Cold-active beta-galactosidase, a method of producing same and use of such enzyme
A novel cold-active beta-galactosidase is enzyme specific for lactose. The enzyme is thus useful in e.g. the food industry for catalyzing at low temperatures the hydrolysis of lactose disaccharide into its constituent monosaccharides, glucose and galactose. A method produces the cold-active beta-galactosidase by recombinant DNA technology.
1. A purified cold-active beta-galactosidase having the amino acid sequence as defined in SEQ ID NO. 1 or one having at least 90% homology to the amino acid sequence as defined in SEQ ID NO. 1, the amino acid sequence being selected so that the enzyme has a stable enzymatic activity at temperatures less than 8° C.
2. A beta-galactosidase according to claim 1 , wherein the amino acid sequence has at least 95%, homology to the amino acid sequence as defined in SEQ ID NO. 1.
3. A beta-galactosidase according to claim 1 , wherein it is produced by a strain of an Alkalilactibacillus.
4. A beta-galactosidase according to claim 1 , wherein it is produced by Alkalilactibacillus ikkense.
5. An isolated DNA sequence comprising a gene which encodes the beta-galactosidase according to claim 1 .
6. An isolated DNA sequence, which a) encodes a protein with an amino acid sequence as given in SEQ ID NO. 2, or b) hybridizes to the sequence of a), at 6×SSC, 0.5% SDS and 50% formamide at 42° C., before being washed in a solution of 0.1% ×SSC, 0.5% SDS at 68° C., or c) is degenerative of the sequence of a) or b).
7. A DNA sequence according to claim 6 , wherein the sequence is as given in SEQ ID NO. 2.
8. A recombinant vector comprising a DNA sequence of claim 5 .
9. A vector of claim 8 , wherein said vector is an expression vector.
10. A host cell transformed with a vector of claim 8 .
11. A cell according to claim 10 , wherein the cell is selected from the group consisting of Escherichia, Bacillus, Bifidobacterium, Lactococcus, Lactobacillus, Streptomyces, Leuconostoc, Streptomyces, Saccharomyces, Kluyveromyces, Candida, Torula, Torulopsis and Aspergillus.
12. A cell of claim 11 , wherein the cell is selected from the group consisting of Bifidobacterium breve, Bifidobacterium longum, Bifidobacteriuminfantis, Bifidobacterium bifidum, Bifidobacterium animalis , and Lactococcus lactis.
13. Use of a cell of claim 10 for producing a product selected from the group consisting of lactose-free milk, low-lactose milk, yoghurt, cheese, fermented milk products, dietary supplement and probiotic comestible product comprising adding the host cell of claim 10 .
14. A method according to claim 13 for producing a dairy product with a lactose concentration at 1% w/v or lower.
15. A method according to claim 14 , wherein the lactose concentration is 0.1% w/v or lower.
16. A method according to claim 15 , wherein the lactose concentration is 0.01% w/v or lower.
17. A method of a beta-galactosidase according to claim 1 for producing a product selected from the group consisting of lactose-free milk, low-lactose milk, yoghurt, cheese, fermented milk products, dietary supplement and probiotic comestible product comprising adding the beta-galactosiadase of claim 1 .
18. A method according to claim 17 for producing a dairy product with a lactose concentration at 1% w/v or lower.
19. A method according to claim 18 , wherein the lactose concentration is 0.1% w/v or lower.
20. A method according to claim 19 , wherein the lactose concentration is 0.01% w/v or lower.
21. A process for producing an enzyme of claim 1 , comprising culturing a cell of any one of claims 10 to 12 in a suitable culture medium under conditions permitting expression of said enzyme, and recovering the resulting enzyme from the culture.
22. A process according to claim 21 , wherein the resulting enzyme is immobilized.
23. A psychrophilic bacterium Alkalilactibacillus ikkense BCCM Accession No LMG P-24866 and variants and mutants derived therefrom capable of producing a cold-active beta galactosidase defined by claim 1 .