WO2024163288A1 - Nouveaux procédés et nouvelles compositions pour améliorer la filtrabilité et le rendement d'une maische dans le brassage - Google Patents

Nouveaux procédés et nouvelles compositions pour améliorer la filtrabilité et le rendement d'une maische dans le brassage Download PDF

Info

Publication number
WO2024163288A1
WO2024163288A1 PCT/US2024/013128 US2024013128W WO2024163288A1 WO 2024163288 A1 WO2024163288 A1 WO 2024163288A1 US 2024013128 W US2024013128 W US 2024013128W WO 2024163288 A1 WO2024163288 A1 WO 2024163288A1
Authority
WO
WIPO (PCT)
Prior art keywords
xylanase
seq
active fragment
polypeptide
glucanase
Prior art date
Application number
PCT/US2024/013128
Other languages
English (en)
Inventor
Jacob Flyvholm Cramer
Tove BLADT WICHMANN
Original Assignee
International N&H Denmark Aps
Danisco Us Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by International N&H Denmark Aps, Danisco Us Inc. filed Critical International N&H Denmark Aps
Publication of WO2024163288A1 publication Critical patent/WO2024163288A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C7/00—Preparation of wort
    • C12C7/04—Preparation or treatment of the mash
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
    • C12H6/00—Methods for increasing the alcohol content of fermented solutions or alcoholic beverages
    • C12H6/02—Methods for increasing the alcohol content of fermented solutions or alcoholic beverages by distillation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)

