Method for the Enzyme-Catalysed Hydrolysis of Polyacrylic Acid Esters, and Esterases Used Therefor

Abstract

The invention relates to a method for the enzyme-catalysed hydrolysis of polyacrylic acid esters. According to said method, at least one polyacrylic acid ester is provided and incubated with at least one enzyme selected from enzymes (EC 3.1) acting on ester bindings, until the ester groups contained in the polyacrylic acid ester are partially or fully hydrolytically split, and optionally the modified polymer obtained thereby is isolated. The invention also relates to the enzymes used and mutants thereof, nucleic acids coding for the enzymes, vectors comprising the nucleic acids, micro-organisms comprising the vectors, and the use of the enzymes, the vectors or the micro-organisms for carrying out a method for the enzyme-catalysed hydrolysis of polyacrylic acid esters. The present application also relates to polymer reaction products that can be obtained by the method, and methods for producing esterases.

1 . A method for the enzyme-catalyzed hydrolysis of polyacrylic acid esters, the method comprising
 a) preparing a polyacrylic acid ester,   b) incubating the polyacrylic acid ester with an enzyme selected from the group consisting of enzymes acting on ester bonds (EC 3.1), whereby at least one ester group of the polyacrylic acid ester is hydrolytically cleaved; and   c) optionally, isolating the modified polymer.   
     
     
         2 . The method of  claim 1 , wherein the enzyme is a carboxylic acid ester hydrolase (EC 3.1.1). 
     
     
         3 . The method of  claim 1 , wherein the polyacrylic acid ester is a homopolymer or a copolymer. 
     
     
         4 . The method of  claim 1 , wherein the polyacrylic acid ester is an alternating copolymer, a random copolymer, a gradient copolymer, a block copolymer or a graft copolymer. 
     
     
         5 . The method of  claim 1 , wherein the modified polymer comprises monomer building blocks of general formula I
   R 1 R 2 C═CR 3 —COOR 4   (I)
   
       wherein
 R 1 , R 2  and R 3  may be identical or different and are selected from the group consisting of H, a linear C 1 -C 20  hydrocarbyl residue and a branched C 3 -C 20  hydrocarbyl residue, and R 4  is selected from the group consisting of H, a linear C 1 -C 20  hydrocarbyl residue, a branched C 3 -C 20  hydrocarbyl residue and a cyclic C 3 -C 20  hydrocarbyl residue, the hydrocarbyl residue optionally is substituted with one or more identical or different groups, selected from hydroxyl, amino, epoxide, thiol groups and halogen atoms, 
 
       and in the polymer, in at least one monomer building block of formula I, R 4  is not H. 
     
     
         6 . The method of  claim 5 , wherein the polyacrylic acid ester contains, additionally to the monomers of formula I, at least one further monomer component different therefrom, in a molar proportion from 0 to 15 mol. %, which is preferably selected from N-vinylformamide, methacrylic acid, methacrylic acid ester, itaconic acid, itaconic acid ester, vinylphosphonic acid, vinylsulfonic acid, vinyl alcohol, N-vinylimidazole, N-vinylformamide, styrene, maleic acid, maleic acid ester, ethylene and/or propylene, and acrylamide and substituted acrylamides, where the substituent is selected from a linear C 1 -C 20  hydrocarbyl residue, a branched C 3 -C 20  hydrocarbyl residue and a cyclic C 3 -C 20  hydrocarbyl residue, the hydrocarbyl residue optionally being substituted with one or more identical or different groups, which are selected from hydroxyl, amino, epoxide, thiol groups and halogen atoms. 
     
     
         7 . The method of  claim 1 , wherein the enzyme is selected from the group consisting of the esterases of family VIII, type C esterases the cutinase of SEQ ID NO:5 and a cutinase derived therefrom, and the triacylglycerol lipase of SEQ ID NO:6 and a triacylglycerol lipase derived therefrom. 
     
     
         8 . The method of  claim 1 , wherein the enzyme is selected from the group consisting of the proteins of SEQ ID NO:1 (esterase B from  Burkholderia gladioli ), SEQ ID NO:2 (esterase C from  Burkholderia gladioli ) and functional mutants derived therefrom. 
     
     
         9 . The method of  claim 8 , wherein the enzyme is a mutant of the esterase of SEQ ID NO:1 or SEQ ID NO:2, and wherein at least one of the activity with respect to the hydrolysis of polyacrylic acid esters is comparable or increased relative to the activity of the esterase of SEQ ID NO:1 or SEQ ID NO:2, the stability is increased relative to the stability of the esterases of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         10 . The method of  claim 9 , wherein the mutant shows increased hydrolytic activity against at least one of polyacrylic acid methyl esters or polyacrylic acid butyl esters compared to the hydrolytic activity of the esterases of SEQ ID NO:1 or SEQ ID NO:2. 
     
     
         11 . The method of  claim 9 , wherein
 (a) the mutant of the esterase of SEQ ID NO:1 has a mutation in one or more of the amino acid residues Ser17, Gly132, Trp134, Arg155, Glu251, Ala311 and Glu316; or   (b) the mutant of the esterase of SEQ ID NO:2 has a mutation in one or more of the amino acid residues Phe138, Val150, Leu160, Thr188 and Leu193.   
     
     
         12 . The method of  claim 9 , wherein the mutant is derived from SEQ ID NO:1 and
 (a) comprises at least one of the mutations Ser17Leu, Gly132Ser, Glu251Gly, Ala311Val and Glu316Lys; or   (b) comprises at least one of the mutations Pro8Leu, Gly132Ser, Trp134Arg, Arg155Cys, Glu251Gly, Ala311Val and Glu316Lys.   
     
