A study of the amylase of Aspergillus Oryzae
by Victor C Bruski
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Chemistry
Montana State University
© Copyright by Victor C Bruski (1951)
Abstract:
A study has been made of the amylase obtained from a submerged culture of Aspergillus oryzae. grown
on a synthetic media of starch and inorganic salts. It was discovered that the amylase produced can be
absorbed and eluted from the mold mycelium by pH adjustments. An initial concentration of 20-40 fold
of the amylase from the filtrate is readily obtainable. The advantages claimed are: elimination of salts,
carbohydrates, proteins, and other extraneous materials; also, the method is simple, rapid, and
inexpensive. Elutes from the mycelium absorptions were subjected to repeated ammonium sulfate
fractionations followed by a bentonite treatment. The preparations thus obtained gave exceedingly high
amylase activities with very little evidence of maltase, limit dex-trinase, and proteolytic activity. A STUDY OF THE AMYLASE
OF
&
ASPERGILLUS ORYZAE
Dy
VICTOR C. BRUSKI
A THESIS
S u b m itte d t o th e G ra d u ate F a c u lty
in
p a r t i a l f u l f i l l m e n t o f th e re q u ire m e n ts
f o r th e d e g re e o f
M a s te r o f S c ie n c e i n C h em istry
at
M ontana S ta te C o lle g e
A pproved:
H ead, M ajo r D epartm ent
C hairm an, S sa m in in g Committee
D ean, G rad u ate D iv is io n
Bozeman, M ontana
J u l y , 1951
IV 3 7 S
-
2
-
TABLE OF CONTENTS
S e c tio n
I.
Page No
A b s tr a c t . . . . . . . . . . . . .
. .
3
II.
I n t r o d u c t i o n ..............................................
. .
4
III.
S ta te m e n t o f P roblem ............................
. .
7
L i t e r a t u r e S u rv ey ..............................
. .
8
IV .
.
V.
A n a ly tic a l P r o c e d u re s . . . . . . .
. .
12
V I.
M ethods and R e s u l t s . . . . . . . .
. .
14
T ab le of R e s u lts . . . . . . . . .
. . 2 3 -2 6
D is c u s s io n . . . . . . . . . . . .
. .
27
S u m m ary .........................................................
. .
31
IX .
L i t e r a t u r e C ite d . . . . . . . . .
. .
32
X.
A cknow ledgem ents . . . . . . . . .
, .
34
VTI.
V III.
137465
- 3 I.
ABSTRACT
A s tu d y h a s "been made o f th e am ylase o b ta in e d from a subm erged c u l­
t u r e o f A s p e r g i llu s o r y z a e , grown on a s y n t h e t i c m edia o f s t a r c h and
i n o r g a n ic s a l t s . I t was d is c o v e r e d t h a t th e am ylase p ro d u c e d can be
a b so rb e d and e l u t e d from th e mold mycelium by pH a d ju s tm e n ts . An i n i t i a l
c o n c e n tr a tio n o f 20-40 f o l d o f th e am ylase from th e f i l t r a t e i s r e a d i l y
o b t a i n a b l e . The a d v a n ta g e s c la im e d a r e : e l i m i n a t i o n o f s a l t s , c arb o ­
h y d r a t e s , p r o t e i n s , and o th e r e x tr a n e o u s m a t e r i a l s ; a l s o , th e method i s
s im p le , r a p i d , and in e x p e n s iv e . E lu te s from th e m ycelium a b s o r p tio n s
w ere s u b je c te d t o r e p e a te d ammonium s u l f a t e f r a c t i o n a t i o n s fo llo w e d by a
b e n to n ite t r e a t m e n t . The p r e p a r a t io n s th u s o b ta in e d gave e x c e e d in g ly
h ig h am ylase a c t i v i t i e s w ith v e ry l i t t l e e v id e n c e o f m a l t a s e , l i m i t d e x t r i n a s e , and p r o t e o l y t i c a c t i v i t y .
- 4 —
II.
INTRODUCTION
B cause o f th e demand f o r a m y lo ly tio enzymes in th e b rew in g , d is ­
t i l l i n g , m i l l i n g , b ak in g, t e x t i l e , pap er, and o th e r i n d u s t r i e s , the
am ylases are one o f th e most im portant c l a s s e s o f enzymes u sed on a
com m ercial b a s i s .
There are two c la s s e s o f am ylases:
alph a-am ylase (th e li q u i f y i n g
enzym e), and b eta-am ylase (th e s a c c h a r ify in g enzym e).
b oth o a se s i s s t a r c h .
The su b s tr a te in
Amylases ( alpha or b e ta ) a ct v ery s lo w ly on raw
s ta r c h (2 0 ) but cooked s t a r c h e s , howev r , are v ery r e a d ily h y d ro ly zed .
S tarch i s b e lie v e d t o c o n s is t o f two fr a c tio n s known as amylose and
am ylopec t i n .
C ereal s ta r c h e s are m ix tu res o f 75 to 80 per c en t amylo-
p e o tin and 20 t o 25 per cen t am ylose.
The g e n e r a l o p in io n i s th a t amy­
lo s e i s th e s t r a ig h t - c h a in component o f s t a r c h , c o n ta in in g g lu c o se u n it s
bound by a lp h a -1 , 4 - g lu e o s id io lin k a g e s .
100 t o 700 g lu c o s e r e s id u e s ( 9 ) ( lO ) .
The s t r a ig h t ch a in s co n ta in
A ccording to Meyer (9 ) (lO ),
amylopec t in i s a la r g e b ranched-ch ain m o le c u le , resem b lin g a t r e e , con­
t a in in g 15 to 18 g lu c o se m o le c u le s betw een b ran ch es.
Most o f th ese g lu ­
co se u n it s are lin k e d by a lp h a -1 , 4 - g lu c o s id i o bonds h a v in g branches
formed by a lp h a -1 , 6 - g lu o o s id ic lin k a g e s .
Am ylopeotin c o n s is t s o f 500
t o 2 ,0 0 0 or more g lu c o s e u n i t s .
The f i n a l p rod u cts o f h y d r o ly s is o f sta r c h by a lp h a -a m y la se , g iv e
no c o lo ^ v ith io d in e -p o ta s s iu m io d id e s o l u t i o n .
T h is i s due to th e ra p id
rupture o f s ta r c h ch a in s in t o low m o lecu la r w eight d e x tr in e ( 2 0 ) .
am ylase s p l i t s th e am ylopeotin o f s ta r c h e s in t o d e x tr in e o f 6 or 7
g lu c o s e u n it s (2 0 ) , w h ereas, the r e s u lt o f b eta —am ylase a c tio n on
Alpha-
- 5 amylopeo t in i s th e p ro d u ctio n o f h ig h -m o le o u la r g lu o o ea n s in which alph a—I ,
6 - g lu c o e id io bonds accu m ulate.
The f i n a l h y d r o ly s is p rod u cts o f b eta -a m y la se on s ta r o h , g iv e a b lu e
c o lo r w it !^ iod in e-p otassiu m io d id e s o l u t i o n .
B eta-am ylase r a p id ly con­
v e r t s over 99 p er cen t o f the amylose f r a c t io n o f s ta r c h t o m altose ( 1 0 ) .
Amylopec t i n , how ever, i s o n ly p a r t i a l l y h yd rolyzed (5 4 per c en t) by b e t a am ylase ( 2 0 ) .
B eta-am ylase appears t o s t a r t at th e n on -red u cin g end o f
th e c h a in , b reak in g o f f m altose u n it s a s i t works toward th e c e n te r ;
how ever, as soon as i t r ea ch es a branch, i t s a c tio n c e a s e s .
T his r e s u l t s
in o b ta in in g a r e l a t i v e l y la r g e n u c le u s o f branched s tr u c tu r e from am ylop e o t in .
S in ce t h i s n u c le u s has more th an th e minimum number o f g lu c o se
u n it s r eq u ir ed t o g iv e the io d in e t e s t , b eta -a m y la se w i l l always g iv e
a purple c o lo r e d end product w ith io d i n e .
Amylases are found in s a l i v a , p a n c r e a tic j u i c e , l i v e r , o th er anim al
o rg a n s, se e d s o f c e r e a ls , m old s, and b a c t e r ia .
The im p ortant commercial
so u r c e s are m old s, m alted c e r e a ls , and b e e f and hog p a n cr e a s.
The am ylase which i s th e s u b je c t o f t h i s stu d y i s produced by th e
mold A s p e r g illu s o r y z a e , and i s c f th e alp h a ty p e .