Definitions

  • the present invention relates to brewing. More particularly, the present invention relates to novel combinations of enzymes for improving mash filterability and yield.
  • the starch present in malt and adjuncts must be gelatinized to achieve high extract yield and to convert carbohydrate polymers to fermentable sugar types.
  • Other components from the raw materials get solubilized as well, e.g., high molecular weight (HMW) B-glucan and HMW arabinoxylan.
  • HMW high molecular weight
  • B-glucan and arabinoxylan are solubilized during the entire mashing process and until mashing-off.
  • B-glucan and arabinoxylan are complex carbohydrates and cause the mash to be highly viscous, rendering subsequent mash separation and beer filtrations steps difficult. In turn, yields of fermentable sugars and, hence, ethanol is lowered.
  • a method for producing a rye whiskey having the steps of: (a) providing a grist having at least 51 w/w % of rye grain; (b) adding water to the grist to provide a mash; (c) pre-liquefying the mash of step (b); (d) gelatinizing the mash of step (c); (e) liquefying the mash of step (d) in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor; (f) saccharifying the mash of step (e); (g) fermenting the mash of step (f) to produce a fermentate containing ethanol; (h) distilling the fermentate of step (g) to provide the rye whiskey.
  • the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by Rye XIP inhibitor.
  • the xylanase has less than 20% inhibition by Rye XIP inhibitor.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID N0:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanasc active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.
  • the rye grain is malted. In other preferred embodiments, the rye grain is unmalted. Optionally, the rye grain is milled.
  • the grist is 60, 70, 80, 85, 90, 95, 99 or 100 w/w % rye.
  • the grist further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oatmeal, or sorghum. More preferably, the grist further comprises malted barley.
  • the method also includes the further step of adding an alphaamylase and/or a beta-glucanase at any of steps (b), (c), (d) and/or (e).
  • a beta- glucanase is added.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8.
  • glucoamylase is added at step (f) and/or a protease is added at step (g).
  • the method further includes the step of adding an alpha-L- arabinofuranosidase at any of steps (b), (c), (d) and/or (e).
  • the alpha- L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidasc active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO:11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with a polypeptide amino acid sequence according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha- L-arabinofuranosidase active fragment thereof.
  • the disclosed method also optionally includes the further step of adding a feruloyl esterase at any of steps (b), (c), (d) and/or (e).
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • a method for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist having rye cereal in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor; (b) optionally filtering the mash to obtain a wort.
  • the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by Rye XIP inhibitor.
  • the xylanase has less than 20% inhibition by Rye XIP inhibitor.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.
  • the rye grain is malted.
  • the rye grain is unmalted.
  • the rye grain is milled.
  • the grist is 60, 70, 80, 85, 90, 95, 99 or 100 w/w % rye.
  • the grist further comprises malted or unmalted barley, wheat, corn, rye, rice, cassava, oatmeal, or sorghum.
  • the grist further comprises malted barley.
  • the method also includes the further step of adding an alpha-amylase and/or a beta-glucanase to the mash.
  • a beta-glucanase is added.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the bcta-glucanasc is an enzyme having bcta-glucanasc activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8.
  • the method further includes the step of adding an alpha-L- arabinofuranosidase to the mash.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidasc active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the disclosed method also optionally includes the further step of adding a feruloyl esterase to the mash.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • a method for identifying a xylanase that has reduced sensitivity to a cereal XIP inhibitor having the step of assaying the activity of the xylanase in the presence and absence of the cereal XIP inhibitor and determining the percent inhibition by the cereal XIP inhibitor.
  • the cereal is barley, wheat, corn, rye, rice, cassava, oatmeal, or sorghum.
  • the cereal is rye or wheat.
  • the cereal is rye.
  • the cereal is wheat.
  • a xylanase having reduced sensitivity to rye XIP inhibitor identified by the method above.
  • the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by rye XIP inhibitor.
  • the xylanase has less than 20% inhibition by rye XIP inhibitor.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID N0:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.
  • a xylanase having reduced sensitivity to wheat XIP inhibitor identified by the method of claim 110.
  • the xylanase has less than 90, 80, 70, 60, 55, 50, 40, 30, 20, 10, 5 or 1% inhibition by wheat XIP inhibitor.
  • the xylanase has less than 55% inhibition by wheat XIP inhibitor.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.
  • a composition for reducing wort viscosity in wheat adjunct brewing having a xylanase having reduced sensitivity to wheat XIP inhibitor and a beta-glue anase.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the bcta-glucanasc is an enzyme having bcta-glucanasc activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3 and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8.
  • a method for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist having wheat grain in the presence of a composition having a xylanase having reduced sensitivity to wheat XIP inhibitor and a beta-glucanase as described above; (b) optionally filtering the mash to obtain a wort.
  • the wheat grain is malted.
  • the wheat grain is unmalted.
  • the wheat grain is milled.
  • the grist is 5, 10, 20, 25, 30, 35, 40, 45, 50, 51, 60, 70, 80, 85, 90, 95, 99 or 100 w/w % wheat.
  • the grist also has malted or unmalted barley, com, rye, rice, cassava, oatmeal, or sorghum.
  • the grist also has malted barley.
  • a composition having a xylanase which has a polypeptide sequence with at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof and a glucanase.
  • the xylanase polypeptide has at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide has at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide has at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide has at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide has at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide has at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide is a sequence according to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide has a sequence according to SEQ ID NO:2.
  • the glucanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase polypeptide has at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase polypeptide has at least 90% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase polypeptide has at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase polypeptide has at least 95% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof.
  • the glucanase polypeptide has at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase polypeptide has at least 99% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase polypeptide is a sequence according to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase polypeptide is a sequence according to SEQ ID NO:8.
  • the xylanasc polypeptide optionally has at least 98, 99 or 100% sequence identity to SEQ ID NO:2.
  • the composition is a liquid.
  • the xylanase is optionally present in an amount of 1,000 to 50,000 NGXU/g and the glucanase is preferably present in an amount of 1,000 to 50,000 BBXU/g.
  • the xylanase is present in an amount of 5,000 to 30,000 NGXU/g and the glucanase is present in an amount of 5,000 to 30,000 BBXU/g.
  • the xylanase is present in an amount of 10,000 to 25,000 NGXU/g and the glucanase is present in an amount of 15,000 to 30,000 BBXU/g.
  • the xylanase is present in an amount of 15,000 to 20,000 NGXU/g and the glucanase is present in an amount of 20,000 to 28,000 BBXU/g.
  • a method is presented of altering filterability of a starch containing material, the method having the step of treating the starch containing material with a composition as described above.
  • a method is presented of reducing pressure built up during lautering in a brewing application, the method having the step of treating a brewing mash having a starch containing material with a composition as described above.
  • a method for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, the method having the step of treating a starch containing material with a composition as described above.
  • a food, feed, or beverage product such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey
  • a method for the production of a brewing mash, the method having the step of treating a starch comprising material with a composition as described above.
  • the starch comprising material comprises a grist.
  • the grist has at least 50, 60, 70, 80 or 90 % malt.
  • the grist further also contains wheat, corn or rye.
  • a method for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor and a beta-glucanase which has increased activity in the presence of at least 10% rye compared to a sample not having rye; and (b) optionally filtering the mash to obtain a wort.
  • the xylanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide according to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide according to SEQ ID NO:2.
  • the glucanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide having at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide having at least 90% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide having at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide having at least 95% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide having at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide having at least 99% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide according to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide according to SEQ ID NO:8.
  • the grist has at least 50, 60, 70, 80 or 90% malt.
  • the grist also has wheat, com or rye.
  • the grist has rye.
  • the grist has at least 10% rye.
  • SEQ ID NO:1 shows the protein sequence of the FveXyn4_l precursor protein.
  • SEQ ID NO:2 shows the protein sequence of FveXyn4_l mature protein.
  • SEQ ID NO:3 shows the protein sequence of FveXyn4_l predicted protein.
  • SEQ ID NO:4 shows the amino acid sequence of PfunXyn GH11 xylanase (E.C. 3.2.1.8) precursor protein.
  • SEQ ID NO:5 shows the amino acid sequence of PfunXyn GH11 xylanase (E.C. 3.2.1.8) mature protein.
  • SEQ ID NO:6 shows the amino acid sequence of TemXyn GH10 xylanase (E.C. 3.2.1.8) precursor protein.
  • SEQ ID NOT shows the amino acid sequence of TemXyn GH10 xylanase (E.C. 3.2.1.8) mature protein.
  • SEQ ID NO:8 shows the amino acid sequence of BsbBglu mature protein (E.C. 3.2.1.6) beta-glucanase from Bacillus subtilis.
  • SEQ ID NO:9 shows the amino acid sequence of AkaXyl mature protein (E.C. 3.2.1.8.) xylanase variant from Aspergillus kawachii.
  • SEQ ID NOTO shows the amino acid sequence of AniAfur precursor protein alpha-L- Arabinofurano sidase .
  • SEQ ID NO: 11 shows the amino acid sequence of BaAfur precursor protein alpha-L- arabinofurano sidase .
  • SEQ ID NO: 12 shows the amino acid sequence of CtFe precursor protein feruloyl esterase.
  • FIG. 1 depicts turbidity quantified by Anton Paar as haze by 90° scattering in EBC units (small particles) on mash supernatants. Haze scattering is shown for trials 3 and 6, 4 and 7, and 5 and 8 respectively with either PfunXyn or FvXyn4_l xylanase as indicated by legends (in combinations with a-L-arabinofuranose and Feruloyl esterase).
  • FIG. 2 depicts turbidity quantified by Anton Paar as haze by 25° scattering in EBC units (larger particles) on mash supernatants. Haze scattering is shown for trials 3 and 6, 4 and 7, and 5 and 8 respectively with either PfunXyn or FvXyn4_l xylanase as indicated by legends (in combinations with a-L-arabinofuranosc and Fcruloyl esterase).
  • FIG. 3 depicts the average wort volume collected after 30 minutes filtration for each xylanase over following dose range: TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist.
  • FIG. 4 depicts the average wort Viscosity at 12°P (mPa.s) after filtration for each xylanase using following dosages: TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist.
  • FIG. 5 depicts the average Original Extract (°P) of wort after filtration for each xylanase using following dosages: TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist.
  • FIG. 6 depicts the effect of increasing xylanase (Viscoferm, PfunXyn and FveXyn4_l ) Dose Rate (g/kg grist) on Ethanol Production in rye-based SSF fermentation.
  • FIG. 7 depicts thermostability of FveXyn4_l, AkaXyl_var, Shearzyme P105 and Ultraflo Max in Malt wort pH 5.6 with 10 minutes incubation time. The residual xylanase activity is shown as function of temperature of wort.
  • FIG. 8 depicts residual xylanase activity through 40%:60% barley:malt mashing programs using a water-to-grist ratio of 3:1 of FveXyn4_l, AkaXyl_var, Shearzyme P105 and Ultrflo Max. Low (A), standard (B) and high (C) temperature mashing diagrams are shown with residual xylanase.
  • FIG. 9 depicts residual beta-glucanase activity (Beta-glucazyme, megazyme) of BsbBglu + FveXyn4_l, Ultraflo Max and Filtrase Fast incubated 24 hours at 5°C with 10% cereal extracts (Rye, Wheat, Com, and Malt).
  • FIG. 10 depicts wort viscosity (mPa*s) (A), HWM b-glucan (mg/L) (B) and HWM pentosan (mg/L) (C) for BsbBglu + FveXyn4_l, Ultraflo Max and BsbBglu + AkaXyl_var in wort produced by 40%:60% barley:malt high temperature mashing.
  • mPa*s wort viscosity
  • B HWM b-glucan
  • HWM pentosan mg/L
  • An “active fragment” of an enzyme is a polypeptide where the enzyme has been deleted either at the C-terminus, the N-terminus and/or internally but still retains some or all of its original activity.
  • An active fragment includes the mature form of an enzyme.
  • variants encompasses variants, homologues, derivatives and fragments thereof.
  • variants is used to mean a nucleotide sequence or amino acid sequence which differs from a wild-type sequence.
  • a variant may include substitutions, insertions, deletions, truncations, transversions and/or inversions at one or more position(s) relative to a wild-type sequence.
  • Variants can be made using methods known in the art for example site scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis and directed-evolution as well as using recombinant methods well known in the art.
