in the absence of using a phospholipase C.
In a preferred embodiment the phospholipase C is a thermostable phospholipase, preferably having a Melting Point (DSC) above 80° C., such as above 82° C., such as above 84° C., such as above 86° C., such as above 88° C., such as above 88° C., such as above 90° C., such as above 92° C., such as above 94° C., such as above 96° C., such as above 98° C., such as above 100° C., such as between 80° C. and 110° C., such as between 82° C. and 110° C., such as between 87° C. and 110° C.
Examples of thermostable phospholipase C of use herein include the phospholipase C shown in SEQ ID NO: 2 herein derived from a strain of Penicillium emersonii; or a polypeptide having phospholipase activity, preferably phospholipase C activity, having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein; the phospholipase C shown in SEQ ID NO: 7 derived from a strain of Trichoderma harzanium; or a polypeptide having phospholipase activity, preferably phospholipase C activity, having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein; the phospholipase C shown in SEQ ID NO: 8 derived from a strain of Trichoderma harzanium; or a polypeptide having phospholipase activity, preferably phospholipase C activity, having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 8 herein; and the phospholipase C shown in SEQ ID NO: 9 derived from a strain of Rasamsonia eburnea or a polypeptide having phospholipase activity, preferably phospholipase C activity, having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 9 herein.
Other enzymes such as endoglucanase, hemicellulases (e.g., xylanases, preferably a thermostable xylanase), carbohydrate source generating enzymes (e.g., glucoamylase, preferably a thermostable glucoamylase), proteases, pullulanases and phytases may also be used in the processes of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the ethanol yield with Pe PLC (SEQ ID NO: 2) addition in liquefaction, and SSF with Glucoamylase SA (GSA).

DETAILED DESCRIPTION OF THE INVENTION

The object of the present disclosure is to provide processes for producing fermentation products, such as ethanol, from starch-containing material that can provide a higher fermentation product yield, or other advantages, compared to conventional processes.
The present disclosure relates to the use of a phospholipase C during the liquefaction step in a fermentation product production process. The use of phospholipase C provides a higher fermentation product yield, such as especially ethanol.

I. METHODS OF INCREASING FERMENTATION PRODUCT YIELD

The inventors have found that increased fermentation product yield, such as especially ethanol yield, is obtained when liquefying starch-containing material using an alpha-amylase in the presence of a thermostable phospholipase C (see Example).
Accordingly, in the first aspect the present disclosure relates to a method of increasing fermentation product yield during a fermentation product production process, wherein a phospholipase C is present and/or added during a liquefaction step of the fermentation product production process.
As used herein, the phrase “present and/or added during” a particular step of a fermentation product production process means that an amount of an enzyme (e.g., phospholipase C) is added before or during the particular step of the fermentation product production process.
In a preferred embodiment the fermentation product is ethanol and the method increases ethanol yield.
In a preferred embodiment the phospholipase C, e.g., one derived from a strain of Penicillium, for example Penicillium emersonii, is the mature part of the sequence shown as SEQ ID NO: 2, or a sequence having a sequence identity thereto of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.
In an embodiment the phospholipase C shown in SEQ ID NO: 2 is present and/or added during liquefaction.
In a preferred embodiment the phospholipase C, e.g., one derived from a strain of Trichoderma, for example Trichoderma harzianum, is the mature part of the sequence shown as SEQ ID NO: 7, or a sequence having a sequence identity thereto of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or the mature part of the sequence shown in SEQ ID NO: 8, or a sequence having a sequence identity thereto of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%
In an embodiment the phospholipase C shown in SEQ ID NO: 7 is present and/or added during liquefaction. In an embodiment the phospholipase C shown in SEQ ID NO: 8 is present and/or added during liquefaction.
In a preferred embodiment the phospholipase C, e.g., one derived from a strain of Rasamsonia, for example Rasamsonia eburnean, is the mature part of the sequence shown as SEQ ID NO: 9, or a sequence having a sequence identity thereto of at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.
In an embodiment the phospholipase C shown in SEQ ID NO: 9 is present and/or added during liquefaction.
The liquefaction is carried out by liquefying a starch-containing material at a temperature above the initial gelatinization temperature using an alpha-amylase, e.g., a bacterial alpha-amylase and the phospholipase C.
In an embodiment, the phospholipase C has a Melting Point (DSC) of at least about 80° C.
Examples of suitable and preferred enzyme can be found below.

II. PROCESS OF PRODUCING FERMENTATION PRODUCTS

In another aspect the present disclosure relates to processes of producing fermentation products, comprising:
(a) liquefying a starch-containing material using an alpha-amylase in the presence of a phospholipase C;
(b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme to form fermentable sugars; and
(c) fermenting the fermentable sugars using a fermenting organism to product the fermentation product.
Liquefaction step (a), saccharification step (b) and fermentation step (c) are carried out sequentially, though saccharification step (b) and fermentation step (c) may be carried out simultaneously (SSF).

