Abstract

Protein engineering has been the most attractive strategy for biologists to redesign enzymes. As the simplest technique of protein engineering, directed evolution has been applied to many fields, such as industry, agriculture and medicine. An experiment of directed evolution comprises mutant libraries creation and screening or selection for enzyme variants with desired properties. Therefore, a successful application of directed evolution depends on whether or not one can generate a quality library and perform effective screening to find the desired properties. Directed evolution is already increasingly used in many laboratories to improve protein stability and activity, alter enzyme substrate specificity, or design new activities. Meanwhile, many more effective novel strategies of mutant library generation and screening or selection have emerged in recent years, and will continue to be developed. Combining computational/rational design with directed evolution has been developed as more available means to redesign enzymes.

Key words: Protein engineering, directed evolution, sequence diversity creation, novel strategy, computational design, rational design.

Abbreviation

Abbreviations: epPCR, Error-prone PCR; RID, random insertion/deletion mutagenesis; RPR, random priming recombination; StEP, staggered extension process; ITCHY, incremental truncation for the creation of hybrid enzyme; PCR, polymerase chain reaction; RACHITT, random chimeragenesis on transient templates; SHIPREC, sequence homology-independent site-directed chimeragenesis; MEGAWHOP, megaprimer polymerase chain reaction of whole plasmid; EP-RCA, error-prone rolling circle amplification; SeSaM, sequence saturation mutagenesis; RAISE, random insertion/deletion strand exchange mutagenesis; RCA, rolling circle amplification; YLBS, Y-ligation-based block shuffling; MUPREC, multiplex-polymerase chain reaction-based recombination; SISDC, sequence-independent site-directed chimeragenesis; NRR, non-homologous random recombination.