  • Polynucleotide sequences encoding variant amino acid sequences may readily be synthesized using methods known in the art.
  • the variant is a naturally occurring nucleotide sequence or amino acid sequence which differs from a wild-type sequence.
  • the variant may be a natural genetic variant.
  • the variant is an engineered variant.
  • the variant may be engineered by recombinant methods.
  • the protein sequences of the instant invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine. Conservative substitutions may be made, for example according to the Table below.
  • Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other as set forth in Table 1.
  • the present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) that may occur i.e., like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalaninc and phcnylglycinc.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine pyriylalanine
  • thienylalanine thienylalanine
  • naphthylalaninc and phcnylglycinc.
  • Replacements may also be made by synthetic amino acids (e.g. unnatural amino acids) include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br- phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, B-alanine*, L-a-amino butyric acid*, L-g- amino butyric acid*, L-a-amino isobutyric acid*, L-e-amino caproic acid # , 7-amino heptanoic acid*, L- methionine sulfone**, L-norleucine*, L-norv aline*, p-nitro-L-phenylalanine*, L- hydroxyproline*, L-
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or b-alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or b-alanine residues.
  • a further form of variation involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the a-carbon substituent group is on the residue’s nitrogen atom rather than the a-carbon.
  • sequences described herein may be obtained for example by probing DNA libraries made from a range of individuals, for example individuals from different populations.
  • other homologues may be obtained and such homologues and fragments thereof in general will be capable of selectively hybridizing to the sequences shown in the sequence listing herein.
  • sequences may be obtained by probing cDNA libraries or genomic DNA libraries made from other animal species and probing such libraries with probes comprising all or part of any one of the sequences in the attached sequence listings under conditions of medium to high stringency. Similar considerations apply to obtaining species homologues and allelic variants of the polypeptide or nucleotide sequences of the invention.
  • Variants and strain/species homologues may also be obtained using degenerate PCR which will use primers designed to target sequences within the variants and homologues encoding conserved amino acid sequences within the sequences of the present invention.
  • conserved sequences can be predicted, for example, by aligning the amino acid sequences from several variants/homologues. Sequence alignments can be performed using computer software known in the art. For example, the GCG Wisconsin PileUp program is widely used.
  • the primers used in degenerate PCR will contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.
  • such polynucleotides may be obtained by site directed mutagenesis of characterized sequences. This may be useful where for example silent codon sequence changes are required to optimize codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be desired in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.
  • the present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology and recombinant DNA, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N. Y.); B. Roe, J. Crabtree, and A.
  • percent (%) sequence identity means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:
  • Gap extension penalty 0.05
  • Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included. For example, a variant with five amino acid deletions of the C-terminus of the mature 617 residue polypeptide would have a percent sequence identity of 99% (612 / 617 identical residues x 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having “at least 99% sequence identity” to a mature polypeptide.
  • proteins, including enzymes, of the present invention exist in multiple forms. Proteins of the instant invention may be clipped or trimmed (i.e., removing amino acids) from the N-terminus and/or the C-terminus, resulting in a shorter protein. Proteins of the instant invention can also have internal deletions. Shorter proteins as described herein can have higher activity or lower activity than longer counterparts.
  • the term precursor protein is a protein, including an enzyme, which has an N-terminal signal peptide that targets the protein for secretion. A precursor protein is sometimes referred to herein as “full length” or “full length protein”. The N- terminal signal peptide is cleaved off in the endoplasmic reticulum to yield a mature protein.
  • Xylanases are classified in EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136 and EC 3.2.1.156.; their activity may be measured e.g., as described in the examples.
  • Suitable xylanases to be used in combination with an enzyme exhibiting endo-l,3(4)-P-glucanase activity according to the invention includes any xylanase classified in EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136 and EC 3.2.1.156, such as anyone disclosed in WO 2010072226, WO 2010072225, WO 2010072224, WO 2005059084, W02007056321, W02008023060A, WO9421785, W02006114095, W02006066582, US 2008233175, and W010059424.
  • Endo-P-1, 4-xylanases also referred herein as xylanases, is the name given to a class of enzymes which degrade the linear polysaccharide beta- 1,4- xylan into xylose, and thus breaking down hemicellulose, one of the major components of plant cell walls.
  • Endo-1, 4-beta xylanase is classified as EC 3.2.1.8. The enzyme causes endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.
  • xylanases of the present invention are superior for solubilization of rye fibers, both un-cxtractablc arabinoxylans (WU-AX) and the water extractable arabinoxylans (WE- AX).
  • xylanases of the instant invention have excellent product properties relevant for e.g., mashing applications and high thermostability.
  • xylanase comprises enzymes with a number of known activities, such as xylanase (EC:3.2.1.8); endo-l,3-beta-xylanase (ECG.2.1.32); cellobiohydrolase (EC:3.2.1.91). These enzymes were formerly known as cellulase family F.
  • GH Family 11 xylanase
  • Glycoside hydrolase (GH) family 11 or simply “GH
  • 11 xylanase refers to an endo- 1 ,4-beta xylanase classified as EC 3.2.1.8, which causes endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans and which is classified as a family 11 xylanase according to B. Henrissat, A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280 (1991), pp. 309-316.
  • a xylanase variant described in the present invention is superior for solubilization of Rye fiber, both un-extractable arabinoxylans (WU-AX) and the water extractable arabinoxylans (WE- AX).
  • WU-AX un-extractable arabinoxylans
  • WE- AX water extractable arabinoxylans
  • the enzymes of the present invention can be produced in host cells, for example, by secretion or intracellular expression.
  • a cultured cell material e.g., a whole-cell broth
  • the enzyme can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final enzyme.
  • Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae or Trichoderma reesei.
  • Other host cells include bacterial cells, e.g., Bacillus subtilis or B. licheniformis. as well as Streptomyces, E. coli.
  • a DNA construct comprising a nucleic acid encoding an enzyme can be constructed to be expressed in a host cell. Because of the well-known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding xylanase can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.
  • the vector may be any vector that can be transformed into and replicated within a host cell.
  • a vector comprising a nucleic acid encoding an enzyme can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector.
  • the vector also may be transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional xylanase.
  • Host cells that serve as expression hosts can include filamentous fungi, for example.
  • the Fungal Genetics Stock Center (FGSC) Catalogue of Strains lists suitable vectors for expression in fungal host cells. See FGSC, Catalogue of Strains, University of Missouri, at www.fgsc.net (last modified January 17, 2007).
  • a representative vector is pJG153, a promoterless Cre expression vector that can be replicated in a bacterial host. See Harrison et al. (June 2011) Applied Environ. Microbiol. 77: 3916-22.
  • pJG153 can be modified with routine skill to comprise and express a nucleic acid encoding a xylanase.
  • a nucleic acid encoding an enzyme can be operably linked to a suitable promoter, which allows transcription in the host cell.
  • the promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • Exemplary promoters for directing the transcription of the DNA sequence encoding a xylanase, especially in a bacterial host, are the promoter of the lac operon of E.
  • the Streptomyces coelicolor agarase gene dagA or celA promoters the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.
  • examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, A. niger acid stable a-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase.
  • TAKA amylase Rhizomucor miehei aspartic proteinase
  • Aspergillus niger neutral a-amylase A. niger acid stable a-amylase
  • A. niger glucoamylase Rhizomucor miehei lipase
  • Rhizomucor miehei lipase Rhizomucor miehe
  • a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter.
  • suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris A0X1 or AOX2 promoters
  • cbhl is an endogenous, inducible promoter from Trichoderma reesei. See Liu el al. (2008) “Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl) promoter optimization,” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.
  • the coding sequence can be operably linked to a signal sequence.
  • the DNA encoding the signal sequence may be the DNA sequence naturally associated with the enzyme gene to be expressed or from a different Genus or species.
  • a signal sequence and a promoter sequence comprising a DNA construct or vector can be introduced into a fungal host cell and can be derived from the same source.
  • the signal sequence is the cbhl signal sequence that is operably linked to a cbhl promoter.
  • An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a variant xylanase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
  • the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell.
  • sequences are the origins of replication of plasmids pUC19, pACYC177, pUBUO, pE194, pAMBl, and pIJ702.
  • the vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • a selectable marker e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xx.sC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art
  • Intracellular expression may be advantageous in some respects, e.g., when using certain bacteria or fungi as host cells to produce large amounts of xylanase for subsequent enrichment or purification.
  • Extracellular secretion of xylanase into the culture medium can also be used to make a cultured cell material comprising the isolated xylanase.
  • the expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes.
  • the expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes.
  • the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the enzyme to a host cell organelle such as a peroxisome, or to a particular host cell compartment.
  • a targeting sequence includes but is not limited to the sequence, SKL.
  • the nucleic acid sequence of the xylanase is operably linked to the control sequences in proper manner with respect to expression.
  • An isolated cell is advantageously used as a host cell in the recombinant production of an enzyme according to the instant invention.
  • the cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
  • suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus', lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis; Lactobacillus sp.
  • Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans
  • strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.
  • a suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, 5. pombe species.
  • a strain of the methylotrophic yeast species, Pichia pastoris can be used as the host organism.
  • the host organism can be a Hansenula species.
  • Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans.
  • strains of a Fusarium species e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism.
  • Other suitable strains include Thermomyces and Mucor species.
  • Trichoderma sp. can be used as a host.
  • a suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023.
  • An enzyme expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety.
  • the glycosylation pattern can be the same or different as present in the wild-type xylanase.
  • the type and/or degree of glycosylation may impart changes in enzymatic and/or biochemical properties.
  • Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation or by any other means that renders a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein.
  • Any gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2. egll, and egl2 genes.
  • Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.
  • Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, e.g., lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion.
  • General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra.
  • the expression of heterologous protein in Trichoderma is described, for example, in U.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991-1001 for transformation of Aspergillus strains.
  • Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding an enzyme is stably integrated into a host cell chromosome. Transformants are then selected and purified by known techniques.
  • a method of producing an enzyme of the instant invention may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the enzyme. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
  • An enzyme secreted from the host cells can be used in a whole broth preparation.
  • the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the ail resulting in the expression of a xylanase. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid- state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the xylanase to be expressed or isolated.
  • the term “spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term “spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.
  • An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • Host cells may be cultured under suitable conditions that allow expression of a xylanase.
  • Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
  • protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose.
  • Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNTTM (Promega) rabbit reticulocyte system.
  • Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare an enzyme polypeptide-containing solution.
  • a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an enzyme solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction, or chromatography, or the like, are generally used.