A. Liquefaction Step (a)

Generally the starch-containing material in step (a) may contain 20-55 wt.-% dry solids (DS), preferably 25-40 wt.-% dry solids, more preferably 30-35% dry solids.
The alpha-amylase and/or the phospholipase C may be added before and/or during liquefaction step (a).
In an embodiment the pH in step (a) is between 4-7, preferably between pH 4.5-6.
Step (a) may be carried out at as a liquefaction step at a temperature above the initial gelatinization temperature.
The term “initial gelatinization temperature” means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 50° C. and 75° C.; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this disclosure the initial gelatinization temperature of a given starch-containing material is the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C, Starch/Starke, Vol. 44 (12) pp. 461-466 (1992).
In an embodiment step (a) is carried out at a temperature between 70 and 100° C. In an embodiment step (a) is carried about at a temperature between 80-90° C. In an embodiment step (a) is carried about at a temperature of about 82° C. In an embodiment step (a) is carried about at a temperature of about 83° C. In an embodiment step (a) is carried about at a temperature of about 84° C. In an embodiment step (a) is carried about at a temperature of about 86° C. In an embodiment step (a) is carried about at a temperature of about 87° C. In an embodiment step (a) is carried about at a temperature of about 88° C.
In an embodiment a jet-cooking step may be carried out before in step (a). Jet-cooking may be carried out at a temperature between 95-140° C. for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes.
In an embodiment a process of the disclosure further comprises, before step (a), and optional jet-cooking step, the steps of:
i) reducing the particle size of the starch-containing material, preferably by dry milling; and
ii) forming a slurry comprising the starch-containing material and water.
The starch-containing starting material, such as whole grains, may be reduced in particle size, e.g., by milling, in order to open up the structure, to increase the surface area and allowing for further processing. Generally there are two types of processes: wet and dry milling. In dry milling whole kernels are milled and used. Wet milling gives a good separation of germ and meal (starch granules and protein). Wet milling is often applied at locations where the starch hydrolysate is used in production of, e.g., syrups. Both dry and wet millings are well known in the art of starch processing. According to the present disclosure dry milling is preferred. In an embodiment the particle size is reduced to between 0.05 to 3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferably at least 50%, more preferably at least 70%, even more preferably at least 90% of the starch-containing material fit through a sieve with a 0.05 to 3.0 mm screen, preferably 0.1-0.5 mm screen. In another embodiment at least 50%, preferably at least 70%, more preferably at least 80%, especially at least 90% of the starch-containing material fit through a sieve with #6 screen.
The aqueous slurry may contain from 10-55 w/w-% dry solids (DS), preferably 25-45 w/w-% dry solids (DS), more preferably 30-40 w/w-% dry solids (DS) of starch-containing material.
The slurry may be heated to above the initial gelatinization temperature, preferably to between 70-95° C., such as between 80-90° C., between pH 5.0-7.0, preferably between 5.0 and 6.0, for 30 minutes to 5 hours, such as around 2 hours.
In an embodiment liquefaction step a) is carried out for 0.5-5 hours at a temperature from 70-95° C. at a pH from 4-6.
In a preferred embodiment liquefaction step a) is carried out for 0.5-3 hours at a temperature from 80-90° C. at a pH from 4-6.
The alpha-amylase and/or phospholipase C may initially be added to the aqueous slurry to initiate liquefaction (thinning). In an embodiment only a portion of the enzymes is added to the aqueous slurry, while the rest of the enzymes are added during liquefaction step a).
The aqueous slurry may in an embodiment be jet-cooked to further gelatinize the slurry before being subjected to liquefaction in step a). The jet-cooking may be carried out at a temperature between 95-160° C., such as between 110-145° C., preferably 120-140° C., such as 125-135° C., preferably around 130° C. for about 1-15 minutes, preferably for about 3-10 minutes, especially around about 5 minutes.
The alpha-amylase used in step (a) may be any alpha-amylase, but is preferably a bacterial alpha-amylase. In a preferred embodiment the bacterial alpha-amylase is derived from the genus Bacillus. A preferred bacterial alpha-amylase may be derived from a strain of Bacillus stearothermophilus, and may be a variant of a Bacillus stearothermophilus alpha-amylase, such as the one shown as SEQ ID NO: 1. Bacillus stearothermophilus alpha-amylases are typically truncated naturally during production. In particular the alpha-amylase may be a truncated Bacillus stearothermophilus alpha-amylase having from 485-495 amino acids, such as one being around 491 amino acids long (SEQ ID NO: 1).
According to the present disclosure the Bacillus stearothermophilus alpha-amylase may be the one shown as SEQ ID NO: 1 or one having a sequence identity thereto of at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96°/h, at least 97%, at least 98%, at least 99%.
In an embodiment the bacterial alpha-amylase may be selected from the group of Bacillus stearothermophilus alpha-amylase variants comprising a deletion of one or two amino acids at any of positions R179, G180, I181 and/or G182, preferably the double deletion disclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to deletion of positions I181+G182 compared to the amino acid sequence of Bacillus stearothermophilus alpha-amylase set forth as SEQ ID NO: 3 disclosed in WO 99/19467 or SEQ ID NO: 1 herein or the deletion of amino acids R179+G180 using SEQ ID NO: 1 herein for numbering.
In a preferred embodiment the Bacillus stearothermophilus alpha-amylase variant comprises one of the following set of mutations:

- R179*+G180*;
- I181*+G182*;
- I181*+G182*+N193F; preferably
- I181*+G182*+N193F+E129V+K177L+R179E;
- I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;
- I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and
- I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1 for numbering).

