Enzymatically catalyzed method of preparing mono-acylated polyols

Abstract

The present invention relates to a biocatalytic method of preparing a mono-acylated polyol catalyzed by triacylglycerol lipase mutants, as for example derived from Candida antarctica lipase B (CALB); a biocatalytic method of enantioselectively preparing an asymmetric mono-acylated polyol, catalyzed by the same enzyme mutants; as well as the use of a mutated triacylglycerol lipase in a method of preparing mono-acylated polyols. The invention also provides novel mutants, coding sequences thereof, and recombinant microorganisms carrying said coding sequences.

1 - 30 . (canceled) 
     
     
         31 . A biocatalytic method of preparing a mono-acylated polyol of the general formula (I): 
       
         
           
           
               
               
           
         
         wherein 
         R 1  represents an optionally substituted, linear or branched, saturated or non-saturated hydrocarbyl residue; and 
         A represents an optionally substituted, linear or branched hydrocarbylene residue having at least two carbon atoms, 
         which method comprises 
         a) reacting a polyol of the formula (II) and an acyl donor compound of the formula (III) 
       
       
         
           
           
               
               
           
         
         wherein R 1  and A are as defined above, and 
         Don represents a donor molecule residue carrying the said acyl group; 
         in the presence of a mutated triacylglycerole lipase (EC 3.1.1.3) until a mono-acylated polyol of the above formula (I) is formed; and 
         b) obtaining a monoacylated polyol product. 
       
     
     
         32 . The method of  claim 31 , wherein said mutated lipase contains at least one amino acid mutation, which increases the selectivity of the lipase for the mono-acylation of said polyol, if compared to the corresponding non-mutated lipase. 
     
     
         33 . The method of  claim 31 , wherein said mutant comprises at least one mutation, which removes a stabilizing functional amino acid from that part of the reactive center of the enzyme which stabilizes an oxyanion transition state of the carbonyl group of the mono-acylated polyol of formula (I) to be formed. 
     
     
         34 . The method of  claim 31 , wherein
 a) a maximum monoester yield is obtained which is at last 1% above the maximum yield as obtained by the corresponding wild-type enzyme;   b) a 3:1 molar ratio of monoester to polyester is reached at a conversion rate of the polyol which is at last 1% above the corresponding conversion rate as obtained by the corresponding wild-type enzyme; and/or   c) the ratio of reaction times (T 90 (mutant)/T 90 (wild-type)) to reach 90% monoacylated polyol based on the total amount of polyol is above 1.   
     
     
         35 . The method of  claim 31 , wherein said enzyme is a mutant of  Candida antarctica  lipase B (CALB) comprising an amino acid sequence of SEQ ID NO: 2, mutated in at least one position. 
     
     
         36 . The method of  claim 35 , wherein said mutant comprises an amino acid sequence of SEQ ID NO:2 wherein the amino acid Thr40 is mutated. 
     
     
         37 . The method of  claim 36 , wherein the mutation is such that substantially no stabilizing interaction between the oxyanion intermediate and the amino acid residue in position 40 occurs. 
     
     
         38 . The method of  claim 37  wherein the mutation comprises the single mutations Thr40Ala, Thr40Val or Thr40Ser. 
     
     
         39 . The method of  claim 38 , wherein said mutant is selected from mutants having an amino acid sequence of SEQ ID NO: 4 or variants of said mutant having a sequence identity of at least 60%, which variants still contain a mutation in an amino acid position corresponding to position Thr40 of SEQ ID NO:4. 
     
     
         40 . The method of  claim 37 , wherein the mutant additionally comprises at least one mutation in one of the amino acid positions Leu 278, Ile 285 and Pro 280 of SEQ ID NO: 2 or 4. 
     
     
         41 . The method of  claim 40 , wherein the mutants and the variants thereof are not mutated in other amino acid positions contributing to the catalytic site of the enzyme. 
     
     
         42 . The method of  claim 41 , wherein the mutants are not mutated in amino acid positions Ser105, Asp187, His224 (catalytic triade) and Gln106 and wherein the variants are not mutated in amino acid positions corresponding thereto. 
     
     
         43 . The method of  claim 35 , wherein in SEQ ID NO:2, or in SEQ ID NO:2 comprising a mutation at amino acid Thr40 according to SEQ ID NO:4, one or more of Leu278, Ala281, Ala282 or Ile285 are mutated. 
     
     
         44 . The method of  claim 43 , wherein the one or more mutations are independently selected from Leu278Ser, Ala281Val or Ala281Glu, and Ala282Leu, Ala282Thr, Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp, Ala282Val, Ala282Met or Ala282Arg. 
     
