Variant alpha-amylases from bacillus subtilis and methods of use, thereof

Patent metadata

Field	Value
Publication number	US-2016010129-A1
Application number	US-201514742519-A
Country	US
Kind code	A1
Filing date	Jun 17, 2015
Priority date	Jun 6, 2008
Publication date	Jan 14, 2016
Grant date	—

Abstract

Alpha-amylases from Bacillus subtilis (AmyE), variants thereof, nucleic acids encoding the same, and host cells comprising the nucleic acids are provided. Methods of using AmyE or variants thereof are disclosed, including liquefaction and/or saccharification of starch. Such methods may yield sugars useful for ethanol production or high fructose corn syrup production. In some cases, the amylases can be used at low pH, in the absence of calcium, and/or in the absence of a glucoamylase.

First claim

What is claimed is:

1 . A method for liquefying and saccharifying starch in a starch conversion process, comprising
contacting a starch substrate with an AmyE polypeptide to form a reaction mixture for liquefying and saccharifying the starch substrate in the reaction mixture to produce glucose, wherein the liquefying and saccharifying are performed in the same reaction mixture without a pH adjustment.

2 . The method of claim 1 , wherein the saccharifying is performed in the absence of an additional polypeptide having glucoamylase activity.

3 . The method of claim 1 , wherein the liquefying is performed at a pH suitable for the activity of a glucoamylase polypeptide.

4 . The method of claim 3 , wherein the pH is 5.0 or lower.

5 . The method of claim 3 , wherein the pH is 4.5 or lower.

6 . The method of claim 3 , wherein the pH is 4.0 or lower.

7 . The method of claim 1 , wherein an additional polypeptide having glucoamylase activity is added to the reaction mixture before contacting the starch substrate with the AmyE polypeptide.

8 . The method of claim 1 , wherein an additional polypeptide having glucoamylase activity is added to the reaction mixture after contacting the starch substrate with the AmyE polypeptide.

9 . The method of claim 1 , wherein an additional polypeptide having glucoamylase activity is added to the reaction mixture simultaneously with contacting the starch substrate with the AmyE polypeptide.

10 . The method of claim 1 , further comprising fermenting the glucose produced by the liquefying and saccharifying to produce an alcohol.

11 . The method of claim 1 , wherein the alcohol is ethanol.

12 . The method of claim 11 , wherein at least a portion of the saccharifying and fermenting occur in the same reaction mixture simultaneously.

13 . The method of claim 1 , further comprising contacting the glucose produced by the liquefying and saccharifying with a glucose isomerase to produce fructose.

14 . The method of claim 1 , wherein exogenous calcium is not added to the reaction mixture.

15 . The method of claim 1 , wherein calcium concentration in the reaction mixture is less than about 8 ppm.

16 . The method of claim 14 , further comprising contacting the glucose produced by the liquefying and saccharifying with a glucose isomerase to produce fructose without reducing the amount of calcium in the reaction mixture.

17 . The method of claim 1 , wherein the AmyE polypeptide has at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1.

18 . The method of claim 1 , wherein the AmyE polypeptide has at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1.

19 . The method of claim 1 , wherein the AmyE polypeptide includes a deletion of the C-terminal starch binding domain.

20 . The method of claim 1 , wherein the AmyE polypeptide is a variant polypeptide having α-amylase activity and at least one altered characteristic that improves enzyme performance, the variant polypeptide comprising:
an amino acid sequence having at least 60% amino acid sequence identity to a parental α-amylase polypeptide selected from AmyE (SEQ ID NO: 1) or a truncated variant of AmyE (SEQ ID NO: 2), and
a modification at one or more positions selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 267, 268, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, and 425.
wherein the modification produces a variant polypeptide having a performance index (PI) greater than 1.0 for at least one characteristic that improves enzyme performance.