In an embodiment the Bacillus stearothermophilus alpha-amylase variant has a sequence identity to SEQ ID NO: 1 of at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100%.
In an embodiment the Bacillus stearothermophilus alpha-amylase variant has from 1-12 mutations, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mutations, compared to the parent alpha-amylase, especially the alpha-amylase shown as SEQ ID NO: 1.
Commercially available bacterial alpha-amylase products and products containing alpha-amylases include TERMAMYL™ SC, LIQUOZYME™ SC, LIQUOZYME™ LpH, AVANTEC™, AVANTEC™ AMP, BAN (Novozymes A/S, Denmark) DEX-LO™, SPEZYME™ XTRA, SPEZYME™ AA, SPEZYME™ FRED-L, SPEZYME™ ALPHA, GC358™, SPEZYME™ RSL, SPEZYME™ HPA and SPEZYME™ DELTA AA (from DuPont, USA), FUELZYME™ (Verenium, USA).
A bacterial alpha-amylase may be added in step (a) in an amount well-known in the art.
In an embodiment the bacterial alpha-amylase, e.g., Bacillus alpha-amylase, such as especially Bacillus stearothermophilus alpha-amylase, or variant thereof, is dosed in liquefaction in a concentration between 0.01-10 KNU-A/g DS, e.g., between 0.02 and 5 KNU-A/g DS, such as 0.03 and 3 KNU-A, preferably 0.04 and 2 KNU-A/g DS, such as especially 0.01 and 2 KNU-A/g DS. In an embodiment the bacterial alpha-amylase, e.g., Bacillus alpha-amylase, such as especially Bacillus stearothermophilus alpha-amylases, or variant thereof, is dosed to liquefaction in a concentration of between 0.0001-1 mg EP (Enzyme Protein)/g DS, e.g., 0.0005-0.5 mg EP/g DS, such as 0.001-0.1 mg EP/g DS.
According to the present disclosure a phospholipase C, preferably a thermostable phospholliase C having a Melting Point (DSC) of at least about 80° C., is present in and/or added to liquefaction step a) in combination with an alpha-amylase, such as a bacterial alpha-amylase (described above).
The thermostability of a phospholipase C may be determined as described in Example 2 of WO 2014/090161 (incorporated herein by reference).
In an embodiment the phospholipase C has a Melting Point (DSC) above 82° C., such as above 84° C., such as above 86° C., such as above 88° C., such as above 88° C., such as above 90° C., such as above 92° C., such as above 94° C., such as above 96° C., such as above 98° C., such as above 100° C., such as between 80° C. and 110° C., such as between 82° C. and 110° C., such as between 87° C. and 110° C.
In a preferred embodiment the phospholipase C has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein, preferably derived from a strain of the genus Penicillium, such as a strain of Penicillium emersonii.
In an embodiment the phospholipase C comprises or consists of the amino acid sequence of SEQ ID NO: 2, or an allelic variant thereof; or is a fragment thereof having phospholipase C activity. In another embodiment, the phospholipase C comprises or consists of the mature polypeptide of SEQ ID NO: 2, or a variant of the mature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion at one or more positions. In another embodiment, the phospholipase C comprises or consists of amino acids 1 to 594 of SEQ ID NO: 2.
In a preferred embodiment the phospholipase C has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein, preferably derived from a strain of the genus Trichoderma, such as a strain of Trichoderma harzianum.
In an embodiment the phospholipase C comprises or consists of the amino acid sequence of SEQ ID NO: 7, or an allelic variant thereof; or is a fragment thereof having phospholipase C activity. In another embodiment, the phospholipase C comprises or consists of the mature polypeptide of SEQ ID NO: 7 (e.g., amino acid residues 19-643 of SEQ ID NO: 7, or a variant of the mature polypeptide of SEQ ID NO: 7 comprising a substitution, deletion, and/or insertion at one or more positions. In another embodiment, the phospholipase C comprises or consists of amino acids 1 to 594 of SEQ ID NO: 7.
In a preferred embodiment the phospholipase C has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 8 herein, preferably derived from a strain of the genus Trichoderma, such as a strain of Trichoderma harzianum.
In an embodiment the phospholipase C comprises or consists of the amino acid sequence of SEQ ID NO: 8, or an allelic variant thereof; or is a fragment thereof having phospholipase C activity. In another embodiment, the phospholipase C comprises or consists of the mature polypeptide of SEQ ID NO: 8 (e.g., amino acid residues 19-645 of SEQ ID NO: 8, or a variant of the mature polypeptide of SEQ ID NO: 8 comprising a substitution, deletion, and/or insertion at one or more positions. In another embodiment, the phospholipase C comprises or consists of amino acids 1 to 594 of SEQ ID NO: 8.
In a preferred embodiment the phospholipase C has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 9 herein, preferably derived from a strain of the genus Rasamsonia, such as a strain of Rasamsonia eburnea.
In an embodiment the phospholipase C comprises or consists of the amino acid sequence of SEQ ID NO: 9, or an allelic variant thereof; or is a fragment thereof having phospholipase C activity. In another embodiment, the phospholipase C comprises or consists of the mature polypeptide of SEQ ID NO: 9 (e.g., amino acid residues 19-611 of SEQ ID NO: 9, or a variant of the mature polypeptide of SEQ ID NO: 9 comprising a substitution, deletion, and/or inseration at one or more positions. In another embodiment, the phospholipase C comprises or consists of amino acids 1 to 594 of SEQ ID NO: 9.
The term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
The term “fragment” means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has phospholipase C activity.
The term “phospholipase C activity” means the activity that catalyzes the reaction: A phosphatidylcholine+H20=1,2-sn-diacylglycerol+choline phosphate. Phospholipase C activity may be determined using a phospholipase C activity assay (see, for example, the procedure described in Example 1 of WO 2014/090161, which is incorporated herein by reference. An enzyme having “phospholipase C activity” may belong to EC 3.1.4.3.
The term “mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one embodiment, the mature polypeptide is amino acids 1 to 594 of SEQ ID NO: 2. Amino acids −16 to −1 of SEQ ID NO: 2 are a signal peptide. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
For purposes of the present disclosure, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the −nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment).
The term “variant” means a polypeptide having phospholipase C activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.
A phospholipase C may be added and/or present in step (a) in an amount effective to increase fermentation product yield, such as especially ethanol yield, during SSF steps (b) and (c) or fermentation step (c).
In an embodiment the phospholipase C, such as especially Penicillium emersonii phoshophlipase C, or variant thereof, is dosed in liquefaction in a concentration of about 0.1-50,000 μg EP (Enzyme Protein)/g DS, such as 10,000 μg EP (Enzyme Protein)/g DS, or especially such as 5-1000 μg EP/g DS.
In an embodiment the phospholipase C, such as especially Trichoderma harzianum phoshophlipase C, or variant thereof, is dosed in liquefaction in a concentration of about 0.1-50,000 μg EP (Enzyme Protein)/g DS, such as 10,000 μg EP (Enzyme Protein)/g DS, or especially such as 5-1000 μg EP/g DS.
In an embodiment the phospholipase C, such as especially Rasamsonia eburnea phoshophlipase C, or variant thereof, is dosed in liquefaction in a concentration of about 0.1-50,000 μg EP (Enzyme Protein)/g DS, such as 10,000 μg EP (Enzyme Protein)/g DS, or especially such as 5-1000 μg EP/g DS.
Optionally, an endoglucanase (e.g., thermostable endoglucanase), hemicellulase (e.g., xylanase, preferably a thermostable xylanase), a protease, a carbohydrate-source generating enzyme, (e.g., glucoamylase, preferably a thermostable glucoamylase), a pullulanase, and/or a phytase may be present and/or added during liquefaction step (a). The enzymes may be added individually or as one or more blend compositions. In some embodiments, liquefaction step (a) is carried out in the absence of a protease.
In some embodiments, the glucoamylase comprises a variant of Penicillium oxalicum glucoamylase having the following mutations: K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering).

