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J. Biol. Chem., Vol. 279, Issue 12, 11495-11502, March 19, 2004

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Improved Catalytic Efficiency and Active Site Modification of 1,4--D-Glucan Glucohydrolase A from Thermotoga neapolitana by Directed Evolution^*

James K. McCarthy, Aleksandra Uzelac, Diane F. Davis, and Douglas E. Eveleigh

From the Department of Biochemistry and Microbiology, Cook College, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901

Thermotoga neapolitana 1,4--D-glucan glucohydrolase A preferentially hydrolyzes cello-oligomers, such as cellotetraose, releasing single glucose moieties from the reducing end of the cello-oligosaccharide chain. Using directed evolution techniques of error-prone PCR and mutant library screening, a variant glucan glucohydrolase has been isolated that hydrolyzes the disaccharide, cellobiose, at a 31% greater rate than its wild type (WT) predecessor. The mutant library, expressed in Escherichia coli, was screened at 85 °C for increased hydrolysis of cellobiose, a native substrate rather than a chromogenic analog, using a continuous, thermostable coupled enzyme assay. The V_max for the mutant was 108 ± 3 units mg^-1, whereas that of the WT was 75 ± 2 units mg^-1. The K_m for both proteins was nearly the same. The k_cat for the new enzyme increased by 31% and its catalytic efficiency (k_cat/K_m) for cellobiose also rose by 31% as compared with the parent. The nucleotide sequence of two positive clones and two null clones identified 11 single base shifts. The nucleotide transition in the most active clone caused an isoleucine to threonine amino acid substitution at position 170. Structural models for I170T and WT proteins were derived by sequence homology with Protein Data Bank code 1BGA from Paenibacillus polymyxa. Analysis of the WT and I170T model structures indicated that the substitution in the mutant enzyme repositioned the conserved catalytic residue Asn-163 and reconfigured entry to the active site.

Received for publication, May 29, 2003 , and in revised form, December 4, 2003.

^* This work was supported by United States Department of Agriculture Competitive Grant 2001-35504-10158, the McIntyre-Stennis Program, a National Institutes of Health and Rutgers University Biotechnology Training Fellowship (to J. K. M.), and the NJ Agricultural Experiment Station Grant K-NJ01133-01-04. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0202.

To whom correspondence should be addressed. E-mail: eveleigh{at}aesop.rutgers.edu.

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