Enzyme immobilization with a disulphide bridge

Claim 1

An insolubilized, enzymatically active substance comprising an enzyme linked by a disulphide group containing covalent bridge to a support carrier, and having the formula (V) ##STR8## in which ##STR9## is the residue of an enzyme having lost a hydrogen atom from an amino group, n and p, which may be the same or different, are integers of 1 to 6; and G represents a support carrier comprising glass beads having a specific pore size of 200A to 1000A and a surface area of 5 to 10 sq. m/g.

Claim 4

A method of making an insolubilized, enzymatically active substance comprising reacting an enzyme having available mercaptan groups, at a temperature of 0° C. to about 15° C., and a pH of 7 to 10 in the presence of an oxidation catalyst, with a support carrier comprising porous glass beads functionalized with mercaptan groups, said beads having a specific pore size of 200A to 1000A and a surface area of 5 to 100 sq.m/g., such that a disulphide group is produced in a covalent bridge linking said enzyme to said carrier; said glass beads having been functionalized with mercaptan groups by reaction at the hydroxyl group of a mercapto alcohol of formula (I) HO--(CH.sub.2).sub.n --SH in which n is an integer of 1 to 6, such that the beads bear mercaptan group-containing radicals of formula HS--(CH.sub.2).sub.n --0-- wherein n is as defined above.

Claim 5

A method of making an insolubilized, enzymatically active substance comprising reacting an enzyme having available mercaptan groups, at a temperature of 0° C. to about 15° C., and a pH of 7 to 10 in the presence of an oxidation catalyst, with a support carrier comprising porous glass beads functionalized with mercaptan groups, said beads having a specific pore size of 200A to 1000A and a surface area of 5 to 100 sq.m/g., such that a disulphide group is produced in a covalent bridge linking said enzyme to said carrier; said glass beads having been functionalized with mercaptan groups by reaction at the hydroxyl group of a mercapto alcohol of formula (I) HO--(CH.sub.2).sub.n --SH in which n is an integer of 1 to 6, such that the beads bear mercaptan group-containing radicals of formula HS--(CH.sub.2).sub.n --0-- wherein n is as defined above.

Claim 9

A method of making an insolubilized, enzymatically active substance comprising reacting an enzyme pre-functionalized by introducing mercapto groups therein, at a temperature of 0° C. to about 15° C., and a pH of 7 to 8.5 in the presence of a catalytic amount of sodium selenate, with a support carrier comprising porous glass beads functionalized with mercaptan groups, such beads having a specific pore size of 200A to 1000A and a surface area of 5 to 100 sq. m/g., such that a disulphide group is produced in a covalent bridge linking said enzyme to said carrier; said glass beads having been functionalized with mercaptan groups by reaction at the hydroxyl group of a mercapto alcohol of formula (I) HO--(CH.sub.2).sub.n --SH in which n is an integer of 1 to 6, such that the beads bear mercaptan group-containing radicals of formula HS--(CH.sub.2).sub.n --O-- wherein n is as defined above.

Claim 10

A method of making an insolubilized, enzymatically active substance comprising reacting an enzyme pre-functionalized by introducing mercapto groups therein, at a temperature of 0° C. to about 15° C., and a pH of 7 to 8.5 in the presence of a catalytic amount of sodium selenate, with a support carrier comprising porous glass beads functionalized with mercaptan groups, such beads having a specific pore size of 200A to 1000A and a surface area of 5 to 100 sq. m/g., such that a disulphide group is produced in a covalent bridge linking said enzyme to said carrier; said glass beads having been functionalized with mercaptan groups by reaction at the hydroxyl group of a mercapto alcohol of formula (I) HO--(CH.sub.2).sub.n --SH in which n is an integer of 1 to 6, such that the beads bear mercaptan group-containing radicals of formula HS--(CH.sub.2).sub.n --O-- wherein n is as defined above.

Claim 14

A method of making an insolubilized, enzymatically active substance comprising functionalizing an enzyme with mercapto groups by reacting amino groups of the enzyme with a mercaptan group containing reagent with formation of a peptide linkage to produce a mercapto functionalized enzyme, reacting the mercaptan groups of the functionalized enzyme, at a temperature of 0° C. to about 15° C., and a pH of 7 to 8.5 in the presence of an oxidation catalyst, with the mercaptan groups of a support carrier comprising porous glass beads bearing about 35 to about 50 micro-equivalents of mercaptan groups per gram of beads, said beads having a specific pore size of 200A to 1000A and a surface area of 5 to 100 sq. m/g. to produce a disulphide group in a covalent bridge linking the enzyme and carrier; said beads having been functionalized with said mercaptan groups by heating the beads in a mercapto alcohol of formula (I) HO--(CH.sub.2).sub.n --SH in which n is an integer of 2 to 4, under reflux conditions for 5 to 18 hours, to react with the mercapto alcohol at the hydroxyl group thereof, such that the beads bear mercaptan group-containing radicals of formula HS--(CH.sub.2).sub.n --0-- wherein n is as defined above.

