Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
1999-10-29
2002-10-29
Saidha, Tekchand (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S069800, C435S832000, C435S252300, C435S320100, C435S325000, C530S412000, C536S023200, C536S023700
Reexamination Certificate
active
06472192
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to cyclodextrin glycosyl transferases (CGTases) EC 2.4.1.19 for producing &ggr;-cyclodextrin, to processes for preparing &ggr;-cyclodextrin glycosyl transferases, and to their use.
2. The Prior Art
As a rule, cyclodextrins are prepared from starch or starch-like substrates. In these preparations, CGTases are used to convert starch enzymically into cyclodextrin (CD). For thermodynamic reasons, the starch is mainly converted into &bgr;-CD, independently of the CGTase used for the reaction, if the reaction is carried out until the thermodynamic equilibrium is reached (maximum CD yield). However, in the initial phase, at the beginning of the starch conversion reaction, the enzymes which are used for the conversion differ in the composition of the primary product mixture. &agr;- &bgr;- or &ggr;-CGTases are differentiated depending upon the product, &agr;, &bgr;-, or &ggr;-CD, which is chiefly formed by the enzyme in this initial phase.
These enzymes, which are suitable, and have also already been used, for the industrial production of CD, have hitherto only been detected in bacteria. &agr;-CGTases have hitherto only been identified
in Bacillus macerans, Bacillus stearothermophilus
and
Klebsiella oxytoca
. &bgr;-CGTases have been detected, for example, in
Bacillus circulans, Bacillus megaterium, Bacillus ohbensis
, Micrococcus sp. and alkalophilic Bacillae which have not been precisely classified taxonomically, such as Bacillus sp. 38-2, 17-1, 1011 or 1-1. Naturally occurring enzymes having an initially high &ggr;-CD-forming activity have been reported in
Bacillus subtilis
313, Bacillus sp. A1-6 and Bacillus sp. 290-3.
Since the CGTases which are used in the industrial preparation of cyclodextrins always yield mixtures of several cyclodextrins when converting starch into cyclodextrins, various processes have been developed for isolating pure cyclodextrins (&agr;, &bgr; or &ggr;). These are described below:
Defined CDs can be separated out chromatographically from the product mixtures, e.g. on the basis of differences in their molecular weights (described, for example, in U.S. Pat. No. 4,808,232).
As a rule, when starch is converted enzymically into cyclodextrins, complexing agents are added which only react with one defined CD and with this CD form an insoluble complex, for example, which can then be separated out from the reaction mixture by physical means. Subsequently, the complex is resolved and the homogeneous CD is isolated (described, for example, in EP 0291067).
When a &ggr;-CGTase is used, the product composition can be displaced in the &ggr;-CD direction by adding an organic solvent, such as ethanol, to the reaction mixture (
J. Ferrm. Bioeng
. (1990) 70 (3), pp. 150-154).
In each of the processes, those CGTases are optimally used which possess an initial product formation preference which is as high as possible for the CD which is to be prepared in pure form.
The specificity of the previously known &agr;- and &bgr;-CGTases is adequate for industrial production of the corresponding cyclodextrins. By contrast, none of the known, naturally occurring &ggr;-CGTases possesses a product specificity which permits a comparable industrial production of &ggr;-CD.
In order to prepare &ggr;-CD, therefore, it was proposed, in CA 115:157165, that &agr;- and/or &bgr;-Cyclodextrins be converted enzymically into &ggr;-CD by adding the &ggr;-CD-specific complexing agent glycosyl glycyrrhizin, maltose and a CGTase.
Another option for preparing &ggr;-CD consists in increasing the &ggr;-CD specificity of &bgr;-CGTase, by means of exchanging defined amino acid residues, to such a degree that the mutagenized enzyme produces &ggr;-CD to an increased. extent and can consequently be used for preparing &ggr;-CD on an industrial scale. Appropriate mutations are known and described, for example, in DE 43 24 650 A1 (corresponds to U.S. Pat. No. 5,474,917),
Biochemistry
(1994) 33 (33), pp. 9929-9936
, Biochemistry
(1995) 34 (10), pp. 3368-3376 and
J. Biotech
. (1994) 32, pp. 283-288.
Such CGTase derivatives, which have been produced by mutagenizing &bgr;-CGTases, possess an increased specificity for &ggr;-CD and are consequently, on the basis of their product spectra, suited, in principle, for the industrial preparation of &ggr;-CD. However, a disadvantage is that the specific activities of the starting enzymes which are used for the mutagenesis are reduced by introducing the relevant mutations. In dependence on the amino acid residues which are introduced, mutated enzymes having an increased specificity for &ggr;-CD only possess between 25% and 50% of the CD-forming activity of the starting enzyme (
Biochemistry
(1994) 33 (33), pp. 9929-9936
, Biochemistry
(1995) 34 (10), pp. 3368-3376).
SUMMARY OF THE INVENTION
It is an object of the present invention to provide cyclodextrin glycosyl transferases (CGTases) which, when converting starch or starch-like substrates into CD, produce &ggr;-CD to an increased extent and which still exhibit at least 60% of the specific total CGTase activity of the starting CGTase which was used for preparing the enzyme concerned.
An additional object of the present invention is to provide processes for preparing the said CGTases.
A further object of the present invention is to provide a process for producing &ggr;-CD.
The first-mentioned object is achieved by CGTases whose amino acid sequence. differs from the amino acid sequence of wild-type CGTases by the deletion of from 3 to 8 amino acids in the region from amino acid position 155 up to and including amino acid position 195, where position 1 of the protein sequence is the beginning of the signal peptide of the CGTase and the deletion increases the &ggr;-CGTase activity of the protein.
Within the meaning of the invention, increases in the &ggr;-CGTase activity is understood to mean that the quotient
quantity
of
γ
-
CD
formed
(
quantity
of
α
-
CD
formed
+
quantity
of
β
-
CD
formed
)
becomes greater in the product mixture which arises when starch or starch-like substrates are reacted with CGTases.
Preferably, the amino acid sequences of CGTases according to the invention differ from the amino acid sequences of known CGTases by between three and eight amino acid residues being deleted in the region between amino acid position 155 and amino acid position 195 of their protein sequence, where position 1 of the protein sequence is the beginning of the signal peptide of the CGTase and the deletion increases the &ggr;-CGTase activity of the protein.
Particularly preferably, the amino acid sequences of CGTases according to the invention differ from the amino acid sequences of known CGTases by five amino acid residues being deleted in the region between amino acid position 155 and amino acid position 195 of their protein sequence, where position 1 of the protein sequence is the beginning of the signal peptide of the CGTase and the deletion increases the &ggr;-CGTase activity of the protein.
It also applies for each of the other amino acid positions mentioned in the application that position 1 of the protein sequence is the beginning of the signal peptide of the CGTase.
In addition, CGTases are in particular preferred whose amino acid sequences differ from the amino acid sequences of the CGTases specified in Table 1 and
FIG. 1
at least by the deletion of the amino acid residues which are in each case printed in bold, with the remaining amino acid sequence of the respective CGTase according to the invention being homologous to the amino acid sequence of the CGTase specified in Table 1 and
FIG. 1
to the extent that the sequence exhibits CGTase activity without the deletion according to the invention.
Examples of CGTases according to the invention are CGTases which are obtained from the CGTases listed in Table 1 and
FIG. 1
, or from other C
Parsiegla Goetz
Schulz Georg E.
Collard & Roe P.C.
Consortium fur elektrochemische Industrie GmbH
Saidha Tekchand
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