A s p e r g illu s oryzae
has been c u lt iv a t e d in th e O rient f o r c e n tu r ie s f o r the p rod u ction o f
enzym es, p a r t ic u la r ly sta r o h h y d r o ly z in g enzym es.
The amylase produced
h as been u sed as a p a r t i a l l y p u r if ie d product in t h i s cou n try s in c e 1898
(1 9 ).
However, s u r p r is in g ly l i t t l e
i s known o f i t s a c tio n and p r o p e r t ie s .
T h is la c k o f q u a n tit a tiv e in fo rm a tio n i s probably due t o th e f a c t th a t
many in v e s t ig a t i o n s have d e a lt
w ith r a th e r crude a lc o h o l p r e c ip it a t e s
o f th e enzyme e x t r a c t s from th e m old.
These co n ta in ed h ig h c o n c e n tr a tio n s
—6 —
o f o th e r enzym es, and s im ila r p r o te in m a t e r ia ls .
Tauher (20) l i s t e d 23
d i f f e r e n t enzymes which have been r e p o r te d fo r t h i s organism .
-
III.
I -
STATEMMT OF PROBLEM
P r e lim in a ry i n v e s t ig a t i o n had in d ic a te d th a t th e am ylase produced
hy A s p e r g illu s oryzae on a media made up o f in o r g a n ic s a l t s and s ta r c h
had somewhat d if f e r e n t p r o p e r tie s than commercial p r e p a r a tio n s o f t h i s
enzyme.
There were even in d ic a t io n s th a t i t might n o t he p r o te in in
nature due t o th e f a c t th a t a n e g a tiv e b iu r e t t e s t was ob ta in ed on some
sam ples ( 8 ) .
Furtherm ore, ty r o s in e appeared to be ab sen t w h ile su r fa c e
c u ltu r e s o f th e same organism grown on o rg a n ic media alw ays gave a pos­
i t i v e ty r o s in e t e s t .
In order t o stu d y t h i s problem and determ ine i f a
more h ig h ly p u r if ie d product would show th e same p r o p e r tie s the fo llo w ­
in g problem s were t o be in v e s t ig a t e d :
1.
E x tr a c tio n and p u r if ic a t io n o f amylase from A s p e r g illu s oryzae
grown on s y n t h e t ic media o f s ta r c h and in o r g a n ic s a l t s .
2.
Work out a fe a s a h le scheme f o r th e p u r if ic a t io n o f the am ylase.
3.
O btain h ig h e s t p o s s ib le a c t i v i t y hy some typ e o f fra ctio n a tio n
from o th e r p r o te in s and carb ohydrates and, i f p o s s i b l e , o b ta in
th e enzyme in c r y s t a l l i n e form.
4.
Determ ine p h y s ic a l and chem ical p r o p e r tie s o f h ig h ly p u r if ie d
enzyme.
T h is in c lu d e s a n a ly s is fo r n itr o g e n and determ in in g i t s
a c tiv ity .
5.
Compare w ith p r o p e r tie s o f am ylases from o th e r s o u r c e s .
—8 —
IV.
LITERATURE SURVEY
In I 8 9 8 , TaJcamine ( 1 9 ) in tro d u ced in t o t h i s cou n try under th e name
T a k a -d ia sta se a p a r t i a l l y p u r if ie d powder prepared by a lc o h o l p r e c ip it a ­
t io n o f th e aqueous e x t r a c t o f "K ojitl f which i s made by c u lt i v a t in g th e
mold A s p e r g illu s oryzae on steam ed wheat bran.
S in ce th e n , the commer­
c i a l p u r if ic a t io n has been im proved, and th e amylase has found many
a p p lic a t io n s in in d u s tr y .
Sherman and Tanberg ( l 8 ) o b ta in ed more co n cen tra ted p r e p a r a tio n s o f
t h i s am ylase in 1916.
They p r e c ip it a t e d th e enzyme from aqueous s o lu t io n s
o f th e com m ercial product ( T a k a -d ia s ta s e ) by use o f ammonium s u l f a t e ,
d is s o lv e d in p r e c ip it a t e in w a ter, d ia ly z e d the s o lu t io n fr e e from s u l­
f a t e , and th en f r a c t i o n a l l y p r e c ip it a t e d i t w ith a lc o h o l in the p resen ce
o f added sodium c h lo r id e .
The most a c t iv e products o b ta in e d in t h i s
manner had 30 tim es th e a c t i v i t y o f th e s t a r t in g m a t e r ia l.
In 1 926, N ishim ura (1 4 ) r ep o rted s t i l l g r e a te r p u r if ic a t io n o f th e
am ylase by a d so r p tio n on alum ina g e l , and e lu t io n w ith phosphate s o l u t i o n s .
The a d so r p tio n was c a r r ie d out in a weak a cid s o lu t io n , and the adsorbed
m a te r ia l e lu t e d w ith a weak a cid -p h o sp h a te s o lu tio n a t a pH o f 8 . 0 .
By
r e p e a tin g t h i s p r o c ess s e v e r a l tim e s , he ob tain ed a product w ith approx­
im a te ly 45 tim e s g r e a te r a c t i v i t y than th e s t a r t in g m a t e r ia l.
C hester o b ta in ed more co n cen tra ted p r e p a r a tio n s o f t h i s amylase in
1933 ( 4 ) .
Her work c o n s is t e d o f f o llo w in g the a d s o r p tio n procedure o f
N ishim ura (1 4 ) alo n g w ith f r a c t io n a t io n w ith ammonium s u l f a t e and a lc o h o l.
Her b e s t p r e p a r a tio n had an a c t i v i t y o f app roxim ately 53 tim es th a t o f
th e o r ig in a l m a t e r ia l.
- 9 B ecause o f th e d if f e r e n t c o n d itio n s used in m easuring the enzyme
a c t i v i t i e s o f th e p u r if ie d p rod u cts in th e r e fe r e n c e s c i t e d above, i t
i s d i f f i c u l t t o make an a ccu ra te com parison o f th e p u r if ie d products
w ith t h i s work.
In th e l i g h t o f th e s t u d ie s th a t have been made, th e
b e s t one can do i s make an approxim ation o f the com parative a c t i v i t i e s .
C a ld w e ll, C h e ster , D oebbeling and V o lz , in 1945 (3 ) prepared an
e x c e e d in g ly p o ten t a m y lo ly tic p r e c ip it a t e which gave no ev id en ce o f
m a lta se a c t i v i t y .
T h eir method o f p u r if ic a t io n c o n s is t e d o f f r a c t io n a l
p r e c ip it a t i o n o f th e aqueous e x t r a c t o f a commercial T a k a -d ia sta se con­
c e n tr a te w ith amcionimum s u l f a t e , su sp e n sio n o f th e p r e c ip it a t e in a
minimum o f d i s t i l l e d w a te r, d i a l y s i s to remove s u l f a t e , and c o n c e n tr a tio n
o f the d ia ly z e d s o lu t io n s .
T his p r o c e ss o f f r a c t io n a t io n and d i a l y s i s
was r ep ea ted on th e most a c tiv e s o lu t io n s u n t i l no fu r th e r in c r e a s e s
occu rred in a c t i v i t i e s e x p r essed on th e b a s is o f t o t a l s o l i d s in th e
d ia ly s e d s o l u t i o n s .
to f iv e .
u n it s
The number o f r e f r a c t io n a t io n s n e c e s s a r y was th r e e
T h e ir most a c t iv e p r ep a ra tio n had an a c t i v i t y o f 16 ,0 0 0 alph a
e r gram.
The am ylase a c t i v i t y i s d e fin e d on page 1 2 .
C r y s ta llin e alp h a-am ylases have been ob tained
s a l i v a , and b a c t e r ia .
from m a lt, p a n c r e a s,
S in ce t h e i r a c tio n and many o f t h e i r p r o p e r tie s
are s im ila r , a sh o r t rev iew o f th e methods used f o r o b ta in in g th e c r y s t ­
a l l i n e m a te r ia l seems a p p rop ria te a t t h i s tim e .
M alt alp h a-am ylase was c r y s t a l l i z e d by Sigmund Sohwimmer and A. K.
