Pullulanase chimeras and polynucleotides encoding same

• US 9,951,323 B2
• Filed: 07/11/2014
• Issued: 04/24/2018
• Est. Priority Date: 07/17/2013
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Matsui, Tomoko
• Primary Examiner(s): Mondesi, Robert B
• Assistant Examiner(s): EPSTEIN, TODD MATTHEW
• Application Number:
  US14/901,953
• Publication Number:
  US 20170051265A1
• Time in Patent Office: 1,383 Days
• Field of Search: None
• CPC Class Codes: C12C 7/04 Preparation or treatment of the mash
• CPC Class Codes: C12N 9/2457 Pullulanase (3.2.1.41)
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/12 Disaccharides
• CPC Class Codes: C12P 19/14 produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase , e.g. by cellulase, hemicellulase
• CPC Class Codes: C12Y 302/01041 Pullulanase (3.2.1.41)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

The present invention relates to hybrid polypeptides having pullulanase activity, and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Claims

1. A polypeptide having pullulanase activity, selected from the group consisting of:

(a) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO;

9 or a polypeptide having at least 93% sequence identity to the mature polypeptide of SEQ ID NO;

11.

2. The polypeptide of claim 1, having at least 95% sequence identity to the mature polypeptide of SEQ ID NO:

9.

3. The polypeptide of claim 1, having at least 95% sequence identity to the mature polypeptide of SEQ ID NO:

11.

4. A composition comprising the polypeptide of claim 1.

5. The composition according to claim 4, comprising one or more enzymes selected from the group consisting of:

glucoamylase, alpha-amylase, beta-amylase, and protease.

6. The composition according to claim 4, comprising the enzymes:

a pullulanase, a glucoamylase, an alpha-amylase and a protease;

or a pullulanase, an alpha-amylase and a protease;

or a pullulanase, a glucoamylase, and an alpha-amylase;

or a pullulanase, and a beta-amylase.

7. The composition according to claim 4, wherein the composition comprises a pullulanase and a glucoamylase and optionally an alpha-amylase, and wherein the pullulanase is selected from a polypeptide having at least 93% sequence identity to the mature polypeptide of SEQ ID NO:

11, and the glucoamylase is selected from i) a variant Gloeophyllum trabeum glucoamylase, which comprises the substitutions S95P+A121P compared to the wild type Gloeophyllum trabeum glucoamylase amino acid sequence set forth in SEQ ID NO;

18;

or ii) a variant having at least 85% sequence identity to SEQ ID NO;

18, and the alpha-amylase is selected from;

i) a variant Rhizomucor pusillus alpha-amylase with an Aspergillus niger glucoamylase linker and starch-binding domain (SBD), which comprises the substitutions G128D+D143N compared to the hybrid Rhizomucor pusillus alpha-amylase amino acid sequence set forth in SEQ ID NO;

14;

or ii) a variant having at least 85% sequence identity to the polypeptide of SEQ ID NO;

14.

8. A process of producing a fermentation product from starch-containing material comprising the steps of:

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

(b) saccharifying the liquefied material in the presence of a glucoamylase; and

(c) fermenting with a fermenting organism;

wherein step (a) and/or step (b) is carried out in the presence of a polypeptide of claim 1.

9. A process of producing a fermentation product from starch-containing material, comprising the steps of:

(a) saccharifying starch-containing material at a temperature below the initial gelatinization temperature of said starch-containing material; and

(b) fermenting with a fermenting organism,

wherein step (a) is carried out using at least a glucoamylase, and a polypeptide of claim 1.

10. A process of producing a syrup product from starch-containing material, comprising the step of:

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

(b) saccharifying the liquefied material in the presence of a glucoamylase, wherein the pullulanase of claim 1 is present during step (b).

11. The process according to claim 8, wherein the starch-containing material is selected from barley, beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof.

12. A polynucleotide encoding the polypeptide of claim 1.

13. A nucleic acid construct or expression vector comprising the polynucleotide of claim 12 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

14. A recombinant host cell comprising the polynucleotide of claim 12 operably linked to one or more control sequences that direct the production of the polypeptide.

15. The recombinant host cell according to claim 14, which is a yeast host cell.

16. The recombinant host cell according to claim 15, which is a yeast host cell selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, and Yarrowia.

17. The recombinant host cell according to claim 16, which is a yeast host cell selected from the group consisting of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Sacccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, and Yarrowia lipolytica.

18. The recombinant host cell according to claim 14, which is a filamentous fungal host cell.

19. The recombinant host cell according to claim 15, which is a filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypodadium, Trametes, or Trichoderma cell.

20. The recombinant host cell according to claim 15, which is a filamentous fungal host cell selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.