Cyclic lipopeptide acylase

Details Cyclic lipopeptide acylase Cyclic lipopeptide acylase

2000-09-12

2002-04-16

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

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

Hydrolase

C435S068100, C435S212000, C435S228000, C514S009100, C514S011400, C530S317000, C530S318000

Reexamination Certificate

active

06372474

ABSTRACT:

TECHNICAL FIELD
This invention is concerned with an enzyme technology.
The present invention relates to a novel acylase which deacylates the acyl side chain of a cyclic lipopeptide compound and to a deacylation process comprising the use thereof.
More particularly, this invention relates to a novel acylase which deacylates the acyl side chain of FR901379 Substance, which is produced by Coleophoma sp. F-11899 (FERM BP-2635) (as described in Japanese Kokai Tokkyo Koho H3-184921), or any analog of FR901379 Substance and to a deacylation process using the same.
BACKGROUND ART
There has been a standing demand for an acylase capable of deacylating the acyl side chain of a cyclic lipopeptide compound, specifically said FR901379 Substance or an analog thereof, with good efficiency.
DISCLOSURE OF THE INVENTION
The inventors of this invention explored in earnest for a new acylase which might be able to deacylate the acyl side chain of a cyclic lipopeptide compound represented by FR901379 Substance, Echinocandin B and Aculeacin A, the latter two being analogs of FR901379 Substance. As a result, they discovered an acylase in the fermentation broth available upon culture of a certain filamentous fungus and succeeded in achieving the objective deacylation with effectiveness.
The characteristics of the above novel cyclic lipopeptide acylase and of the deacylation process using the enzyme are now described in detail.
The cyclic lipopeptide acylase-producing strain of the invention is first described. The filamentous fungus as a novel cyclic lipopeptide acylase producer specifically includes but is not limited to Oidiodendron sp. No. 30084,
Oidiodendron echinulatum
IFO 31963,
Oidiodendron tenuissimum
IFO 6798,
Oidiodendron truncatum
IFO 9951 and
Oidiodendron truncatum
IFO 31812, all of which belong to the genus Oidiodendron, and Verticillium sp. No. 30085 which belongs to the genus Verticillium.
The mycological characteristics of Oidiodendron sp. No. 30084 and Verticillium sp. No. 30085 are described below.
The fungus strain No. 30084 was isolated from a soil sample collected in Jouhoku-machi, Higashi Ibaraki-gun, Ibaraki Prefecture. This strain grew repressively on various media, forming colonies varying in color, e.g. greenish gray, brownish orange, yellowish white, etc., according to different kinds of culture media. On several media, strain No. 30084 formed anamorphs showing a conidial structure consisting of a dendritic conidiophore rising up from the surface of the medium and arthroconidia formed at its branches.
The detailed mycological characteristics of strain No. 30084 are as follows.
The cultural characteristics of this fungus on various agar media are summarized in Table 1. Colonies on malt extract agar grew repressively and spread to attain diameters from 1.5 to 2.0 cm after 2 weeks of incubation at 25° C. The colony was circular and either raised as a whole or elevated peripherally and depressed in the center. The strain formed anamorphs in abundance, which presented with a powdery surface. The colony was greenish gray with a yellowish gray peripheral zone. The reverse side was light yellow with a yellowish white peripheral zone. On potato dextrose agar, too, the colony grew repressively and spread to attain diameters from 1.5 to 2.0 cm under the same cultural conditions as above. The surface of the colony was centrally elevated or convex, wrinkled, and somewhat felt-like, forming a small amount of anamorphs. The colony was brownish orange to light brown with an orange white peripheral zone. The reverse side was brown with an orange white peripheral zone.
