Carlsberg A/s

Heineken Supply Chain B.v.

Carlsberg Breweries A/s

Novo Nordisk A/s

Board of Regents of the University of Nebraska

Bionebraska, Inc.

De Forenede Bryggerier A/s

Carlsberg Biotechnology Ltd. A/s

Carlsberg Laboratory

Klaus Breddam

Ole Olsen

Birgitte Skadhauge

Fred W Wagner

Søren Knudsen

Finn Lok

Jack T Johansen

Morten C Kielland-Brandt

Kjeld Ove Olesen

Uffe Hasbo Mortensen

Henning Ralf Stennicke

Lene Molskov Bech

Soren Knudsen

Thomas R Coolidge

Sven Branner

Jay S Stout

Dwane E Wylie

Sheldon M Schuster

Dennis B Henriksen

William Lewis

Pia Vaag

Fred Widmer

Lene Mølskov Bech

Marianne Muldbjerg

Uffe Mortensen

Kjeld Olesen

Henning Stennicke

Steen B So Slashedrensen

Thorkild Beenfeldt

Robert Leah

Lene Mo Bech

Steen Bech So Slashedrensen

Ole Buchardt

Hanne Groen

Lene M Bech

Gustav Hambraeus

Steen Bech Sørensen

Lette Mølskov Bech

Steen Bech SØRENSEN

Bech Sorensen Steen

According to the invention, there is provided barley-derived beverages characterized by notably reduced levels of both dimethyl sulfide (DMS) and/or its precursor S-methyl-L-methionine (SMM), or lacking said compounds. In addition, the invention relates to methods for producing the above-mentioned beverages&#x2014;and also to barley plants useful in the preparation of such beverages, as well as other plant products prepared from said plants. Utilization of the invention clears the way for making improved production procedures of beverages with improved taste profiles, and promises also for notable reductions in the thermal energy input for production of beer.

Barley and malt-derived beverages with low DMS level

According to the invention, there is provided barley with total loss of functional lipoxygenase (LOX)-1 and LOX-2 enzymes, and plant products produced thereof, such as malt manufactured by using barley kernels defective in the synthesis of the fatty acid-dioxygenating enzymes LOX-1 and LOX-2. Said enzymes account for the principal activities related to dioxygenation of linoleic acid into 9- and 13-hydroperoxy octadecadienoic acid, respectively. 9-Hydroperoxy octadecadienoic acid represents a LOX pathway metabolite, which&#x2014;through further enzymatic or spontaneous reactions&#x2014;may lead to the appearance of trans-2-nonenal (T2N). The invention enables brewers to produce a beer having insignificant levels of stale, T2N-specific off-flavors, even after prolonged storage of the beverage.

Barley with reduced lipoxygenase activity and beverage prepared therefrom

According to the invention, there is provided null-LOX-1 barley and plant products produced thereof, such as malt manufactured by using barley kernels defective in synthesis of the fatty acid-converting enzyme lipoxygenase-1. Said enzyme accounts for the principal activity related to conversion of linoleic acid into 9-hydroperoxy octadecadienoic acid, a lipoxygenase pathway metabolite, which-through further enzymatic or spontaneous reactions-may lead to the appearance of trans-2-nonenal. The invention enables brewers to produce a beer devoid of detectable trans-2-nonenal-specific off flavors, even after prolonged storage of the beverage.

Barley for production of flavor-stable beverage

According to the invention, there is provided null-LOX-1 barley and plant products produced thereof, such as malt manufactured by using barley kernels defective in synthesis of the fatty acid-converting enzyme lipoxygenase-1. Said enzyme accounts for the principal activity related to conversion of linoleic acid into 9-hydroperoxy octadecadienoic acid, a lipoxygenase pathway metabolite, which&#x2014;through further enzymatic or spontaneous reactions&#x2014;may lead to the appearance of trans-2-nonenal. The invention enables brewers to produce a beer devoid of detectable trans-2-nonenal-specific off-flavors, even after prolonged storage of the beverage.