B. Saccharification Step (b)

Liquefaction step (a) is followed by saccharification of dextrins in step (b).
In an embodiment a process of the disclosure may comprise a pre-saccharification step, i.e., after step (a), but before saccharification step (b), carried out for 40-90 minutes at a temperature between 30-65° C.
According to the present disclosure saccharification step (b) may be carried out at a temperature from 20-75° C., preferably from 40-70° C., such as around 60° C., and at a pH between 4 and 5.
In a preferred embodiment fermentation step (c) or simultaneous saccharification and fermentation (SSF) (i.e., combined steps (b) and (c)) may be carried out at a temperature between 20-60° C., preferably between 25-40° C., such as around 32° C. In an embodiment fermentation step (c) or simultaneous saccharification and fermentation (SSF) are ongoing for 6 to 120 hours, in particular 24 to 96 hours.
According to the present disclosure a carbohydrate-source generating enzyme, preferably a glucoamylase, is present and/or added during saccharification step (b) and/or fermentation step (c) or simultaneous saccharification step (b) and fermentation step (c) (SSF).
The term “carbohydrate-source generating enzyme” includes any enzymes generating fermentable sugars. A carbohydrate-source generating enzyme is capable of producing one or more carbohydrates that can be used as an energy source by the fermenting organism(s) in question, for instance, when used in a process of the disclosure for producing ethanol. The generated carbohydrates may be converted directly or indirectly to the desired fermentation product, preferably ethanol. According to the disclosure a mixture of carbohydrate-source generating enzymes may be used.
Specific examples of carbohydrate-source generating enzyme activities include glucoamylase (being glucose generators), beta-amylase and maltogenic amylase (being maltose generators). A “maltogenic alpha-amylase” (glucan 1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration. A maltogenic amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S. Maltogenic alpha-amylases are described in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference. The maltogenic amylase may in a preferred embodiment be added in an amount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/g DS.
In a preferred embodiment the carbohydrate-source generating enzyme is a glucoamylase.
The process of the disclosure, including steps (b) and/or (c), may be carried out using any suitable glucoamylase. The glucoamylase may be of any origin, in particular of fungal origin.
Contemplated glucoamylases include those from the group consisting of Aspergillus glucoamylases, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylase disclosed in WO 84/02921, A. oryzae glucoamylase (AgriC. Biol. Chem. (1991), 55 (4), p. 941-949), or variants or fragments thereof. Other Aspergillus glucoamylase variants include variants with enhanced thermal stability: G137A and G139A (Chen et al. (1996), Prot. Eng. 9, 499-505); D257E and D293E/Q (Chen et al. (1995), Prot. Eng. 8, 575-582); N182 (Chen et al. (1994), Biochem. J. 301, 275-281); disulphide bonds, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and introduction of Pro residues in position A435 and S436 (Li et al. (1997), Protein Eng. 10, 1 199-1204.
Other glucoamylases contemplated include glucoamylase derived from a strain of Athelia, preferably a strain of Athelia rolfsii (previously denoted Corticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and (Nagasaka, Y. et al. (1998) “Purification and properties of the raw-starch-degrading glucoamylases from Corticium rolfsii, Appl Microbial Biotechnol 50:323-330), Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No. 4,587,215). Also contemplated are Trichoderma reesei glucoamylases including the one disclosed as SEQ ID NO: 4 in WO 2006/060062 and glucoamylases being at least 80% or at least 90% identical thereto (hereby incorporated by reference).
In an embodiment the glucoamylase is derived from a strain of Aspergillus, preferably A, niger, A. awamori, or A. oryzae; or a strain of Trichoderma, preferably T. reesei; or a strain of Talaromyces, preferably T. emersonii.
In an embodiment the glucoamylase present and/or added during saccharification step (b) and/or fermentation step (c) is of fungal origin, such as from a strain of Pycnoporus, or a strain of Gloephyllum. In an embodiment the glucoamylase is derived from a strain of the genus Pycnoporus, in particular a strain of Pycnoporus sanguinous described in WO 2011/066576 (SEQ ID NOs 2, 4 or 6), such as the one shown as SEQ ID NO: 4 in WO 2011/066576.
In a preferred embodiment the glucoamylase is derived from a strain of the genus Gloeophyllum, such as a strain of Gloeophyllum sepiarium or Gloeophyllum trabeum, in particular a strain of Gloeophyllum as described in WO 2011/068803 (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16). In a preferred embodiment the glucoamylase is the Gloeophyllum sepiarium shown in SEQ ID NO: 2 in WO 2011/068803.
Other contemplated glucoamylases include glucoamylase derived from a strain of Trametes, preferably a strain of Trametes cingulata disclosed as SEQ ID NO: 34 in WO 2006/069289 (which is hereby incorporated by reference).
Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831).
Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL, SPIRIZYME™ ACHIEVE, SPIRIZYME™ B4U and AMG™ E (from Novozymes A/S); OPTIDEX™ 300 (from Genencor Int.); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR (from Genencor Int).
Glucoamylases may in an embodiment be added in an amount of 0.02-20 AGU/g DS, preferably 0.05-5 AGU/g DS (in whole stillage), especially between 0.1-2 AGU/g DS.
Glucoamylase may be added in an effective amount, preferably in the range from 0.001-1 mg enzyme protein per g DS, preferably 0.01-0.5 mg enzyme protein per g dry solid (DS).
Optionally an alpha-amylase (EC 3.2.1.1) may be added during saccharification step (b) and/or fermentation step (c). The alpha-amylase may be of any origin, but is typically of filamentous fungus origin. According to the disclosure an alpha-amylases adding during saccharification and/or fermentation is typically a fungal acid alpha-amylase.
The fungal acid alpha-amylases may be an acid fungal alpha-amylase derived from a strain of the genus Aspergillus, such as Aspergillus oryzae and Aspergillus niger.
A suitable fungal acid alpha-amylase is one derived from a strain Aspergillus niger. In a preferred embodiment the fungal acid alpha-amylase is the one from A. niger disclosed as “AMYA_ASPNG” in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in more detail in WO 89/01969 (Example 3), The acid Aspergillus niger acid alpha-amylase is also shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes) which is hereby incorporated by reference. Also variants of said acid fungal amylase having at least 70% identity, such as at least 80% or even at least 90% identity, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 in WO 2004/080923 are contemplated. A suitable commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark).
The fungal acid alpha-amylase may also be a wild-type enzyme comprising a carbohydrate-binding module (CBM) and an alpha-amylase catalytic domain (i.e., a non-hybrid), or a variant thereof. In an embodiment the wild-type fungal acid alpha-amylase is derived from a strain of Aspergillus kawachii.
A specific example of a contemplated hybrid alpha-amylase includes the Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and starch-binding domain (SBD) (which is disclosed in Table 5 as a combination of amino acid sequences SEQ ID NO: 20, SEQ ID NO: 72 and SEQ ID NO: 96 in U.S. application Ser. No. 11/316,535) (hereby incorporated by reference), In another embodiment the hybrid fungal acid alpha-amylase is a Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in U.S. 60/638,614) (hereby incorporated by reference). Other specific examples of contemplated hybrid alpha-amylases include those disclosed in U.S. Patent Publication no. 2005/0054071, including those disclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylase with Aspergillus kawachii linker and starch binding domain.
In a preferred embodiment the fungal acid alpha-amylase is one disclosed in WO 2013/006756 including the following variants: Rhizomucor pusillus alpha-amylase variant having an Aspergillus niger glucoamylase linker and starch-binding domain (SBD) which further comprises at least one of the following substitutions or combinations of substitutions: D165M; Y141W; Y141R; K136F; K192R; P224A; P224R; S123H+Y141W; G20S+Y141W; A76G+Y141W; G128D+Y141W; G128D+D143N; P219C+Y141W; N142D+D143N; Y141W+K192R; Y141W+D143N; Y141W+N383R; Y141W+P219C+A265C; Y141W+N142D+D143N; Y141W+K192R V410A; G128D+Y141W+D143N; Y141W+D143N+P219C; Y141W+D143N+K192R; G128D+D143N+K192R; Y141W+D143N+K192R+P219C; G128D+Y141W+D143N+K192R; or G128D+Y141W+D143N+K192R+P219C.
An acid alpha-amylase may according to the present disclosure be added in an amount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5 AFAU/g DS, especially 0.3 to 2 AFAU/g DS.