The morphological characteristics of the strain was recorded by observing its growth on Miura's medium (Miura, H. and M. Kudo: Trans. Mycol. Soc. Japan, 11: 116-118; 1970). The conidiophore of strain No. 30084 stood erect from the surface of the medium and consisted of a tan-colored linear trunk and colorless intricate branches. This dendritic structure made the conidiophore clearly distinguishable from the vegetative hypha. The conidiophore was 90 to 220 (or 240) &mgr;m in height, with its trunk being 2 to 3 (or 3.5) &mgr;m wide. The branches were bifurcated or trifurcated in succession and spread to occupy the space 30 to 50 &mgr;m in height and 40~60 &mgr;m in width over top of the conidiophore. Each branch was fragmented along a plurality of septae, forming conidia each in the form of a rod or an ellipsoid truncated at one end or both ends. The conidium was colorless, smooth-surfaced, unicellular, and varied in size from 2~4×1.5~2 &mgr;m. Individual conidia were linked through vestiges of cell walls of the branch. The vegetative hypha was smooth-surfaced, septate, colorless, and branched. The hyphal cell was cylindrical and 1.0~2.5 &mgr;m in width. No clamydospore was observed.
Strain No. 30084 was able to grow at 3 to 32° C. and the optimum temperature for growth was 24 to 28° C. Those data were generated on potato dextrose agar “Nissui” (Nissui Pharmaceutical).
The foregoing characteristics of strain No. 30084 were compared with the descriptions in the taxonomic reference books on fungi such as (G. R. Barron: The Genera of Hyphomycetes from Soil, pp. 239-241, Williams & Wilkins, Baltimore, 1968), (J. A. von Arx: The Genera of Fungi, Sporulating in Pure Culture, pp. 180-184, J. Cramer, Vaduz, 1974) and (K. H. Domsch, W. Gams & T. H. Anderson: Compendium of Soil Fungi, pp. 517-524, Academic Press, London 1980). As a result, the above characteristics were found to agree with the descriptions of the genus Oidiodendron (Oidiodendron Robak 1932). Therefore, the strain was identified to be a strain belonging to the genus Oidiodendron and named Oidiodendron sp. No. 30084.
TABLE 1
Cultural Characteristics of Strain No. 30084
Medium
Cultural Characteristics
Malt extract agar*
Growth: repressible, diameters
1.5~2.0 cm.
Surface: circular, elevated to
centrally concave, powdery,
abundant anamorphs. Colonies
are greenish gray (1B2-1C2) with
yellowish gray (4B2) peripheral
zone.
Reverse: pale yellow (4A3) with
a yellowish white (4A2)
peripheral zone.
Potato dextrose agar
Growth: repressible, diameters
(Difco 0013)
1.5~2.0 cm.
Surface: circular, elevated to
centrally convex, wrinkled,
slightly felt-like, scanty
anamorphs. Brownish orange
(6C3) to light brown (6D6) with
an orangish white (6A2)
peripheral zone.
Reverse: brown (7E6) with an
orangish white (6A2) peripheral
zone
Czapek's solution agar*
Growth: very
repressible, diameters 0.5~1.0
cm.
Surface: circular to amorphous,
flat, powdery, abundant
anamorphs. Greenish gray (29C2)
with a light gray (1B1)
peripheral zone.
Reverse: Light gray (1B1) to
greenish gray (25D2).
Sabouraud's dextrose
Growth: repressible,
agar medium
diameters
(Difco 0190)
1.5~2.0 cm.
Surface: circular, elevated to
centrally convex, slightly
felt-like, wrinkled. No
anamorph formed. Grayish orange
(6B3) to brownish orange (6C7)
with an orangish white (6A2)
peripheral zone.
Reverse: light brown (6D6), with
an orangish white (5A2)
peripheral zone.
Emerson YpSs
Growth: very repressible,
diameters
0.5~1.0 cm.
agar medium
Surface: circular, flat, no
(Difco 0739)
rise-up of aerial hypha. No
anamorph formed. Yellowish
white (4A2)
Reverse: Yellowish white (4A2)
Corn meal agar
Growth: very repressible,
diameters
1.0~1.5 cm.
(Difco 0386)
Surface: circular, flat, no
rise-up of aerial hypha. Scanty
anamorphs. White (1A1) to
yellowish white (4A2)
Reverse: yellowish white (4A2)
MY20 agar*
Growth: very repressible,
diameters 1.0~1.5 cm.
Surface: circular, elevated to
convex, powdery, wrinkled.
Abundant anamorphs. Greenish
gray (27D2) in center, with a
light gray (1B1) peripheral zone.
Reverse: yellowish white (4A2) to
pale yellow (4A3).
*The compositions of malt extract agar, Czapek's solution agar, and MY20 agar are based on JCM Catalog (Nakase, T., 5th ed., 503 p., Japan Collection of Microorganisms and Life Science Research Information Section of the Institute of Physical and Chemical Research, Saitama, 1992).
The above data are the results of observation after 14 days of incubation at 25° C.

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