  • the enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved by any of the techniques discussed herein. Exemplary methods of enrichment and purification include but are not limited to rotary vacuum filtration and/or ultrafiltration.
  • xylanases having reduced sensitivity to xylanase inhibitors in adjuncts such as rye and wheat can be employed in distilling and brewing to overcome longstanding problems.
  • U.S. law requires that spirits sold as rye whiskey must be produced from malts composed of at least 51% rye grains. It is known to those of skill in the art that such malts are exceedingly difficult to work with on an industrial scale.
  • the present invention relates to a rye mashing and an optional filtration step in a process for the production of an alcoholic beverage, such as beer or distilling applications such as whiskey, and to a composition useful in the mashing and optional filtration step in such a process.
  • xylanases to improve filterability of malt-based mashes.
  • specific proteinaceous inhibitors of xylanases are present in non-malt adjuncts such as rye and wheat.
  • Methods are provided in accordance with the instant invention for screening xylanases to find those that are less inhibited by wheat and rye factors.
  • the present invention discloses a xylanase with very low rye and wheat XIP inhibition that show excellent properties in decreasing the viscosity or rye and wheat based mashes.
  • it is common to filter the mash to prepare a liquid wort for subsequent operations.
  • the lower viscosity provides greater filterability.
  • distilling and in some beer brewing the mash goes to fermentation without filtration.
  • lower mash viscosity results in less gumming of fermentation and distilling equipment and a higher yield of ethanol.
  • a method for producing a rye whiskey having the steps of: (a) providing a grist comprising 51 w/w % of rye grain; (b) adding water to the grist to provide a mash; (c) pre-liquefying the mash of step (b); (d) gelatinizing the mash of step (c); (e) liquefying the mash of step (d) in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor; (f) saccharifying the mash of step (e); (g) fermenting the mash of step (f) to produce a fermentate containing ethanol; (h) distilling the fcrmcntatc of step (g) to provide the rye whiskey.
  • the xylanasc has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by Rye XIP inhibitor. More preferably, the xylanase has less than 20% inhibition by Rye XIP inhibitor.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.
  • the rye grain is malted. In other preferred embodiments, the rye grain is unmaltcd. Preferably, the rye grain is milled.
  • the grist is 60, 70, 80, 85, 90, 95, 99 or 100 w/w % rye.
  • the grist further comprises malted or unmalted barley, wheat, com, rye, rice, cassava, oatmeal or sorghum. More preferably, the grist further comprises malted barley.
  • the method also includes the further step of adding an alpha-amylase and/or a beta-glucanase at any of steps (b), (c), (d) and/or (e). More preferably, a beta-glucanase is added.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof. Still more preferably, the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof. Yet more preferably, the beta-glucanase is an enzyme having beta- glucanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. More preferably, the beta-glucanase is an enzyme having beta- glucanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof. Still more preferably, the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8.
  • a glucoamylase is added at step (f) and/or a protease is added at step (g).
  • the method further includes the step of adding an alpha-L- arabinofuranosidase at any of steps (b), (c), (d) and/or (e).
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 or an alpha-L- arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 or an alpha-L- arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L- arabinofurano sidasc active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the disclosed method also preferably includes the further step of adding a feruloyl esterase at any of steps (b), (c), (d) and/or (e).
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. More preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Yet more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Yet more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. More preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Most preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • a method for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist having rye cereal in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor; (b) optionally filtering the mash to obtain a wort.
  • the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by Rye XIP inhibitor. More preferably, the xylanase has less than 20% inhibition by Rye XIP inhibitor.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. Yet more preferably, an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.
  • the rye grain is malted. In other preferred embodiments, the rye grain is unmalted. Preferably, the rye grain is milled. Preferably, the grist is 60, 70, 80, 85, 90, 95, 99 or 100 w/w % rye. In other preferred embodiments, the grist further comprises malted or unmalted barley, wheat, com, rye, rice, cassava, oatmeal or sorghum. More preferably, the grist further comprises malted barley.
  • the method also includes the further step of adding an alpha-amylase and/or a beta-glucanase to the mash. More preferably, a beta-glucanase is added.
  • the beta- glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof. More preferably, the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. Yet more preferably, the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof. In still more preferred embodiments, the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof. Still more preferably, the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof. Yet more preferably, the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8 or a beta-glucanase active fragment thereof. Most preferably, the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8.
  • the method further includes the step of adding an alpha-L- arabinofuranosidase to the mash.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L- arabinofuranosidase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L-arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10 or an alpha-L- arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10 or an alpha-L- arabinofurano sidasc active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L- arabinofuranosidase active fragment thereof.
  • the alpha-L- arabinofuranosidase is an enzyme having alpha-L-arabinofuranosidase activity with a polypeptide amino acid sequence according to SEQ ID NO: 10 or an alpha-L-arabinofuranosidase active fragment thereof or SEQ ID NO: 11 or an alpha-L-arabinofuranosidase active fragment thereof.
  • the disclosed method also preferably includes the further step of adding a feruloyl esterase to the mash.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. More preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Yet more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Yet more preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. More preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof. Most preferably, the feruloyl esterase is an enzyme having feruloyl esterase activity with a polypeptide amino acid sequence according to SEQ ID NO: 12 or a feruloyl esterase active fragment thereof.
  • a method for identifying a xylanase that has reduced sensitivity to a cereal XIP inhibitor having the step of assaying the activity of the xylanase in the presence and absence of the cereal XIP inhibitor and determining the percent inhibition by the cereal XIP inhibitor.
  • the cereal is barley, wheat, corn, rye, rice, cassava, oatmeal, or sorghum. More preferably, the cereal is rye or wheat. Still more preferably, the cereal is rye. In other preferred embodiments, the cereal is wheat.
  • a xylanase having reduced sensitivity to rye XIP inhibitor identified by the method above.
  • the xylanase has less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% inhibition by rye XIP inhibitor.
  • the xylanase has less than 20% inhibition by rye XIP inhibitor.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. Yet more preferably, an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.
  • a xylanase having reduced sensitivity to wheat XIP inhibitor identified by the method of claim 110.
  • the xylanase has less than 90, 80, 70, 60, 55, 50, 40, 30, 20, 10, 5 or 1% inhibition by wheat XIP inhibitor. More preferably, the xylanase has less than 55% inhibition by wheat XIP inhibitor.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. Yet more preferably, an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof. More preferably, the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3.
  • a composition for reducing wort viscosity in wheat adjunct brewing having a xylanase having reduced sensitivity to wheat XIP inhibitor and a beta-glucanase.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof
  • the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 70% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 85% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence having at least 90% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta- glucanase activity with a polypeptide amino acid sequence having at least 92% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta- glucanase activity with a polypeptide amino acid sequence having at least 95% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta- glucanase activity with a polypeptide amino acid sequence having at least 98% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta- glucanase activity with a polypeptide amino acid sequence having at least 99% sequence identity to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or a xylanase active fragment thereof or SEQ ID NO:3 or a xylanase active fragment thereof and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8 or a beta-glucanase active fragment thereof.
  • the xylanase is an enzyme having xylanase activity with a polypeptide amino acid sequence according to SEQ ID NO:2 or SEQ ID NO:3 and wherein the beta-glucanase is an enzyme having beta-glucanase activity with a polypeptide amino acid sequence according to SEQ ID NO:8.
  • a method for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist having wheat grain in the presence of a composition having a xylanase having reduced sensitivity to wheat XTP inhibitor and a bcta-glucanasc as described above; (b) optionally filtering the mash to obtain a wort.
  • the wheat grain is malted. In other preferred embodiments, the wheat grain is unmalted. Preferably, the wheat grain is milled.
  • the grist is 5, 10, 20, 25, 30, 35, 40, 45, 50, 51, 60, 70, 80, 85, 90, 95, 99 or 100 w/w % wheat.
  • the grist also has malted or unmalted barley, corn, rye, rice, cassava, oatmeal, or sorghum. More preferably, the grist also has malted barley.
  • a composition having a xylanase which has a polypeptide sequence with at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof and a glucanase.
  • the xylanase polypeptide has at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. More preferably, the xylanase polypeptide has at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide has at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Yet more preferably, the xylanase polypeptide has at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. In yet more preferably embodiments, the xylanase polypeptide has at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Still more preferably, the xylanase polypeptide has at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide is a sequence according to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase polypeptide has a sequence according to SEQ ID NO:2.
  • the glucanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. More preferably, the glucanase polypeptide has at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Still more preferably, the glucanase polypeptide has at least 90% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof. In yet more preferred embodiments, the glucanase polypeptide has at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase polypeptide has at least 95% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Yet more preferably, the glucanase polypeptide has at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Still more preferably, the glucanase polypeptide has at least 99% sequence identity to SEQ ID NO: 8 or a glucanase active fragment thereof. In yet more preferred embodiments, the glucanase polypeptide is a sequence according to SEQ ID NO: 8 or a glucanase active fragment thereof. In the most preferred embodiments, the glucanase polypeptide is a sequence according to SEQ ID NO:8.
  • the xylanase polypeptide preferably has at least 98, 99 or 100% sequence identity to SEQ ID NO:2.
  • the composition is a liquid.
  • the xylanase is preferably present in an amount of 1,000 to 50,000 NGXU/g and the glucanase is preferably present in an amount of 1,000 to 50,000 BBXU/g. More preferably, the xylanase is present in an amount of 5,000 to 30,000 NGXU/g and the glucanase is present in an amount of 5,000 to 30,000 BBXU/g. Still more preferably, the xylanase is present in an amount of 10,000 to 25,000 NGXU/g and the glucanase is present in an amount of 15,000 to 30,000 BBXU/g. In the most preferred embodiments, the xylanase is present in an amount of 15,000 to 20,000 NGXU/g and the glucanase is present in an amount of 20,000 to 28,000 BBXU/g.
  • a method is presented of altering filterability of a starch containing material, the method having the step of treating the starch containing material with a composition as described above.
  • a method is presented of reducing pressure built up during lautering in a brewing application, the method having the step of treating a brewing mash having a starch containing material with a composition as described above.
  • a method for the production of a food, feed, or beverage product, such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey, the method having the step of treating a starch containing material with a composition as described above.
  • a food, feed, or beverage product such as an alcoholic or non-alcoholic beverage, such as a cereal- or malt-based beverage like beer or whiskey
  • a method for the production of a brewing mash, the method having the step of treating a starch comprising material with a composition as described above.
  • the starch comprising material comprises a grist.
  • the grist has at least 50, 60, 70, 80 or 90 % malt. More preferably, the grist further also contains wheat, corn or rye. In the above methods, preferably 0.01 to 1 grams of the composition is added per Kg of grist. More preferably, 0.02 to 0.1 grams of the composition is added per Kg of grist.