C. Fermenting Organisms

The term “fermenting organism” refers to any organism, including bacterial and fungal organisms, especially yeast, suitable for use in a fermentation process and capable of producing the desired fermentation product.
Examples of fermenting organisms used in fermentation step (c) or simultaneous saccharification and fermentation (i.e., SSF) for converting fermentable sugars in the fermentation medium into fermentation products, such as especially ethanol, include fungal organisms, such as especially yeast. Preferred yeast includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae.
Suitable concentrations of the viable fermenting organism during fermentation, such as SSF, are well known in the art or can easily be determined by the skilled person in the art. In one embodiment the fermenting organism, such as ethanol fermenting yeast, (e.g., Saccharomyces cerevisiae) is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 10⁵to 10¹², preferably from 10⁷to 10¹⁰, especially about 5×10⁷.
“Fermentation medium” refers to the environment in which fermentation is carried out. The fermentation medium includes the fermentation substrate, that is, the carbohydrate source that is metabolized by the fermenting organism. According to the present disclosure the fermentation medium may comprise nutrients and growth stimulator(s) for the fermenting organism(s). Nutrient and growth stimulators are widely used in the art of fermentation and include nitrogen sources, such as ammonia; urea, vitamins and minerals, or combinations thereof.
Examples of commercially available yeast includes, e.g., RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM AFT and XR (available from NABC—North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

D. Starch-Containing Materials

Any suitable starch-containing material may be used as starting material according to the present disclosure. Examples of starch-containing materials, suitable for use in a process of the disclosure, include whole grains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, or sweet potatoes, or mixtures thereof or starches derived there from, or cereals. Contemplated are also waxy and non-waxy types of corn and barley.
In a preferred embodiment the starch-containing material, used for fermentation product production, such as especially ethanol production, is corn or wheat.

E. Fermentation Products

The term “fermentation product” means a product produced by a process including a fermentation step using a fermenting organism. Fermentation products contemplated according to the invention include alcohols (e.g., ethanol, methanol, butanol; polyols such as glycerol, sorbitol and inositol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. Preferably processes of the present disclosure are used for producing an alcohol, such as ethanol. The fermentation product, such as ethanol, obtained according to the present disclosure, may be used as fuel, which is typically blended with gasoline. However, in the case of ethanol it may also be used as potable ethanol.

F. Recovery

Subsequent to fermentation, or SSF, the fermentation product may be separated from the fermentation medium. The slurry may be distilled to extract the desired fermentation product (e.g., ethanol), Alternatively the desired fermentation product may be extracted from the fermentation medium by micro or membrane filtration techniques. The fermentation product may also be recovered by stripping or other method well known in the art.

III. USE OF PHOSPHOLIPASE C DURING LIQUEFACTION FOR INCREASING FERMENTATION PRODUCT YIELD

In yet another aspect, the present disclosure relates to the use of a phospholipase C during liquefaction in a fermentation product production process for increasing yield of a fermentation product (e.g., ethanol yield).
Any phospholipase C, for example a phospholipase C described above, can be used in a liquefaction step of an ethanol production process to increase ethanol yield. In preferred embodiments, the phospholipase C used in the liquefaction step has at least 60%, such as at least 70%, such as at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein, preferably derived from a strain of the genus Penicillium, such as a strain of Penicillium emersonii.

IV. EXAMPLES OF PREFERRED EMBODIMENTS OF THE DISCLOSURE

In a preferred embodiment the present disclosure relates to a process for producing ethanol from starch-containing material comprising the steps of:
(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature in the range from 70-100° C. using:

- an alpha-amylase derived from Bacillus stearothermophilus;
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C.;
  (b) saccharifying using a glucoamylase enzyme; and
  (c) fermenting using a fermenting organism.

In a preferred embodiment the process of the disclosure comprises the steps of:
(a) liquefying the starch-containing material at a pH in the range between 4.5-6.2 at a temperature above the initial gelatinization temperature using:

- an alpha-amylase, preferably derived from Bacillus stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂of at least 10;
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C.;
  (b) saccharifying using a glucoamylase enzyme; and
  (c) fermenting using a fermenting organism.

