C. antarctica lipase variants

Details C. antarctica lipase variants C. antarctica lipase variants

1994-12-22

2000-06-13

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

4352523, 4353201, 435134, 536 232, C12N 920, C12N 1563, C12P 764, C07H 2104

Patent

active

06074863&

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates to novel lipase enzyme variants with improved properties, DNA constructs coding for the expression of said variants, host cells capable of expressing the variants from the DNA constructs, as well as a method of producing the variants by cultivation of said host cells. Furthermore, the present invention relates to a recombinant essentially pure Candida antarctica lipase and variants thereof as well as a DNA sequence encoding the said lipase or variants thereof.


BACKGROUND OF THE INVENTION

A wide variety of lipases of microbial and mammalian origin are known. The amino acid sequence of many of these lipases have been elucidated and analyzed with respect to structural and functional elements important for their catalytic function, see, for instance, Winkler et al., 1990 and Schrag et al., 1991. It has been found that the lipase enzyme upon binding of a lipid substrate and activation undergoes a conformational change, which inter alia, results in an exposure of the active site to the substrate. This conformational change together with the presumed interaction between enzyme and substrate have been discussed by, inter alia, Brady et al., 1990, Brzozowski et al., 1991, Derewenda et al., 1992.
Based on the knowledge of the structure of a number of lipases, it has been possible to construct lipase variants having improved properties by use of recombinant DNA techniques. Thus, WO 92/05249 discloses the construction of certain lipase variants, in which the lipid contact zone has been modified so as to provide the variants with different substrate specificities and/or an improved accessibility of the active site of the lipase to a lipid substrate. The modifications involve changing the electrostatic charge, hydrophobicity or the surface conformation of the lipid contact zone by way of amino acid substitutions.
Although the structural and functional relationship of lipases have been the subject of a number of studies as described in the above cited references, the research has mainly focused on the macroscopic characteristics of the lipases upon substrate binding and activation, whereas the identity of the amino acids actually involved in the substrate binding and catalytic activity has been discussed only to a lesser extent.


SUMMARY OF THE INVENTION

By sequence alignment analysis combined with analysis of the structure and activity of a number of lipases, the present inventors have now surprisingly found that the presence of certain amino acids, especially tryptophan, in a critical position of the lipase seems to be important for optimal catalytic activity.
It is consequently an object of the present invention to modify lipases which do not comprise such an amino acid residue in the critical position (which lipases in the present context are termed parent lipases) by replacing the amino acid residue located in this position with an amino acid residue which gives rise to a variant having an increased specific activity.
More specifically, in one aspect the present invention relates to a lipase variant of a parent lipase comprising a trypsin-like catalytic triad including an active serine located in a predominantly hydrophobic, elongated binding pocket of the lipase molecule and, located in a critical position of a lipid contact zone of the lipase structure, an amino acid residue different from an aromatic amino acid residue, which interacts with a lipid substrate at or during hydrolysis, in which lipase variant said amino acid residue has been replaced by an aromatic amino acid residue so as to confer to the variant an increased specific activity as compared to that of the parent lipase.
In the present context, the term "trypsin-like" is intended to indicate that the parent lipase comprises a catalytic triad at the active site corresponding to that of trypsin, i.e. the amino acids Ser, His and one of Asp, Glu, Asn or Gln.
Lipases degrade triglycerides down to fatty acids, glycerol and di- and/or monoglycerides. The lipase action depends on interfacial activati

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