21 . The method of claim 20 , wherein the modification is a substitution of one or more amino residues present in the parental polypeptide to different amino acid residues, at one or more positions selected from the group consisting of 1A, 1C, 1D, 1E, 1F, 1G, 1H, 1K, 1M, 1N, 1Q, 1R, 1S, 1T, 1V, 1W, 1Y, 2A, 2C, 2D, 2E, 2F, 2G, 2H, 21, 2K, 2L, 2M, 2N, 2P, 2Q, 2R, 2S, 2V, 2W, 2Y, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3K, 3L, 3M, 3N, 3P, 3Q, 3R, 3S, 3V, 3W, 3Y, 4C, 4D, 4E, 4F, 4G, 4H, 4I, 4K, 4L, 4M, 4N, 4Q, 4S, 4T, 4V, 4W, 4Y, 5A, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5K, 5L, 5N, 5R, 5V, 5W, 5Y, 6C, 6D, 6E, 6H, 6K, 6L, 6M, 6N, 6P, 6Q, 6R, 6S, 6T, 6V, 6W, 7A, 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7L, 7M, 7N, 7P, 7Q, 7R, 7S, 7T, 7W, 7Y, 8A, 8C, 8E, 8F, 8G, 8H, 8I, 8K, 8L, 8M, 8N, 8P, 8Q, 8R, 8T, 8V, 8W, 8Y, 9A, 9C, 9D, 9E, 9F, 9H, 9I, 9K, 9M, 9N, 9P, 9R, 9S, 9T, 9V, 9W, 9Y, 10A, 10I, 10L, 10M, 10N, 10P, 10Q, 10S, 10y, 11A, 11F, 11G, 11H, 11M, 11S, 11V, 11W, 11Y, 12I, 12M, 12V, 13A, 13C, 13D, 13E, 13F, 13G, 13I, 13L, 13M, 13Q, 13T, 13V, 13W, 13Y, 14C, 14F, 14G, 14M, 14N, 14S, 14T, 14V, 15A, 15F, 16A, 16D, 16E, 16F, 16G, 16H, 16I, 16L, 16M, 16Q, 16S, 16T, 16V, 17A, 17F, 171, 17M, 17Q, 17Y, 18A, 18C, 18D, 18E, 18G, 18H, 18M, 18N, 18Q, 18R, 18T, 19A, 19C, 19H, 19L, 19M, 19N, 19S, 19W, 19Y, 20A, 20C, 20D, 20F, 20G, 20H, 20I, 20K, 20L, 20M, 20P, 20Q, 20R, 20S, 20T, 20V, 20W, 20Y, 21A, 21C, 21D, 21E, 21H, 21I, 21K, 21L, 21M, 21N, 21Q, 21R, 21S, 21V, 22I, 22M, 22Q, 22S, 22T, 22V, 23A, 23C, 23D, 23E, 23F, 23G, 23H, 23I, 23L, 23M, 23N, 23R, 23S, 23T, 23V, 23W, 23Y, 24A, 24C, 24D, 24F, 24G, 24L, 24N, 24P, 24Q, 24R, 24S, 24T, 24V, 24Y, 25A, 25C, 25D, 25E, 25F, 25G, 25H, 25I, 25K, 25L, 25R, 25S, 25T, 25V, 25W, 25Y, 26A, 26F, 26I, 26L, 26V, 27A, 27C, 27D, 27E, 27F, 27G, 27H, 27I, 27L, 27M, 27N, 27P, 27Q, 27R, 27S, 27T, 27V, 27W, 27Y, 28A, 28C, 28F, 28G, 28H, 28I, 28K, 28L, 28M, 28N, 28P, 28Q, 28R, 28S, 28T, 28V, 28W, 28Y, 29A, 29C, 29F, 29L, 29M, 29T, 29V, 30A, 30C, 30D, 30E, 30F, 30G, 30I, 30K, 30L, 30M, 30N, 30P, 30Q, 30R, 30S, 30T, 30V, 30W, 30Y, 31A, 31C, 31E, 31F, 31G, 31H

Assignees

• Danisco Us Inc

Inventors

• Cuevas William A
• Lee Sang-Kyu
• Ramer Sandra W
• Shaw Andrew
• Toppozada Amr R
• Estell David A
• Wallace Louise
• Chin Regina
• Requadt Carol A
• Power Scott D
• Pepsin Michael J

Classifications

• C12N9/2408

  acting on alpha -1,4-glucosidic bonds · CPC title

• C12P7/14

  Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms · CPC title

• C11D3/386

  Preparations containing enzymes {, e.g. protease or amylase} · CPC title

• C12N9/2417 Primary

  from microbiological source · CPC title

• G01N11/00

  Investigating flow properties of materials, e.g. viscosity, plasticity; Analysing materials by determining flow properties · CPC title