In a preferred embodiment the process of the disclosure comprises the steps of:
(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature between 70-100° C. using:

- a bacterial alpha-amylase, preferably derived from Bacillus stearothermophilus, having a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of at least 10;
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C.;
  (b) saccharifying using a glucoamylase enzyme; and
  (c) fermenting using a fermenting organism.

In a preferred embodiment the process of the disclosure comprises the steps of:
(a) liquefying the starch-containing material at a pH in the range between 4.0-6.5 at a temperature above the initial gelatinization temperature using:

- an alpha-amylase shown in SEQ ID NO: 1 having a double deletion in positions R179+G180 or I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:
- E129V+K177L+R179E;
- V59A+089R+E129V+K177L+R179E+H208Y+K220P+N224 L+Q254S;
- E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;
- V59A+Q89R+E129V+K177L+R179E+0254S+M284V (using SEQ ID NO: 1 herein for numbering);
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C., such as a phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein;
  (b) saccharifying using a glucoamylase enzyme;
  (c) fermenting using a fermenting organism.

- an alpha-amylase shown in SEQ ID NO: 1 having a double deletion in positions R179+G180 or I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:
- E129V+K177L+R179E;
- V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;
- E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;
- V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C., such as a phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein;
  (b) saccharifying using a glucoamylase enzyme;
  (c) fermenting using a fermenting organism.

- an alpha-amylase shown in SEQ ID NO: 1 having a double deletion in positions R179+G180 or I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:
- E129V+K177L+R179E;
- V59A+089R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;
- E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;
- V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C., such as a phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 8 herein;
  (b) saccharifying using a glucoamylase enzyme;
  (c) fermenting using a fermenting organism.

- an alpha-amylase shown in SEQ ID NO: 1 having a double deletion in positions R179+G180 or I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:
- E129V+K177L+R179E;
- V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;
- E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;
- V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C., such as a phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 9 herein;
  (b) saccharifying using a glucoamylase enzyme;
  (c) fermenting using a fermenting organism.

- an alpha-amylase derived from Bacillus stearothermophilus having a double deletion in positions I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:
- V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or
- V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C.; such as an phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 2 herein; and
- optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 3 herein, preferably having substitutions selected from the group of:
- K79V; or
- K79V+P11F+T65A+Q327F; or
- K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering);
  (b) saccharifying using a glucoamylase enzyme;
  (c) fermenting using a fermenting organism.

- an alpha-amylase derived from Bacillus stearothermophilus having a double deletion in positions I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:
- V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or
- V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering);
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C.; such as an phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein; and
- optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 3 herein, preferably having substitutions selected from the group of:
- K79V; or
- K79V+P11F+T65A+Q327F; or
- K79V+P2N+P45+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering);
  (b) saccharifying using a glucoamylase enzyme;
  (c) fermenting using a fermenting organism.

- an alpha-amylase derived from Bacillus stearothermophilus having a double deletion in positions I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:
- V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or
- V59A+089R+E129V+K177L+R179E+0254S+M284V (using SEQ ID NO: 1 herein for numbering);
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C.; such as an phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 8 herein; and
- optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 3 herein, preferably having substitutions selected from the group of:
- K79V; or
- K79V+P11F+T65A+Q327F; or
- K79V+P2N+P45+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering);
  (b) saccharifying using a glucoamylase enzyme;
  (c) fermenting using a fermenting organism.

- an alpha-amylase derived from Bacillus stearothermophilus having a double deletion in positions I181+G182, and optional substitution N193F; and optionally further one of the following set of substitutions:
- V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S; or
- V59A+Q89R+E129V+K177L+R179E+Q254S+M284V (using SEQ ID NO: 1 herein for numbering):
- a phospholipase C, preferably having a Melting Point (DSC) of at least about 80° C.; such as an phospholipase C having at least 60%, such as at least 70%, such as at least 75% identity, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, even more preferably at least 93%, most preferably at least 94%, and even most preferably at least 95%, such as even at least 96%, at least 97%, at least 98%, at least 99%, such as 100% identity to the mature part of the polypeptide of SEQ ID NO: 9 herein; and
- optionally a Penicillium oxalicum glucoamylase in SEQ ID NO: 3 herein, preferably having substitutions selected from the group of:
- K79V; or
- K79V+P11F+T65A+Q327F; or
- K79V+P2N+P4S+P11F+T65A+Q327F (using SEQ ID NO: 3 herein for numbering);
  (b) saccharifying using a glucoamylase enzyme;
  (c) fermenting using a fermenting organism.

In another preferred embodiment the disclosure relates to processes of producing ethanol, comprising:
(a) liquifying a starch-containing material using the alpha-amylase shown as SEQ ID NO: 1 or an alpha-amylase having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 1;
(b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme, in particular a glucoamylase, to form fermentable sugars;
(c) fermenting the fermentable sugars into ethanol using a fermenting organism;
wherein the phospholipase C shown as SEQ ID NO: 2 or a phospholipase C having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 2 is present and/or added during step (a).
In another preferred embodiment the disclosure relates to processes of producing ethanol, comprising:
(a) liquifying a starch-containing material using the alpha-amylase shown as SEQ ID NO: 1 or an alpha-amylase having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 1;
(b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme, in particular a glucoamylase, to form fermentable sugars;
(c) fermenting the fermentable sugars into ethanol using a fermenting organism;
wherein the phospholipase C shown as SEQ ID NO: 7 or a phospholipase C having at least 60%, at least 70%, at least 80%; at least 90%, at least 95%, at least 97%; at least 99% sequence identity to SEQ ID NO: 7 is present and/or added during step (a).
In another preferred embodiment the disclosure relates to processes of producing ethanol, comprising:
(a) liquifying a starch-containing material using the alpha-amylase shown as SEQ ID NO: 1 or an alpha-amylase having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 1;
(b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme; in particular a glucoamylase, to form fermentable sugars;
(c) fermenting the fermentable sugars into ethanol using a fermenting organism;
wherein the phospholipase C shown as SEQ ID NO: 8 or a phospholipase C having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 8 is present and/or added during step (a).
In another preferred embodiment the disclosure relates to processes of producing ethanol, comprising:
(a) liquifying a starch-containing material using the alpha-amylase shown as SEQ ID NO: 1 or an alpha-amylase having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 1;
(b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme, in particular a glucoamylase, to form fermentable sugars;
(c) fermenting the fermentable sugars into ethanol using a fermenting organism;
wherein the phospholipase C shown as SEQ ID NO: 9 or a phospholipase C having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% sequence identity to SEQ ID NO: 9 is present and/or added during step (a).
In a preferred embodiment a cellulase or cellulolytic enzyme composition is present and/or added during fermentation or simultaneous saccharification and fermentation.
In a preferred embodiment a cellulase or cellulolytic enzyme composition derived from Trichoderma reesei is present and/or added during fermentation or simultaneous saccharification and fermentation (SSF).
In a preferred embodiment a cellulase or cellulolytic enzyme composition and a glucoamylase are present and/or added during fermentation or simultaneous saccharification and fermentation.
In an embodiment the cellulase or cellulolytic enzyme composition is derived from Trichoderma reesei, Humicola insolens, Chrysosporium lucknowense or Penicillium decumbens.
The disclosure described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the disclosure. Any equivalent embodiments are intended to be within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. Various references are cited herein, the disclosures of which are incorporated by reference in their entireties. The present disclosure is further described by the following examples which should not be construed as limiting the scope of the disclosure.

