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2.2. G. oxydans
it with 50% aqueous glycerol solution (74 mL/L) in erlenmeyer flask
and stored at −80 ◦C for 1–2 days. pH of the solution before addi-
tion of glycerol should be maintained between 5 and 7 using dilute
NaOH and dilute orthophosphoric acid solution. A G. oxydans DSM
2003 seed I was first prepared by inoculating and incubating G.
oxydans DSM 2003 glycerol stock in sterilized media containing
100 mL/L of sorbitol solution (70%), 10 g/L of peptone (bacterio-
logical grade), 7.5 g/L of yeast extract and 1.5 g/L of potassium
dihydrogen phosphate in purified water (900 mL) in an Erlenmeyer
flask at 25–30 ◦C for 1 day at 150–200 rpm. pH of the solution
before addition of glycerol should be maintained between 5 and
7. A G. oxydans DSM 2003 seed II was prepared in an Erlenmeyer
flask by inoculating and incubating G. oxydans DSM 2003 seed I
in sterilized media containing 100 mL/L of sorbitol solution (70%),
10 g/L of peptone (bacteriological grade), 7.5 g/L of yeast extract
and 1.5 g/L of potassium dihydrogen phosphate in purified water
(900 mL) 25–30 ◦C for 1 day while maintaining pH between 5 and 7
at 150–200 rpm. A G. oxydans DSM 2003 biomass was prepared in a
fermentor by inoculating and incubating G. oxydans DSM 2003 seed
II in media containing 100 mL/L of sorbitol solution (70%), 10 g/L
of peptone (bacteriological grade), 7.5 g/L of yeast extract, 1.5 g/L
potassium dihydrogen phosphate, 10 g/L of dipotassium hydrogen
phosphate, 3.75 mL of UCON(TM) LB 625 lubricant in purified water
(13.5 L) and maintaining at 25–30 ◦C and pH between 5 and 7 for 1
day at 500 rpm and finally separating G. oxydans DSM 2003 whole
cell biomass by centrifugation. 50–100 g of G. oxydans DSM 2003
biomass was isolated.
G. oxydans DSM 2003 was purchased from Leibniz-Institut
DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul-
turen, GmbH, Germany.
2.3. Chemicals
All the chemicals used were of commercial grade.
2.4. Preparation of 1-amino-1-deoxy-d-galactitol (2)
Anhydrous d-galactose (100 g) and benzylamine (73 mL) were
added to a pressure reactor containing methanol (2000 mL) under
nitrogen atmosphere. To this, 15 g of activated Raney-Nickel
was added with moisture content less than 1% under nitro-
gen atmosphere and the reaction mixture was hydrogenated at
10–12 kg/cm2 for 65–70 h at 50 ◦C under stirring. After completion
of reaction with the formation of N-benzyl-1-amino-1-deoxy-d-
galactitol (1), the hydrogen pressure was released under nitrogen
atmosphere. The reaction mixture was cooled to 45 ◦C and dem-
ineralized water (2000 mL) was added. The reaction mixture was
again cooled to 35 ◦C and filtered through hiflow and 5 m filter
under nitrogen atmosphere. The filtrate was collected and added
once again to a pressure reactor. To this 15 g of 10% palladium on
carbon (50% wet) was added and hydrogenated at 10–12 kg/cm2 for
24–30 h at 50 ◦C under stirring. After the completion of reaction, the
hydrogen pressure was released under nitrogen atmosphere. The
reaction mixture was cooled to ambient temperature and filtered
through hiflow and 5 m filter under nitrogen atmosphere. The
filtrate was collected and the reaction mixture was concentrated
under high vacuum at temperature below 40 ◦C. To the concen-
trated oily reaction mass, 600 mL of isopropyl alcohol was added
and stirred at 50 ◦C for 30 min. The reaction was concentrated under
reduced vacuum at temperature below 40 ◦C till moisture content
reaches below 5%. To the concentrated reaction mass, 750 mL of
isopropyl alcohol was added, stirred for 30–40 min. The reaction
mixture was again stirred at 3 ◦C for 60–90 min and filtered under
nitrogen atmosphere. The solid obtained was dried under vacuum
till moisture content reaches below 3%. Yield: 80–95%; purity >95%
by HPLC. MASS m/z = 182 (M+H), 1H NMR (D2O, 300 MHz) 2.77–2.91
(2H, m), 3.61–3.70 (4H, m), 3.89–3.99 (2H, m), 13C NMR (D2O,
300 MHz) 46.35, 66.09, 72.32, 73.04, 73.16, 73.03, specific optical
rotation [˛]20 −2.232 (c = 1, H2O).
