Methods for production of hyaluronic acid, and isolated strains of supercapsulated streptococci
View Patent ↗Isolated strains of supercapsulated streptococci band at a density of no greater than 1.03 g/cm 3 in a Percoll gradient and are capable of producing hyaluronic acid with molecular weight exceeding 6 million Da. Methods of producing high molecular weight hyaluronic acid employ a supercapsulated strain of streptococcus which bands at a density of no greater than 1.03 g/cm 3 in a Percoll gradient. Methods of selecting streptococcus strains capable of producing hyaluronic acid with a molecular weight exceeding 6 million Da comprise, inter alia, cultivating supercapsulated strains of streptococci which band at a density of no greater than 1.03 g/cm 3 in a Percoll gradient.
1. An isolated strain of supercapsulated streptococci , wherein supercapsulated members of the strain have a density of no greater than 1.03 g/cm 3 and are capable of producing hyaluronic acid with molecular weight exceeding 6 million Da.
2. The isolated strain of supercapsulated streptococci according to claim 1 , wherein the supercapsulated strain is selected from the group consisting of supercapsulated group A streptococci and supercapsulated group C streptococci.
3. A method of producing high molecular weight hyaluronic acid, comprising the steps of:
(i) selecting the isolated strain of supercapsulated streptococcus of claim 1 ;
(ii) cultivating said strain at a temperature of 30 to 35° C. in a reactor under agitation conditions substantially free from shear forces and in a culture medium which is free of metal ions promoting the degradation of hyaluronic acid, does not release from the reactor metal ions promoting degradation of hyaluronic acid and has a pH in the range of 5.6 to 6.2, whereby hyaluronic acid is formed; and
(iii) purifying the hyaluronic acid formed in step (ii) from the culture medium to give hyaluronic acid with a molecular weight above six million Da.
4. The method according to claim 3 , wherein the culture medium is free of iron and copper.
5. The method according to claim 3 , wherein the culture medium has a pH in the range of 5.6 to 5.95.
6. The method according to claim 3 , wherein the supercapsulated strain is selected from the group consisting of supercapsulated group A streptococci and supercapsulated group C streptococci.
7. The method according to claim 6 , wherein the culture medium is free of iron and copper.
8. The method according to claim 6 , wherein the culture medium has a pH in the range of 5.6 to 5.95.
9. The method according to claim 3 , wherein the supercapsulated strain has been obtained by mutagenesis.
10. The method according to claim 9 , wherein the culture medium is free of iron and copper.
11. The method according to claim 9 , wherein the culture medium has a pH in the range of 5.6 to 5.95.
12. A method of selecting streptococcus strains capable of producing hyaluronic acid with a molecular weight exceeding six million Da, comprising the steps of:
(i) subjecting streptococci strains to mutagenesis, whereby mutated strains are obtained; and
selecting a mutated strain wherein supercapsulated members of the strain have a density of no greater than 1.03 g/cm 3 .
13. The method according to claim 12 , wherein the mutagenesis is chemical mutagenesis.
14. The method according to claim 13 , wherein the streptococci strains which are subjected to mutagenesis are selected from the group consisting of group A streptococci and group C streptococci.
15. The method according to claim 12 , wherein the supercapsulated members of the strain are capable of producing hyaluronic acid having a molecular weight exceeding 8 million Da but not greater than about 9.5 million Da.
16. The method according to claim 12 , wherein the supercapsulated members of the strain are capable of producing hyaluronic acid having a molecular weight exceeding 9 million Da but not greater than about 9.5 million Da.
17. The method according to claim 12 , wherein the streptococci strains which are subjected to mutagenesis are selected from the group consisting of group A streptococci and group C streptococci.