The present study was initiated with an aim to identify an organism having a lipopeptide, acylase, with high specificity towards Echinocandin B. Therefore, a rapid screening protocol was undertaken with bacteria belonging to Actinomycetes, isolated from tropical soil. The selected species was evaluated for its efficiency to bio convert ECB to ECB nucleus and the cultural and environmental parameters for the bioconversion was optimized.

MATERIALS AND METHODS

Chemicals and reagents

All the chemicals and reagents used were of analytical grade and were purchased from Merck. Echinocandin B was prepared in-house by the fermentation of Aspergillus nidulans [13].

Microorganisms and culture conditions

All the cultures used for screening were isolated from natural sources (Soil, water or endophytes) across Karnataka, India. The cultures thus isolated were stored as glycerol stocks at the Biocon India Culture Collection (BICC). Totally, 140 Actinomycetes were cultured on ISP-4 (from Difco medium) plates at 28°C for one week. The control organism Actinoplanes utahensis (NRRL 12052) a type strain obtained from Agricultural research service culture collection (NRRL), Peoria, Illinois.

Enzyme screening studies

Qualitative plate assay for screening of Acylase producing strains: In the first round of screening, all the 140 Actinomycetes were inoculated onto fresh ISP-4 plates containing 1 mg/ml of Echinocandin B. The plates were incubated at 30°C for 7 days and at the end of the incubation, the plates were overlaid with 5 mL of soft agar containing 10³ CFU/mL of Candida albicans. [14]. The overlaid plates were further incubated at 30°C for 24 h and the plates with the growth of C. albicans were considered as positive for acylase.

Quantitative assay for production of acylase under submerged fermentation (SMF): Fifty-three strains positive for acylase were selected from quantitative plate assay. These strains were further inoculated into Streptomyces seed media (4% glucose, 1% yeast extract, 0.1% CaCO₃, pH adjusted to 6.5 and 25 mL of the media was dispensed into 250 mL flask) and incubated at 28°C for 3 days in an orbital shaker at 230 rpm. Three mL of the inoculum was transferred into production medium (4% glucose, 1% Soya peptone, 1% yeast extract, 1% KH₂PO₄, 0.5% K₂HPO₄, 0.1% KCL, 0.01% calcium carbonate, pH adjusted to 7.0 and 30 mL of the media was dispensed into 250 mL flask) containing filter sterilized Echinocandin B (dissolved in methanol) at a concentration of 3.0 g/L and incubated at 30°C for 3 days in an orbital shaker at 230 rpm. Samples were withdrawn at an interval of 24 h from the time of inoculation, centrifuged at 3000 rpm for 15 min and the supernatant was analysed for the formation of Echinocandin B nucleus by HPLC. Agilent Poroshell EC18 (150*4.6 mm, 2.7 µm pore size) column with KH₂PO₄ buffer and HPLC grade Acetonitrile mobile phases used for HPLC analysis using Echinocandin B and Echinocandin B nucleus from standard commercial sources [15].

The production of Acylase in Streptomyces seed and production media (devoid of the substrate – Echinocandin B) were analysed by separating the cells by centrifugation at 3000 rpm for 15 min. 1 g/L of Echinocandin B (in 50 mM phosphate buffer at pH 7) was added to the cell free supernatant and incubated for 24 h at 30°C. The reaction mixture was centrifuged at 3000 rpm for 15 min and the supernatant was analysed in HPLC (as described above) to detect Echinocandin B nucleus.

Optimisation studies for bioconversion of Echinocandin B to Echinocandin B nucleus

For the optimisation studies different parameters which affect the bioconversion of Echinocandin B to Echinocandin B nucleus were studied which include pH, temperature, substrate concentration, feeding time and media volume. Two strains positive for acylase production coded as BICC-8848, BICC-8547 with a control organism Actinoplanes utahensis in the production media were studied under submerged fermentation conditions. These strains were inoculated into Streptomyces seed media and incubated at 28°C for 3 days in an orbital shaker at 230 rpm. Three mL of the inoculum was transferred into production medium and incubated at 30°C for 3 days in an orbital shaker at 230 rpm. Samples (1 mL) were withdrawn at an interval of 24 h from the time of inoculation till end of incubation, centrifuged at 3000 rpm for 15 min and the supernatant was analysed for the formation of Echinocandin B nucleus by HPLC (as described in section 2.2.2) [15].

Effect of pH

The production medium was adjusted to pH ranges (5.5, 6.0, 6.5, 7.0 and 7.5) with 10% H₃PO₄/10% NaOH before sterilization. The sterile medium was supplemented with the substrate Echinocandin B (3 g/L) before they were inoculated with the cultures.

Effect of temperature

The inoculated production medium was incubated at different temperature (22°C, 26°C, 30°C and 34°C) for 72 h in an orbital shaker at 230 rpm. Samples were withdrawn at regular interval of time (every 24 h) and analysed by HPLC.

Effect of substrate concentration

The inoculated production medium was fed with Echinocandin B at different concentrations (2, 3, 4, 5 g/L) incubated at 26°C for 72 h in an orbital shaker at 230 rpm. Samples were withdrawn at regular interval of time (every 24 h) and analysed by HPLC.

Effect of feeding time of substrate

The inoculated production medium was incubated at 26°C in an orbital shaker at 230 rpm and fed with Echinocandin B (3 g/L) at regular intervals from the time of inoculation (0, 24, 48, 72, 96, 120 h). Samples were withdrawn at regular interval of time (every 24 h) and analysed by HPLC.

Effect of media volume on bioconversion

In general, the acylase activity varies depending on the volume of the medium used in the shake flasks. The pre-grown culture in the seed medium was inoculated into production media (500 mL in 2000 mL conical flask adjusted to pH 7.0) and incubated at 30 C for 72 h. Echinocandin B was fed at a concentration of 3.0 g/L and incubated at 26 C in an orbital shaker and samples were withdrawn at 24 h interval and processed for HPLC analysis.

Molecular level Identification of the selected Actinomycete

Two strains (BICC-8848 and BICC-8547) were identified to produce the enzyme. Genomic DNA from the selected Actinomycetes was extracted; 16S rRNA gene was amplified using 16 S universal primers. The amplified PCR product was sequenced, the obtained sequence was checked in NCBI BLAST analyser and identification is made based on the maximum homology to the available sequence in the NCBI database [16].

RESULTS

Qualitative plate assay for screening for Echinocandin B acylase producing strains

A total of 140 isolates belonging to Actinomycetes were screened by plate assay method and 53 isolates (37%) were found to be positive for converting the Echinocandin B, an antifungal compound, into a non-effective molecule, Echinocandin B Nucleus. Growth of Candida albicans around the Actinomycete colony in the soft agar was an indication of bioconversion. Satoshi et al. [14] screened 3300 Actinomycetes and 500 fungal strains and were able to select 22 Actinomycetes (0.6% only) and 2 fungal strains (0.06% only) positive for the bioconversion.

Quantitative assay for production of acylase under submerged fermentation (SMF)

Actinomycetes isolates (53 in number) selected from the plate assay method of screening were subjected to second round of screening by submerged fermentation, for the selection of strains with higher Acylase activity. Two Streptomyces species were selected, coded as BICC 8547 and BICC 8848. Among the two, BICC 8547 showed acylase activity equivalent to that of the control micro-organism (Figure 2). However, BICC 8848 showed higher activity (25 to 30% more) of Acylase compared to the control organism and therefore selected for further studies. The cell free supernatant fed with 1 g/L Echinocandin B showed very poor conversion.