The present invention relates to a variant pullulanase, wherein the pullulanase comprises at least the following combination of substitutions: N368G+N393A+Q431E+L432F+A492A,S+N610R+G624S+T631S+S632C, and optionally further comprises N222P+Q252A+Q256R; wherein the variant has pullulanase activity, and wherein the variants have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3. Further aspect the present invention relates to a process for liquefying starch-containing material at a temperature above the initial gelatinization temperature using an alpha-amylase and a thermo-stable pullulanase of the invention.

A method for making a mutated pullulanase enzyme that hydrolyzes an -1,6-glycosidic linkage is provided. The method includes obtaining the amino acid sequence of a pullulanase enzyme having an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 13; identifying an amino acid to be mutated in the pullulanase enzyme of step (1), wherein the amino acid to be mutated corresponds to the amino acid at position Phe476 of SEQ ID NO: 2; constructing a mutated amino acid sequence by substituting the amino acid to be mutated with another amino acid or deleting the amino acid to be mutated, thereby making a mutated pullulanase enzyme having the mutated amino acid sequence that has increased affinity for pullulan and that hydrolyzes an -1,6-glycosidic linkage.

A method of preparing a wort with an increased level of free amino nitrogen (FAN) comprising: a) preparing a mash from a grist comprising malt and/or adjunct; and b) adding a M4 mettalloprotease obtainable from Actinobacteria; and wherein the amount of free amino nitrogen (FAN) in the wort is increased as compared to a wort produced in the absence of the M4 metalloprotease.

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.

A method of preparing a wort with an increased level of free amino nitrogen (FAN) comprising: a) preparing a mash from a grist comprising malt and/or adjunct; and b) adding a protease having at least 80% sequence identity to the polypeptide of SEQ ID NO: 1.

The present invention relates to a new vaccine delivery system. In particular, the present invention includes compositions and methods of integrally transformed non-pathogenic, commensal bacteria that can express a nucleic acid molecule of a foreign polypeptide, wherein the nucleic acid molecule that encodes the foreign polypeptide is stably integrated into genomic DNA of the bacteria. The foreign polypeptide includes a vaccine antigen that elicits an immunogenic response, an inhibitor of a pathogen, or an immune booster or modulator.

A process of recovering oil, comprising (a) converting a starch-containing material into dextrins with an alpha-amylase; (b) saccharifying the dextrins using a carbohydrate source generating enzyme to form a sugar; (c) fermenting the sugar in a fermentation medium into a fermentation product using a fermenting organism; (d) recovering the fermentation product to form a whole stillage; (e) separating the whole stillage into thin stillage and wet cake; (e) optionally concentrating the thin stillage into syrup; (f) recovering oil from the thin stillage and/or optionally the syrup, wherein a protease and a phospholipase are present and/or added during steps (a) to (c). Use of a protease and a phospholipase for increasing oil recovery yields from thin stillage and/or syrup in a fermentation product production process.

The present invention relates to processes for producing fermentation products from starch-containing material, wherein a bacterial alpha-amylase, a raw starch hydrolyzing alpha-amylase and a carbohydrate-source generating enzyme are present and/or added during liquefaction. The invention also relates to compositions suitable for use in processes of the invention.

The present invention relates to processes for producing fermentation products from starch-containing material, wherein a bacterial alpha-amylase, a raw starch hydrolyzing alpha-amylase and a carbohydrate-source generating enzyme are present and/or added during liquefaction. The invention also relates to compositions suitable for use in processes of the invention.

The present invention relates to a method of making glucose syrup from liquefied starch comprising, (a) contacting the liquefied starch with a glucoamylase, a pullulanase, and optionally an alpha-amylase wherein the ratio of pullulanase dose expressed as NPUN/gDS, to alpha-amylase dose expressed as FAU(A)/gDS is at least 60, particularly at least 75, particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 250, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 800 or if no alpha-amylase is present the pullulanse is present in a dose of at least 0.5, particularly at least 0.75, particularly at least 1.0, particularly at least 1.5 NPUN/gDS, and (b) saccharifying the liquefied starch.