Production of lactase enzymes using altered regulation strains
The present invention relates to new improved methods for expressing native lactases in their native hosts. Methods for homologous as well as heterologous expression of lactase in lactic acid bacteria with altered expression dynamics are comprised by present invention.
1 . A method for producing an enzyme having lactase activity in a lactic acid bacterium, wherein the lactic acid bacterium carries a mutation in a glck DNA sequence encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein, comprising:
(a) inoculating a lactic acid bacterium into a growth medium comprising lactose, wherein the lactic acid bacterium expresses an enzyme having lactase activity;
(b) growing the lactic acid bacterium to a desired density to obtain a first solution comprising the enzyme having lactase activity and bacterial cells of the lactic acid bacterium;
(c) inactivating and/or disrupting the bacterial cells of the lactic acid bacterium, such that the bacterial cells are broken;
(d) removing cellula debris to obtain a second solution which comprises the enzyme having the lactase activity; and
(e) optionally processing the second solution to obtain a third solution which comprises the enzyme having the lactase activity,
wherein the lactase activity and amount of protein produced per liter of the first solution is higher from a lactic acid bacterium carrying a mutation in a glck DNA sequence encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein, compared to the lactase activity and amount of protein produced per liter from an otherwise identical strain which does not carry a mutation in a glck DNA sequence.
2 . The method of claim 1 , wherein the second solution of step (d) or the third solution of step (e) is further formulated to produce the purified enzyme in a suitable formulation.
3 . A method for producing an enzyme having lactase activity in a lactic acid bacterium, wherein the lactic acid bacterium carries a mutation in a glck DNA sequence encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein, comprising:
(a) introducing a DNA sequence encoding the enzyme having lactase activity into a lactic acid bacterium, wherein the DNA sequence encoding the enzyme is introduced into the lactic acid bacterium under control of a regulatory element of a lac-operon;
(b) inoculating the lactic acid bacterium of step (a) into a growth medium comprising lactose, wherein the lactic acid bacterium expresses an enzyme having lactase activity;
(c) growing the lactic acid bacterium to a desired density to obtain a first solution comprising the enzyme having lactase activity and bacterial cells of the lactic acid bacterium;
(d) inactivating and/or disrupting the bacterial cells of the lactic acid bacterium, such that the bacterial cells are broken;
(e) removing cellula debris to obtain a second solution which comprises the enzyme having the lactase activity; and
(f) optionally processing the second solution to obtain a third solution which comprises the enzyme having the lactase activity,
wherein the lactase activity and amount of protein produced per liter of the first solution is higher from a lactic acid bacterium carrying a mutation in a glck DNA sequence encoding a glucokinase protein, wherein the mutation inactivates the glucokinase protein, compared to the lactase activity and amount of protein produced per liter from an otherwise identical strain which does not carry a mutation in a glck DNA sequence.
4 . The method of claim 3 , wherein the second solution of step (d) or the third solution of step (e) is further formulated to produce the purified enzyme in a suitable formulation.
5 . The method of claim 3 , wherein the lac-operon is Carbon control Protein A (CcpA).
6 . The method of claim 3 , wherein the DNA sequence encoding the enzyme encodes a dimeric peptide exhibiting beta-galactosidase enzyme activity, wherein the dimeric peptide consist of two peptides having amino acid sequences selected from SEQ ID NOs. 2 and 3; SEQ ID NOs. 5 and 6; SEQ ID NOs. 20 and 21; SEQ ID NOs. 23 and 24; SEQ ID NOs. 26 and 27, and SEQ ID NOs. 28 and 29, and enzymatically active fragments thereof, and variants thereof having not more than 22 amino acid substitutions, additions or deletions.
7 . The method of claim 3 , wherein the DNA sequence encoding the enzyme encodes an amino acid sequence selected from any one of SEQ ID NOs. 1-33, and enzymatically active fragments thereof, and variants thereof having not more than 22 amino acid substitutions, additions or deletions.
8 . The method of claim 1 , wherein the enzyme having lactase activity expressed by the lactic acid bacterium has an amino acid sequence selected from any one of SEQ ID NOs. 34-38, and variants thereof having not more than 22 amino acid substitutions, additions or deletions.
9 . The method of claim 1 , wherein the lactic acid bacterium is a strain of Lactobacillus delbreuckii subspecies bulgaricus.
10 . The method of claim 1 , wherein the lactic acid bacterium is a strain of Lactobacillus delbreuckii subspecies bulgaricus selected from strain CHCC16159 deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH1 (DSMZ) (Braunschweig, Germany) under Accession Number DSM 26420 and mutants thereof, and strain CHCC16160 deposited at DSMZ under Accession Number DSM 26421 and mutants thereof.
11 . The method of claim 1 , wherein the lactic acid bacterium is a galactose fermenting strain of Streptococcus thermophilus.
12 . The method of claim 1 , wherein the lactic acid bacterium is a galactose fermenting strain of Streptococcus thermophilus selected from the strains deposited at DSMZ under Accession Numbers DSM 28889, DSM 25850, DSM 25851, DSM 26722 and DSM 32227, and Streptococcus thermophilus strains sharing the functional characteristics of said deposited strains.