Enzyme catalysts for a biotechnology-based chemical industry. Quarterly progress report, April 1--June 28, 1996 Page: 3 of 8
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The goal of this research is to engineer enzymes to be efficient and economically
attractive catalysts for the chemical industry. We are attempting to demonstrate generally-
applicable approaches to enzyme improvement as well as develop specific catalysts for
potential industrial application.
Progress during quarter April 1 -June 28, 1996
1. Random mutagenesis of pNB esterase: improved activity and stability.
A major goal of this DOE contract is to extend our basic enzyme engineering work
to developing specific catalysts of industrial importance. Thus part of our effort includes
a collaboration with Eli Lilly & Co. to engineer an esterase for use in antibiotics
production. This quarter has been devoted to further efforts to evolve a thermostable
esterase.
The thermostability of the wild type pNB esterase was measured by incubating the
enzyme at various temperatures for 1 hour and then chilling promptly in a ice bath.
Residual esterase activity was assayed at 23 "C using para-nitrophenyl acetate (pNPA).
The results show that the enzyme is half-deactivated around 50 C. The loss of enzyme
activity was irreversible under the conditions employed in this experiment. The kinetics of
thermal denaturation at 55 *C are shown in Figure 1. In this experiment we also compared
the inactivation rate of the purified protein with the protein inside the E. coli cells. Fig. 1
shows that inactivation rates inside and outside the cell are indistinguishable. This is an
important result since the goal is to eventually find enzyme variants that are more
thermostable outside the cell in purified form, while the screening reaction takes place
intracellularly. The irreversible thermoinactivation of the pNB esterase obeys first-order
kinetics, with a half-life of inactivation of 1.44 minutes. The screening assay described in
the previous report was developed on the basis of this information.
We have screened 690 pNB esterase mutants created by shuffling the genes of four
fourth-generation variants evolved for increased pNB esterase activity in 15% DMF. The
result of screening these mutants for thermostability is shown in Figure 2, where the clones
have been ordered in decreasing order of their Ra/la. A significant fraction of the shuffled
fourth-generation pNB esterases are more thermostable than the wild-type enzyme. These
have been isolated. During the next quarter we will attempt to recombine these mutations
in a single, improved enzyme.
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Arnold, F.H. Enzyme catalysts for a biotechnology-based chemical industry. Quarterly progress report, April 1--June 28, 1996, report, July 22, 1996; Pasadena, California. (https://digital.library.unt.edu/ark:/67531/metadc686692/m1/3/: accessed April 19, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.