Claims
- 1. A process for preparing purified Echinocandin B deacylase, an enzyme that is a heterodimer of approximately 81-kilodaltons molecular weight; whose approximately 63-kilodalton subunit has the amino-terminal sequence: Ser-Asn-Ala-Tyr-Gly-Leu-Gly-Ala-Gln-Ala-Thr-Val-Asn-Gly-Ser-Gly-Met-Val-Leu-Ala-Asn-Pro-His-Phe-Pro; whose approximately 18-kilodalton subunit has the amino-terminal sequence: His-Asp-Gly-Gly-Tyr-Ala-Ala-Leu-Ile-Arg-Arg-Ala-Ser-Tyr-Gly-Val: and whose optimal catalytic activity for deacylation of echinochandin B is at about pH 6, at 60.degree. C.; which comprises the steps;
- (a) solubilizing the enzyme from Actinoplanes utahensis cells to produce a soluble extract;
- (b) heating the soluble extract of step (a), buffered at about pH 6, for about one hour at a temperature of about 60.degree. C.; and removing precipitate;
- (c) adding to the heat-treated extract of step (b) (NH.sub.4).sub.2 SO.sub.4 to a final concentration of about 14% (weight/volume) and KCl to a final concentration of about 1.2M KCl;
- (d) loading the solution of step (c) onto a hydrophobic interaction chromatography resin equilibrated with pH 6 buffer containing about 14% (weight/volume) (NH.sub.4).sub.2 SO.sub.4 and 1.2M KCl, and eluting bound ECB deacylase activity with a simultaneous linear gradient of KCl from 1.2 to 0.1M and (NH.sub.4).sub.2 SO.sub.4 from 14 to 0% in pH 6 buffer, to produce a hydrophobic interaction eluate;
- (e) adding to the hydrophobic interaction eluate of step (d) (NH.sub.4).sub.2 SO.sub.4 to a concentration of about 10% of saturation, removing precipitate, adding (NH.sub.4).sub.2 SO.sub.4 to a final concentration of about 36% of saturation, and recovering the precipitate to produce a 10-36% (NH.sub.4).sub.2 SO.sub.4 precipitate;
- (f) dissolving the 10-36% (NH.sub.4).sub.2 SO.sub.4 precipitate of step (e) in pH 6 buffer containing 0.8M KCl, gel filtering the dissolved 10-36% (NH.sub.4).sub.2 SO.sub.4 precipitate using a gel chromatography resin equilibrated with pH 6 buffer containing 0.8M KCl, and combining eluate containing deacylase activity;
- (g) chromatographing the eluate of step (f) on cation-exchange chromatography resin equilibrated in pH 5.6 buffer containing 0.05M KCl, eluting a first and a second peak of deacylase activity from said cation-exchange chromatography resin using a linear gradient of KCl from 0.05M to 0.5M in pH 5.6 buffer, and combining cation-exchange chromatography eluate from the first peak containing deacylase activity;
- (h) adjusting the KCl concentration of the combined elute of step (g) to about 0.05M, loading the combined eluate onto a dye-ligand chromatography resin, wherein the dye-ligand is Procion Red, equilibrated in pH 6 buffer containing 0.05M KCl, eluting said dye-ligand chromatography resin using a step-wise gradient of KCl from 0.05M to 2M to 3.3M, and combining eluate containing deacylase activity;
- (i) gel filtering the dye-ligand chromatography eluate of step (h) using a gel chromatography resin equilibrated with pH 6 buffer containing 0.2M KCl;
- (j) adjusting the KCl concentration of the gel filtration eluate of step (i) to about 0.04M KCl and about pH 7, chromatographing the gel filtration eluate using a cation-exchange chromatography resin equilibrated in pH 7 buffer containing 0.04M KCl, and eluting with successive steps of KCl concentration of 0.04M, 0.5M, and 2M; and
- (k) recovering purified Echinocandin B deacylase by combining eluate from the cation-exchange chromatography resin of step (j) containing deacylase activity.
Parent Case Info
This application is a division of application Ser. No. 07/534,394 filed Jun. 7, 1990.
Non-Patent Literature Citations (2)
Entry |
Boeck et al., Journal of Antibiotics 41(8), 1085-1092 (Aug. 1988). |
Takeshima et al., Journal of Biochem. 105(4), 606-610 (Apr. 1989). |
Divisions (1)
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Number |
Date |
Country |
Parent |
534394 |
Jun 1990 |
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