  • a method for preparing a low viscosity mash having the steps of: (a) preparing a mash from a grist in the presence of a xylanase having reduced sensitivity to rye XIP inhibitor and a beta-glucanase which has increased activity in the presence of at least 10% rye compared to a sample not having rye; and (b) optionally filtering the mash to obtain a wort.
  • the xylanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. More preferably, the xylanase is a polypeptide having at least 85% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Still more preferably, the xylanase is a polypeptide having at least 90% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Yet more preferably, the xylanase is a polypeptide having at least 92% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof.
  • the xylanase is a polypeptide having at least 95% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. More preferably, the xylanase is a polypeptide having at least 98% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Still more preferably, the xylanase is a polypeptide having at least 99% sequence identity to SEQ ID NO:2 or a xylanase active fragment thereof. Yet more preferably, the xylanase is a polypeptide according to SEQ ID NO:2 or a xylanase active fragment thereof. Most preferably, the xylanase is a polypeptide according to SEQ ID NO:2.
  • the glucanase is a polypeptide having at least 80% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. More preferably, the glucanase is a polypeptide having at least 85% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Yet more preferably, the glucanase is a polypeptide having at least 90% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. In still more preferred embodiments, the glucanase is a polypeptide having at least 92% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof.
  • the glucanase is a polypeptide having at least 95% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Still more preferably, the glucanase is a polypeptide having at least 98% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. Yet more preferably, the glucanase is a polypeptide having at least 99% sequence identity to SEQ ID NO:8 or a glucanase active fragment thereof. In still more preferred embodiments, the glucanase is a polypeptide according to SEQ ID NO:8 or a glucanasc active fragment thereof. Most preferably, the glucanase is a polypeptide according to SEQ ID NO:8.
  • the grist has at least 50, 60, 70, 80 or 90% malt. More preferably, the grist also has wheat, corn or rye. More preferably, the grist has rye. Still more preferably, the grist has at least 10% rye.
  • FveXyn4_l is a mature xylanase variant from Fusarium verticilliodes having the amino acid sequence shown in SEQ ID NO:2.
  • a preparation of the GH11 xylanase (E.C. 3.2.1.8) from Penicillium funiculosum was utilized (PfunXyn) having the mature amino acid sequence shown in SEQ ID NO:5.
  • This preparation of PfunXyn (having an activity of 3150 CMC-DNS U/g) was provided from Dupont Nutrition Bioscience Aps (Denmark).
  • Another example of an efficient solubilizing xylanase used in brewing a preparation of the GH10 xylanase (E.C.
  • TemXyn having the mature amino acid sequence shown in SEQ ID NO:7.
  • This preparation of TemXyn (having an activity of 5525 units/g) was provided from Dupont Nutrition Bioscience Aps (Denmark).
  • Esmark as an example of an enzyme complex hydrolyzing betaglucans and non-starch polysaccharides like arabinoxylans, containing a (E.C. 3.2.1.6) beta- glucanase from Bacillus subtilis (BsbBglu) having the mature amino acid sequence shown in SEQ ID NO:8 and a (E.C.
  • Xylanase variant from Aspergillus kawachii (AkaXyl_var) having the mature amino acid sequence shown in SEQ ID NO:9 was utilized.
  • This preparation of BsbBglu + AkaXyl_var (having a xylanase activity of 13680 - 16720 XBU/g and having beta- glucanase activity of 9090 - 12500 BBU/g) was provided from Dupont Nutrition Bioscience Aps (Denmark).
  • TrXyl As an example of an enzyme complex having various cellulytic activities including bcta-glucosidasc (EC3.2.1.21) and xylanasc produced by Trichoderma reesei (Hypocrea jecorind) TrXyl was obtained from Dupont Nutrition Bioscience Aps (Denmark), beta- glucanase from Bacillus subtilis (BsdBglu) (beta-glucanase activity of 51000 U/g) was provided from Dupont Nutrition Bioscience Aps (Denmark).
  • BsdBglu Bacillus subtilis
  • a-L-Arabinofuranosidase with potential hydrolysis of hydrolysis of terminal, non-reducing a-L-arabinofuranose from singly substituted xylose residues in arabinoxylan (a- 1,2 > a- 1,3), AniAfur, an a-L-Arabinofuranosidase from Aspergillus nidulans (89U/mg 500U) was sourced from Megazyme International, Ireland. AniAfur would have a pH optimum of 4.5 and temperature optimum of 40°C.
  • BaAfur an a-L-Arabinofuranosidase from Bifidobacterium adolescentis, (102U/mg 400U) was sourced from Megazyme International, Ireland. BaAfur would have a pH optimum of 6.0 and temperature optimum of 50°C. This enzyme would be expected to be highly specific hydrolysis of a-l,3-linked L-arabinofuranose residues from doubly substituted D- xylosyl or L-arabinosyl residues of arabinoxylans and branched arabinans, respectively.
  • CtFe a Feruloyl esterase from Clostridium thermocellum (0.5U/mg 100U) was sourced from Megazyme International, Ireland.
  • CtFe would have a pH optimum of 6.0 and a temperature optimum of 60°C.
  • CtFe is expected to catalyse the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is usually arabinose in "natural" substrates.
  • Genomic DNA isolated from a strain of Fusarium verticillioides was used for amplifying a xylanase gene.
  • the sequence of the cloned gene, called the FveXyn4 was recombinantly changed as described in WO2015/114108.
  • the protein encoded by the FveXyn4_l gene is depicted in SEQ ID No. 1.
  • the protein product of gene FveXyn4 belongs to glycosyl hydrolase family 10 (GH10) based on the PF AM search (http://pfam.sanger.ac.uk/).
  • FveXyn4 protein has a 25 amino acid signal peptide predicted by SignalP-NN (Emanuels son et al., Nature Protocols, 2:953-971, 2007). This indicates that FveXyn4 is a secreted glycosyl hydrolase.
  • Example 3 Expression of Fusarium verticillioides xylanase variant FveXyn4_l
  • the FvcXyn4_l gene was amplified from genomic DNA of Fusarium verticillioides variant 1 using the following primers: Primer 1 5'-caccATGAAGCTGTCTTCTTTCCTCTA-3', and Primer 2 5'-TTTTTAGCGGAGAGCGTTGACAACAGC-3'.
  • the PCR product was cloned into pENTR/D-TOPO vector (invitrogen K2400) to generate the FveXyn4_l pEntry plasmid.
  • the expression plasmid pZZH254 was obtained by Gateway cloning reaction between the FveXyn4_l pEntry plasmid and pTrex3gM expression vector (described in US 2011/0136197 Al) using Gateway® LR Clonase® II enzyme kit (Invitrogen 11791). The sequence of the FveXyn4 gene was confirmed by DNA sequencing.
  • the plasmid pZZH254 was transformed into a quad deleted Trichoderma reesei strain (described in WO 05/001036) using biolistic method (Te'o VS et al., J Microbiol Methods, 51:393-9, 2002).
  • protoplasts of a quad deleted T. reesei strain were transformed with the expression plasmid pTTT-Ate CAI using the PEG protoplast method (Penttila et al, Gene, 61:155-164, 1987).
  • spores were grown for about 10 hours at 24°C in Trichoderma Minimal Medium MM (20 g/L glucose, 15 g/L KH2PO4, pH 4.5, 5 g/L (NH4)2SO4, 0.6 g/L MgSO4x7H2O, 0.6 g/L CaC12x2H2O, 1 ml of 1000X T.
  • reesei Trace elements solution (175 g/L Citric Acid anhydrous, 200 g/L FeSO4x7H2O, 16 g/L ZnSO4x7H2O, 3.2 g/L CuSO4, 1.4 g/L MnSO4xH2O, and 0.8 g/L Boric Acid).
  • Germinating spores were harvested by centrifugation and treated with 30 mg/mL Vinoflow FCE (Novozymes, AG Switzerland) solution for from 7 hours to overnight at 30°C at 100 rpm to lyse the fungal cell walls.
  • Protoplasts were washed in 0.1 M Tris HC1 buffer (pH 7) containing 0.6 M sorbitol and resuspended in 10 mM Tris HC1 buffer (pH 7.5) containing 1.2 M sorbitol and 10 mM calcium chloride.
  • 10 mM Tris HC1 buffer pH 7.5/10 mM CaC12 solution.
  • Transformants were selected on a medium containing acetamide as a sole source of nitrogen (acetamide 0.6 g/L; cesium chloride 1.68 g/L; glucose 20 g/L; potassium dihydrogen phosphate 15 g/L; magnesium sulfate heptahydrate 0.6 g/L; calcium chloride dihydrate 0.6 g/L; iron (II) sulfate 5 mg/L; zinc sulfate 1.4 mg/L; cobalt (II) chloride 1 mg/L; manganese (11) sulfate 1.6 mg/L; agar 20 g/L; pH 4.25). Transformed colonies (about 50-100) appeared in about 1 week.
  • the spores were collected and reselected on acetamide plates. After 5 days, the spores were collected using 10% glycerol, and 1 x 108 spores were inoculated in a 250 ml shake flask with 30 ml Glucosc/Sophorosc defined medium for protein expression. Protein expression was confirmed by SDS-PAGE. The spore suspension was subsequently grown in a 7 L fermentor in a defined medium containing 60% gluco se-sophorose feed.
  • Glucose/Sophorose defined medium (per liter) consists of (NH4)2SO4 5 g, PIPPS buffer 33 g, Casamino Acids 9 g, KH2PO4 4.5 g, CaC12 (anhydrous) 1 g, MgSO4.7H2O 1 g, pH to 5.5 adjusted with 50% NaOH with Milli-Q H2O to bring to 966.5 mL. After sterilization, the following were added: 26 mL 60% Glucose/Sophrose, and 400X T. reeselTrace Metals 2.5 mL.
  • FveXyn4_l was purified from concentrated fermentation broth of a 7L fermentor culture using two chromatography columns. Concentrated fermentation broth buffered in 20 mM sodium phosphate buffer pH 6.0 containing 1 M ammonium sulfate was loaded on a hydrophobic interaction chromatography column (Sepharose Phenyl FF, 26/10). The protein was eluted from the column using a linear gradient of equilibration/wash buffer to 20 mM sodium phosphate buffer pH 6.0.
  • the fraction containing FveXyn4_l protein was loaded onto a gel filtration column (HiLoad Superdex 75 pg 26/60), and the mobile phase used was 20 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl.
  • the purified protein was concentrated using a 3K Amicon Ultra- 15 device and the concentrated protein fraction was used in further studies.
  • Reagents used in the assay Concentrated (2x) Laemmli Sample Buffer (Bio-Rad, Catalogue #161-0737); 26-well XT 4-12% Bis-Tris Gel (Bio-Rad, Catalogue #345-0125); protein markers “Precision Plus Protein Standards” (Bio-Rad, Catalogue #161- 0363); protein standard BSA (Thermo Scientific, Catalogue #23208) and SimplyBlue Safestain (Invitrogen, Catalogue #LC 6060.
  • the assay was carried out as follow: In a 96well-PCR plate 50pL diluted enzyme sample were mixed with 50 pL sample buffer containing 2.7 mg DTT. The plate was sealed by Microseal ‘B’ Film from Bio-Rad and was placed into PCR machine to be heated to 70°C for 10 minutes. After that the chamber was filled by running buffer, gel cassette was set. Then 10 pL of each sample and standard (0.125-1.00 mg/mL BSA) was loaded on the gel and 5 pL of the markers were loaded. After that the electrophoresis was run at 200 V for 45 min. Following electrophoresis, the gel was rinsed 3 times 5 min in water, then stained in Safestain overnight and finally destained in water.
  • Example 5 Mature polypeptide sequence of Fusarium verticillioides xylanase variant FveXyn4_l
  • the amino acid sequence of the predicted form of FveXyn4_l protein is set forth as SEQ ID No. 3. This was expected to be the active form of the enzyme. However, we surprisingly identified the mature and active form of FveXyn4_l protein to be identical with the sequence set forth in SEQ ID No. 2, likely due to the host generated post-translation modifications and endogenous activities during fermentation influencing cleavage of the signal peptide.
  • FveXyn4_l was unexpectedly found to be less proteolytically modified in the final broth at the end of fermentation as compared to the predicted variant shown in SEQ ID NO:3.
  • the variant generated was verified using mass spectrometry as described in detail below.
  • the final expressed variant was less processed in the N-terminus as compared to the predicted 25 amino acids cleavage by SignalP version 4.0, resulting in a final N-terminal truncation of 23 amino acids corresponding to a mature version of FveXyn4_l having the polypeptide sequence of SEQ ID NO:2.
  • This mature variant of FveXyn4_l has been used in all examples discussed herein.
  • the protein band of the protein was cut from an SDS-PAGE gel and digested using three different enzymes (Trypsin, A-Chymotrypsin and Glu-C) to prepare the sample for mass spectrometry analysis. Trypsin hydrolyzes peptide bonds specifically at the carboxyl side of arginine I and lysine (K) residues except when a proline (P) is on the carboxyl side.
  • A-Chymotrypsin hydrolyzes peptide bonds specifically at the carboxyl side of tyrosine (Y), phenylalanine (F), tryptophan (W) and leucine (L) except when a proline (P) is on the carboxyl side.
  • Glu-C preferentially cleaves at the carboxyl side of glutamyl (E) in ammonium bicarbonate buffer pH 8, but also cleaves at the carboxyl side of aspartyl (D) if the hydrolysis is carried out in a phosphate buffer pH 8.
  • the protein of interest was prepared for analysis using IFF procedure for protein characterization (A2963), with one change using 40% 180-water in the digestion buffer.
  • the proteolytic cleavage will hereby incorporate both 180-water and 160- water in the resulting peptides, which consequently will appear as doublets in MS spectra.
  • the protein C-terminal though will only appear as a single peptide with 160- water since it is not cleaved but just the “last peptide” left of the protein. In this way the C-terminal is mapped using MS/MS analysis.
  • the intact protein is labeled with acetylation of the N-terminal before proteolytic digestion (IFF A-manual 3448).
  • lysine converts lysine to homoarginine and protects lysine (side chain) from being acetylated. Only the peptide originating from the protein N-terminal will be acetylated and hereby unambiguously identified.
  • Example 6 Viscosity reduction and filterability of rye-based mashing trials with different xylanases in combination with a-L-Arabinofuranosidase and Feruloyl esterase
  • the objective of this example was to demonstrate the benefit of having an un-inhibited xylanase present during a 100% rye based mashing process. Enzymes was tested in a mashing operation model system for wort production using milled Rye (Supplied by Hiram Walker, US., Material Code: 202109-2201) milled at a Buhler Miag malt mill 0.5 mm setting, position 4).
  • Rye grist (60.0g milled Rye grains) was mixed in beakers with 203g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid., resulting in a water to grist ratio of 3.38:1.
  • the FveXyn4_l xylanase variant was added based on mg protein determined according to example 4 in the following setup:
  • Trial 1 1,89 CMC-DNS U PfunXyn per g Rye; Trial 2, 6.8 pg FveXyn4_l protein per g Rye; Trial 3, 1,89 CMC-DNS U PfunXyn per g Rye + 0.3 U AniAfur per g Rye + 0.3 U BaAfur per g Rye; Trial 4, 1,89 CMC-DNS U PfunXyn per g Rye + 0.3 U AniAfur per g Rye + 0.3 U CtFe per g Rye; Trial 5, 1,89 CMC-DNS U PfunXyn per g Rye + 0.3 U BaAfur per g Rye + 0.3 U CtFe per g Rye Trial 6, 6.8 pg FveXyn4_l protein per g Rye + 0.3 U AniAfur per g Rye + 0.3 U BaAfur per g Rye; Trial 7, 6.8 pg FvcXyn4_l protein per g Rye
  • the 0.730 mg AMYLEX® 6T per g rye grist was added to each trial.
