Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes

Billingham, J.

Lyn, J.

English

NASA Technical Reports Server

November 1971	 Brief 71-10443 
NASA TECH BRIEF 
Ames Research Center
	
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IIM 
Insolubili zation Process Increases Enzyme Stability 
phate dehydrogenase (GAPiY) and fructose-1,6-
diphosphatase (FDP-ase) may be attached to poly-
meric matrices and that the enzymes retain a portion 
of their catalytic activities. Three different carrier 
materials were used to form these enzyme-polymer 
adducts: ethylene maleic anhydride (EMA), p-ami-
nobenzyl cellulose (PAB), and aminoethyl cellulose 
(A EC). 
The stability of ins olubilized enzyme derivatives 
is markedly greater than that of soluble enzymes. 
For example, aldolase-EMA in either powder or 
suspension form was found to be stable for a period 
of 20 days at 4° C; in contrast, soluble aldolase lost 
more than 50% of its activity in 15 days. A suspen-
sion of aldolase-AEC was found to be stable over a 
period of 21 days. The soluble form of GAPD be-
came completely deactivated overnight; in contrast, 
the GADP-AEC retained most of its enzymatic 
activity after 1 day. 
Studies of the range of optimum pH values re-
vealed that the enzyme-polymer adducts were sig-
nificantly different from their progenitors. In 
general, the attachment of enzymes to basic poly-
mers resulted in shifts of optimal pH to lower 
values, and the attachment to acidic polymers resulted 
in shifts to higher values. For instance, soluble 
aldolase exhibited optimum activity at pH 8, but 
the modified forms were different: aldolase-PAB, 
pH 6.0; aldolase-AEC, pH 6.5; and aldolase-EMA, 
ph 9.0. Soluble GAPD exhibited optimum activity 
at ph 9.5, but the GAPD-AEC exhibited an opti-
mum at p1-I 7.0. The optimum of soluble FDP-ase, 
ph 9.2, was shifted to p1-I 8.5 when attached to AEC. 
Studies of the reaction between the insolubilized 
adducts and their substrates demonstrated that the 
Km value was determined by the type of polymer 
(continued overleaf) 
Nur
Enzymes rendered insoluble by attachment to 
polymeric matrices have properties which suggest 
novel application to enzyme-controlled reactions. 
The modified enzymes may have properties that are 
significantly different from those of the original 
enzymes; in particular, the stability of several modi-
fied enzymes is greater. Moreover, the physical form 
of the insolubilized enzymes makes them useful in 
column processes where the substrate is passed 
through a bed; in batch processes, the enzymes can 
easily be recovered for reuse. More importantly, it 
is possible to exercise control over enzymatic func-
tions by the use of appropriate polymeric matrices, 
because the p1-I optima and Km (equilibrium con-
stant) values of insolubilized enzymes are determined 
by the type of polymers to which they are attached. 
In recent years, a large number of enzymes have 
been complexed with polymeric matrices by a va-
riety of synthetic techniques. Almost exclusively, 
however, only low molecular weight, hydrolytic 
enzymes of relatively simple structure have been 
modified by these techniques. It has been difficult 
to synthesize polymer adducts with enzymes of 
complex structure, particularly the enzymes of the 
Calvin cycle, which are sensitive to chemical rea-
gents. 
Three of the large-molecule enzymes of the Calvin 
cycle have been successfully incorporated into 
polymeric matrices by the optimization of synthetic 
techniques. Extension of these techniques to other 
enzymes of the Calvin cycle should lead to the 
structuring of enzyme-packed columns capable of 
converting metabolic wastes into edible products; 
e.g., carbon dioxide into fructose or glucose. 
Experimental results indicate that large enzyme 
molecules such as aldolase, glyceraldehyde-3-phos-
This document was prepared under the sponsorship of the National 
Aeronautics and Space Administration. Neither the United States 
Government nor any person acting on behalf of the United States
Government assumes any liability resulting from the use of the 
information contained in this document, or warrants that such use 
will be free from privately owned rights.
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to which the enzyme was attached. When the en-
zyme was attached to basic polymers, the K, values 
were lower than those of the soluble enzymes. Con-
versely, the Km values of the enzyme-acidic polymer 
adducts were higher than those of the soluble en-
zymes. 
The insolubilized enzyme adducts formed between 
aminoethyl cellulose and each of the three enzymes 
contained the largest amounts of protein and re-
tained most of the original enzyme activities. These 
adducts were remarkably stable at room tempera-
ture. A sequential reaction using columns of aldo-
lase and GAPD was demonstrated. The substrate 
for the aldolase column was fed at the top, and the 
eluate became the substrate for the GAPD column. 
The result of the reaction was visually demon-
strated by the disappearance of the blue color of 
methylene in the presence of diaphorase activity. 
Notes: 
I. The following documentation may be obtained 
from: 
National Technical Information Service 
Springfield, Virginia 22151 
Single document price $3.00 
(or microfiche $0.95)
Reference: 
NASA CR-73354 (N70-23428), Studies on 
the Optimization of Techniques for 
Enzyme Insolubilization. 
2. Requests for further information may be directed 
to:
Technology Utilization Officer 
Ames Research Center 
Moffett Field, California 94035 
Reference: B71-10443 
Patent status: 
No patent action is contemplated by NASA. 
Source: J. Billingham
Ames Research Center, and
J. Lyn of
Battelle Memorial Institute 
under contract to

Ames Research Center

(ARC-10314) 
Brief 71-10443	 Category 04


Insolubilization process increases enzyme stability

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