B a lls (1 7 ) •
The procedure c o n s is t s e s s e n t i a l l y o f h e a tin g co n cen tra ted
m alt e x t r a c t t o d e s tr o y th e b eta -a m y la se a c t i v i t y , p r e c ip it a t in g th e
rem aining p r o te in w ith ammonium s u l f a t e , ad sorb in g th e enzyme from an
- 10 -
a lo o h o lio s o l u t i o n on w heat s t a r c h g r a n u l e s , and c r y s t a l l i z i n g th e e l u t e d
enzyme w ith ammonium s u l f a t e .
The c o n d itio n s f o r th e a d s o r p tio n were
a d ju s tm e n t o f pH t o 5 ,9 - 6 .0 w ith 0 .1 H ammonium h y d ro x id e i n th e p re s e n c e
o f c a lc iu m io n s a t a te m p e ra tu re o f 300C.
P a n c r e a t i c a lp h a -a m y la s e was c r y s t a l l i z e d by M eyer, F i s h e r , and B e m fe ld ( l l )
(1 2 ).
T h e ir m ethod c o n s is te d o f th e d i a l y s i s o f e x t r a c t s o f
hog p a n c r e a s , f r a c t i o n a l p r e c i p i t a t i o n w ith ammonium s u l f a t e (pH 6 . 9 ) ,
rem oval o f d e n a tu r e d p r o t e i n by s h a k in g w ith c h lo ro fo rm i n th e p re s e n c e
o f ammonium h y d r o x id e , and th e n c r y s t a l l i z e d from w a te r a t 3°C.
S a liv a r y a lp h a -a m y la s e was c r y s t a l l i z e d by M eyer, F i s h e r , B e m f e l d ,
and S ta u b ( 1 3 ) .
c ard ed .
Human s a l i v a was c e n t r i f u g e d , and th e p r e c i p i t a t e d i s ­
Then two f r a c t i o n a l p r e c i p i t a t i o n s w ith a c e to n e , and two f r a c t i o n a l
p r e c i p i t a t i o n s w ith ammonium s u l f a t e were c a r r i e d o u t .
I n th e n e x t s t e p ,
th e s u l f a t e io n s w ere r e p la c e d by a c e t a t e by u s in g a b a s i c io n exchange
re s in .
An a d d i t i o n a l p r e c i p i t a t i o n was made w ith a c e to n e .
i t a t e was ta k e n up i n w a te r and c o o le d .
The p r e c ip ­
C r y s ta l s form ed i n 48 h o u rs a t
a pH o f 7 . 0 .
When t h i s p r o j e c t was s t a r t e d , no r e p o r t had been made o f c r y s t a l l i ­
z a t i o n o f fu n g a l a lp h a - a m y la s e .
H ow ever, i n F e b ru a ry o f t h i s y e a r ,
U n d e rk o fle r and Eoy a t Iow a S ta te C o lle g e , p u b lis h e d an o u tlin e d p ro c e d u re
f o r o b ta in in g c r y s t a l s o f th e a lp h a -a m y la s e from subm erged c u l t u r e s o f
A s p e r g i llu s o ry z a e ( 2 l ) .
I n b r i e f , th e method c o n s i s t s o f s i x ammonium
s u l f a t e p r e c i p i t a t i o n s , w ith th r e e b e n t o n i t e a d s o r p tio n s t o remove l i m i t
d e x trin a s e .
C r y s t a l l i z a t i o n was co m p le ted in o n e - h a lf s a t u r a t e d ammonium
s u l f a t e a t a r e f r i g e r a t e d te m p e r a tu r e .
11
-
The au th ors o f th e above procedure o b tain ed o n ly 10 mg o f c r y s t a l s .
T h is amount was i n s u f f i c i e n t t o a llo w p h y s ic a l p r o p e r tie s to be d eterm in ed .
As a r e s u lt o n ly a p ic tu r e was made o f th e c r y s t a ls and th e a m y lo ly tio
a c t i v i t y d eterm in ed .
I t was le a rn ed through correspondence th a t th e a u th o rs
o f t h i s procedure had g r e a t d i f f i c u l t y in r e p e a tin g th e above p r o c e s s .
SI/
12 V.
AUALTTIOAL PROCEDURES
T hroughout th e c o u rse o f t h i s i n v e s t i g a t i o n th e fo llo w in g a n a l y t i c a l
m ethods w ere u s e d .
A lp h a-am y lase was d e te rm in e d "by a m o d ifie d m ethod o f S a n d s te d t, K een,
and B lis h ( l 6 ) , th e a c t i v i t y b e in g e x p re s s e d a s " a lp h a u n i t s . "
An " a lp h a
u n i t " r e p r e s e n t s th e number o f gram s o f L in tn e r s s o lu b le s t a r c h d e x t r i n iz e d i n one h o u r , a t 30°C , by one m i l l i l i t e r o f enzyme s o l u t i o n , u n l e s s
o th e rw is e i n d i c a t e d .
The m o d if ic a tio n i s th e e l i m i n a t i o n o f th e p r e lim in a r y
tr e a tm e n t o f th e s t a r c h w ith b e ta - a m y la s e s in c e b e ta -a m y la s e i s n o t p ro ­
duced by t h i s p a r t i c u l a r m old.
One a lp h a u n i t , a s d e s c r ib e d h e r e ,
r e p r e s e n t s 8 .8 S a n d s te d t, KjS e n , and B lis h " a lp h a u n i t s . "
L im it d e x tr in a s e was d e te rm in e d by th e m ethod o f B ack, S t a r k , and
S c a lf ( 2 ) ,
The u n i t o f m easurem ent i s e x p re s s e d a s p e r c en t h y d r o l y s i s
o f l i m i t d e x t r i n t o fe rm e n ta b le s u g a r i n one h o u r , a t 30°C, by one m i l l i ­
l i t e r enzyme s o l u t i o n u n le s s i n d i c a t e d o th e r w is e .
M a lta s e a c t i v i t y was d e te rm in e d by th e m ethod o f Gorman and L an g ly k k e
(5 ).
The a c t i v i t y i s e x p re s s e d a s p e r c e n t h y d r o ly s is o f m a lto se t o g l u ­
cose i n two h o u rs a t 30°C.
P r o t e o l y t i c a c t i v i t y was ru n by th e method o f N o rth ro p ( 1 5 ) .
A
change o f one p e r c e n t p e r m inu te i n s p e c i f i c v i s c o s i t y o f a s ta n d a r d
g e l a t i n s o l u t i o n i s d e f in e d a s one u n i t o f p r o t e o l y t i c a c t i v i t y .
The K je ld a h l n itr o g e n d e te r m in a tio n u sed i s an a d a p ta tio n o f th e
o f f i c i a l m ethod o f th e A. 0 . A. 0 . ( l ) , u se d by th e C h em istry R e se a rc h
D epartm ent o f th e A g r i c u l t u r a l E x p erim en t S ta tio n a t M ontana S ta te
C o lle g e .
The c a t a l y t i c m ix tu re c o n ta in s 1 ,0 0 0 gram s o f p o ta ssiu m
-1 3 s u l f a t e , and $0 grams o f m ercu ric o x id e .
A mixed in d ic a t o r (2 2 ) was u sed
in th e t i t r a t i o n .
A ll pH d e te r m in a tio n s were made w ith a Beckman pH m e te r, which em ploys
th e g la s s e le c t r o d e .
— 14 —
V I.
METHODS MD RESULTS
I n d e te r m in in g th e ty p e o f f e r m e n ta tio n m edia to be u s e d , a s e r i e s
o f s y n t h e t i c p r e p a r a t io n s made u p o f in o r g a n ic s a l t s and c o r n s ta r c h were
trie d .
T h is was done by v a ry in g th e s a l t c o n c e n tr a tio n i n th e m e d ia , b u t
k e e p in g th e b a s i c c o n s t i t u e n t s th e sam e.
As a r e s u l t o f t h i s p r e lim in a r y
e x p e r im e n t, i t was c o n clu d e d t h a t th e medium p r e p a r a t io n t h a t w ould s e rv e
th e n e e d s o f t h i s i n v e s t i g a t i o n m ost f u l l y was o f th e f o llo w in g n a t u r e i
In g re d ie n ts
$ by W eight
C o rn s ta rc h
1 .5
KNO3
0 .4
CaCC3
0 .1
KEgPO^
0 .0 5
MgSO4
0 .0 2 5
KCl
0.0 2 3 5
Ife S04 »THgO
5 .0 p . t . m.
ZnSO4 .TH2O
5 .0 P .P .M .
CuSO4 e SH2O
5 .0 P .P .M .