Claim 15

A method of making an insolubilized, enzymatically active substance comprising functionalizing an enzyme with mercapto groups by reacting amino groups of the enzyme with a mercaptan group containing reagent with formation of a peptide linkage to produce a mercapto functionalized enzyme, reacting the mercaptan groups of the functionalized enzyme, at a temperature of 0° C. to about 15° C., and a pH of 7 to 8.5 in the presence of an oxidation catalyst, with the mercaptan groups of a support carrier comprising porous glass beads bearing about 35 to about 50 micro-equivalents of mercaptan groups per gram of beads, said beads having a specific pore size of 200A to 1000A and a surface area of 5 to 100 sq. m/g. to produce a disulphide group in a covalent bridge linking the enzyme and carrier; said beads having been functionalized with said mercaptan groups by heating the beads in a mercapto alcohol of formula (I) HO--(CH.sub.2).sub.n --SH in which n is an integer of 2 to 4, under reflux conditions for 5 to 18 hours, to react with the mercapto alcohol at the hydroxyl group thereof, such that the beads bear mercaptan group-containing radicals of formula HS--(CH.sub.2).sub.n --0-- wherein n is as defined above.

Claim 17

A method of regenerating a mercaptan group bearing support carrier from a spent insolubilized enzyme substance of formula ##STR17## wherein ##STR18## is the residue of a spent enzyme having lost a hydrogen atom from an amino group n and p, which may be the same or different, are integers of 1 to 6 and G represents an inorganic support carrier comprising porous glass beads having a specific pore size of 200A to 1000A, prepared by reacting an enzyme having available mercaptan groups, at a temperature of 0° C. to about 15° C., and a pH of 7 to 10 in the presence of an oxidation catalyst with the support carrier after it has been prefunctionalized with mercaptan groups by reacting at the hydroxyl group of a mercapto alcohol of the formula HO--(CH 2 ) n --SH wherein n is as defined above, said enzyme being prefunctionalized with the mercapto groups by reacting the enzyme with a mixed anhydride of formula (IV) ##STR19## in which R 3 is a lower alkyl group of 1 to 6 carbon atoms and R is an alkylene radical of formula --(CH 2 ) n --, in which n is an integer of 1 to 6, to form an enriched enzyme of formula (III) ##STR20## in which ##STR21## is the residue of an enzyme having lost a hydrogen atom from an amino group, and n is an integer of 1 to 6, reducing the disulphide group in the spent insolubilized enzyme substance with a thioalcohol of formula (I) HO--(CH.sub.2).sub.n --SH in which n is an integer of 1 to 6, and recovering the insoluble support carrier bearing mercaptan groups.

Claim 18

A method of regenerating a mercaptan group bearing support carrier from a spent insolubilized enzyme substance of formula ##STR17## wherein ##STR18## is the residue of a spent enzyme having lost a hydrogen atom from an amino group n and p, which may be the same or different, are integers of 1 to 6 and G represents an inorganic support carrier comprising porous glass beads having a specific pore size of 200A to 1000A, prepared by reacting an enzyme having available mercaptan groups, at a temperature of 0° C. to about 15° C., and a pH of 7 to 10 in the presence of an oxidation catalyst with the support carrier after it has been prefunctionalized with mercaptan groups by reacting at the hydroxyl group of a mercapto alcohol of the formula HO--(CH 2 ) n --SH wherein n is as defined above, said enzyme being prefunctionalized with the mercapto groups by reacting the enzyme with a mixed anhydride of formula (IV) ##STR19## in which R 3 is a lower alkyl group of 1 to 6 carbon atoms and R is an alkylene radical of formula --(CH 2 ) n --, in which n is an integer of 1 to 6, to form an enriched enzyme of formula (III) ##STR20## in which ##STR21## is the residue of an enzyme having lost a hydrogen atom from an amino group, and n is an integer of 1 to 6, reducing the disulphide group in the spent insolubilized enzyme substance with a thioalcohol of formula (I) HO--(CH.sub.2).sub.n --SH in which n is an integer of 1 to 6, and recovering the insoluble support carrier bearing mercaptan groups.