Biotechnologically Relevant Enzymes and Proteins

Applied Microbiology and Biotechnology

, Volume 79, Issue 1, pp 69-75

Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR

  • Moon-Soo KimAffiliated withDepartment of Animal Science and Graduate field of Food Science, Cornell UniversityDepartment of Medicinal Chemistry and Molecular Pharmacology, Purdue University
  • , Xin Gen LeiAffiliated withDepartment of Animal Science and Graduate field of Food Science, Cornell UniversityDepartment of Animal Science, Cornell University Email author 

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Abstract

Phytases are used to improve phosphorus nutrition of food animals and reduce their phosphorus excretion to the environment. Due to favorable properties, Escherichia coli AppA2 phytase is of particular interest for biotechnological applications. Directed evolution was applied in the present study to improve AppA2 phytase thermostability for lowering its heat inactivation during feed pelleting (60–80°C). After a mutant library of AppA2 was generated by error-prone polymerase chain reaction, variants were expressed initially in Saccharomyces cerevisiae for screening and then in Pichia pastoris for characterizing thermostability. Compared with the wild-type enzyme, two variants (K46E and K65E/K97M/S209G) showed over 20% improvement in thermostability (80°C for 10 min), and 6–7°C increases in melting temperatures (T m). Structural predictions suggest that substitutions of K46E and K65E might introduce additional hydrogen bonds with adjacent residues, improving the enzyme thermostability by stabilizing local interactions. Overall catalytic efficiency (k cat / K m) of K46E and K65E/K97M/S209G was improved by 56% and 152% than that of wild type at pH 3.5, respectively. Thus, the catalytic efficiency of these enzymes was not inversely related to their thermostability.

Keywords

Thermostability Phytase Error-prone PCR Melting temperature Enzyme