1 s t e r i l i z e d i n 10 l i t e r q u a n t i t i e s
L isp e rs in g can v as b a g s .
The medium
on s u c c e s s iv e days t o in s u r e h ig h e s t p o s s ib le s t e r i l i t y .
Each b o t t l e was
in o c u la te d w ith 600 ml o f 48 h o u r p u re c u ltu r e o f A s p e r g i llu s o r y z a e .
T h is
in o cu lu m was c u l t i v a t e d i n 300 ml q u a n t i t i e s o f th e same medium a s l i s t e d
a b o v e , e x c e p t f o r th e a d d i t i o n o f 0 .2 4 p e r c en t by w eig h t b e n t o n i t e .
The
b e n to n ite was added t o k e ep th e mold m ycelium grow ing i n s m a ll p a r t i c l e s .
T hese se e d c u l t u r e s were in o c u la te d from a s l a n t c u l t u r e o f th e mold grow n
- 15 on th e same m edia e x c e p t f o r th e a d d i t i o n o f 2 .5 p e r c e n t a g a r , and th e n
sh ak en f o r 40 h o u rs t o in s u r e g ro w th u n d e r a e r o b ic c o n d i t i o n s .
The medium,
a f t e r i n o c u l a t i o n , was in c u b a te d a t a te m p e ra tu re o f 32-34°C w ith c o n tin ­
u ous a e r a t i o n .
The a i r was s u p p lie d b / com pressed a i r from th e a i r l i n e
w hich was p a s s e d th ro u g h a s t e r i l e
d i s t i l l e d w a te r .
c o tto n f i l t e r , and th e n th ro u g h s t e r i l e
A p p ro x im a te ly o n e - t h i r d volume o f a i r p e r volume o f m ed ia
p e r m in u te was u s e d .
A f te r 72-9 6 h o u rs o f f e r m e n ta tio n , th e alpha am ylase
a c t i v i t y re a c h e d a v a lu e o f 1 .0 t o 4 .0 u n i t s p e r ml w ith an a v erag e o f
a p p ro x im a te ly 1 .5 u n i t s p e r m l.
The mold f i l t r a t e
p er cent i s s a l t s .
c o n s i s t s o f 0 .8 p e r c e n t s o l i d m a t t e r , o f w hich 40
The o th e r com ponents a re c a r b o h y d ra te s (w hich a re
p ro d u c ts o f enzyme a c ti o n on s t a r c h ) , p r o t e i n s (w h ich d i f f u s e from mold
c e l l s ) , and enzymes (T a u b e r l i s t s 23) ( 2 0 ) .
A number o f p ro c e d u re s were t r i e d f o r th e c o n c e n tr a tio n and p u r i f i ­
c a ti o n o f th e a lp h a -a m y la s e from th e mold f i l t r a t e .
1.
These in v o lv e d I
C o n c e n tra tio n by vacuum d i s t i l l a t i o n and f r a c t i o n a l p r e c i p i t a t i o n
w ith a lc o h o l .
2.
C o n c e n tra tio n by vacuum d i s t i l l a t i o n and f r a c t i o n a l p r e c i p i t a t i o n
w ith ammonium s u l f a t e .
3.
C o n c e n tr a tio n by vacuum d i s t i l l a t i o n and a d s o r p tio n o f a lp h a am ylase on s t a r c h and p u re am ylop e c t i n .
4.
The T Jnderkoflex m ethod.
5.
A d so rp tio n o f th e a lp h a -a m y la s e from mold f i l t r a t e
on th e mold
m ycelium , e l u t i o n and f r a c t i o n a l p r e c i p i t a t i o n w ith ammonium s u l ­
fa te
— 16 —
M ethod number f iv e was th e m ost s u c c e s s f u l .
Tho f i r s t f o u r m ethods
a tte m p te d w i l l be d e s c r ib e d in b r i e f , and method f i v e d e s c r ib e d i n d e t a i l .
1.
C o n c e n tra tio n by Vacuum D i s t i l l a t i o n
and F r a c t i o n a l P r e c i p i t a t i o n w ith Is o p r o p y l A lc o h o l.
The vacuum d i s t i l l a t i o n was c a r r i e d o u t a t te m p e r a tu r e s helow 35°C»
th e c o n c e n tr a tio n b e in g 8 -1 0 f o l d .
The l o s s in a c t i v i t y i n t h i s s t e p was
from 0 -4 0 p e r c e n t d e p e n d in g on th e tim e and c o n ta m in a tio n in th e medium.
A f te r a c o n c e n tr a tio n o f 8 -10 f o l d , th e medium was f i l t e r e d th ro u g h a mat
o f c e I i t e , p la c e d i n d i a l y z i n g b a g s made o f c e l l u l o s e a c e t a t e , and d ia ly z e d
a t r e f r i g e r a t e d te m p e r a tu r e s u n t i l f r e e o f s a l t s .
was th e n p r e c i p i t a t e d w ith a lc o h o l .
T h is s a l t - f r e e s o l u t i o n
The b e s t c o n d itio n s f o r t h i s p r e c i p ­
i t a t i o n w ere found t o be th e u s e o f two p a r t s a lc o h o l t o one p a r t o f enzyme
s o l u t i o n a t 0°C and pH 7 . 0 .
Twice p r e c i p i t a t e d m a t e r i a l , upon d r y in g ,
gave a maxiumu a lp h a a c t i v i t y o f 6 ,8 0 0 u n i t s p e r gram .
I n o rd e r t o check
th e amount o f f r a c t i o n a t i o n from o th e r com ponents, j u s t b e fo re p r e c ip ­
i t a t i n g , a Sm-iI l amount o f th e d ia ly z e d enzyme s o l u t i o n was ta k e n t o d ry ­
n e s s in a d ia l y z i n g b ag by h a n g in g i n a s tre a m o f warm a i r .
o f t h i s m a t e r i a l was 7 ,5 0 0 a lp h a u n i t e p e r ,gram.
The a c t i v i t y
T h is i n d i c a t e d t h a t p a r t
o f th e enzyme a c t i v i t y was d e s tr o y e d b y th e a l c o h o l , and v e ry l i t t l e
t i o n a t i o n h ad ta k e n p l a c e .
fra c ­
T h e r e f o r e , t h i s ap p ro ach t o th e p u r i f i c a t i o n
was ab and oned .
2.
C o n c e n tra tio n by Vacuum D i s t i l l a t i o n
and F r a c t i o n a l P r e c i p i t a t i o n W ith Ammonium S u lf a t e
T h is p ro c e d u re was c a r r i e d o u t e x a c t l y th e same a s above e x c e p t
ammonium s u l f a t e f r a c t i o n a t i o n was u s e d i n s t e a d o f a l c o h o l .
The enzyme
- 17 s c I u t io n w hich was p r e c i p i t a t e d f o u r tim e s w ith t h r e e - f o u r t h s s a t u r a t e d
A
ammonium s u l f a t e , d ia ly z o d and to k e n t o d ry n e e o , y ie ld e d an a c t i v i t y o f
8 ,0 0 0 alphc u n i t s p e r gram .
3.
C o n c e n tra tio n By Vacuum D i s t i l l a t i o n
and A d so rp tio n o f Enzyme on S ta ro h and A m y lo p ectin
C o n c e n tr a tio n , f i l t e r i n g , and d i a l y s i s was c a r r i e d o u t a s in th e
above two p r o c e d u r e s .
S in ce one o f th e in te r m e d ia te s t e p s in th e p u r i ­
f i c a t i o n o f m a lt am ylase ( l ? ) was t o a d s o rb th e enzyme on s t a r c h i n th e
p re s e n c e o f 40 p e r c e n t a lc o h o l , t h i s m ethod was t r i e d .
The b e s t r e s u l t s
t h a t co u ld he o b ta in e d i n t h i s m anner w ere 20 p e r c e n t a d s o r p tio n o f th e
enzyme on c o r n e ta r c h and 40 p e r c e n t a d s o r p tio n on am ylopec t i n .
B ecause
o f th e la r g e l o s s e s , t h i s p ro c e d u re was d is c a r d e d .
4«
The U n d o rk o fle r K ethod
T h is m ethod was c a r r i e d ou t a s o u tlin e d by U n d e r k o f le r ( 2 l ) .
c r y s t a l s c o u ld ha o b ta in e d by t h i s m eth o d .
Ho
On rem oval o f the ammonium
s u l f a t e by d i a l y s i s and ta k in g t o d ry n e s s by h a n g in g i n a stre a m o f warm
a i r , t h i s m a t e r i a l y ie ld e d an a lp h a a c t i v i t y o f 1 0 ,0 0 0 u n i t s p e r gram .
