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PROCEDURE FOR PRODUCTION OF GLUCOSE ISOMERASE

I . Preparation of Stock Culture Plates :

A. Medium - (C-3-6)

3 gm (60% concentrate) O .M . HAP (Amber Labs)


6 gm - (NH4)zHPO4
2 gm - 1'112P04
1 liter - distilled water

Mix thoroughly and then add :

1 .5 gm - BYF-100 (Amber Labs)

Adjust pH to 7 .1 with concentrated H3P04 ;

Add 21 .25 gm Bacto agar

Sterilize by autoclaving 25 minutes at 15 psig .

Solutions of dextrose and MgS04-7H2O are prepared to add to the C-3-6


medium . Granular dextrose (100 gm) is added to 120 nil of distilled water .
The solution is gently heated and stirred until the dextrose is dissolved .
A final volume of 200 nil is obtained by adding distilled water . MgS0y .7H2O
(5 gm) is dissolved in 100 nil of distilled water . The two solutions con-
tained in 500-m1 flasks are autoclaved for 20 minutes at 15 psi .

The dextrose solution (2 ml) and the MgSO4 solution (0 .4 ml) are added
and mixed in a 500-m1 Erlenmeyer flask containing 200 ml of the C-3-6 medium .
The medium is then poured into sterile standard culture plates (15-18 ml each) .
The plates are stored at 25° C . for at least 24 hours before using .

One colony from a C-3-6 plate (age : 3-5 days) is transferred by streaking
to a fresh C-3-6 plate . All plates are incubated at 25° C . Plates are con-
tinuously observed by the use of a stereoscope to ensure that colony character-
istics remain uniform .

II . Preparation of Inoculum for 30-Gallon Fermentation

A . Preparation of Medium

O .M . HAP (6 gm of 60% concentrate) is added and mixed with one liter of


tap water . The pH of 5 .3-5 .4 is adjusted to pH 7 .0-7 .3 by addition of KOH
solution . BYF-100 (1 gm) is then added and mixed . The pH of 6 .6-6 .7 is adjusted
to pH of 7 .1 by addition of KOH solution . The 61 medium (100 ml/500-ml flask
or 200 ml/1-L flask) is autoclaved for 25 minutes at 15 psi . Just prior to
inoculation, the sterile 50% dextrose solution (1 nil) and the sterile 5% 1"IgSO,,
solution (0 .25 m1) are added to a flask containing 100 nil of the cold 61 medium .
-2-

B . Inoculation Procedure

The medium is then inoculated using one loop of growth from a 48-hour
C-3-6 plate . The shake flask is incubated at 30° C . on a reciprocal shaker
rotating at 250 rpm with a 1-inch radius .

After 24 hours on the shaker, the whole culture at a pH of 7 .6-7 .8 is S~I


transferred by 1% to fresh cold 6I medium (200 ml/1-L flask) containing 0 .5%
dextrose and 0 .0125% MgS04-7H20 . This second serial shake is incubated on
the shaker in the same manner as the first serial shake .

At the 24-hour incubation period, the whole culture at a pH of 7 .7-8 .0)


is transferred by 1% to a fermentor containing about 113 liters of cell-
production medium . Microscopic examinations of cells f rom both 24-hour
shake cultures show that they are avoids or spheres .

III . Production of Cells

A . Preparation of Medium

The basal portion of the cell production medium consists of 0 .6%


(NH4)2HP04, 0 .2% KH2P04, 0 .6% O .M . HAP, and tap water . The constituents
are added separately to the tap water in the order as given . Each is
dissolved before adding another . The 30 gallons of basal medium is
sterilized in place by autoclaving for 45 minutes at 15 psi . During
the entire period of autoclaving, a small amount of air is passed through
the medium and the propeller is rotated at 125 rpm . The medium is then
cooled to 30° C .

Constituents to add to the basal are prepared separately . Dextrose


(2280 gm) is dissolved in 3300 ml of warm distilled water . Addition of
distilled water ives a final volume of 5200 ml . Magnesium sulfate (14 .3
gm of M S04•7H20g is dissolved in 500 ml of distilled water . BYF-100
(114 gmq) is mixed with 2000 ml of tap water containing 4 gm of pellet KOH .
The pH of 6 .4 is raised to pH 6 .6 by addition of KOH . These solutions
in separate vessels are autoclaved for 30 minutes at 15 psi . After
cooling, the MgS04 is added to the dextrose solution and this solution
is added aseptically to sterile basal medium in the fermentor . The
sterile BYF-100 solution is then added to the medium in an aseptic
manner . The volume at a pH 6 .7-6 .8 is then about 113 liters .

B . Inoculation Procedure

Inoculation with the 24-hour whole-culture inoculum (1140 ml) gives


a final volume of about 114 liters (30 gallons) . The fermentor propeller
is adjusted to rotate at 300 rpm and an aeration rate of 0 .45 cubic feet/
minute/114 liters is employed . A temperature of 30° C . is maintained by
continuous circulation of water through the jacket . Foam is controlled
by the use of a sterile solution of antifoam G .E .-60 (100 ml G .E .-60 per
250 ml of distilled water) .
-3-

The pH of the fermentation broth decreases to 5 .1-5 .3 during 40-43


hours of incubation . An increase in pH to 5 .4-5 .7 usually occurs with
a continuation of incubation through 48-53 hours . Samples are taken
periodically and the cells are assayed for D-glucose isomerase activity .
Cells produced during 48-53 hours of fermentation time show about 800
uUni ts per nil .

The cells that are recovered by centrifuging at 15,000 rpm are


placed in polyethylene bags and sealed for freezing .
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