  • the grist was mashed with the program; heated to 45°C and kept for 40 minutes for mashing in; heated to 65°C for 10.0 minutes by increasing temperature with l°C/minute; kept at 65°C for 40 minutes; heated to 74°C for 10 minutes by increasing temperature with l°C/minute; kept at 74°C for 160 minutes and mashing off.
  • each beaker was adjusted to 263 g with tap water and cooled to 25°C before centrifugation. Separation was performed by centrifugation; 4000 rpm for 5 minutes (5°C) in swing bucket centrifuge (Thermo Scientific SL 8R Centrifuge). The volumes of supernatant were measured in mL (measuring glass) after centrifugation and result are given in table 3. It is clear’ that the un-inhibited FveXyn4_l resulted in much larger supernatant volume, being equivalent to an increased filtration yield. Table 3, Mash supernatant volume after centrifugation. Trial 1 , 6.8 pg FveXyn4_l protein per g Rye and Trial 2, 1,89 CMC-DNS U PfunXyn per g rye.
  • Turbidity of the mash supernatant samples after mashing was measured using an Anton Paar (DMA 5000, HazeQC ME module) according to Dupont Standard Instruction Brewing, 23.8580-B28 and shown very different depending on the xylanase use in experiments.
  • the turbidity (S90/S25 EBC) was measured at a 90° scatter angle to detect the presence of small particles and turbidity measured at 25° scatter angle was included as additional information on larger particles.
  • the turbidity data were shown in Figure 1 and 2 with S90 and S25 turbidity respectively.
  • shear (flow) viscosity of samples were measured at 20°C using an Anton Paar rheometer type MCR301 or MCR302e with measuring system “Double Gap (DG26.7)”. The viscosity was measured over shear rates from 0.1 to 100 s-1 using a logarithmic ramp with integration times from 20 to 1 s. Viscosity results was obtained at the specific shear rate at 10 s-1 and are shown in table 5.
  • Example 7 Application of xylanases for Rye mashing in brew analysis with determination of real degree of fermentation (RDF) and alcohol content Rye mashes were prepared similar to example 6 with following differences.
  • Rye grist (65.0g milled Rye grains) was mixed in beakers with 220g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid., resulting in a water to grist ratio of 3.38:1.
  • the FveXyn4_l xylanase variant was added based on mg protein determined according to example 4 in the following setup:
  • 0.184 mg AMYLEX® 6T per g rye grist was added to at mashing in 66°C and 0.184 mg AMYLEX® 6T per g rye grist was added to main mashing in 74°C at each trial.
  • the grist was mashed with the program; heated to 66°C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 66°C for 40 minutes; heated to 74°C for 8.0 minutes by increasing temperature with l°C/minute; kept at 74°C for 55 minutes; second dose of AMYLEX® 6T and kept at 74°C for another 105 minutes; content of each sample was adjusted to 285g; cool to 35°C.
  • each mash was weighed into a 500 mL conical flask for fermentation adding 15 mL of yeast slurry (0.5% yeast, SafSpirit HG-1 dry yeast for distilling from Fermentis) to the mash having 35 °C; mash was fermented on the grain.
  • yeast slurry (0.5% yeast, SafSpirit HG-1 dry yeast for distilling from Fermentis
  • the remaining of the mash was separated by centrifugation; 4000 rpm for 5 minutes (5°C) in swing bucket centrifuge (Thermo SCIENTIFIC SL 8R Centrifuge). The supernatant was used for analysis, see below.
  • the samples were fermented at 35°C and 150 rpm after yeast addition. All sample were added 0.262 mg DIAZYME® 480 per g rye to facilitate saccharification during fermentation. Analysis was performed when fermentation had finished.
  • Supernatant analysis Original Extract (OE), Extract in the wort samples after mashing was measured using Anton Paar (Lovis) following Standard Instruction Brewing, 23.8580-B28.
  • FAN Free Alpha- Amino Nitrogen
  • Beer analysis was measured using an Anton Paar (DMA 5000) following Standard Instruction Brewing, 23.8580-B28 and alcohol by Standard Instruction Brewing, 23.8580-B28.
  • Real degree of fermentation (RDF) value may be calculated according to the equation below:
  • RDF Real degree of fermentation
  • E(r) is the real extract in degree Plato (°P) and OE is the original extract in °P.
  • RDF Real degree of fermentation
  • extract and alcohol content of the fermented beer are shown in table 6. Both the obtained RDF values, extract and alcohol values were higher of fermented beer with FveXyn4_l applied in mashing as compared to PfunXyn.
  • the extract average of three ferments with PfunXyn was 16.71°P and three ferments with FveXyn4_lwas 17,04°P.
  • the RDF average of three ferments with PfunXyn was 77.50% and three ferments with FveXyn4_lwas 77.58%.
  • the alcohol content average of three ferments with PfunXyn was 8.70% and three ferments with FveXyn4_lwas 8.89%.
  • Example 8 Arabinoxylane analysis of Rye wort samples prepared with different xylanases in combination with a-L-Arabinofuranosidase and Feruloyl esterase.
  • the content of soluble arabinoxylans was quantified in the Rye mash produced as described in example 6.
  • LC-MS reverse phase chromatography was used to characterize and quantify the content of the different soluble arabinoxylan were fractionated by DP (extending from AXl/mono to AX7+) on an Agilent 1290 BinPump system coupled to a Bruker maXis QTOF 4G.
  • the chromatographic separations was performed on a Acquity UPEC® HSS T3 column (100 x 2,1 mm id, 1,8 pm) using a water:Methanol:TBA:Acetic acid 980:20:2.4:10 (A) MethanokTB A: Acetic Acid 1000:2.4:10 (B) gradient run for 16 min, Injection volume 10 pl, flow rate of 200 pl/min.
  • the effluent from the column was coupled via an clcctrospray ionisation interface (Ionisation: ESI (electrospray) in Negativ mode) to the high-resolution maXis mass spectrometer.
  • the following spectrophotometer setting was applied: Nebulizer: 2.0 Bar, dry gas: 8 E/min and dry heater: 200 °C.
  • AX fractions were determined for the two samples applied cither PfunXyn or FvcXyn4_l with combinations of a-L- Arabinofuranosidase and Feruloyl esterase. The method was limited to determine the soluble arabinoxylan which means AX9 or lower.
  • Example 9 - Xylanase rye and wheat inhibition assay.
  • xylanase action on various cereals may be the presence of specific proteinaceous inhibitors of xylanases in grains. Proteinaceous soluble inhibitors of xylanases been found in barley, rye, com and other cereals.
  • the example describes a simple and novel sensitivity assay to rye and wheat xylanase-inhibitor-protein-like (XIP-like) inhibitors. The inhibition degree of rye and wheat was determined for various xylanase products.
  • Rye and wheat XIP inhibitor extracts were prepared by the following procedure.
  • Dilution series ranging from 500 - 1000000 times were prepared using 0.1 M Na-acetate buffer, pH 5.6, Rye XIP inhibitor extract pH 5.6 and Wheat XIP inhibitor extract pH 5.6 with the following xylanase samples; BsbBglu + AkaXyl_var, TrXyl, PfunXyn, TemXyn and FveXyn4_l all obtained from Dupont Nutrition Bioscience Aps, Denmark, Shearzyme P105, Viscoferm and Ultraflo Max from Novozymes, Denmark.
  • the reaction solution was filtered by paper filter (Whatman, Cytiva Grade 597 Plus Qualitative Filter Paper, Circles) and absorbance at 590nm was determined using a Spectrophotometer (Genesys Spectrophotometer 10S UV-Vis).
  • the %-Rye XIP and %-Wheat XIP inhibition were calculated using the following formulas below, only using the enzyme sample dilution resulting in absorbance values ranging between 0.0 and 1.0: % Rye XIP inhibition
  • Abs 590 nm (enzyme diluted in buffer) — Abs 590nm (enzyme diluted in rye extract) Abs 590 nm (enzyme diluted in buffer)
  • Abs 590 nm (enzyme diluted in buffer) — A Abs 590 nm (enzym
  • Example 10 Rye mashing trials with dose-response test of inhibited and un-inhibited xylanases and their effect on viscosity reduction and filterability.
  • the objective of this example was to test potential benefits of a Rye XIP un-inhibited vs inhibited xylanase present during a 100% rye based mashing process, using large dosage range. Enzymes was tested in a mashing operation model system for wort production using milled Rye (Supplied by Hiram Walker, US., Material Code: 202109-2201) milled at a Buhler Miag malt mill 0.5 mm setting, position 4).
  • Rye grist (60.0g milled Rye grains) was mixed in beakers with 203g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid., resulting in a water to grist ratio of 3.38:1.
  • the FveXyn4_l xylanase variant was added based on mg protein determined according to example 4. PfunXyn was dosed 0.787 to 15.7 CMC-DNS U per g Rye and FveXyn4_l dosed 5.7 to 113.3 g FveXyn4_l protein per g Rye for comparison.
  • Mash separation was performed by centrifugation; 4000 rpm for 5 minutes (5°C) in swing bucket centrifuge (Thermo SCIENTIFIC SE 8R Centrifuge). The volumes of the wort were measured in mF (measuring glass) after centrifugation. The wort samples were boiled for 60 minutes in a boiling bath and samples were cooled down to 17°C. All the samples were filtered and used for analysis, sec below.
  • Wort sample extract, density and flow viscosity were determined as described in example 6 and the results are shown in table 9 and 10.
  • Example 11 Malt mashing trials with dose-response test of Rye inhibited and un-inhibited xylanases and their effect on viscosity reduction and filterability.
  • the objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase present during a 100% malt based mashing process, using various dosages. Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Fuglsang, Denmark, Batch Number 12.10.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4).
  • Malt grist (60.0g milled Malt grains) was mixed in beakers with 203g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid., resulting in a water to grist ratio of 3.38:1.
  • the FveXyn4_l xylanase variant was added based on mg protein determined according to example 4. PfunXyn was dosed 0.315 to 1.570 CMC-DNS U per g Rye and FveXyn4_l dosed 2.27 to 11.3 pg FveXyn4_l protein per g Malt for comparison.
  • the grist was mashed as described in example 10 also regarding mash separation and following mash analysis.
  • Wort sample extract, density and flow viscosity were determined as described in example 6 and the results are shown in table 11 and 12.
  • the objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase present during a 50% malt - 50% rye based mashing process, using various xylanase dosages.
  • Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Sophus Fuglsang, Denmark, Batch Number 10.12.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4) and milled Rye (Wey Rye 3-8 EBC from Maltbazaren, Denmark, Batch: 12.03.2020, DK20-00066) milled at a Buhler Miag malt mill 0.5 mm setting, position 4).
  • PfunXyn was dosed 3.15 to 9.45 CMC-DNS U per g Grist
  • TemXyn was dosed 5.52 to 16.57 U per g Grist
  • FveXyn4_l dosed 22.7 to 68.0 pg FveXyn4_l protein per g Grist for comparison.
  • the grist was mashed with the program; heated to 52°C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 52°C for 10 minutes; heated to 65 °C for 13 minutes by increasing temperature with l°C/minute; kept at 65 °C for 45 minutes; heated to 72°C for 7 minutes by increasing temperature with l°C/minute; and kept at 72°C for another 15 minutes; heated to 78°C for 6 minutes by increasing temperature with l°C/minute; and kept at 78 °C for another 30 minutes - mashing off; content of each sample was adjusted to 350g and filtered.
  • the volumes of the wort collected after 5, 10, 15 and 30 minutes were measured, and pH was adjusted to pH 5.2 with 2.5 M sulphuric acid.
  • One pellet of hops was added to each flask.
  • the wort samples were boiled for 60 minutes in a boiling bath and samples were cooled down to 17°C. All the samples were filtered used for analysis, see below.
  • Wort sample extract, density and flow viscosity were determined as described in example 6 and the results are shown in table 13 and 14.
  • Example 13 Comparison of xylanase performance in Malt mashing trials with high Rye, Wheat and Barley inclusion
  • the objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase present during a 60% malt: 40% rye, a 60% malt: 40% wheat and a 60% malt: 40% barley based mashing process, using various xylanase dosages.
  • Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Sophus Fuglsang, Denmark, Batch Number 10.12.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4, milled Rye (Wey Rye 3-8 EBC from Maltbazaren, Denmark, Batch: 12.03.2020, DK20-00066) milled at a Buhler Miag malt mill 0.5 mm setting, position 4, milled barley (Raw Barley from Sophus Fuglsang, Denmark, Batch Number 09.03.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4 and milled wheat (Wheat Whole kernel organic from XXX, Batch Number 17.11.2022-1) at a Buhler Miag malt mill 0.5 mm setting, position 4.
  • Rye, Wheat or Barley grist (28.0g milled Rye grains) and Malt grist (42.0g milled Malt grains) were mixed in beakers with 210g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid, resulting in a water to grist ratio of 3.1:1.
  • the FveXyn4_l xylanase variant was added based on mg protein determined according to example 4. TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist for comparison. To all trials BsdBglu was added to support the xylanase in a varying dose of 2-5U/ kg grist, identical for both xylanases used.
  • the grist was mashed with the program; heated to 60°C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 60°C for 5 minutes; heated to 65°C for 5 minutes by increasing temperature with l°C/minute; kept at 65°C for 45 minutes; heated to 72°C for 7 minutes by increasing temperature with l°C/minute; and kept at 72°C for another 20 minutes; heated to 78°C for 6 minutes by increasing temperature with l°C/minute; and kept at 78°C for another 10 minutes - mashing off; content of each sample was adjusted to 350g and filtered.
  • Wort sample extract and viscosity were determined as described in example 6 and the results shown as an average over all dosages (TemXyn was dosed 0.27 to 0.69 U per g Grist and FveXyn4_l dosed 9.0 to 27.2 pg FveXyn4_l protein per g Grist) for each xylanase are presented in figure 4 and 5.
  • Example 14 Effect of xylanase on ethanol production in Rye-Malt SSF trials with high Rye inclusion.
  • the objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase present during a rye based mashing and fermentation process, using various xylanase dosages.
  • Enzymes was tested in a mashing operation model system for wort production using milled Malt (Distiller’s malt: DK19-01017, Denmark) at a Buhler Miag malt mill 0.5 mm setting, position 4) and milled Rye (Briess Raw rye: Denmark, Batch: DK 20-00053) milled at a Buhler Miag malt mill 0.5 mm setting, position 4).
  • Mash was kept at 65°C for 30 minutes and saccharification of starch was tested with iodine after 30 minutes. Then cooled to 32°C, kept for 5 minutes and each beaker was adjusted to 280 g with tap water. The mash was filled into a 500- ml conical flask for fermentation adding 0.5 % yeast (1.4 g for 280 g mash) to the wort having 32°C/ 90°F. Then yeast (White Labs Distilling Yeast: DK 202009-0301) was added for fermentation. No enzyme was added, and Mash was fermented on the grain. Temperature was set to 35°C/ 95°F and the fermentation lasted for 3 days.