Materials & Methods

Alpha-Amylase 369 (AA369): Bacillus stearothermophilus alpha-amylase with the mutations: I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V truncated to 491 amino acids (SEQ ID NO: 1 herein).
Phospholipase C (PePLC): Penicillium emersonii phospholipase C (SEQ ID NO: 2 herein).
Glucoamylase SA (GSA): Blend comprising Talaromyces emersonii glucoamylase disclosed as SEQ ID NO: 34 in WO99/28448 or SEQ ID NO: 4 herein, Trametes cingulata glucoamylase disclosed as SEQ ID NO: 2 in WO 06/69289 or SEQ ID NO: 5 herein, and Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and starch binding domain (SBD) disclosed in SEQ ID NO: 6 herein having the following substitutions G128D+D143N (activity ratio in AGU:AGU:FAU-F is about 20:5:1).
Yeast: ETHANOL RED™ available from Red Star/Lesaffre, USA.

Determination of Td by Differential Scanning Calorimetry.

The thermostability of the phospholipase C (PePLC) was determined at pH 4.0, pH 5.5 and pH 7.0 by Differential Scanning calorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter (MicroCal Inc., Piscataway, N.J., USA) at a protein concentration of approximately 0.5 mg/ml. The thermal denaturation temperature, Td (° C.), was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions in buffer at a constant programmed heating rate of 200 K/hr. Sample- and reference-solutions (approx. 0.2 ml) were loaded into the calorimeter (reference: buffer without enzyme) from storage conditions at 10° C. and thermally pre-equilibrated for 20 minutes at 20° C. prior to DSC scan from 20° C. to 100° C. Denaturation temperatures (Td) were determined at an accuracy of approximately +/−1° C. Tds obtained under these conditions for PePLC were 90 (pH 4.0), 88 (pH 5.5), and 83 (pH 7.0).
The thermostability of Harzianum (U49A3) was determined by Differential Scanning calorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter (MicroCal Inc., Piscataway, N.J., USA). The thermal denaturation temperature, Td (° C.), was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions (approx, 0.5 mg/ml) in buffer (50 mM acetate buffer pH 5.0) at a constant programmed heating rate of 200 K/hr.
Sample- and reference-solutions (approx. 0.2 ml) were loaded into the calorimeter (reference: buffer without enzyme) from storage conditions at 10 deg C. and thermally pre-equilibrated for 20 minutes at 20° C. prior to DSC scan from 20° C. to 100° C. Denaturation temperatures were determined at an accuracy of approximately +1-1° C., Td obtained under these conditions for U49A3 was 79 deg C.
The thermostability of Rasamsonia (U4BCJ) was determined by Differential Scanning calorimetry (DSC) using a VP-Capillary Differential Scanning calorimeter (MicroCal Inc., Piscataway, N.J., USA). The thermal denaturation temperature, Td (° C.), was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating enzyme solutions (approx. 0.5 mg/ml) in buffer (50 mM acetate buffer pH 5.5) at a constant programmed heating rate of 200 K/hr.
Sample- and reference-solutions (approx. 0.2 ml) were loaded into the calorimeter (reference: buffer without enzyme) from storage conditions at 10 deg C. and thermally pre-equilibrated for 20 minutes at 20° C. prior to DSC scan from 20° C. to 100° C. Denaturation temperatures were determined at an accuracy of approximately +/−1° C. Td obtained under these conditions for U4BCJ was 82 deg C.

EXAMPLE

Use of Thermostable Phospholipase C in Liquefaction Increases Ethanol Yield Liquefactions were performed on a 100 g scale using ground corn and backset obtained from different industrial corn ethanol plants to be used for the experiment. The dry solids (% DS) of the corn flour was 85.70% and the dry solids of the backset was 8.67%, both determined by Mettler-Toldeo HB43 halogen moisture balance. For liquefaction of the corn flour, 34.89 g corn flour was weighed into 200 mL Lab-O-Mat canisters along with 30 g backset and 35.11 g tap water to target a % DS of 32.5%. The pH of the corn slurry was adjusted to 5.0 for liquefaction.
The alpha-amylase (AA369) was used for liquefaction. In addition, thermostable Phospholipase C (PLC) from P. emersonii was evaluated at two doses, 10 and 50 μg EP/g DS. Each canister was dosed with the appropriate amount of diluted enzyme shown in Table 1 below. Alpha-amylase was dosed based on weight of ground corn and PLC was dosed based on total weight of corn slurry and % DS. After enzymes were added, the canisters were closed and sealed tightly, shaken thoroughly, and then placed in a Mathis Lab-O-Mat. The program used starts out at room temperature and ramps up 6° C./min for 12 minutes until it reaches 80° C. The temperature is then controlled at 85° C. for 105 minutes resulting in a total run of 1 hour and 57 minutes. The canisters are rotated back and forth at 45 rpm during the program.

