(263e) Process and Enzyme Engineering of Aminotransferases for Improved Activity and Thermostability

Mártin-García, A. R., Michigan technological University
Shonnard, D. R., Michigan Technological University
Pannuri, S., Cambrex North Brunswick



Process and Enzyme Engineering of Aminotransferases for
Improved Activity and Thermostability


Abraham Rogelio Mártin García(1)(a),
David R. Shonnard(1)(b),
and Sachin Pannuri(2)(c)

1. Department of Chemical Engineering, Michigan Technological University

 2. Cambrex, North Brunswick, NJ

(a) armartin@mtu.edu





The production by biosynthesis of optically active amino
acids and amines satisfies the pharmaceutical industry in its demand for chiral
building blocks for the synthesis of various pharmaceuticals. Among several
enzymatic methods that allow the synthesis of optically active aminoacids and
amines, the use of aminotransferase is a promising one due to its broad
substrate specificity and no requirement for external cofactor regeneration.
The synthesis of chiral compounds by aminotransferases can be done either by
asymmetric synthesis starting from keto acids or ketones, and by kinetic
resolution starting from racemic aminoacids or amines.


The asymmetric synthesis of substituted (S)-aminotetralin,
an active pharmaceutical ingredient (API) has shown to have two major factors
that contribute to increasing the cost of production. These factors are the raw
material cost of biocatalyst used to produce it and product loss during biomass
separation. To minimize the cost contribution of biocatalyst and to minimize
the loss of product, two routes have been chosen in this research: 1. To engineer
the biocatalyst have greater specific activity 2. Improve the engineering of
the process by immobilization of biocatalyst in calcium alginate and addition
of cosolvents.


An (S)-aminotransferase was immobilized and used to produce
substituted (S)-aminotetralin at 50 °C and pH 7 in experiments where the
immobilized enzyme was recycled. Initial rate for cycle 1 (6 hr duration) was
determined, for cycle 2 (20 hr duration) it decreased by ~50% compared to cycle
1, and for cycle 3 (20 hr duration) it decreased by ~90% compared to cycle 1
(immobilized preparation consisted of 50 mg of spray dried cells per gram of
calcium alginate). Total product accumulation for each cycle decreased as well,
from 100% in cycle 1, 80% in cycle 2, and 30% after cycle 3. This enzyme was
determined to be deactivated at elevated temperatures during the reaction cycle
and was not stable enough to allow multiple cycles in its immobilized form.


A new enzyme was isolated by means of error-prone polymerase
chain reaction (PCR) and screening. This enzyme showed a significant
improvement in thermostability in comparison to previous enzyme. The new enzyme
was immobilized and tested under similar conditions.  Initial rate
remained fairly constant over four cycles (each cycle with a duration of about
20 hours) with the enzyme retaining almost 80% of initial rate in the fourth
cycle.  The final product concentrations after each cycle did not decrease
during recycle experiments. Thermostability of the new enzyme was much improved
compared to the previous enzyme. 


Under the same conditions as stated above, the addition of
co-solvents was studied. Toluene and sodium dodecyl-sulfate (SDS) were
used.   SDS at 0.01% (w/v) allowed four recycles of the immobilized
enzyme, always reaching higher product concentration than the system with
toluene at 3% (v/v). The initial rate of immobilized enzyme in a system with
SDS 0.01% (w/v) at 50 °C, pH 7 was retained for three cycles but dropped
precipitously in the fourth cycle. The final product concentrations for each
cycle also followed the same pattern although significant improvement of
immobilized enzyme productivity and stability were observed by one round of
mutagenesis; another observation demonstrated the limitations of an
immobilization strategy on reducing process economics.  After analyzing
the results of this experiment it was seen that a sudden drop occurred in
activity after the third recycle. This was due to a byproduct imine
accumulation inside the immobilized preparation (a reaction of product amine
with reactant ketone). In order to improve the economics of the process
research was focused on developing an enzyme with an even higher activity thus
reducing raw material cost as well as improve biomass separation.


A new enzyme was obtained using error-prone PCR and
screening derived from the previous improved enzyme. This enzyme was determined
to have three times the initial rate as the previous enzyme and had a higher
temperature optimum. This new enzyme was able to reduce enzyme loading in the
reaction by three-fold. In addition, the decrease in enzyme loading allows for
a better biomass separation leading to smaller product loss.   


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