The q u a n t i t y o f ammonium s u l f a t e n eed ed f o r t h i s p u r i f i c a t i o n scheme was
to o g r e a t t o w a rra n t f u r t h e r a tte m p ts .
5.
C n o e n tr a tio n and P u r i f i c a t i o n by A d so rp tio n
o f A lp h a-am y lase From Mold F i l t r a t e on Mold M ycelium ,
E l u tio n and F r a c t i o n a l P r e c i p i t a t i o n With Ammonium S u lf a t e
I t was d is c o v e re d i n th e c o u rse o f t h i s i n v e s t i g a t i o n t h a t by p r o p e r
a d ju s tm e n t o f te m p e r a t u r e , pH, s a l t c o n c e n tr a tio n , and s e l e c t i o n o f
m ycelium o f th e p ro p e r a g e , th e a lp h a -a m y la s e p ro d u ced b y A s p e r g illu s
— 13 oryzr.e " v s c o m p le te ly a d so rb e d on on - f i f t h o f th e m ycelium p r e s e n t in
th e g ro w th medium.
By e l u t i o n from t h i s m ycelium , a c o n c e n tr a tio n o f 2 0 -
40 f o l d was o b ta in e d and a t th e same tim e th e enzyme was s e p a r a te d from
th e c o n ta m in a tin g c a r b o h y d r a te s , s a l t s , and o v e r 80 p e r c e n t o f th e p r o t e i n
p re s e n t.
A seco n d a d s o r p tio n a llo w e d an a d d i t i o n a l 2 -3 f o l d c o n c e n tra ­
tio n .
T h is p ro c e d u re e lim in a te d th e u s u a l m ethods o f c o n c e n tr a tio n u n d e r
re d u c e d p r e s s u r e and p r e c i p i t a t i o n by s a l t s , a lc o h o l , o r o th e r o rg a n ic
s o lv e n t s th u s a llo w e d f o r an e a s y and in e x p e n s iv e m ethod o f c o n c e n tra ­
tio n .
I n t h i s p r o c e d u r e , j u s t as i n th e o t h e r s , th e c u l t u r e was grown u n d e r
th e u s u a l c o n d itio n s o f fo r c e d a e r a t i o n on a s t a r c h and n itr o g e n c o n ta in ­
in g medium.
When th e optimum a m y lo ly tic a c t i v i t y was o b ta in e d , th e
m ycelium was rem oved from th e f e r m e n ta tio n l i q u o r by f i l t r a t i o n , a f t e r
f i r s t a d j u s t i n g th e pH t o 8 .5 w ith d i l u t e sodium h y d ro x id e .
T h is was
n e c e s s a r y t o a llo w com plete s e p a r a t i o n o f m ycelium and enzym e.
A p p ro x i­
m a te ly o n e - f i f t h o f th e m ycelium was w ashed and th e n re s u s p e n d e d b ack i n t o
th e mold f i l t r a t e .
The pH o f th e medium was a d ju s te d t o 5 .0 w ith g l a c i a l
n o e t i c a c i d , th e te m p e ra tu re a d ju s t e d t o 3 7 °0 , and th e e n t i r e m ix tu re
g e n t l y og i t a t e d f o r one h o u r.
th e n washed
At th e end o f t h i s tim e th e mycelium was
I t h w a te r w ith th e pH a d ju s te d to 5 » 0 .
The m ycelium was
re s u s p e n d e d in a minimum o f w a te r ( . 0 5 - .0 2 5 o f th e o r i g i n a l volume o f
f e r m e n ta tio n ) t o w hich had been added 1 .0 p e r c e n t sodium c h l o r i d e , and
25 p e r c e n t h„, volume b o r a te b u f f e r p re p a re d in th e f o llo w in g m anner;
(5 0 ml 0 .2 H F C l; 50 ml 0 .2 m
8 .5 ml 0 ,2 M TTaOH; mixed and
- 19 d i l u t e d t o 200 m l ) ,
The pH o f e l u t i o n l i q u i d was th e n a d ju s t e d t o 8 .5
w ith sodium h y d ro x id e w ith c a r e f u l s t i r r i n g .
A f te r o n e - h a l f h o u r, th e
m ycelium was removed from th e enzyme s o l u t i o n by f i l t r a t i o n , and a con­
c e n t r a t i o n o f 20-40 f o ld was o b ta in e d .
T h is p r o c e s s was r e p e a te d , th u s a llo w e d a f u r t h e r c o n c e n tr a tio n o f
2 -3 f o l d .
from a b o v e t
H ow ever, one o f two t h i n g s m ust be done t o th e e l u t i o n l i q u i d
I.
d i l u t i o n u n t i l th e t o t a l s a l t c o n c e n tr a tio n i s below
o n e -h a lf p er c e n t, o r
2.
d i a l y s i s t o remove th e s a l t s .
T f a more con­
c e n t r a t e d l i q u o r i s d e s i r e d , th e m a t e r i a l from th e seco n d a d s o r p tio n
can be c o n c e n tr a te d by vacuum d i s t i l l a t i o n o v e r 6 f o l d more w ith l i t t l e
o r no l o s s in a c t i v i t y .
S in ce a t t h i s s ta g e th e t o t a l volume h a n d le d
i s a p p ro x im a te ly one h u n d re d th o f th e o r i g i n a l , th e le n g th o f tim e r e ­
q u ir e d f o r e v a p o r a tio n i s much l e s s , a llo w in g l e s s tim e f o r c o n ta m in a tio n
and l o s s o f a c t i v i t y .
W ith t h i s t h i r d s t e p , c o n c e n tr a tio n s o f 400-600
tim e s were r e a d i l y o b t a i n a b l e .
A f te r p r e lim in a r y e x p e rim e n ts i n d i c a t e d t h a t t h i s m ethod m ight he
f e a s a h l e , th e optimum pH, and te m p e ra tu re f o r th e a d s o r p tio n were d e t e r ­
m ined as i n d i c a t e d i n T a b le s I and T I .
The same so u rc e o f media and
m ycelium was u s e d f o r a l l a d s o r p tio n a n a ly s e s .
I t can r e a d i l y be se e n from o b s e r v in g th e two t a b l e s , t h a t th e b e s t
c o n d itio n s o f a d s o r p tio n a re pH o f 5«0 a t a te m p e ra tu re o f 37°C.
T here was no problem o f e l u t i n g th e enzyme from tV - m ycelium .
As
was m e n tio n e d p r e v i o u s l y , th e pH o f th e e l u t i o n l i q u i d was a d ju s te d t o
8 . 5 , w hich a llo w e d th e enzyme t o s e p a r a t e from th e m ycelium .
E lu tio n s
o f 98-100 p e r c e n t oan be o b ta in e d e v e ry tim e in th e p re s e n c e o f 1 .0 p e r
— 20 —
cen t sodium c h lo r id e .
The b u ffe r was found n e c e ssa r y t o avoid lo c a l con­
c e n tr a tio n o f sodium hydroxide on th e pH a d ju stm en t, s in c e the a c t i v i t y i s
d e str o y ed a t once in an a lk a lin e c o n d it io n .
A fte r t h i s p r e lim in a r y work had in d ic a te d a d so r p tio n and e lu t i o n o f
th e enzyme b
t h i s p r o c e ss t o he f e a s a b l e , the n ex t s te p was to determ ine
th e l i m i t s o f c o n c e n tr a tio n , the amount o f f r a c t io n a t io n from o th er p r o te in s
and enzymes and f i n a l l y , i f t h i s co n c en tr a ted enzyme s o lu t io n o b ta in ed by
a d so r p tio n and e lu t i o n procedures could be fu r th e r c o n c e n tr a te d , by vacuum
d is tilla tio n .
The l a t t e r would be u s e f u l fo r prep arin g c o n c e n tr a te s o f
th e enzymes f o r commercial p u rp o ses.
The d ata o b ta in ed in b e h a lf o f th e s e
q u e s tio n s i s s e t fo r th in Table I I I .
From th e r e s u l t s l i s t e d on T able I I I , i t can be observed th a t a concen­
t r a t i o n o f 29 tim es th a t o f the o r ig in a l was o b tain ed on th e f i r s t a d so r p tio n
and e lu t i o n w ith an e lim in a tio n o f ap p roxim ately 84 per c en t o f th e com­
pounds which co n ta in ed K jeld a h l n it r o g e n .
