Process for the deacylation of cyclic lipopeptides

Details Process for the deacylation of cyclic lipopeptides Process for the deacylation of cyclic lipopeptides

2000-09-06

2003-03-25

Marx, Irene (Department: 1651)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S253500, C435S068100, C435S196000

Reexamination Certificate

active

06537789

ABSTRACT:

TECHNICAL FIELD
The present invention relates to enzyme technology.
The present invention relates to a novel acylase capable of deacylating the acyl side chain of a cyclic lipopeptide compound and to a deacylation method using the same.
More particularly, the invention relates to a novel acylase adapted to deacylate the acyl side chain of Substance FR901379 (described in Japanese Kokai Tokkyo Koho H3-184921), which is produced by the microorganism Coleophoma sp. F-11899 (FERM BP-2635), or an analog of Substance FR901379 and to a deacylation method using the same.
BACKGROUND ART
There has been a standing need for an acylase capable of deacylating the acyl side chain of a cyclic lipopeptide compound, specifically said Substance FR901379 or analog, with good efficiency.
DISCLOSURE OF THE INVENTION
The inventors of the present invention did an extensive research in search of a novel acylase capable of deacylating the acyl side chain of a cyclic lipopeptide represented by Substance FR901379 or its analogs such as Echinocandin B and Aculeacin A. As a result, they discovered an acylase produced by
Streptomyces anulatus
and succeeded in effective achievement of the objective deacylation.
The above novel cyclic lipopeptide acylase and the deacylation method using the same are now described with reference to their salient features.
First, the cyclic lipopeptide acylase-producing microorganism is described.
The novel cyclic lipopeptide acylase-producing microorganism includes but is not limited to
Streptomyces anulatus
No. 4811,
Streptomyces anulatus
No. 8703, and Streptomyces sp. 6907, all of which belong to the genus Streptomyces.
The characteristics of those strains are now described.
The novel cyclic lipopeptide acylase-producing strain named
Streptomyces anulatus
No. 4811 (herein after referred to briefly as Strain No. 4811) was isolated for the first time from a soil sample collected in Fukushima Prefecture. The bacteriological characteristics of this Strain No. 4811 are now described.
Cultural Characteristics
The cultural characteristics of Strain No. 4811 on yeast extract-malt extract agar, oatmeal agar, inorganic salts-starch agar, glycerin-asparagine agar, peptone-yeast extract-iron agar, and tyrosine agar after incubation at 30° C. for 14 days and the light and scanning electron microscopic observation of the respective growths are summarized in Table 1. The color descriptions given below correspond to the nomenclature defined in Methuen Handbook of Colour, Methuen, London, 1978.
TABLE 1
Cultural characteristics of Strain NO. 4811
Medium
Cultural characteristics
Yeast extract-
G: good
malt extract agar
A: abundant, yellowish gray (2B2)
(ISP-2)
R: brown (7F4)
S: scanty, brown
Oatmeal agar
G: good-moderate
(ISP-3)
A: abundant, yellowish gray (2C3)
R: brown (7F4)
S: trace, brown
Inorganic salts-
G: good
Starch agar
A: abundant, yellowish gray (2C3)
(ISP-4)
R: yellowish brown (5E4)
S: none
Glycerin-asparagine agar
G: good
(ISP-5)
A: abundant, yellowish gray (2C3)
R: brown (6E4)
S: none
Peptone-yeast extract-iron
G: moderate
agar
A: poor, white
(ISP-6)
R: light brown (6D5)
S: none
Tyrosine agar
G: moderate
(ISP-7)
A: moderate, yellowish gray (2B2)
R: brown (7F4)
S: none
Codes: G: growth, A: aerial mycelium, R: reverse side color, S: soluble pigment
The color of the aerial mycelium was yellowish gray to greenish gray, the reverse side color of growth was yellowish brown to brown, the soluble pigment was light brown, and neither intracellular pigments nor soluble pigments were pH-sensitive. No melanoid pigments were produced.
Physiological Characteristics
The physiological characteristics of Strain No. 4811 are summarized in Table 2.
TABLE 2
Physiological characteristics of Strain No. 4811
Test item
Result
Temperature range for growth