2.7. Preparation of 1-deoxygalactonojirmycin hydrochloride
using G. oxydans DSM 2003 whole cells
100 g of N-formyl-1-amino-1-deoxy-d-galactitol (3) solution
was dissolved in 1000 mL of DM water and pH was adjusted to
4.5 using dil. phosphoric acid. The reaction mixture was filtered
through 0.22 micron filter to remove undissolved particles and
microbial contaminants. To this 20% of G. oxydans DSM 2003 cell
paste was added and stirred at 200 rpm for 20 days at 25–30 ◦C
along with oxygen purging. The progress of reaction was moni-
tored by TLC and HPLC analysis. After 20 days, a 50% conversion was
observed by HPLC. The reaction mixture was first filtered through
hiflow followed by 0.22 micron filter. To filtrate, was slowly added,
210 mL of 10% sodium hydroxide solution at 5 ◦C and stirred at
20 ◦C for 2–5 h. The reaction mixture was again cooled to 5 ◦C and
a 120 mL of sodium borohydride solution (12.5 g sodium borohy-
dride in 10% NaOH) was added slowly at 5 ◦C. The temperature of
reaction mixture was raised to 20 ◦C and stirred at 20 ◦C for 2–5 h.
The reaction mixture was again cooled and adjusted pH to 7.0
using dilute hydrochloric acid. The cooled neutral reaction mixture
was loaded into a column containing pre-activated Indion 225 H+
resin. The column was washed with water till NaCl was removed
completely (The complete removal of NaCl was checked by silver
nitrate test). The desired product was eluted with 6% chilled ammo-
nia solution. The eluant containing desired product was collected,
degasified under nitrogen and concentrated under high reduced
vacuum at 40 ◦C till ∼5 volumes with respect to batch size. The
reaction was then cooled to 5 ◦C and pH was adjusted to 2 with 1:1
HCl solution. To the reaction mixture was added Ceca C and stirred
for 30 min. The reaction mixture was stirred at 5 ◦C for 30 min and
filtered through hiflow bed followed by 0.2 micron filter at ambi-
ent temperature. The filtrate mLs was concentrated under reduced
vacuum at 40 ◦C till moisture content below 1%. The reaction was
then stirred in ethanol for 20–30 min at 40 ◦C and concentrated
under reduced vacuum at 40 ◦C. A thick residue is obtained. To the
thick residue, was added, 70 mL of water and 700 mL of ethanol
at 40 ◦C and stirred for 3 h. The reaction mixture was filtered.
The residue obtained was washed with 200 mL of ethanol and
2.5. Preparation of N-formyl-1-amino-1-deoxy-d-galactitol (3)
100 g of Amino-1-deoxy-d-galactitol (2) was charged into a
reactor containing (64 mL) methyl formate and (700 mL) methanol.
The reaction mixture was refluxed for 6 h at 60 ◦C. After the com-
pletion of reaction, the reaction mixture was cooled to ambient
temperature and filtered off. The solid obtained was dried under
vacuum at temperature below 35 ◦C to give N-formyl-1-amino-1-
deoxy-d-galactitol (3). Yield: 90–95%; purity >95% by HPLC. MASS
m/z = 232.0 (M+Na), 1H NMR (D2O, 300 MHz) 3.36–3.51 (2H, m),
3.64–3.70 (4H, m), 3.98–4.03 (2H, m), 8.013–8.16 (1H, d), 13C NMR
(D2O, 300 MHz) 43.98, 66.09, 70.95, 72.22, 72.73, 73.01, 167.52,
specific optical rotation [˛]20 −20.094 (c = 1, H2O).
2.6. Preparation of G. oxydans DSM 2003 whole cells
A Glycerol stock of G. oxydans DSM 2003 was prepared by inoc-
ulating and incubating G. oxydans DSM 2003 in sterilized media
containing 70% sorbitol solution (11.1 mL/L), 11.1 g/L peptone
(bacteriological grade), yeast extract (8.3 g/L) and potassium dihy-
drogen phosphate (1.66 g/L) in purified water (1000 mL) and mixing