  • Adjunct Liquefaction Temperature was adjusted to 55°C for 1 minute for mashing in, the total grist of 66.5 g rye + 1.75 g malt was added to 210.0 grams water. pH adjustment to 5.5 if needed. After correct pH was measured, alpha amylase (0.15 kg/t grist) was added to ensure efficient liquefaction (AMYLEX 5T, 1.050g dissolved in lOOmL provided from Dupont Nutrition Bioscience Aps Denmark). Temperature was increased to 95 °C with l°C/minute and held for 40 minutes. Saccharification was tested with iodine test at 10, 20, and 30 min at 95°C. Main Mashing: Temperature for mashing in was adjusted to 65°C.
  • Xylanase was dosed on g sample /kg grist according to table 16 below.
  • the mash was cooled to 65 °C and added conversion 1.75 g malt.
  • Mash was kept at 65°C for 30 minutes and saccharification of starch was tested with iodine after 30 minutes.
  • the mash was filled into a 500-ml conical flask for fermentation adding 0.5 % yeast (1.4 g for 280 g mash) (White Labs Distilling Yeast: DK 202009-0301) to the wort having 32°C/ 90°F.
  • yeast 1.750g dissolved in lOOmL provided from Dupont Nutrition Bioscience Aps Denmark.
  • Mash was fermented on the grain. Temperature was set to 35°C/ 95°F and the fermentation lasted for 3 days.
  • Wort sample density, specific gravity and viscosity were determined as described in example 6 and the results are shown in table 17. The density and specific gravity were found to be very similar in all samples, viscosity of the control was simply too high to enable analysis.
  • FveXyn4_l provided the highest levels of ethanol in addition to having the lowest viscosity as seen on figure 6. The results clearly showed that an increasing dose rate of FveXyn4_l also resulted in increasing ethanol yields and more than what was obtained by PfunXyn and FveXyn4_l in a rye-based SSF fermentation.
  • thermostability was tested in malt-based wort
  • a 100% malt-based wort was prepared from malt extract as follows: 1100 g Munton's Light Malt Extract (Batch XB 35189, expiry date 01-2024) was dissolved in 3000 ml warm tap water (45°C). This slurry was stirred for about 10 min until the liquid was homogeneous and the pH was adjusted to 5.6 with 2.5 M sulphuric acid. To the slurry was added 10 pellets of Bitter hops from Hopfenveredlung, St. Johann, Germany: Alpha content of 16,0 % (EBC 7.7 0 specific HPLC analysis, 01.10.2023), then split in 500mL blue-cap bottles and boiled for 1 hour to ensure
  • the final wort had an initial Specific Gravity of 1048 (i.c. 12 °Plato). 200g of the filtered wort was added to a 500 ml and frozen until use.
  • the xylanase activity was determined colorimetricly by monitoring the rate of degradation of 4,6-O-(3-Ketobutylidene)-4-nitrophenyl-0-D-45-glucosyl-xylopentaoside substrate (XylX6).
  • the release of the substrate's 4-nitrophenol directly related to the hydrolysis of the XylX6 by the endo-xylanase activity and is measured at 405nm.
  • the colorimetric substrate contained in the XylX6 assay kit K-XylX6, Megazymes
  • K-XylX6, Megazymes is combined with a 0- xylosidase which allows for the release of the colorimetric group 4-nitrophenol.
  • the blocked XylX6 substrate exhibits the distinct advantage over commonly employed colorimetric oligosaccharide substrates in that the ketobutylidene acetal on the non-reducing terminal residue acts as a “blocking group” preventing hydrolysis by exo-acting enzymes including b-xylosidase and b-glucosidase which commonly occur in crude sample extracts.
  • the XylX6 Substrate used was “Bottle 1” of Megazyme kit K-XylX6-2V purchased from Megazyme Ireland. 0,1 M Sodium Acetate Buffer pH 5.0 (for Substrate Prep only) was prepared by dissolving 9.57g of Sodium Acetate (trihydrate) in 800 mL of Milli-Q water. Adjustment of pH to 5.0 with concentrated acetic acid while stirring.
  • the XylX6 Stock Substrate was prepared by addition of 5mL of Milli-Q water to 1 bottle of XylX6 substrate. The solution was mixed well until thoroughly dissolved. Aliquot 0.75mL of substrate stock into 4mL amber vials and stored at -20°C until use. The XylX6 working Substrate was prepared by 1 vial of frozen stock substrate, 0.75mL, was added 3mL of 12 plato all-malt wort pH 5.6 (as described above) and mixed thoroughly.
  • Mcllvaine Buffer pH 5.0 (Assay Buffer) was prepared by dissolving 10.19g of citric acid (monohydrate) and 18.33g of disodium hydrogen phosphate dihydrate into 900 mL of Milli-Q water. pH was adjusted if needed by IM HC1 or ImNaOH. 2% Tris Stop Solution was prepared by dissolving 20g of Trizma base into 900 mL of Milli-Q water. No pH adjustment was needed.
  • Xylanase samples Xylanase variant from Aspergillus kawachii (AkaXyl_var) having the mature amino acid sequence shown in SEQ ID NO:9.
  • a preparation of BsbBglu + AkaXyl_var (having a xylanase activity of 13680 - 16720 XBU/g and having an 0- glucanase activity of 9090 - 12500 BBU/g) and FveXyn4_l as set in SEQ IS NO 1 were both obtained from Dupont Nutrition Bioscience Aps, Denmark, Shearzyme P105 and Ultraflo Max from Novozymes, Denmark.
  • All xylanase samples were diluted 1g in 9g 12°Plato wort pH 5.6.
  • ImL of the diluted xylanase samples was in Eppendorf tubes incubated ranging from 20 to 90°C for 10 minutes using an Eppendorf Thermomixer C (Eppendorf, Hamburg) and followingly immediately put on ice before determination of the residual xylanase activity.
  • Xylanase activity was determined in MTP format with liquid handling using Biomek robots (Beckman Coulter, USA). lOOpI of the freshly prepared reaction substrate XylX6 was transferred (using Sodium Acetate buffer pH 5.0, working substrate) to the microtiter plate. 15pl of the enzyme sample appropriately diluted in Mcllvaine Buffer pH 5.0 was added to ensure Abs405nm between 0.0- 1.0 for all temperatures. The same enzyme dilution is used across various temperature tests. The enzyme is mixed with substrate and plate is sealed with tape. Incubation of each plate for 1800 sec at 30°C with 200 rpm shaking is applied and reaction is stopped using lOOpL 2% Tris Stop Solution.
  • the absorbance was measured at 405 nm in a MTP-reader (Molecular Devices Spectramax 190) was determined for reactions. Blank reaction is constructed using Mcllvaine Buffer pH 5.0 instead of enzyme and Abs405nm for each sample is subtracted Abs405nm for blank. All reactions are determined as average of duplicates. The relative activity (Abs405nm) of a given temperature is plotted against the highest Abs405nm obtained for the given temperatures.
  • thermostability test in the 100% malt-based wort pH 5.6 is shown in figure 7. It is clear that FveXyn4_l show significant higher thermostability than the tested xylanase samples known for mashing use (AkaXyl_var, Shearzyme Pl 05 and Ultraflo Max). FveXyn4_l showed 100% residual activity at 70°C and 63% residual activity at 80°C indicative of optimal malt based mashing extending from 50°C to 78°C.
  • XIP un-inhibited xylanase FveXyn4_l was applied and as an example of a XIP inhibited xylanase AkaXyl_var, Shearzyme P105 and Ultraflo Max were applied.
  • Xylanase variant from Aspergillus kawachii AkaXyl_var having the mature amino acid sequence shown in SEQ ID NO:9.
  • 3-glucanase activity of 9090 - 12500 BBU/g) and FveXyn4_l as set in SEQ IS NO 1 were both obtained from Dupont Nutrition Bioscience Aps, Denmark and Ultraflo Max from Novozymes, Denmark
  • Barley grist (28.0g milled Barley grains) and Malt grist (42.0g milled Malt grains) were mixed in beakers with 210g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid, resulting in a water to grist ratio of 3.0:1.
  • the FveXyn4_l xylanase variant was added based on mg protein determined according to example 4 and dosed at 68.0 pg FveXyn4_l protein per g Grist for comparison.
  • AkaXyl_var was dosed 4,3 XBU per g Grist and 0.3mg Ultraflo Max per g Grist.
  • the grist was mashed with three mashing programs (1-3) listed in table 18 to 20;
  • Mash samples was taking out (5mL) from Main mash at each individual temperature indicated in table 18 to 20 and immediately cooled to 5°C, followingly centrifuged 12000 rpm in tabletop centrifuge, and supernatant of mash sample stored at -20°C for later xylanase activity analysis.
  • Residual xylanase activity was determined in mash samples using XylX6 substrate as described in example 15. Blank reaction is constructed using tap water instead of enzyme addition to the mash and Abs405nm of blank for each sample was subtracted Abs405nm for each mash samples. Xylanase activity of all mash samples were determined as average of duplicates. The relative activity (Abs405nm) of a given mash sample was set relative to the activity of xylanase activity of the initial activity (0 min) and the relative xylanase activity was plotted against sampling during the various mashing programs.
  • the residual xylanase activity during mashing programs 1 , 2 and 3 are shown in figure 8 A, B and C respectively. It can be seen, that FveXyn4_l has significant higher residual xylanase through various mashing profiles, maintaining at least 75% xylanase activity at end of mash off (78°C) compared to no determined activity of AkaXyl_var, and Ultraflo Max (0%) at mash off. This may enable better mash filtration by xylanase activity in end mashing to reduce viscosity.
  • Example 17 Influence of cereal extracts on Beta-glucanase activity
  • a limiting factor for overall mash filtration performance is both efficiency of xylanase and Beta-glucanase action on various cereals that may be the present.
  • beta-glucanase inhibition by raw material inhibitors we tested beta-glucanase performance in presence of milled grain used in mashing for beer production.
  • Proteinaceous soluble inhibitors of xylanases been found in barley, rye, com and other cereals.
  • the example describes a simple and novel assay for Beta-glucanase inhibition/action by presence wheat, corn, rye and malt. The inhibition degree of wheat, com, rye and malt was determined for various Beta-glucanase products.
  • Cereal extracts were prepared by the following procedure. A weighted amount of either rye, or wheat flour (Valsempllen, Denmark) (20 g) or fine milled com grist or Malt (Buhler Miag malt mill 0.5 mm setting, position 4) (20g) was suspended in 200 mL (10% w/v, DS) of 0.1 M Na-acetate buffer, pH 5.6, and the mixture agitated on a shaker at 300 rpm for 3.0 hr. The insoluble portion was separated by centrifugation at 5,000 rpm for 20 min in a swing-bucket centrifuge. The supernatants were treated as Rye, Wheat, Corn and Malt extracts pH 5.6 respectively.
  • BsbBglu + FveXyn4_l having a xylanase activity of 15969 - 19517 NGXU/g and having a Beta-glucanase activity of 22356 - 27324 BBXU/g
  • the reaction solution was filtered by paper filter (Whatman, Cytiva Grade 597 Plus Qualitative Filter Paper, Circles) and absorbance at 590nm was determined using a Spectrophotometer (Genesys Spectrophotometer 10S UV-Vis).
  • the relative Beta-glucanase activity was calculated as % compared to the buffer without any cereal extracts (Rye, Wheat, Com, and Malt) only using the enzyme sample dilution resulting in absorbance values ranging between 0.0 and 1.0 in assay.
  • Example 18 Combination of beta-glucanase and xylanase performance in Malt mashing trials with Barley inclusion
  • the objective of this example was to test a Rye XIP un-inhibited vs inhibited xylanase in combination with un-inhibited Beta-glucanase present during a 60% malt: 40% barley based mashing process, using various xylanase dosages.
  • Enzymes was tested in a mashing operation model system for wort production using milled Malt (Pilsner Malt from Sophus Fuglsang, Denmark, Batch Number 10.12.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4, milled barley (Raw Barley from Sophus Fuglsang, Denmark, Batch Number 09.03.2019) at a Buhler Miag malt mill 0.5 mm setting, position 4.
  • BsbBglu + FveXyn4_l having a xylanase activity of 15969 - 19517 NGXU/g and having a Beta-glucanase activity of 22356 - 27324 BBXU/g
  • BsbBglu + AkaXyl_var having a xylanase activity of 13680 - 16720 XBU/g and having an [3-glucanase activity of 9090 - 12500 BBU/g
  • Barley grist (28.0g milled Barley grains) and Malt grist (42.0g milled Malt grains) were mixed in beakers with 210g of tap water in mashing bath (Lockner, LG-electronics) cups and pH adjusted to pH 5.5 with 2.5M sulphuric acid, resulting in a water to grist ratio of 3.1 : 1 .
  • the enzyme materials were all added based on g per kg grist for comparison (0.025, 0.05 and O.lg/kg).
  • the grist was mashed with the program; heated to 60°C for mashing in and pH adjustment before enzymes were applied and hereafter kept at 60°C for 5 minutes; heated to 65°C for 5 minutes by increasing temperature with l°C/minute; kept at 65°C for 45 minutes; heated to 72°C for 7 minutes by increasing temperature with l°C/minute; and kept at 72°C for another 20 minutes; heated to 78°C for 6 minutes by increasing temperature with l°C/minute; and kept at 78°C for another 10 minutes - mashing off; content of each sample was adjusted to 350g and filtered.
  • the volumes of the wort collected after 5, 10, 15 and 30 minutes were measured, and pH was adjusted to pH 5.2 with 2.5 M sulphuric acid.
  • One pellet of hops was added to each flask.
  • the wort samples were boiled for 60 minutes in a boiling bath and samples were cooled down to 17°C. All the samples were filtered used for analysis, see below.
  • Wort sample viscosity were determined as described in example 6 and the results shown for all dosages (0.025, 0.05 and O.lg/kg) for all Beta-glucanase and xylanase samples.
  • the wort sample viscosity is shown in Figure 10 A.
  • HMW B-glucan The content of High Molecular Weight (HMW) B-glucan was measured in the wort using a Microtitre plate reader from BMG Labtech (FLUOstar Omega) following Standard Instruction Brewing, 23.8580-B30, Based on Microtitre method, from EBC 8.13.2. Wort sample B-glucan content is shown in Figure 10 B).
  • HMW pentosan The content of High Molecular Weight (HMW) pentosan was measured in wort samples following Standard Instruction Brewing, 23.8580-B39. (Based on Advanced, A3605 precipitation with 96 (w/w)% ethanol followed by acid hydrolysis). Wort sample HMW pentosan content is shown in Figure 10 C).
  • an un-inhibited xylanase and un-inhibited Beta- glucanase such as BsbBglu + FveXyn4_l to obtain low wort viscosity and very efficient mash performance of malt with other cereals such as Barley, Rye, Wheat and Corn.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne des techniques de brassage améliorées. Plus particulièrement, la présente invention concerne des compositions et des procédés pour améliorer la filtrabilité et le rendement de la maische.
PCT/US2024/013128 2023-02-01 2024-01-26 Nouveaux procédés et nouvelles compositions pour améliorer la filtrabilité et le rendement d'une maische dans le brassage WO2024163288A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363482611P 2023-02-01 2023-02-01
US63/482,611 2023-02-01

