Acetolactate decarboxylase variants having improved specific activity

Abstract

Compositions and methods are provided comprising acetolactate decarboxylase (ALDC) enzyme variants having higher specific activity. Composition and method are provided where the ALDC variants are usedin combination with metal ions to further increase stability and / or activity.

Description

Background technique

[0001] Diacetyl is sometimes an undesirable by-product of the fermentation of carbohydrates containing substances such as wort or grape juice. The formation of diacetyl is the most detrimental because it has a strong and unpleasant smell and in the case of beer even small amounts of diacetyl around 0.10mg / liter to 0.15mg / liter will have a negative effect on the flavor and taste of beer Negative impact. During beer maturation, diacetyl is converted to acetoin by reductases in yeast cells. Higher concentrations of acetoin than diacetyl are acceptable for beer flavor and taste.

[0002] Acetolactate decarboxylase (ALDC) can also be used as an enzyme to prevent diacetyl formation. During fermentation, alpha-acetolactate can be converted to acetoin by adding ALDC enzyme. However, ALDC may be unstable under fermentation conditions, especially those in which worts are fermented with low maltose content.

[0003] Compositions and methods related to the use of...

Examples

example

[0201] The present disclosure is described in further detail in the following examples, which are not intended to limit the scope of the claimed disclosure in any way. The accompanying drawings are intended to be considered an integral part of the specification and description of this disclosure. The following examples are offered to illustrate but not limit the claimed disclosure.

example 1

[0203] Heterologous expression of acetolactate decarboxylase ALDB

[0204] The Brevibacillus brevis (possibly called Bacillus brevis) acetolactate decarboxylase (ALDC) aldB gene was previously identified (Diderichsen et al., J Bacteriol. (1990) 172(8): 4315), whose sequence is shown in UNIPROT accession number P23616.1. The sequence number of this gene, aldB, is described in SEQ ID NO:1. Nucleotides highlighted in bold and underlined are those encoding the signal peptide. The aldB gene and the corresponding encoded zymogen are also referred to as wild type (WT).

[0205] SEO ID NO: 1 lists the nucleotide sequence of the aldB gene:

[0206]

[0207] The zymogen encoded by the aldB gene is described in SEQ ID NO:2. At the N-terminus, the protein has a signal peptide of 24 amino acids in length as predicted by SignalP-NN (Emanuelsson et al., Nature Protocols (2007) 2:953-971). The signal peptide sequence is underlined and shown in bold in SEQ ID NO:2. The presence of a s...

example 2

[0236] Protein Assay Method

[0237] Protein Assay by Stain Free Imager Criterion

[0238] Using Gel Doc TM The EZ Imaging System quantifies proteins by SDS-PAGE gels and densitometry. Reagents used in the assay: Concentrated (2x) Laemmli sample buffer (Bio-Rad, catalog #161-0737); 26-well XT4-12% Bis-Tris gel (Bio-Rad , catalog #345-0125); protein markers "Precision Plus Protein Standards (Precision Plus Protein Standards)" (Bio-Rad, catalog #161-0363); protein standards BSA (Thermo Scientific , catalog #23208) and SimplyBlue Safestain (Invitrogen, catalog #LC 6060). The assay was performed as follows: 50 μL of the diluted enzyme sample was mixed with 50 μL of sample buffer containing 2.7 mg DTT in a 96-well PCR plate. The plate was sealed with Microseal 'B' membrane from Bio-Rad and placed in a PCR machine heated to 70°C for 10 minutes. Afterwards, fill the chamber with running buffer and set up the gel cassette. Then 10 μL of each sample and standard (0.125-1.00 mg...

Background technique

[0001] Diacetyl is sometimes an undesirable by-product of the fermentation of carbohydrates containing substances such as wort or grape juice. The formation of diacetyl is the most detrimental because it has a strong and unpleasant smell and in the case of beer even small amounts of diacetyl around 0.10mg / liter to 0.15mg / liter will have a negative effect on the flavor and taste of beer Negative impact. During beer maturation, diacetyl is converted to acetoin by reductases in yeast cells. Higher concentrations of acetoin than diacetyl are acceptable for beer flavor and taste.

[0002] Acetolactate decarboxylase (ALDC) can also be used as an enzyme to prevent diacetyl formation. During fermentation, alpha-acetolactate can be converted to acetoin by adding ALDC enzyme. However, ALDC may be unstable under fermentation conditions, especially those in which worts are fermented with low maltose content.

[0003] Compositions and methods related to the use of...

Examples

example

[0201] The present disclosure is described in further detail in the following examples, which are not intended to limit the scope of the claimed disclosure in any way. The accompanying drawings are intended to be considered an integral part of the specification and description of this disclosure. The following examples are offered to illustrate but not limit the claimed disclosure.

example 1

[0203] Heterologous expression of acetolactate decarboxylase ALDB

[0204] The Brevibacillus brevis (possibly called Bacillus brevis) acetolactate decarboxylase (ALDC) aldB gene was previously identified (Diderichsen et al., J Bacteriol. (1990) 172(8): 4315), whose sequence is shown in UNIPROT accession number P23616.1. The sequence number of this gene, aldB, is described in SEQ ID NO:1. Nucleotides highlighted in bold and underlined are those encoding the signal peptide. The aldB gene and the corresponding encoded zymogen are also referred to as wild type (WT).

[0205] SEO ID NO: 1 lists the nucleotide sequence of the aldB gene:

[0206]

[0207] The zymogen encoded by the aldB gene is described in SEQ ID NO:2. At the N-terminus, the protein has a signal peptide of 24 amino acids in length as predicted by SignalP-NN (Emanuelsson et al., Nature Protocols (2007) 2:953-971). The signal peptide sequence is underlined and shown in bold in SEQ ID NO:2. The presence of a s...

example 2

[0236] Protein Assay Method

[0237] Protein Assay by Stain Free Imager Criterion

[0238] Using Gel Doc TM The EZ Imaging System quantifies proteins by SDS-PAGE gels and densitometry. Reagents used in the assay: Concentrated (2x) Laemmli sample buffer (Bio-Rad, catalog #161-0737); 26-well XT4-12% Bis-Tris gel (Bio-Rad , catalog #345-0125); protein markers "Precision Plus Protein Standards (Precision Plus Protein Standards)" (Bio-Rad, catalog #161-0363); protein standards BSA (Thermo Scientific , catalog #23208) and SimplyBlue Safestain (Invitrogen, catalog #LC 6060). The assay was performed as follows: 50 μL of the diluted enzyme sample was mixed with 50 μL of sample buffer containing 2.7 mg DTT in a 96-well PCR plate. The plate was sealed with Microseal 'B' membrane from Bio-Rad and placed in a PCR machine heated to 70°C for 10 minutes. Afterwards, fill the chamber with running buffer and set up the gel cassette. Then 10 μL of each sample and standard (0.125-1.00 mg...

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