Production of lactase enzymes using altered regulation strains
AssigneeChr. Hansen A/S
InventorsHans Raj, Charlotte Elisabeth Grüner Schöller, Kim Ib Soerensen, Johannes Maarten Van Den Brink, Ahmad Zeidan, Martin Holm Rau, Paula Gaspar, George Nabin Baroi
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Abstract
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.
Claims12
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.
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.
Dependencies are inferred from the claim text.