TABLE 1

Enzyme Dosage In Liquefaction

	Amylase	PLC

	AA369 0.016% w/w corn	0 μg EP/g DS
	AA369 0.016% w/w corn	10 μg EP/g DS
	AA369 0.016% w/w corn	50 μg EP/g DS

After the Lab-O-Mat incubation, the canisters were removed and cooled in a room temperature water bath for 15 minutes. Once canisters were cooled, a spatula was used to remove as much material as possible into the corresponding labelled beaker. Liquefact weights were obtained to prepare each mash to 200 ppm urea and 3 ppm penicillin, and adjusted to pH 5 using 40% H₂SO₄if necessary. The % DS of the alpha-amylase-only liquefact was measured on the Mettler-Toldeo moisture balance at 33.90% DS. This % DS was used to calculate doses for all treatments, which were run in triplicates. To prepare for fermentation, approximately 5 grams of liquefact was aliquoted into the appropriate number of 15 mL Nunc flip top tubes. Exact weight of mash added was calculated by weighing the tubes before and after mash addition. Each tube was dosed with the appropriate amount of diluted Glucoamylase SA (GSA). The actual glucoamylase dosage was 0.6 AGU/g DS with the addition of DI water to normalize the % DS of all the tubes for direct comparison. Each tube was dosed with 100 μl of Ethanol Red rehydrated yeast (rehydrated with tap water), vented by drilling a 1/64″ hole in the cap, and then vortexed. Tubes were placed in a 32° C. waterbath for 52 hours but removed twice a day for vortexing.
After 52 hours of fermentation, samples were collected for HPLC analysis. This process began with stopping the enzyme and yeast reactions by adding 50 μL of 40% H₂SO₄(10 μL/g corn mash) and vortexing to distribute the acid. Samples were then centrifuged at 1430×g for 10 minutes and the supernatant was filtered through a 0.45 μm filter into a HPLC vial. Samples were stored at 4° C. until analysis. The system used to determine ethanol and oligosaccharides concentration was the Agilent 1100/1200 HPLC system coupled with an RI detector. The separation column was a BioRad Aminex HPX-87H ion exclusion column (300 mm×7.8 mm).

RESULTS

The final ethanol and percentage of ethanol increase by addition of Phospholipase C is summarized in Table 2 below.

TABLE 2

Summarized Ethanol Yield and Precent Change Results

	Ethanol	Ethanol
Liquefaction Treatment	(% w/v)	Increase (%)

AA369 only	Control	12.13	—
AA369 plus	10 μg PePLC	12.22	0.74%
AA369 plus	50 μg PePLC	12.31	1.48%

Claims

1. A process for increasing fermentation product yield during a fermentation product production process, wherein a phospholipase C is present and/or added during a liquefaction step of the fermentation product production process.

2. A process for producing a fermentation product, comprising the steps of:

(a) liquefying a starch-containing material using an alpha-amylase in the presence of a phospholipase C;

(b) saccharifying the liquified starch-containing material using a carbohydrate-source generating enzyme to form fermentable sugars; and

(c) fermenting the fermentable sugars using a fermenting organism to product the fermentation product.

3. The process of claim 1, wherein the phospholipase C is a thermostable phospholipase C having a Melting Point (DSC) of at least about 80° C.

4. The process of claim 1, wherein the phospholipase C has:

(i) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 2;

(ii) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 7;

(iii) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, most preferably at least 94%, and at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 8; or

(iv) at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the mature part of the polypeptide of SEQ ID NO: 9.

5. The process of claim 1, wherein

an alpha-amylase; and

a phospholipase C having a Melting Point (DSC) above 80° C.;

are present and/or added in liquefaction step (a).

6. The process of claim 1, further comprises, prior to the liquefaction step a), the steps of:

i) reducing the particle size of the starch-containing material; and

ii) forming a slurry comprising the starch-containing material and water.

7. The process of claim 1, wherein at least 50% of the starch-containing material fit through a sieve with #6 screen.

8. The process of claim 1, wherein the pH during liquefaction is between 4.0-6.5.

9. The process of claim 1, wherein the temperature during liquefaction is in the range from 70-100° C.

10. The process of claim 1, wherein a jet-cooking step is carried out before liquefaction in step a).

11. The process of claim 10, wherein the jet-cooking is carried out at a temperature between 95-160° C. for about 1-15 minutes.

12. The process of claim 1, wherein saccharification and fermentation is carried out sequentially or simultaneously.

13. The process of claim 1, wherein saccharification is carried out at a temperature from 20-75° C., and at a pH between 4 and 5.

14. The process of claim 1 wherein fermentation or simultaneous saccharification and fermentation (SSF) is carried out at a temperature from 25° C. to 40° C. for 6 to 120 hours.

15. The process of claim 1, wherein fermentation product is recovered after fermentation.

16. The process of claim 1, wherein the fermentation product is an alcohol.

17. The process of claim 1 wherein the starch-containing starting material is whole grains.

18. The process of claim 1, wherein the starch-containing material is derived from corn, wheat, barley, rye, milo, sago, cassava, manioc, tapioca, sorghum, rice or potatoes.

19. The process of claim 1, wherein the fermenting organism is yeast.

20. The process of claim 1, wherein the alpha-amylase is a bacterial alpha-amylase.

21. The process of claim 20, wherein the alpha-amylase is a Bacillus stearothermophilus alpha-amylase having the amino acid sequence of SEQ ID NO: 1 or having a sequence identity to SEQ ID NO: 1 of at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and is truncated to have from 485 to 495 amino acids.

22. The process of claim 20, wherein the Bacillus stearothermophilus alpha-amylase has one or more of the following sets of mutations:

I181*+G182*;

I181*+G182*+N193F;

I181*+G182*+E129V+K177L+R179E;

I181*+G182*+N193F+E129V+K177L+R179E;

I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S

I181*+G182*+N193F+V59A Q89R+E129V+K177L+R179E+Q254S+M284V; and

I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ ID NO: 1 for numbering).

23. The process of claim 20, wherein the Bacillus stearothermophilus alpha-amylase variant has a sequence identity to SEQ ID NO: 1 of at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%.

24. (canceled)

25. (canceled)

26. The process of claim 19, wherein the fermenting organism is a strain of Saccharomyces.

27. The process of claim 19, wherein the fermenting organism is a strain of Saccharomyces cerevisiae.