tudiea~qn,~Pto~lucin ells of P-`futant Posscssin D-GlucosQ Tso*meras e

ACtiVi ty ,'H;,

in the MRR for February, efforts .were being made to fermen t

an~finexpAnsive,medium
. .,_ .. M. .. .to,obtainArthrohactercells :wi,th high glucoseisomeras e.
activi.ty .,;Investigations by Dr . Lee showed that cells of a double-mutant
(F-mutant) :'whichhe had obtained possessed high enzymatic activity when culture d
in&'a`-'rnedi,um 'with'2X dextroseas the c3rbori source : Therefore, production of
6011Sof F-•mutant was examined .

An inaeulum of F-mutant was prepared by either of two methods . Th e

mutantwas transferred by streaking from"a~stock culture to plates of nutrient aga r
(Aifco) :, ;,,The;plates :were incubated at 30°C for 24 hours . Sterile water (10 ml)
was added,;to~-oneplate and with gentle agitation, a cell suspension as prepared .
Production .media :`were .then inoculated by'a`0 .5% transfer of the cell suspension ,
inoculum A : To'prepare inoculum B, the mutantwas cultured for 24 hours (30°C) on
nutrientagar, and the growth`(1/2 standardloop) was transferred to a medium
.(100 in1 at ;;pHk.7 .l5,before autoclyving/500-m1_1flasks) :that contained 2% dextrose ,
0 .6% O .M : HAP,'0 .1% BFY 100, 0 .6% (%'H4)2HP04', 0 .2% KH2P04 ; 0 .0125% MgSO4•71120 ,
. and_ ;distilled,water :, After24-27 hours on the shaker (30°C), produciton medi a
ti•ere"inoculated by a0 .5 - 1Z transfer from the shake .

The media (100 ml at pH 7 .15 before autoclaving 500-m1 flask) used fo r

cell'production contained 2% dextrose, 0 .6%, 0 .r1 .llAP, 0 .1 - 0 .175% BYF 100,
0 .6% (N114)2HP04, 0 .2% K112P04, 0 .01 - 0 .025% 1-1gSO4•71120, and distilled water .
The flasks were incubated at 30°C on the shaker rotating at 250 rpm with a
2-inch stroke .

Assays of the cell for glucose isomerase activity showed activities of

as much as 470 - 630 u units/ml after 64 to 112 hours of incubation (Table I) .
The yields were considered good but not as high as expected .

In co-operation with Dr . Lee, two 30-gallon fermentations (Nos . 46 and 47)

were conducted . Data on those will be reported by Dr . Lee .

One 30-gallon fermentation (No .48) with the 2% dextrose medium as used in
shakes was inoculated with 1% of B-type inoculum . The propeller was rotate d
at 300 rpm and an aeration rate of 0 .45 cu . feet/r :inute/30 gallons was e ;,p]oyed .
The results showed a naximum yield of 480 p units/ml after 64 licurs of incubation .
The yield (440) was slightly lower at 67 hours . The final p1i w : s 5 .4 -Ind on
centrifrigation, 3333 bms of cells were obtained .

Presently, methods to consistently produce cells of F-mutant with high

enzymatic activity (7001•) are being studied .
 :~~Asslbtud Dr . W . C . Squires. in''propari'ng' .thrcio, ;`repo :,ts . ' Two ; ;Bio-
syntheFis of Compound 350 and Treatnent of Air'-Conditioning Sj•stcirls,' .wo're
R~It rcports and one, Proparation of 1 .ectono of`3-Sucrinoylpyridonc-6, was
an KUM .

Pir .' :-MAtthews assisted,Tlise Long with,foo4 ;studies and antibiotic studies ;;'
ral :~tn~l tn tha F..mntnnt_ . ,.
 ~ . ~ .. . . ., . . .. .
.
~;,'~1 . .. .- . ~

f'

t . ` : A F) I .fi

..,_, . ;J;IG t'hi,},~,

. ,.:l~iT
OY'1'•`'~U1' . IACONhUi~[1r i'~1
: - ;",C551\R
• .,. . ,«f~-r~l U1.-.f15K IS0:11 RASh: At:~~1.V11'Y
. .-~.-.- .

py~y .5* y1• 'i~*~ p. }: ~

Constituents (X) In riediu m Yield (u unita/m1 )

.~,~^•~-----,-----
~ f .~.y , . . , . .

. M . ~r{ ., }3YF~ MgSO aftera .

~AextrasQ 4` Y}1AP~ 00~ NH Z}PO K}1 PO % 7H 0 64 ' 88 " 109-112 hrs t
~_>;t;,{

.R

~;..:~._~ -- . .. . .
0 .6 0 .15 : - .0 .6 0 .2 0.0125~• ~

0 .6 0.2 0.0125

0:6 0.2 0.0125 .

6 0 .2 -0 .0125 ;
.6 : . 0 .2 0.0125 : 397

: 6 = 0. 2 0 . 025 _, .. ' .

0 .02 5

0 .6 : ' 0 .125 551 626

0.6 0 .1 0 .6 0 .2 0 .0125 B 395 34 5

0 .6 0 .125 0.6 0.2 0.0125 A 421 20 4

0 .6 0 .175 0 .6 0 .2 0 .0125 A 432 25 5

0.6 0 .1 0 .6 0.2 0.01 A 418 155

0 .6 0 .1 0 .6 0 .2 0 .01 A 421 17 2

0.6 0 .1 0 .6 0 .2 0 .0125 A 399 25 3

2 0 .6 0 .1 0 .6 0 .2 0 .0125 A 412 35 3
 4 y,` alr.~ . '•'rotri,n :

c
As :`1
t,6$ :~}~`~~G+Y'/!~ : rq 1•. . a. . .

y?

*Incculum A culturod' on ;:nutrient agar plate for 24 hours at 30°C . added

10 ml ateril o.: ;distilicd`wAter'.~ ,Transferred Q,.S ml ` cell : cuspansiQnto - :
productionmedia, 100 m1/500 ml" f1ASk .f' Inc+culum`$ ; p Cu .itured' on tiutrient
agar plato ; ;for 24 hours at 30'C . .transfcrred :;growth'(1/2 stAnc~Ard loop) to
medium containing 2Xidaxtroso, 100, :0 .6x~',(NH~)21iP04,
0,2X hH2P0y'0125Y ;1~1gSO4dlstilled'h~aker .+j Incubated on'shaker
for 24 houra,-;_and then inoculated media'by 0,.5 on ;1%''transfer .
~,

**2X, dext`rose ;content~`w~as obtailled ;by adding 51 .8% dextrose syrup that w,as
~preadfom'strchbyRJposnela

Lawrence E . Hayes ~'