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Saccharification Enzyme Composition with Bacillus Subtilis Alpha-Amylase

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Saccharification Enzyme Composition with Bacillus Subtilis Alpha-Amylase (19) TZZ _ _T (11) EP 2 291 526 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 9/28 (2006.01) C12N 9/34 (2006.01) 13.08.2014 Bulletin 2014/33 C12P 7/00 (2006.01) (21) Application number: 09759442.8 (86) International application number: PCT/US2009/046296 (22) Date of filing: 04.06.2009 (87) International publication number: WO 2009/149283 (10.12.2009 Gazette 2009/50) (54) Saccharification enzyme composition with Bacillus subtilis alpha-amylase Zusammensetzung von Verzuckerungsenzymen enthaltend Bacillus subtilis alpha-Amylase Composition d’enzymes de saccharification comprenant une alpha-amylase de Bacillus subtilis (84) Designated Contracting States: (56) References cited: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR • KONSOULA ET AL: "Alpha-amylases and HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL glucoamylases free or immobilized in calcium PT RO SE SI SK TR alginate gel capsules for synergistic hydrolysis of crude starches" AMINO ACIDS, vol. 33, 2007, (30) Priority: 06.06.2008 US 59535 P page XIII, XP002552827 01.04.2009 US 165856 P • YEESANG ET AL: "Sago starch as a low-cost carbon source for exopolysaccharide production (43) Date of publication of application: by Lactobacillus kefiranofaciens" WORLD 09.03.2011 Bulletin 2011/10 JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 24, 15 November 2007 (73) Proprietor: Danisco US Inc. (2007-11-15), pages 1195-1201, XP019617015 Palo Alto, CA 94304 (US) • DE MORAES ET AL: "Development of yeast strains for the efficient utilisation of starch: (72) Inventors: evaluation of constructs that express alpha- • BRENEMAN, Suzanne amylase and glucoamylase separately or as Orfordville bifunctional fusion proteins" APPLIED WI 53576 (US) MICROBIOLOGY AND BIOTECHNOLOGY, vol. • LEE, Sung, Ho 43, 1995, pages 1067-1076, XP008033102 Palo Alto • HAYASHIDA ET AL: "Production and CA 94304 (US) characteristics of raw-potato-starch-digesting • SHARMA, Vivek alpha-amylase from Bacillus subtilis 65" Palo Alto APPLIED AND ENVIRONMENTAL CA 94304 (US) MICROBIOLOGY, vol. 54, 1988, pages 1516-1522, • SHETTY, Jayarama, K. XP008022975 Palo Alto • LIU ET AL: "A novel raw starch digesting alpha- CA 94304 (US) amylase from a newly isolated Bacillus sp. YX- 1: Purification and characterization" (74) Representative: Kiddle, Simon John et al BIORESOURCE TECHNOLOGY, vol. 99, 24 Mewburn Ellis LLP October 2007 (2007-10-24), pages 4315-4320, 33 Gutter Lane XP022526243 London EC2V 8AS (GB) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 291 526 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 291 526 B1 • SODHI ET AL: "Production of a thermostable • DATABASE UniProt pages 1-5 1986, VARIOUS alpha-amylase from Bacillus sp. PS-7 by solid AUTHORS: "Various titles" XP002552679 state fermentation and its synergistic use in the Database accession no. P00691 cited in the hydrolysis of malt starch for alcohol production" application PROCESS BIOCHEMISTRY, vol. 40, 2005, • DATABASE UniProt page 1 January 2008 XP004646977 (2008-01), XIN ET AL: "Cloning and expression of • FUJIMOTO ET AL: "Crystal structure of a Bacillus subtilis FS321 alpha-amylase gene" catalytic-site mutant alpha-amylase from XP002552680 Database accession no. A8W7J1 Bacillus subtilis complexed with maltopentaose" cited in the application JOURNAL OF MOLECULAR BIOLOGY, vol. 277, • KUNAMNENI ET AL: "Response surface 1998, pages 393-407, XP004456037 cited in the optimization of enzymatic hydrolysis of maize application starch for higher glucose production", • DATABASE UniProt page 1 1998, OHDAN ET AL: BIOCHEMICALENGINEERING JOURNAL, vol. 27, "Characteristics of two forms of alpha- amylases 2005, pages 179-190, XP027849191, and structural implication" XP002552678 Database accession no. O82953 cited in the application 2 EP 2 291 526 B1 Description FIELD OF THE INVENTION 5 [0001] A composition comprising a glucoamylase and a Bacillus subtilis alpha-amylase (AmyE) or variant thereof is useful in producing fermentable sugars from starch substrate, for example. Methods of using a glucoamylase and an AmyE or variant thereof to produce ethanol from starch, for example, are also disclosed. BACKGROUND 10 [0002] Vegetable starches, e.g., cornstarch, are widely used in the industrial manufacture of products such as syrups and biofuels. For example, high fructose corn syrup (HFCS) is a processed form of corn syrup having high fructose content and a sweetness comparable to sugar, making HFCS useful as a sugar substitute in soft drinks and other processed foods. HFCS production currently represents a billion dollar industry. Similarly, the production of ethanol from 15 vegetable starches is a rapidly expanding industry. Ethanol has widespread applications as an industrial chemical, a gasoline additive, or a liquid fuel by itself. The use of ethanol as a fuel or fuel additive significantly reduces air emission s while maintaining or even improving engine performance. On the other hand, ethanol is a renewable fuel, so that its use may reduce dependence on finite fossil fuel sources. Furthermore, use of ethanol may decrease the net accumulation of carbon dioxide in the atmosphere. 