92 per cen t o f lim it d e x tr in a s e .
T his a d so r p tio n a ls o e lim in a te d
On th e second a d so r p tio n and e l u t i o n , a
c o n c e n tr a tio n o f 73 tim es th a t o f th e o r ig in a l was o b ta in e d , how ever, th e
amount o f l i m i t d e x tr in a s e and th o se compounds which co n ta in ed K je ld a h l
n itr o g e n and th a t were f r a c tio n a t e d were v e ry s m a ll.
per cen t f o r e a c h .
I t was l e s s than 3 .0
In order to t e s t th e s u i t a b i l i t y o f fu r th e r concen­
t r a t i n g , th e co n c en tr a te (E , Table I I I ) by vacuum d i s t i l l a t i o n th e
f o llo w in g experim ent was run*
90 ml o f th e HO u n it m a te r ia l was con­
c e n tr a te d b, vacuum d i s t i l l a t i o n t o 14 ml (a l i t t l e more than 6 t im e s ) .
The c o n c en tr a te was an alyzed and found t o have an a c t i v i t y o f 700 u n it s
per m l.
R ecovery was 100 per cen t w ith in th e l i m i t s o f ex p erim en ta l
- 21 e r r o r on the m easuring o f volum es o f th e s e co n cen tra ted s o lu t io n s .
There
i s no q u e s tio n but a t th e se c o n c e n tr a te s could be c o n cen tra ted even more
i f n ecessary.
Q u a lita tiv e t e s t s a ls o in d ic a t e th e rem oval o f the o r ig in a l
s a l t s and most o f the carbohydrate by t h i s a d so r p tio n proced u re.
A number o f a d d itio n a l runs o f a d so r p tio n and e l u t i o n o f th e enzyme
have been made.
The r e s u l t s o f some o f th e s e are found i n Table IV .
o f th e a d so r p tio n s was c a r r ie d out in 10 l i t e r q u a n t i t i e s .
Each
The mycelium
and medium were grown in sep a ra te b o t t l e s a t d if f e r e n t tim e s .
The a d so r p tio n p r o c e ss i s v e ry s e l e c t i v e , s in c e A s p e r g illu s oryzae
mycelium f a i l s t o absorb th e am ylase from m alt e x t r a c t and A s p e r g illu s
n i£ e r .
The most s u c c e s s f u l p u r if ic a t io n procedure i s o u t lin e d on Table V.
T h is r e s u lt e d in a f i n a l dry co n c en tr a te which had h igh amylase a c t i v i t y
and o n ly tr a c e s o f m a lta s e , lim it d e x t r in a s e , and p r o t e o ly t ic a c t i v i t y .
The p u r if ic a t io n in v o lv e d c o n c e n tr a tin g th e enzyme by mycelium a d so r p tio n
and th en u s in g t h r e e -fo u r th s s a tu r a te d ammonium s u lf a t e f r a c t io n a t io n ,
a lo n g w ith b e n to n ite a d so r p tio n as was u sed by TJnderkofler ( 2 1 ) ,
The
i n i t i a l c o n c e n tr a tio n ( f r a c t io n la b e le d A and B) was c a r r ie d out in te n
d if f e r e n t s t e p s , spread over a p erio d o f f i v e w eek s.
The e lu t e s were
s to r e d a t r e f r ig e r a t e d tem p eratu res u n t i l the d e s ir e d q u a n tity needed
to con tin u e fu r th e r p u r if ic a t io n had been o b ta in e d .
C r y s t a lliz a t io n was
attem p ted fo u r d if f e r e n t tim es w ith f r a c t io n la b e le d H, tw ic e a t r e f r i g ­
e r a te d tem p eratu res and tw ic e a t room tem p era tu res.
C r y s t a lliz a t io n a t
room tem perature was attem pted because m alt alpha-am ylaee was c r y s t a l l i z e d
in th a t manner.
Ammonium s u lf a t e was added in each ca se over a p e r io d o f
- 22 24 hours u n t i l o n e -h a lf s a tu r a tio n was rea ch ed .
The pH was a d ju sted to
5 .5 - 6 .0 .
A fte r c r y s t a ls o f th e amylase f a i l e d to appear, th e enzyme s o lu t io n
was d ia ly z e d fr e e o f ammonium s u l f a t e , and th en ta k en t o d ryn ess in a
d ia ly z in g h ag.
The enzyme s o lu t io n , b e fo r e d r y in g , had a t o t a l a c t i v i t y
o f 5 8 ,0 0 0 alp h a u n i t s .
T h is y ie ld e d 3 .4 grams o f dry enzyme p r e p a r a tio n ,
t h e r e f o r e , th e enzyme p r e p a r a tio n had an a c t i v i t y o f 1 7 ,3 5 0 u n it s per gram
b efo re d r y in g .
The dry m a te r ia l had an a c t i v i t y o f 1 4 ,3 0 0 u n it s per gram,
a l o s s o f 3 ,0 5 0 u n it s on d r y in g .
K je ld a h l n itr o g e n a n a ly s is o f th e dry
co n c en tr a te in d ic a te d 1 2 .1 5 per cen t n it r o g e n .
- 23 TABLE I
OPTIMUM TEMfEHATUEE FOR ABSORPTION
( 5 £ wet m ycelium p e r 100 ml o f m edia)
Assumed, o p t. pH5 from p r e lim in a r y r e s u l t s
T em p eratu re 0C
o f T o ta l Amylase
A dsorbed
77
25
30
35
40
86
97
96
TABLE I I
(5
OPTIMUM pH FOE ADSORPTION
w et m ycelium p e r 100 ml o f m edia)
T em perature o f a d s o r p tio n 37vO
pH
4 .5 0
4 .7 5
5 .0 0
5 .5 0
6 .0 0
56 o f T o ta l Amylase
A dsorbed
66
66
93
72
70
TABLE I I I
INITIAL PURIFICATION SCHEME
AMYLASE
FRACTION
u n it s / ml
(A) Mold f i l t r a t e
as grown (20 l i t e r s )
1 .5 0
(B ) F i l t r a t e from
A a f t e r mycelium
a d so r p tio n (2 0 l i t e r s )
0 .2 2
(C) E lu tio n liq u id
from mycelium adsorp­
t io n (550 ml)
(D) F i l t r a t e from C
a f t e r second mycelium
a d so r p tio n (5 5 0 ml)
4 3 .0
% AMYLASE
OF ORIG.
CONC. BY
ACTIVITY
100
KJELDAHL
NITROGEN
mg/ ml
0 .1 4 7 5
% KJELDAHL
NITROGEN
OF ORIG.
100
0 .1 3 8 0
7 8 .5
2 9 .0
0 .3 2
0 .8 5 5
1 1 0 .0
7 3 .5
7 3 .0
(F ) Vacuum d i s t i l l e d
con e. 6 .4 more t im e s ;
Uo m easurable l o s s in
a c tiv ity
7 0 0 .0
7 3 .5
4 6 5 .0
1 .8 0
2 .0 1
% LIMIT
DElBTRINASE
OF ORIG.
100
1 .9 0
1 5 .9
0 .2 5 5
(E) E lu tio n liq u id
from second mycelium
a d so r p tio n D (200 ml)
LIMIT
DEXTRINASE
HYDROL/ML
5 .6 7
7 .7 5
4 .5 0
1 2 .2
1 2 .5
6 .2
— 25 —
TABLE IV
ADDITIONAL ADSORPTION DATA
Performed on Separate Grown B o t t le s
(lO l i t e r q u a n t it ie s )
Bun N
o
.
% B eoovery
C oncen tration
(tim e s by a c t i v i t y)
I.
75
15
2.
80
16
3.
60
19
4.
75
37
5.
74
32
6
8
—
7.
41
1 5 .5
8.
27
14
9.
O
—
10.
46
2 2 .3
11.
52
26
12.
62
31
13.
90
25
14.
78
34
15.
