BIG-GRANT

(BIRAC/KIIT0660/BIG-14/19)

  OBJECTIVES

  1. Scale-up of 24-desmethylrifamycin to increase the yield from genetically modified strain DCO34/36 of Amycolatopsis mediterranei S699: We could achieve an increase of concentration from 20 mg/L to 700mg/L of the desired analog.
  2. Upscaled purification of 24-desmethylrifamycin B from the fermentation broth: By using a special nylon mesh cloth we have developed a strategy to avoid centrifugation and purification has also been improved by using a special resin (amberlite).
  3. Conversion of 24-desmethyl rifamycin B to 24-desmethylrifamycin S and synthesis of 24-desmethyl rifampicin: Semisynthetic derivatives of 24-desmethyl rifamycin B were prepared and the activity of the derivatives was confirmed.

We standardized all the fermentation parameters in 5L fermenter and the results are briefly described below (Table 1 and Table 2):

S. No

Fermentation Media

1 litre (g)

1

D-Glucose

50

2

Soyabean Meal

50

3

Propylene Glycol

10

4

Ammonium sulphate

5

5

Potassium dihydrogen phosphate

1

6

Calcium carbonate

5

7

Magnesium sulphate

1

8

Ferrous sulphate

0.01

9

Copper Sulphate

0.0033

10

Zinc Sulphate

0.05

11

Mangenese sulphate

0.004

12

Cobalt Chroride

0.002

13

Ammonium molybdate

0.001

14

Silicon anti-foam agent (ml)

4

Table 1: Fermentation media standardized for the production of 24 desmethylrifamycin B. We specifically used Soyabean meal as a nitrogen source, which made the production economical and helped increase the production of 24 desmethylrifamycin B compared with Yeast and Malt extract, usually used in shake flask media. The media was also supplemented with vital micronutrients that increased the biomass and provided proper growth conditions in large-volume fermentation.

Table 2: Optimum conditions for enhanced production of 24 desmethylrifamycin B. Dissolved oxygen was always kept above 30% by modulating the agitation and air-flow.

S. No

Parameter

Set

1

Temperature

28⁰C

2

pH

6.8-7.2

3

Agitation

200-300 rpm

4

Air Flow

1.5 L/min to 10 L/ min

5

Dissolved Oxygen

100%*

 

Figure 1 (A): Table top fermenters used for carrying out 5L fermentation. The fermenters had clear vessel that helped in monitoring the production of yellow-colored rifamycin analogs produced after 72 h of incubation (B) Production of rifamycin analogue as observed by the change in yellow coloration. Production starts after 72 h of incubation and maxima was reached around 144 h (C) Improved extraction and purification using amberlite resin. The broth containing rifamycin was incubated with white amberlite beads for 24 h. Amberlite adsorbed the rifamycin that can be separated using 250-micron mesh size strainers.

By using the modified fermentation protocols as proposed in the previous BIG grant we produced the analogue around 700mg/L (from 20 mg /L).