4.0-35.0° C.
Liquefaction of gelatin
+
Coagulation of milk
±
Peptonization of milk
+
Hydrolysis of starch
+
Production of Melanoid pigments
−
Carbon utilization:
D-glucose
+
L-arabinose
+
D-xylose
+
Inositol
−
Mannitol
+
D-fructose
+
L-rhamnose
±
Sucrose
−
Raffinose
−
+: positive, ±: weakly positive, −: negative
The vegetative mycelium of Strain No. 4811 developed well and branched irregularly but not fragmented. The aerial mycelium extending from the vegetative mycelium branched monopodially to form elongated spore chains. The spore chain morphology of the aerial mycelium was straight-flexuous, thus belonging to the RF type according to the classification of Pridham et al. (Pridham, T. G. et al.: Appl. Microbiol., 6:54, 1958). Each spore chain consisted of 20 or more spores. The spores were smooth-surfaced (glabrous) and cylindrical. The spore size was 0.5~0.7×0.7~1.1 &mgr;m.
None of sclerotium, sporangium, and zoospore was observed.
Cell Wall Type
As to the cell wall amino acid composition, the whole-cell lysate was analyzed by the method of Becker et al. (Becker, B., M. P. Lechevalier, R. E. Gordon and H. A. Lechevalier: Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole-cell hydrolysates: Appl. Microbiol., 12:421-423, 1964)and the method of Yamaguchi (Yamaguchi, T.: Comparison of the cell wall composition of morphologically distinct actinomycetes: J. Bacteriol. 89:444-453, 1965) The result indicated the existence of LL-diaminopimelic acid. Therefore, the cell wall of this strain was considered to be of Type I.
Based on the above morphological observation and chemical analysis, Strain No. 4811 was considered to belong to the genus Streptomyces according to the taxonomic classification of Pridham et al. (Pridham, T. G. et al: Appl. Microbiol., 6:54, 1958). Accordingly, the characteristics of this strain were compared with those of Streptomyces species as described in the literature, namely Shirling et al. (Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 2. Species descriptions from first study, Intern. J. Syst. Bacteriol., 18:69-189, 1968; Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 3. Additional species descriptions from first and second studies, Intern. J. Syst. Bacteriol., 18:279-392, 1968; Shirling, E. B. and D. Gottlieb: Cooperative Description of Type Culture of Streptomyces. 4. Species descriptions from second, third and forth studies, Intern. J. Syst. Bacteriol., 19:391-512, 1969); Skerman et al. (Skerman, V. B., V. McGowan and P. H. A. Sneath: Approved List of Bacterial Names, Amended Edition, American Society for Microbiology, Washington D. C., 1989); and Moore et al. (Moore, W. E., E. P. Cato and L. V. H. Moore: Index of Bacterial and Yeast Nomencultural Changes, American Society for Microbiology, Washington D. C., 1992). The comparison indicated that the characteristics of
Streptomyces anulatus
so described were substantially identical to the characteristics of this strain. Accordingly, this Strain No. 4811 was identified as
Streptomyces anulatus
and named
Streptomyces anulatus
No. 4811.
This
Streptomyces anulatus
No. 4811 was originally deposited with National Institute of Bioscience and Human Technology (NIBH, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) (ZIP code 305) on Dec. 27, 1995 under the accession number of FERM P-15377 and subsequently converted to deposit according to the Budapest Treaty as of Feb. 3, 1997 under the accession number of FERM BP-5808.
The novel cyclic lipopeptide acylase-producing strain named
Streptomyces anulatus
No. 8703 (herein after referred to briefly as Strain No. 8703) was isolated for the first time from a soil sample collected in Fukushima Prefecture. The bacteriological characteristics of this Strain No. 8703 are now described.
Cultural Characteristics The cultural characteristics of Strain No. 8703 on yeast extract-malt extract agar, oatmeal agar, inorganic salts-starch agar, glycerin-asparagine agar, peptone-yeast extract-iron agar, and tyrosine agar after incubation at 30° C. for 14 days and the light and scanning electron microscopic observations o

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