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)zHPO42 gm - 1'112P041 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-3-6medium . 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 addedand 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 streakingto 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 oftap water . The pH of 5 .3-5 .4 is adjusted to pH 7 .0-7 .3 by addition of KOHsolution . BYF-100 (1 gm) is then added and mixed . The pH of 6 .6-6 .7 is adjustedto pH of 7 .1 by addition of KOH solution . The 61 medium (100 ml/500-ml flaskor 200 ml/1-L flask) is autoclaved for 25 minutes at 15 psi . Just prior toinoculation, 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 .

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B . Inoculation Procedure

The medium is then inoculated using one loop of growth from a 48-hourC-3-6 plate . The shake flask is incubated at 30° C . on a reciprocal shakerrotating 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~Itransferred 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 onthe 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-hourshake 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 constituentsare added separately to the tap water in the order as given . Each isdissolved before adding another . The 30 gallons of basal medium issterilized in place by autoclaving for 45 minutes at 15 psi . Duringthe entire period of autoclaving, a small amount of air is passed throughthe medium and the propeller is rotated at 125 rpm . The medium is thencooled 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 ofdistilled water ives a final volume of 5200 ml . Magnesium sulfate (14 .3gm 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 solutionsin separate vessels are autoclaved for 30 minutes at 15 psi . Aftercooling, the MgS04 is added to the dextrose solution and this solutionis added aseptically to sterile basal medium in the fermentor . Thesterile BYF-100 solution is then added to the medium in an asepticmanner . 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) givesa final volume of about 114 liters (30 gallons) . The fermentor propelleris 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 bycontinuous circulation of water through the jacket . Foam is controlledby the use of a sterile solution of antifoam G .E .-60 (100 ml G .E .-60 per250 ml of distilled water) .

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The pH of the fermentation broth decreases to 5 .1-5 .3 during 40-43hours of incubation . An increase in pH to 5 .4-5 .7 usually occurs witha continuation of incubation through 48-53 hours . Samples are takenperiodically and the cells are assayed for D-glucose isomerase activity .Cells produced during 48-53 hours of fermentation time show about 800uUni ts per nil .

The cells that are recovered by centrifuging at 15,000 rpm areplaced in polyethylene bags and sealed for freezing .

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