85
35
— 26 —
TABLE V
MOST SUCCESSFUL PURIFICATION SCHEME
FRACTION
AMYLASE j % SMYLA3E
OF ORIG.
u n it s / ml
( a ) Mold F i l t r a t e (100 l i t e r s )
1 .4 4
CONC. BY ‘
ACTIVITY
100
——
(B ) E lu tio n L iq u id 2 .5 l i t e r s )
4 1 .5
72
29
(C ) F i r s t Ammonium S u lf a t e P r e c ip ­
i t a t e D is s o lv e d i n D i s t i l l e d
W ater t o Volume o f (950 ml)
9 2 .5
61
64
(D) Second Ammonium S u lf a t e P r e c ip ­
i t a t e D is s o lv e d i n D i s t i l l e d
W ater t o Volume o f ( l8 0 ml)
458
5 7 .2
318
(E ) T h ird Ammonium S u lf a t e P r e c ip ­
i t a t e D is s o lv e d i n D i s t i l l e d
W ater t o Volume o f (5 0 m l)
1 ,5 6 0
54
1 ,3 8 0
48
(F ) A f te r B e n to n ite A d s o rp tio n t o
Remove L im it D e x trin a s e (50 m l)
1 ,0 8 0
"I
(G) F o u rth Ammonium S u lf a t e P r e c ip ­
i t a t e D is s o lv e d i n D i s t i l l e d
W ater t o Volume (5 0 ml)
1 ,3 5 0
47
(H) F i f t h Ammonium S u lf a t e P r e c ip ­
i t a t e D is s o lv e d i n D i s t i l l e d
W ater t o Volume (5 0 ml)
1 ,1 6 0
4 0 .2
( l ) F r a c tio n (H) D ia ly z e d F ree o f
Ammonium S u l f a t e , Taken to Dry­
n e s s i n D ia ly z in g Bag ( 3 .4 gram s)
——
17,3 5 0 U n its /g r a m , wet B ases
14,300 U n its /g r a m , d ry B ases
ANILYSIS OF FRACTION (H)
P r o te o ly tio A c tiv ity
T race o f O r ig in a l
M a lta se A c t i v i t y
0 .1 # o f O r ig in a l
L im it D e x trin a s e A c t i v i t y
0 .0 5 # o f O r ig in a l
- 27 V II.
DISCUSSION
The em phasis o f th e in v e s t ig a t i o n on th e p resen t problem was co n fin ed
t o ex p erim en ta l work on methods o f c o n c e n tr a tio n and p u r if ic a t io n o f th e
amylase ob tain ed from a submerged c u ltu r e o f A s p e r g illu s o r y z a e .
One o f the most im portant f a c t o r s in t h i s i n v e s t ig a t i o n th a t d e s e r v e s
a t t e n t io n and s t i l l i s u n so lv e d , i s th e la r g e v a r ia tio n in the amount o f
am ylase th a t th e mold mycelium ad sorb ed , (0 -9 0 $ , Table I V ).
For a tim e
t h i s a d so rp tio n appeared v e ry uniform and averaged 73 per c e n t, th en f o r
no apparent reason poor r e s u l t s were o b ta in e d .
The m edia, ra te o f a ir
flo w , and growth c o n d itio n s were in each case as uniform a s p o s s ib le .
T his b r in g s up th e q u e s tio n , "Why do th e s e v a r ia tio n s occur?"
As th e in ­
v e s t ig a t io n p r o g r essed s e v e r a l p o s s i b i l i t i e s p resen ted th e m s e lv e s :
I.
S a lt c o n c e n tr a tio n
At th e b e g in n in g o f t h i s p r o j e c t , a s a l t m ixture was prepared which
co n ta in ed 50# KH2PO4 $ 25# MgSO4t 2 3 .5 # KCl; 0 .5 # FeSO4 .TBgO; 0 .5 # ZnSO4 .TEgO;
0 .5 # CuSO4 . 5H20 ,
t h i s s a l t m ix tu re.
The medium used was made up o f 0 .1 per cen t by w eigh t o f
S in ce t h i s m ixture was not b a ll m ille d , i t seems v e ry
l i k e l y th a t th e elem en ts p resen t in tr a c e amounts (FeSO4 eTH2O; ZnSO4 eTH2O;
CuSO4 *SHgO) may n ot have been d is t r ib u t e d hom ogeneously.
F o ster (6 )
s t a t e s th a t th e s e elem en ts are probab ly t i e d up w ith some enzyme system
o f th e mold.
Too much or to o l i t t l e
o f th e s e elem en ts may have i n t e r ­
fe r e d w ith th e p rod u ction o f the c h a r a c t e r is t ic p ro p erty o f the mold
mycelium th a t ta k e s p art in the a d s o r p tio n .
formed on s e v e r a l b a tch es o f mycelium .
A p r o te in a n a ly s is was per­
I t was found th a t th e good
a d sorb in g mycelium had much low er p r o te in content than th e myoeIium
— 28 —
t h a t d id n o t a d s o r b .
F o s te r (6 ) s a i d t h a t th e p r o t e i n c o n te n t o f th e my­
c e liu m i s i n v e r s e l y p r o p o r tio n a l t o th e amount o f z in c p r e s e n t .
T h e re fo re ,
a s e r i e s was ru n by v a ry in g th e z in c c o n c e n t r a t i o n s , k e e p in g a l l th e o t h e r
s a l t c o n c e n tr a tio n s c o n s t a n t .
The r e s u l t s o b ta in e d a t t h i s tim e i n d i c a t e
t h a t t h i s m ight be th e l i m i t i n g f a c t o r b u t checks must be ru n on t h i s io n .
The p o s s i b i l i t y o f FeSO^eTHgO and CuSO^e5Hg0 have n o t b een ch eck ed .
2.
B e n to n ite
A n o th er p o s s i b i l i t y f o r e x p la i n in g th e v a r i a t i o n i n th e a d s o r p tio n
i s th e b e n to n ite c o n c e n tr a tio n o f th e s e e d c u l t u r e s u s e d t o in o c u la te th e
m e d ia .
A lth o u g h th e b e n to n ite was alw ays added in c o n s ta n t am ounts by
w e ig h t, i t was n o t o f u n ifo rm p a r t i c l e s i z e .
I t was n o t i c e d t h a t d i f f e r ­
e n t am ounts o f b e n to n ite had a d e c id e d e f f e c t on th e sh ap e o f th e m ycel­
ium p a r t i c l e s .
L arge am ounts made th e m ycelium ta k e on a s t r i n g y a p p e a r­
a n c e , w h ile s m a l le r am ounts made th e m ycelium ta k e on th e shape o f t i n y
b a l l s w ith t e n t i c l e s coming out o f th e c e n t e r .
The amount o f s u rfa c e
a v a i l a b l e f o r a d s o r p tio n may he t i e d up w ith t h i s f a c t o r .
I t a p p e a rs
a s th o u g h an in te r m e d ia te form betw een s t r i n g s and b a l l s g iv e s th e b e s t
a d s o r p tio n .
F u r th e r work must he done on t h i s to e s t a b l i s h th e in f lu e n c e
o f b e n to n ite .
3.
pH
I t was o b se rv e d by k e e p in g a r e c o r d o f th e pH o f th e grow ing m edia
t h a t whan th e pH tu r n e d a l k a l i n e i n a s h o r t p e rio d o f tim e (2 4 -2 8
h o u r s ) , th e m ycelium a d so rb e d th e am ylase on alm o st e v e r y o c c a s io n , how­
e v e r , i f th e m edia s ta y e d on th e a c id s id e from 96-120 h o u r s , no a d so rp ­
t i o n to o k p l a c e .
T h is i n d i c a t e s t h a t th e r e must be some r e a c t i o n ta k in g
-2 9 p la c e in th o g ro w in g myoelim w hich d e c id e s th e c o u rse e a r l y in th e g ro w th .
A r t i f i c i a l l y c o n t r o l l e d pH d id n o t p ro v e s u c c e s s f u l ,
4.
M u ta tio n
The p o s s i b i l i t y o f m u ta tio n c an n o t he o v e rlo o k e d .
F o s t e r (6 ) h a s
s t a t e d t h a t th e e le m e n ts o f z in c and c o p p e r in d u ce m u ta tio n s t o fu n g i
u n d er c e r ta in c o n d itio n s .
H ow ever, t h i s p o s s i b i l i t y f o r p ro d u c in g my­
c e liu m w ith p r o p e r t i e s w ith d i f f e r e n t c h a r a c t e r i s t i c s i s u n l i k e l y .
C ul­
t u r e s p r e s e r v e d f o r tw e lv e months u n d e r p a r a f f i n o i l d id n o t g iv e any
d i f f e r e n t r e s u l t s th a n th o s e r e p e a te d ly t r a n s f e r r e d a t r e g u l a r i n t e r v a l s .
5.