20 [0003] Syrups and biofuels can be produced from starch by an enzymatic process that catalyzes the breakdown of starch into glucose. This enzymatic process typically involves a sequence of enzyme-catalyzed reactions: [0004] (1) Liquefaction: Alpha-amylases (EC 3.2.1.1) first catalyze the degradation of a starch suspension, which may contain 30-40% w/w dry solids (ds), to maltodextrans. Alpha-amylases are endohydrolases that catalyze the random cleavage of internal α-1,4-D-glucosidic bonds. Because liquefaction typically is conducted at high temperatures, e.g., 25 90-100°C, thermostable alpha-amylases, such as an alpha-amylase from Bacillus sp., are preferred for this step. Alpha- amylases currently used for this step,e.g., alpha-amylases from B. licheniformis (AmyL), B. amyloliquefaciens, and Geobacillus stearothermophilus (AmyS), do not produce significant amounts of glucose. Instead, the resulting liquefact has a low dextrose equivalent (DE) and contains maltose and sugars with high degrees of polymerization (DPn). [0005] (2) Saccharification: Glucoamylases and/or maltogenic alpha-amylases catalyze the hydrolysis of non-reducing 30 ends of the maltodextrans formed after liquefaction, releasing D-glucose, maltose and isomaltose. Saccharification produces either glucose-rich or high-maltose syrups. In the former case, glucoamylases typically catalyze saccharification under acidic conditions at elevated temperatures, e.g., 60°C, pH 4.3. Glucoamylases used in this process typically are obtained from fungi, e.g., Aspergillus nigger glucoamylase used in Optidex® L400 or Humicola grisea glucoamylase. De-branching enzymes, such as pullulanases, can aid saccharification. 35 [0006] Maltogenic alpha-amylases alternatively may catalyze saccharification to form high-maltose syrups. Maltogenic alpha-amylases typically have a higher optimal pH and a lower optimal temperature than glucoamylase, and maltogenic amylases typically require Ca 2+. Maltogenic alpha-amylases currently used for this application include B. subtilis alpha- amylases, plant amylases, and the alpha-amylase from Aspergillus oryzae,the active ingredient ofClarase® L. Exemplary saccharification reactions used to produce various products are depicted below: 40 45 50 55 3 EP 2 291 526 B1 5 10 15 20 25 [0007] (3) Further processing: A branch point in the process occurs after the production of a glucose-rich syrup, shown on the left side of the reaction pathways above. If the final desired product is a biofuel, yeast can ferment the glucose-rich syrup to ethanol. On the other hand, if the final desired product is a fructose-rich syrup, glucose isomerase can catalyze the conversion of the glucose-rich syrup to fructose. 30 [0008] Saccharification is the rate-limiting step in the production of a glucose-rich syrup. Saccharification typically occurs over 48-72 hours, by which time many fungal glucoamylases lose significant activity. Moreover, it takes a significant portion, e.g., more than 70%, of saccharification time for the glucose yield to increase from 85% to 96%. This is mainly due to the inefficient hydrolysis of low molecular weight oligosaccharides by the glucoamylase. Accordingly, maximization of glucose production would require a relatively high dose of glucoamylase and/or a longer saccharification period: 35 Furthermore, although maltogenic alpha-amylases and glucoamylases both can catalyze saccharification, the enzymes typically operate at different optimal pH and temperatures, as shown above. If both enzymes are used sequentially, the difference in reaction conditions between the two enzymes necessitates adjusting the pH and temperature, which slows down the overall the process and may give rise to the formation of insoluble amylose aggregates. [0009] Accordingly, there is a need in the art for an improved starch processing method to make industrial products. 40 In particular, there is a need for improved efficiencies in a saccharification step. [0010] Konsoula et al., Amino Acids, 33: XIII, Abstract
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