Abstract:
A polypeptide with pyruvate decarboxylase activity, derived from the pyruvate decarboxylase from  Zymomonas mobilis  by substitution of an amino acid in position 553.

Description:
FIELD OF THE INVENTION 
     The invention relates to a process for preparing acyloins by the enzymic conversion of alpha-ketocarboxylic acids and/or aldehydes in the presence of pyruvate decarboxylase (PDC), and to a novel PDC, which is suitable for this process, and its preparation. 
     DESCRIPTION OF THE BACKGROUND 
     Acyloins, or alpha-hydroxyketones, are compounds which possess an optically active C atom and which play a considerable role in the synthesis of more complex compounds, for example, and in particular, (R)-(−)-phenylacetylcarbinol (PAC), which is of great economic interest for producing ephedrine. The R enantiomer, which is formed by using  Saccharomyces cerevisiae  to fermentatively convert pyruvate in the presence of benzaldehyde (DE-PS 548 459 dated 1932), is required for this purpose. 
     In this synthesis of PAC using yeast cells, a large number of byproducts are formed as a result of the activity of the majority of the enzymes present in the yeast, and the growth of the cells is inhibited by the presence of benzaldehyde. 
     Even when it has been isolated from the yeast, the pyruvate decarboxylase (PDC) gives rise, in this reaction, to substantial proportions of 2-hydroxypropiophenone, which is an isomer of PAC. 
     Thiamine diphosphate-dependent and Mg ++ -dependent PDC (E.C. 4.1.1.1) is widely distributed and is found in many plants, yeasts and other fungi and in some bacteria. It catalyzes the non-oxidative decarboxylation of pyruvate to acetaldehyde, and an acyloin condensation, with the formation of alpha-hydroxyketones, takes place as a side reaction. 
     An enzymic reaction of this nature also takes place when an aldehyde is used as the starting compound, instead of alpha-ketocarboxylic acids, and the aldehyde which is formed by the decarboxylation can also take part in the condensation reaction as a “cosubstrate”, with the formation of homoacyloins R—CHOH—CO—R′, in which R=R′. 
     WO 96/37620 describes the preparation of acyloins from acetaldehyde and benzaldehyde by catalysis using a recombinantly modified pyruvate decarboxylase obtained from  Zymomonas mobilis . Those enzymes in which the tryptophan residue in position 392 has been replaced with a sterically smaller residue, such as alanine, glycine, phenylalanine, leucine, isoleucine, arginine, histidine, serine or threonine, are in particular described as being suitable. However, it is reported that acetaldehyde inactivates all these enzymes. 
     SUMMARY OF THE INVENTION 
     It was, therefore, an object of the present invention to provide novel pyruvate decarboxylases which do not exhibit the abovementioned disadvantages at all, or only exhibit them to a substantially lesser degree, without, however, the synthetic capacity of the novel enzymes being reduced. 
     We have found that this object is achieved by a pyruvate decarboxylase which, as compared with the  Zymomonas mobilis  PDC, carries an amino acid substitution at position 553, with the lysine at this position being replaced with an amino acid exhibiting similar space-filling and/or charge properties, in particular with the amino acid lysine or alanine. 
     Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the invention. 
    
    
     
       DESCRIPTION OF THE DRAWING 
         FIG. 1 . Depiction of loss of activity of protein R553. 
     
    
    
     DESCRIPTION OF THE INVENTION 
     These novel enzymes (PDC) can be prepared from the known  Zymomonas mobilis  PDC gene sequence using recombinant methods which are known to the skilled person, such as site-directed mutagenesis. In this regard, the experimental section in WO 96/37620, which describes the recombinant construction, expression and purification of a mutant PDC from  Zymomonas mobilis , is hereby expressly incorporated by reference. The  Zymomonas mobilis  nucleic acid sequence encoding the PDC is shown, in the 5′-3′ direction, on page 14 in WO 96/37620, with the first nucleotides Nos. 1 to 3 (ATG) constituting the start codon (methionine, amino acid position +1) for the translation and nucleotides 1705–1707 (TAG) forming the stop codon as shown in SEQ ID NO: 1. The amino acid sequence of the  Zymomonas mobilis  PDC can be obtained from this nucleotide sequence by translating it in accordance with the genetic code. 
     A preferred embodiment of the invention relates to a PDC in which the lysine (K) at position 553 has been replaced with arginine (R) (R553). 
     As compared with the starting molecule (K553), this enzyme exhibits superior stability toward acetaldehyde ( FIG. 1 ), i.e. R553 loses substantially less activity in the presence of acetaldehyde (+ACA) than does K553. 
     A further advantage of this enzyme is that its synthetic performance (measurable as space/time yield) is substantially greater in a fed batch experiment than is that of K553. 
     Another object of the invention is a process for preparing enantiomerically pure phenylacetylcarbinols of the formula (I) 
                                
where R is H, F, Cl or Br,
 
from acetaldehyde or pyruvate and benzaldehydes of the formula (II)
 
                                
in the presence of the novel pyruvate decarboxylase.
 
     Particularly suitable embodiments of this novel process are those in which acetaldehyde or pyruvate is subsequently metered in continuously or discontinuously, during the course of the biotransformation, such that the concentration of acetaldehyde or pyruvate in the reaction medium is between 20 and 50 mMol/l. 
     The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention. 
     As far as further details of this process are concerned, the reader is referred to WO 99/9195, which is hereby expressly incorporated by reference. The conduct of the novel process is essentially identical to that of the process described in WO 99/9195 apart from the catalyst employed (pyruvate decarboxylase). 
     EXAMPLE 1 
     Inactivating the PDC with Acetaldehyde 
     PDC 553R was treated with acetaldehyde under the following conditions:
     30 mM acetaldehyde   50 mM morpholinoethanesulfonic acid   20 mM MgCl 2      20 μg cell-free crude protein extract dissolved in 100 μl of H 2 O, pH 7.0   

     The incubation was carried out at 4° C. in order to prevent the enzyme-catalyzed acetoin formation reducing the concentration of acetaldehyde. 
       FIG. 1  depicts the loss of activity of the novel protein R553, as compared with the starting molecule K553, in the presence of acetaldehyde (+ACA). In the absence of acetaldehyde (−ACA), the two enzymes are equally stable over the observed period of 30 hours.