     
         13 . The method of  claim 9 , wherein the mutant is derived from SEQ ID NO:2 and comprises one of the following mutations or combinations of mutations:
 (a) Phe138Ala   (b) Phe138Ala, Thr188Ser   (c) Phe138Ala, Leu160Ala, Thr188Ser   (d) Leu193Ala   (e) Leu193Ala, Phe138Ala, Thr188Ser, Val150Ala   (f) Leu193Ala, Phe138Ala, Thr188Ser   (g) Leu193Ala, Phe138Ala, Thr188Ser, Leu160Ala, Val150Ala   (h) Val150Ala   (i) Val150Ala, Thr188Ser   (j) Leu193Ala, Phe138Val   (k) Leu193Ala, Phe138Val, Thr188Ser, Val150Ala   (l) Leu193Ala, Thr188Ser   (m) Leu193Ala, Phe138Val, Thr188Ser   (n) Leu193Ala, Phe138Val, Thr188Ser, Leu160Ala   (o) Phe138Val, Val150Ala, Thr188Ser   (p) Phe138Val   (q) Phe138Val, Thr188Ser   
     
     
         14 . The method of  claim 7 , wherein the mutant is a deletion mutant of an esterase of family VIII or of a type C esterase. 
     
     
         15 . The method of  claim 14 , wherein the deletion mutant has at least one loop shortening. 
     
     
         16 . The method of  claim 15 , wherein the deletion mutant has an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO:37 and SEQ ID NO:38. 
     
     
         17 . A functional esterase mutant
 a) selected from the protein of SEQ ID NO:1 (esterase B from  Burkholderia gladioli ), SEQ ID NO:2 (esterase C from  Burkholderia gladioli ) or functional mutants derived therefrom;   b) that is the esterase of SEQ ID NO:1 or SEQ ID NO:2, and wherein at least one of the activity with respect to the hydrolysis of polyacrylic acid esters is comparable or increased relative to the activity of the esterase of SEQ ID NO:1 or SEQ ID NO:2, the stability is increased relative to the stability of the esterases of SEQ ID NO:1 or SEQ ID NO:2;   c) that shows increased hydrolytic activity against at least one of polyacrylic acid methyl esters or polyacrylic acid butyl compared to the hydrolytic activity of the esterases of SEQ ID NO:1 or SEQ ID NO:2;   d) that is the esterase of SEQ ID NO:1 having a mutation in one or more of the amino acid residues Ser17, Gly132, Trp134, Arg155, Glu251, Ala311 and Glu316; or the esterase of SEQ ID NO:2 has a mutation in one or more of the amino acid residues Phe138, Val150, Leu160, Thr188 and Leu193;   e) that is derived from SEQ ID NO:1 and comprises at least one of the mutations Ser17Leu, Gly132Ser, Glu251Gly, Ala311Val and Glu316Lys; or comprises at least one of the mutations Pro8Leu, Gly132Ser, Trp134Arg, Arg155Cys, Glu251Gly, Ala311Val and Glu316Lys;   f) that is derived from SEQ ID NO:2 and comprises one of the following mutations or combinations of mutations: (a) Phe138Ala; (b) Phe138Ala, Thr188Ser; (c) Phe138Ala, Leu160Ala, Thr188Ser;   (d) Leu193Ala; (e) Leu193Ala, Phe138Ala, Thr188Ser, Val150Ala; (f) Leu193Ala, Phe138Ala, Thr188Ser; (g) Leu193Ala, Phe138Ala, Thr188Ser, Leu160Ala, Val150Ala; (h) Val150Ala; (i) Val150Ala, Thr188Ser; (j) Leu193Ala, Phe138Val;   (k) Leu193Ala, Phe138Val, Thr188Ser, Val150Ala;   (l) Leu193Ala, Thr188Ser; (m) Leu193Ala, Phe138Val, Thr188Ser; (n) Leu193Ala, Phe138Val, Thr188Ser, Leu160Ala; (o) Phe138Val, Val150Ala, Thr188Ser; (p) Phe138Val; or (q) Phe138Val, Thr188Ser;   g) that is a deletion mutant of an esterase of family VIII or of a type C esterase;   h) that is a deletion mutant of an esterase of family VIII or of a type C esterase and has at least one loop shortening; or   i) that has an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO:37 and SEQ ID NO:38.   
     
     
         18 . A nucleic acid, which
 a) codes for the esterase mutant of  claim 17 , or   b) represents a nucleic acid complementary to a), or   c) hybridizes to a nucleic acid according to a) or b) under stringent conditions, has a sequence identity of at least 80% and codes for a mutant of an esterase of family VIII or for a type C esterase mutant, which hydrolyzes polyacrylic acid esters.   
     
     
         19 . A vector comprising the nucleic acid of  claim 18 . 
     
     
         20 . The vector of  claim 19 , wherein the nucleic acid is linked operatively to a promoter. 
     
     
         21 . A microorganism comprising the vector of  claim 19 , wherein the nucleic acid optionally is linked operatively to a promoter. 
     
     
         22 . A method of producing the esterase mutant of  claim 17 , the method comprising
 a) cultivating a host organism capable of expressing an esterase of claim  26 ;   b) optionally, inducing expression of the esterase is induced, and   c) optionally, isolating the esterase from at least one of the host organism or the culture medium.   
     
     
         23 . A polymeric reaction product, obtained by the method of  claim 1 . 
     
     
         24 . The method of  claim 2 , wherein the carboxylic acid ester hydrolase (EC 3.1.1) is selected from the group consisting of carboxyl esterases (E.C. 3.1.1.1), triacylglycerol lipases (EC 3.1.1.3) and cutinases (3.1.1.74).