I n c r e a s i n g th o am ounts o f m ycelium
I t was o b se rv e d t h a t when th e p r o p o r tio n o f m yoelim was in c r e a s e d ,
th e amount o f enzyme a d so rb e d a ls o i n c r e a s e d .
C a l c u la tio n s were n o t
made to d e te rm in e i f th e law s o f a d s o r p tio n h o ld t r u e .
The em phases o f
t h i s p a r t o f th e i n v e s t i g a t i o n was t o c o n c e n tr a te th e enzyme as much as
p o s s i b l e , t h e r e f o r e , c o n c e n tr a tio n o f f i f t y tim e s by volume was a r b i t r a r i l y
ta k e n and a l l a d s o r p tio n s c a r r i e d o u t and c a l c u l a t e d on t h a t b a s i s .
T here
i s no q u e s tio n h u t t h a t t h i s oould he in c r e a s e d c o n s id e r a b ly i f concen­
t r a t i o n s o f f i f t y f o ld by volume were n o t d e s i r e d .
I n g e n e r a l , an i n ­
c r e a s e o f $0 p e r c e n t i n a d s o r p tio n was o b ta in e d when th e amount o f
m ycelium u s e d was t r i p l e d .
However, t h i s would mean t h a t i t oould o n ly
he c o n c e n tr a te d a p p ro x im a te ly 18 tim e s a n d , t h e r e f o r e , th e s e c o n c e n tra ­
t i o n s were n o t r u n .
I t i s b e lie v e d t h a t c r y s t a l s o f th e enzyme o o u ld he o b ta in e d by th e
p ro c e d u re o u t l i n e d , p ro v id e d t h a t a la r g e enough q u a n t i t y o f mold f i l ­
t r a t e was a v a i l a b l e a t one tim e .
T h is w ould e lim in a te s t o r i n g u n t i l
137465
- 30 th e d e s i r e d q u a n t i t y was a v a i l a b l e .
t i o n and a , w e r e
L o sse s in a c t i v i t y due to c o n tam in a­
c o n s id e r a b le o v e r lo n g p e r io d s o f tim e .
F u rth e rm o re ,
th e p r o p e r t i e s o f th e enzyme m ig h t change on e x te n d e d s t o r a g e .
-3 1 V III.
1.
SUUMAEY
A m ethod waa p r e s e n te d f o r c o n c e n tr a tin g th e am ylase from a subm erg­
ed c u l t u r e by a d s o r b in g on o n e - f i f t h th e m ycelium p r e s e n t , e l u t i n g
and r e c o v e r in g i n a c o n c e n tr a te d form from 20 -4 0 tim e s th e o r i g i n a l
a c t i v i t y i n onu s t e p , th u s e l i m i n a t i n g th e c o s t l y p ro c e d u re s u s u a l l y
e n c o u n te re d .
2.
A m ethod was p r e s e n te d w hereby c o n c e n tr a te d fu n g a l a m y lo ly tic l i q u o r s
can be f u r t h e r c o n c e n tr a te d from 2 -3 tim e s ( y i e l d i n g a t o t a l concen­
t r a t i o n o f 60-120 tim e s t h a t o f th e o r i g i n a l ) by a sim p le p ro c e d u re
o f a sec o n d a d s o r p tio n on th e m ycelium fo llo w e d by e l u t i o n .
3.
A m ethod was p r e s e n te d w hereby th e f u n g a l c o n c e n tr a te (6 0 -1 2 0 tim e s
o r i g i n a l ) can be f u r t h e r c o n c e n tr a te d 6-10 tim e s g i v in g a f i n a l con­
c e n t r a t i o n o f 360-1200 tim e s th e o r i g i n a l c u ltu r e l i q u o r "by a p r o c e s s
o f vacuum d i s t i l l a t i o n .
4.
A m ethod was p r e s e n te d w hereby c o n c e n tr a te d fu n g a l am ylase can be
f u r t h e r c o n c e n tr a te d and p u r i f i e d by f r a c t i o n a l p r e c i p i t a t i o n w ith
ammonium s u l f a t e , a lo n g w ith a b e n to n ite a d s o r p tio n .
- 32 IX.
LITERATURE CITED
(1 )
A .O .A .C ., p . 2 6 , 2 .2 4 s i x t y e d i t i o n , (1 9 4 5 )•
(2)
B aok, T . I I . , S ta r k , W Ht f and S h a l f , R. E . , A n a l. Ohero 2 0 . 50-60
(1 9 4 3 ) .
( 3)
C a ld w e ll, C h e s te r , D o e h h e lin g , and T b l z . , J . B i o l . Chem. I o l .
351 (1 9 4 5 ).
(4)
C h e s te r R .M ., D i s s e r t a t i o n , C olum bia U n iv e r s ity (1 9 3 3 )•
(5 )
Gorman, J . , and Langlyklce, A -F ., C e re a l Chem. 2J1; 190-201 ( 1948) .
(6 )
F o s t e r , J . Vi., Chem ioal A c t i v i t i e s o f F u n g ie . , Hew York Academic
P r e s s , (1 9 4 9 ) .
(7 )
H a s s id , 17.Z ., and M oCready, E .1 I., J . Am. Chem. S o o ., o 5 . 1157 ( 1 9 4 3 ) .
(8 )
LeM ense, E .H ., Enzymes I n c o r p o r a te d , U n p u b lish e d w ork
(9 )
M eyer, K .H ., A dvances i n C o llo id S o i . ,
143
(1 9 4 2 ) .
(1 0 )
M eyer, K. H. A dvances i n Euzym ol.
(1 1 )
M eyer, K. H ., F i s h e r , E .H ., and B e r n f e l d , P . , E e iv . Chim. A c ta , 3 0 .
64-78
(1 2 )
109
(1 9 4 9 )•
(1 9 4 3 ) .
(1 9 4 7 ) .
M eyer, K .H ., F i s h e r , E .H ., and B ern fe I d , P . , E x p e r i e n t i a l , 106
(1 9 4 7 ).
(1 3 )
M eyer, K .H ., F i s h e r , E.H . B e rn fe I d , P . , and S ta u b , A ., E x p e r ie n tia
A , 455
(1 9 4 7 ).
(1 4 )
N is h im u ra , A .S ., B u l l , A g ri0 . Chem. S o c .
(1 5 )
N o rth r o p , J . H^ J . Gen. P h y s io l. 1 6 , 41-58
(1 6 )
S a n d a te d t, K neen, and B lis h C e re a l Chem. 16. 712-732 (1 9 3 9 ).
(1 7 )
Schwimmer, S . , and B a l l s , A -K ., J . B i o l . Chem., 1 7 9 . 1063-1074
(1 9 4 9 ) .
J a p a n Zmt
129
(1 9 2 6 ).
(1 9 3 2 ).
- 33 (1 8 )
Shermzm, H .O ., and Timber®, A . ? . , J . Am. Chem. S o o ., _^8, 1638
(1 9 1 6 ) .
(1 9 )
Ta!;m i n e , J o k i o h i , ,
(2 0 )
T a u b e r, C hem intry and T echn o lo g y o f Snzymee, New Y o rk , Jo h n W iley
and S o n s, I n o .
J . S o o ., I n d . I ^ ,
118
( 1898) .
(1 9 4 0 ) .
(2 1 )
U n d a r k o f le r , L .A ., and Eoy, D .E, C e re a l C h e m istry , 2 8 , 18-29
(2 2 )
Z u azag a, Q ., and Ma, T .S ., I n d . Eng. Chem. A n a l. Ed. 14*2, 281
(1 9 4 2 ).
(1 9 5 1 )•
- 34 X.
AGKHOlMpOBMSIiTS
The a u th o r w ish e s t o ta k e t h i s o p p o r tu n ity t o e x p r e s s h i s s i n c e r e
a p p r e c i a t i o n and th a n k s t o B r. K enneth J . O o erin g f o r h ia p e rs o n a l
g u ld e n oe
.nd i n s p i r a t i o n d u r in g t h i s r e s e a r c h , and t o th e s t a f f o f th e
C h e m istry D ep artm en t f o r t h e i r many h e l p f u l s u g g e s tio n s .
He a ls o w ish e s
t o th a n k th e E e e e a ro h F o u n d a tio n f o r th e g r a n t w hich made t h i s i n v e s t i ­
g a tio n p o s s ib le .
M O N TA N A S T A T E U N IV E R SIT Y L IB R A R IE S
3
762 10004966 5
137465
B ru sk i, V. C.
amylase o f
t ** i
v /
\3
.
•'