Continuous processes for enzymatic production of carbohydrates from glucose are discussed. Key reactant in process is identified as phosphorylase which catalyzes reversible formation or degradation of polysaccharide. Chemical compounds and reactions to synthesize polysaccharides are analyzed

Marshall, D. L.

English

NASA Technical Reports Server

September 1972	 B72-10550 
S	 NASA TECH BRIEF 
Ames Research Center 	 -4r 
NASA Tech Briefs announce new technology derived from the U.S. space program. They are issued to encourage 
commercial application. Tech Briefs are available on a subscription basis from the National Technical Information 
Service, Springfield, Virginia 22151. Requests for individual copies or questions relating to the Tech Brief program may 
be directed to the Technology Utilization Office, NASA, Code KT, Washington, D.C. 20546. 
Immobilized Phosphorylase for Synthesis of

Polysaccharides from Glucose 
Continuous processes for the enzymatic production 
of carbohydrates from glucose require that enzymes 
be immobilized or rendered insoluble so that they 
can be readily separated from process streams. A 
number of enzymes have been successfully immobil-
ized, but not of the type which form high molecular 
weight polysaccharides. 
One of the key reactants in a system leading to 
carbohydrate synthesis is a phosphorylase which cat-
alyzes the reversible formation or degradation of 
polysaccharide (or starch-like) material; in a contin-
uous process, the enzyme is preferably immobilized 
by attachment to a water-insoluble support. For a 
systematic program of research, colloidal silica—
cellulose and a porous "alkylamine glass" were se-
lected as the insoluble substrates for immobilization 
of phosphorylase. Two different methods were devel-
oped for immobilizing phosphorylase on the sub-
strates, and it was found that the products have widely 
different binding capacities as well as physical prop-
erties' It was also found that the amount of enzyme 
that can be bound to a unit weight of porous glass is 
less than can be bound to a silica—cellulose support; 
of greater interest, however, is the finding that the 
physical properties of the alkylamine glass product 
are superior for the flow-through operations involved 
in a practical process for forming polysaccharides. 
Fumed silica was treated with polyethyleneimine 
to form an intermediate layer which could absorb 
purified potato phosphorylase; after treatment with 
the enzyme, glutaraldehyde was crosslinked to the 
product, and the composite was treated with an
active-ester cellulose to form an immobilized phos-
phorylase—siica—cellulose complex. 
Alkylamine glass, with an essential surface charac-
ter consisting of (H2N-R-Si-O—) groupings, was 
treated with glutaraldehyde until all active groupings 
had been derivatized. The glutaraldehyde—glass 
product was washed with water and then treated 
with a solution of phosphorylase to form an immobil-
ized complex. 
The activity of each of the immobilized phospho-
rylase products was determined in buffered solutions 
of glucose-i-phosphate containing a specially fraction-
ated amylopectin (primer). The primer is a polysac-
charide onto which the enzyme adds successive glu-
cose residues; the concentration and size of the primer 
controls the molecular weight of the polysaccharide 
formed by the immobilized phosphorylase. The silica 
—cellulose—phosphorylase complex is about five times 
more active than the corresponding glass complex. 
The phosphorylase—glass complex was made into 
a column and then used to catalyze the formation of 
polysaccharide from glucose on a continuous basis. 
The column was maintained at 45°C to enhance the 
reaction rate and solubility of the polysaccharide 
product. When the enzyme column was operated 
under conditions which produced a product too high 
in molecular weight, a polysaccharide coating formed 
on the glass surface and decreased the apparent 
activity of the column. This coating could be removed 
by a starch-hydrolyzing enzyme (glucoamylase); the 
treatment restored the column efficiency nearly to the 
original level.
(continued overleaf) 
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.
https://ntrs.nasa.gov/search.jsp?R=19720000549 2020-03-17T03:34:44+00:00Z
Notes: 
1. The alkylamine glass can be treated with 2-amino-
4,6-dichloro-s-triazine followed by phosphorylase 
in borate buffer to give another form of an im-
mobilized phosphorylase—glass complex, but it is 
not as active as the complex described above. 
2. Requests for further information may be directed 
to:
Technology Utilization Officer 
Ames Research Center 
Moffett Field, California 94035 
Reference: TSP 72-10550
Patent status: 
No patent action is contemplated by NASA. 
Source: David L. Marshall of 
Battelle Memorial Institute/Columbus Laboratories 
under contract to 
Ames Research Center 
(ARC-10680)
. 
B72-10550	 Category 04


Immobilized phosphorylase for synthesis of polysaccharides from glucose

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