Publications (1)

Publication Number Publication Date
WO2024163288A1 true WO2024163288A1 (fr) 2024-08-08

Family

ID=90105337

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2024/013128 WO2024163288A1 (fr) 2023-02-01 2024-01-26 Nouveaux procédés et nouvelles compositions pour améliorer la filtrabilité et le rendement d'une maische dans le brassage
PCT/US2024/013130 WO2024163289A1 (fr) 2023-02-01 2024-01-26 Production améliorée de boissons alcoolisées à base de seigle

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US2024/013130 WO2024163289A1 (fr) 2023-02-01 2024-01-26 Production améliorée de boissons alcoolisées à base de seigle

Country Status (1)

Country Link
WO (2) WO2024163288A1 (fr)

Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0238023A2 (fr) 1986-03-17 1987-09-23 Novo Nordisk A/S Procédé de production de produits protéiniques dans aspergillus oryzae et promoteur à utiliser dans aspergillus
WO1991017243A1 (fr) 1990-05-09 1991-11-14 Novo Nordisk A/S Preparation de cellulase comprenant un enzyme d'endoglucanase
WO1994021785A1 (fr) 1993-03-10 1994-09-29 Novo Nordisk A/S Enzymes derivees d'aspergillus aculeatus presentant une activite de xylanase
US6022725A (en) 1990-12-10 2000-02-08 Genencor International, Inc. Cloning and amplification of the β-glucosidase gene of Trichoderma reesei
WO2005001036A2 (fr) 2003-05-29 2005-01-06 Genencor International, Inc. Nouveaux genes de trichoderma
WO2005059084A1 (fr) 2003-12-19 2005-06-30 Novozymes A/S Processus d'empatage
WO2006066582A1 (fr) 2004-12-22 2006-06-29 Novozymes A/S Procedes de production de produits de fermentation
WO2006114095A1 (fr) 2005-04-26 2006-11-02 Novozymes A/S Hydrolyse d'arabinoxylane
WO2007056321A1 (fr) 2005-11-08 2007-05-18 Novozymes North America, Inc. Deshydratation d’un residu
WO2008023060A1 (fr) 2006-08-25 2008-02-28 Novozymes A/S Procédé de fermentation
US20080233175A1 (en) 2003-07-02 2008-09-25 Brian Steer Glucanases, Nucleic Acids Encoding Them And Methods For Making And Using Them
WO2010059424A2 (fr) 2008-11-18 2010-05-27 Novozymes, Inc. Procédés et compositions pour dégrader un matériau cellulosique
WO2010072224A1 (fr) 2008-12-23 2010-07-01 Danisco A/S Polypeptides dotés d'une activité xylanase
WO2010072225A1 (fr) 2008-12-23 2010-07-01 Danisco A/S Polypeptides dotés d'une activité xylanase
WO2010072226A1 (fr) 2008-12-23 2010-07-01 Danisco A/S Polypeptides dotés d'une activité xylanase
US20110136197A1 (en) 2008-03-07 2011-06-09 Timothy C Dodge Expression of Catalase in Trichoderma
US20120270263A9 (en) * 2009-05-07 2012-10-25 Danisco A/S Enzyme complex from trichoderma reesei and p. funiculosum enzymes
WO2015114108A1 (fr) 2014-01-31 2015-08-06 Dupont Nutrition Biosciences Aps Compositions et procédés comportant une variante d'une enzyme xylanase
US10165787B2 (en) * 2014-01-31 2019-01-01 Dupont Nutrition Biosciences Aps GH10 family xylanase

Patent Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0238023A2 (fr) 1986-03-17 1987-09-23 Novo Nordisk A/S Procédé de production de produits protéiniques dans aspergillus oryzae et promoteur à utiliser dans aspergillus
WO1991017243A1 (fr) 1990-05-09 1991-11-14 Novo Nordisk A/S Preparation de cellulase comprenant un enzyme d'endoglucanase
US6022725A (en) 1990-12-10 2000-02-08 Genencor International, Inc. Cloning and amplification of the β-glucosidase gene of Trichoderma reesei
WO1994021785A1 (fr) 1993-03-10 1994-09-29 Novo Nordisk A/S Enzymes derivees d'aspergillus aculeatus presentant une activite de xylanase
WO2005001036A2 (fr) 2003-05-29 2005-01-06 Genencor International, Inc. Nouveaux genes de trichoderma
US20080233175A1 (en) 2003-07-02 2008-09-25 Brian Steer Glucanases, Nucleic Acids Encoding Them And Methods For Making And Using Them
WO2005059084A1 (fr) 2003-12-19 2005-06-30 Novozymes A/S Processus d'empatage
WO2006066582A1 (fr) 2004-12-22 2006-06-29 Novozymes A/S Procedes de production de produits de fermentation
WO2006114095A1 (fr) 2005-04-26 2006-11-02 Novozymes A/S Hydrolyse d'arabinoxylane
WO2007056321A1 (fr) 2005-11-08 2007-05-18 Novozymes North America, Inc. Deshydratation d’un residu
WO2008023060A1 (fr) 2006-08-25 2008-02-28 Novozymes A/S Procédé de fermentation
US20110136197A1 (en) 2008-03-07 2011-06-09 Timothy C Dodge Expression of Catalase in Trichoderma
WO2010059424A2 (fr) 2008-11-18 2010-05-27 Novozymes, Inc. Procédés et compositions pour dégrader un matériau cellulosique
WO2010072224A1 (fr) 2008-12-23 2010-07-01 Danisco A/S Polypeptides dotés d'une activité xylanase
WO2010072225A1 (fr) 2008-12-23 2010-07-01 Danisco A/S Polypeptides dotés d'une activité xylanase
WO2010072226A1 (fr) 2008-12-23 2010-07-01 Danisco A/S Polypeptides dotés d'une activité xylanase
US20120270263A9 (en) * 2009-05-07 2012-10-25 Danisco A/S Enzyme complex from trichoderma reesei and p. funiculosum enzymes
WO2015114108A1 (fr) 2014-01-31 2015-08-06 Dupont Nutrition Biosciences Aps Compositions et procédés comportant une variante d'une enzyme xylanase
US10165787B2 (en) * 2014-01-31 2019-01-01 Dupont Nutrition Biosciences Aps GH10 family xylanase

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
"Catalogue of Strains", 17 January 2007, UNIVERSITY OF MISSOURI
"Oligonucleotide Synthesis: A Practical Approach", 1984, IRI PRESS
AUSUBEL, F. M. ET AL.: "Current Protocols in Molecular Biology", 1995, JOHN WILEY & SONS
B. HENRISSAT: "A classification of glycosyl hydrolases based on amino acid sequence similarities", BIOCHEM. J., vol. 280, 1991, pages 309 - 316
B. ROEJ. CRABTREEA. KAHN: "DNA Isolation and Sequencing: Essential Techniques", 1996, JOHN WILEY & SONS
CAO ET AL., SCIENCE, vol. 9, 2000, pages 991 - 1001
D. M. J. LILLEYJ. E. DAHLBERG: "Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology", 1992, ACADEMIC PRESS
EMANUELSSON ET AL., NATURE PROTOCOLS, vol. 2, 2007, pages 953 - 971
HARRISON ET AL., APPLIED ENVIRON. MICROBIOL., vol. 77, June 2011 (2011-06-01), pages 3916 - 22
HORWELL DC, TRENDS BIOTECHNOL., vol. 13, no. 4, 1995, pages 132 - 134
J. SAMBROOKE. F. FRITSCHT. MANIATIS: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
LIU ET AL.: "Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl) promoter optimization", ACTA BIOCHIM. BIOPHYS. SIN (SHANGHAI, vol. 40, no. 2, 2008, pages 158 - 65, XP002595560, DOI: 10.1111/J.1745-7270.2008.00388.X
PENTTILA ET AL., GENE, vol. 61, 1987, pages 155 - 164
SAMBROOK ET AL.: "MOLECULAR CLONING: A LABORATORY MANUAL", 2001, COLD SPRING HARBOR
SIMON RJ ET AL., PNAS, vol. 89, no. 20, 1992, pages 9367 - 9371
TE'O VS ET AL., J MICROBIOL METHODS, vol. 51, 2002, pages 393 - 9
THOMPSON ET AL., NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 4680

Also Published As

Publication number Publication date
WO2024163289A1 (fr) 2024-08-08

Similar Documents

Publication Publication Date Title
CN105722989B (zh) 发酵中的海藻糖酶
JP5221347B2 (ja) β−グルコシダーゼ活性を有するポリペプチドおよびそれをコードするポリヌクレオチド
US7510857B2 (en) Thermostable cellulase and methods of use
CA3032736A1 (fr) Polypeptides de glucoamylase modifies par une sequence de tete et souches de levure manipulees presentant une production de produits biologiques amelioree
JP7069201B2 (ja) 熱安定性グルコアミラーゼ及びその使用方法
WO2010148148A2 (fr) Variants de β-glucosidase
KR20150079676A (ko) 마그나포르테 그리세아로부터의 베타-글루코시다제
EP2748188A1 (fr) Polypeptides ayant une activité glucoamylase et polynucléotides codant pour ces polypeptides
CN110651048A (zh) 葡糖淀粉酶变体和编码它们的多核苷酸
WO2015066669A1 (fr) Protéases utilisables dans le cadre du traitement du maïs
AU2019364262A1 (en) Enzymes for infusion mashing in adjunct brewing technical field
US20240228917A1 (en) Proteases for beer haze reduction
WO2015021601A1 (fr) Liquéfaction et malto-saccharification simultanées
AU2022202136A1 (en) Trehalase in fermentations
US11873470B2 (en) Brewing method
WO2015094714A1 (fr) Protéases dans le traitement des grains
CN111757931A (zh) 变体g6p g7p葡糖淀粉酶组合物及方法
WO2024163288A1 (fr) Nouveaux procédés et nouvelles compositions pour améliorer la filtrabilité et le rendement d'une maische dans le brassage
CN111989400B (zh) α-淀粉酶、组合物和方法
US20200216787A1 (en) Riboflavinase enzymes and their use to prevent off flavor in brewing
WO2006079346A1 (fr) Enzyme nouvelle a activite alpha-xylosidase
WO2015066667A1 (fr) Protéases utilisées dans le traitement du blé
WO2013034097A1 (fr) Polypeptides ayant une activité glucoamylase et polynucléotides codant pour ceux-ci
Nascimento Production of recombinant α-amylase from Bacillus subtilis by Kluyveromyces lactis
AU2014342553A1 (en) Trehalase in fermentations

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24710913

Country of ref document: EP

Kind code of ref document: A1