Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes

Marshall, D. L.

English

NASA Technical Reports Server

May 1974	 B74-10057 
o	
NASA TECH BRIEF 
Ames Research Center 
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commercial application. Tech Briefs are available on a subscription basis from the National Technical Information 
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be directed to the Technology Utilization Office, NASA, Code KT, Washington, D.C. 20546. 
Enzymatic Regeneration of Adenosine Triphosphate Cofactor 
. 
is
The problem: 
Recent advances in enzyme technology have pro-
vided a new class of catalysts which may be of great 
use in industrial processes. Current applications of 
the new technology are limited to simple enzyme 
processes, where the cost of the expended cofactor is 
of little concern; however, if more complex enzymatic 
reactions are to be given consideration as candidates 
for large-scale continuous processes, it is first neces-
sary to develop techniques for regenerating the ex-
pensive cofactors in order to reduce catalyst costs 
to acceptably low values. 
The solution: 
Regenerate adenosine triphosphate (ATP) from 
adenosine diphosphate (ADP) by an enzymatic pro-
cess which utilizes carbamyl phosphate as the phos-
phoryl donor. 
How it's done: 
The reaction between ADP and carbamyl phos-
phate to form ATP is catalyzed by carbamyl phos-
phokinase; the overall reaction may be represented 
essentially as:
carbamyl 
ADP + NH2CO2PO,H 
phosphokinase 
ATP + [NH,CO2H]. 
A novel feature of the process is the extremely simple 
means of forming the carbamyl phosphate donor in 
the reaction medium. 
The enzyme, carbamyl phosphokinase, is isolated 
from Streptococcus faecalis and partially purified. To 
permit its reuse, it is immobilized by attachment to 
porous glass: Porous glass, obtained as the alkyl-
amine derivative, is treated for 30 minutes with a 2-
percent aqueous solution of glutaraldehyde and then 
washed thoroughly to remove excess glutaraldehyde.
The derivatized glass is added to a solution of the 
enzyme, the mixture stirred gently for 2 hours, and 
then the enzyme—glass product is washed free of Un-
reacted enzyme. The activity of the immobilized en-
zyme is 200 to 250 units per gram of glass (1 unit = 
1 micromole of ATP formed per minute at room 
temperature). 
The conversion of ADP to ATP takes place in a 
medium which contains potassium cyanate and potas-
sium phospate. Under these conditions, carbamyl 
phosphate forms spontaneously at room temperature 
and is utilized before decomposition can occur. Thus, 
when phosphate is used as buffer in the enzymatic 
reactions, only cyanate need be added along with 
ADP to regenerate ATP. 
A small column containing 0.5 gram of the im-
mobilized enzyme product was operated continuously 
for a period of 14 days. The solution pumped through 
the column was formed continuously from two stock 
solutions, one containing ADP, magnesium ions, and 
phosphate, and the other containing cyanate. The 
stock solutions were maintained at ice temperature 
and brought together in a mixing coil prior to enter-
ing the enzyme column. The column effluent was 
monitored periodically for ATP concentration by an 
enzymatic assay method. During the 14-day period, 
the activity of the column decreased by about 16 
percent. 
Note: 
No additional documentation is available. Specific 
questions, however, may be directed to: 
Technology Utilization Officer 
Ames Research Center 
Moffett Field, California 94035 
Reference: B74-10057
(continued overleaf) 
This document was prepared under the sponsorship of the National	 Government assumes any liability resulting from the use of the 
Aeronautics and Space Administration. Neither the United States	 information contained in this document, or warrants that such use 
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https://ntrs.nasa.gov/search.jsp?R=19740000057 2020-03-17T14:03:32+00:00Z
Patent status: 
NASA has decided not to apply for a patent. 
Source: David L. Marshall of 
Battelle Memorial Institute - Columbus Laboratories 
under contract to

Ames Research Center 
(ARC-10837)
. 
. 
B74-10057
	 Category 04


Enzymatic regeneration of adenosine triphosphate cofactor

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