     
         45 . The method of  claim 44 , wherein SEQ ID NO:2 comprises one mutation, selected from Ala281Val, Ala281Glu, Ala282Leu, Ala282Thr, Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp, Ala282Val, Ala282Met, Ala282Arg and Ile285Phe, or wherein SEQ ID NO:2 comprises the double mutation Leu278Ser and Ala282Leu. 
     
     
         46 . The method of  claim 31 , wherein the reaction is performed in the presence of the isolated enzyme mutant or a recombinant microorganism functionally expressing said mutant. 
     
     
         47 . The method of  claim 31 , wherein the polyol is a compound of formula (II)
 wherein A is selected from the groups
   —(CH 2 ) n — and —(CH 2 ) m —CR 2 R 3 —(CH 2 ) m′ —
 
   wherein   n is an integer of 2-6,   m and m′ independently of each other are integers of 1-3   R 2  and R 3  independently of each other are selected from H, OH, SH, NH 2 , optionally substituted carbo- or heterocyclic rings and hydrocarbyl-residues, provided that R 2  and R 3  are not simultaneously H.   
     
     
         48 . The method of  claim 31 , wherein the donor of formula (III) is selected from compounds wherein R1 is C1-C6-alkyl and Don is an —OR residue, wherein R is selected from C1-C6-alkyl and C2-C4-alkenyl. 
     
     
         49 . An enzymatically catalyzed method of enantioselectively preparing an asymmetric mono-acylated polyol of the general formula (I): 
       
         
           
           
               
               
           
         
         wherein 
         R 1  represents an optionally substituted, linear or branched, saturated or non-saturated hydrocarbyl residue; and 
         A* represents an optionally substituted, linear or branched, asymmetric hydrocarbylene residue having at least two carbon atoms; 
         which method comprises 
         a) reacting a stereoisomeric mixture of a polyol of the formula (II′) and an acyl donor compound of the formula (III) 
       
       
         
           
           
               
               
           
         
         wherein R 1  and A* are as defined above, and 
         Don represents a donor molecule residue carrying the said acyl group; 
         in the presence of a mutated triacylglycerole lipase (EC 3.1.1.3) until a mono-acylated polyol of the above formula (I) is formed; and 
         b) obtaining an asymmetric monoacylated polyol product. 
       
     
     
         50 . The method of  claim 49  wherein an enzyme mutant as defined in anyone of the claims  2  to  15  in the form of an isolated enzyme mutant or a recombinant microorganism functionally expressing said mutant is applied. 
     
     
         51 . The method of  claim 49 , wherein the polyol is a compound of formula (II′) wherein A* is selected from the groups
   —(CH 2 ) m —CHR 2 —(CH 2 ) m′ —
 
 wherein m, m′ and R 2  are as defined above. 
 
     
     
         52 . The use of a mutated triacylglycerol lipase (EC 3.1.1.3) in a method of preparing a mono-acylated polyol of the general formula (I) or (I′) as defined above. 
     
     
         53 . A  Candida antarctica  lipase B (CALB) mutant showing a pattern of at least two mutation of the amino acid sequence of SEQ ID NO:2, which pattern is selected from the pattern as shown in Table A. 
     
     
         54 . The mutant of  claim 53  showing additionally one mutation selected from Val210Ile, Ala281Glu, Val221Asp. 
     
     
         55 . A  Candida antarctica  lipase B (CALB) mutant, having one or more mutations in the amino acid sequence of SEQ ID NO:2, which are independently selected from Leu278Ser, Ala281Val or Ala281Glu, and Ala282Leu, Ala282Thr, Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp, Ala282Val, Ala282Met or Ala282Arg. 
     
     
         56 . The mutant of  claim 55 , having one mutation in SEQ ID NO:2, selected from Ala281Val, Ala281Glu, Ala282Leu, Ala282Thr, Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp, Ala282Val, Ala282Met, Ala282Arg and Ile285Phe, or having in SEQ ID NO:2 the double mutation Leu278Ser and Ala282Leu. 
     
     
         57 . The mutant of  claim 53 , additionally having at least one of the mutations as defined in  claim 55 . 
     
     
         58 . A nucleic acid molecule encoding a mutant of  claim 50 . 
     
     
         59 . An expression vector, comprising, optionally under the control of a regulatory nucleic acid sequence, at least one coding sequence of  claim 58 . 
     
     
         60 . A microbial host carrying at least one expression vector of  claim 59  or coding sequence of  claim 58 .