The invention provides customized proteases (i.e., mutant enzymes), methods of making customized proteases, as well as methods of using customized proteases. The customized proteases of the invention are derived from the known proteases. Altered transacylation reactions include the capability to perform transacylation reactions not substantially catalyzed by the known protease or the capability to perform transacylation reactions with improved yields, or both. The methods of the invention provide for customized proteases through site specific or random mutagenesis of the active site amino acids of the known proteases. The invention also provides for methods of using the customized proteases to prepare a preselected transacylation products. The preselected transacylation products produced can be modified by substitution at the N-or C-terminal with nucleophiles such as L-amino acids, D-amino acids, amino acid amides, and radioactive amino acids.

Customized proteases

Beverage and a method of preparing it

A method for transamidating a peptide substrate having a P.sub.1 amino acid residue with a positively charged side chain. According to the invention, carboxypeptidase Y is modified to substitute at least one amino acid having a negatively charged side chain in an S.sub.1 subsite. Additionally, the modified carboxypeptidase Y can include substituted amino acid residues in an S.sub.1 ', S.sub.2 and/or S.sub.3 subsite to accommodate a specific peptide substrate.

Modified carboxypeptidase

The invention provides customized proteases (i.e., mutant enzymes), methods of making customized proteases, as well as methods of using customized proteases. The customized proteases of the invention are derived from the known proteases. Altered transacylation reactions include the capability to perform transacylation reactions not substantially catalyzed by the known protease or the capability to perform transacylation reactions with improved yields, or both. The methods of the invention provide for customized proteases through site specific or random mutagenesis of the active site amino acids of the known proteases. The invention also provides for methods of using the customized proteases to prepare a preselected transacylation products. The preselected transacylation products produced can be modified by substitution at the N- or C-terminal with nucleophiles such as L-amino acids, D-amino acids, amino acid amides, and radioactive amino acids.

A process for preparing C-terminally amidated peptides, Peptide-NH.sub.2, is presented. In a first step, a substrate component is reacted with a nucleophile component in the presence of trypsin or a carboxypeptidase using as nucleophile a compound NH.sub.2 -R to form a first reaction product Peptide-NH-R. In a second step, the first reaction product is non-enzymatically chemically cleaved to form the C-terminally amidated product, Peptide-NH.sub.2. The substrate component is selected from a) peptide derivatives Peptide-X-Y, where X is an amino acid or peptide residue and Y is OH, OMe or C-terminal modification and c) C-terminally esterified peptides, Peptide-OR', where R' is alkyl, aryl, heteroaryl, or aralkyl. The nucleophile component is selected from ##STR1## wherein A-F and A'-E' are carbon atoms or up to two hetero atoms, Y is H, alkyl, aryl, aralkyl, oxo or carboxy, X.sup.1 -X.sup.5 are H or various substituents. The cleavage may be induced by photolysis, solvolysis, reduction, rearrangement elimination, or oxidation. The process may be adapted to enzymatic synthesis and lends itself to C-terminal amidation of many types of peptides.

Process for the preparation of C-terminally amidated peptides

An auxiliary substance such as a label, support, or bioactive agent is attached to a protein at a site that is remote from the active site of the protein by the use of exopeptidase and a nucleophile which is an amino acid, amino acid derivative, amine or alcohol. In one embodiment, the nucleophile is attached to the carboxy terminus of a protein by catalysis with exopeptidase to form an adduct and then the adduct or its combination with a linker arm is bound to the auxiliary substance. In another embodiment, the auxiliary substance or its combination with a linker arm is bound to the nucleophile to form an intermediate substance which is then coupled by catalysis with exopeptidase to the carboxy terminus of a protein.

Exopeptidase catalyzed site-specific bonding of supports, labels and

Novel chemically modified detergent enzymes are provided, wherein one or more methionines have been mutated into cysteines, said cysteines subsequently being chemically modified in order to confer the enzyme improved stability towards oxidative agents. A novel process for stabilizing detergent enzymes against oxidation is also provided. Furthermore, there are provided detergent compositions comprising these novel oxidation stable detergent enzymes.

Oxidation stable detergent enzymes

Process for enzymatic replacement of the B-30 amino acid in insulins

An insulin such as porcine insulin is reacted enzymatically with an L-amino acid, amide, or ester in the presence of L-specific serine carboxypeptidase modified by reaction with divalent metal ions in aqueous solution or dispersion containing F.sup.-, Cl.sup.-, Br.sup.-, I.sup.-, CN.sup.-, or SCN.sup.-.

Growing community of inventors

Glostrup, Denmark

Klaus Breddam