Enzyme engineering allows to explore sequence diversity in search for new properties. The scientific literature is populated with methods to create enzyme libraries for engineering purposes, however, choosing a suitable method for the creation of mutant libraries can be daunting, in particular for the novices. Here, we address both novices and experts: how can one enter the arena of enzyme library design and what guidelines can advanced users apply to select strategies best suited to their purpose? Section I is dedicated to the novices and presents an overview of established and standard methods for library creation, as well as available commercial solutions. The expert will discover an up-to-date tool to freshen up their repertoire (Section I) and learn of the newest methods that are likely to become a mainstay (Section II). We focus primarily on in vitro methods, presenting the advantages of each method. Our ultimate aim is to offer a selection of methods/strategies that we believe to be most useful to the enzyme engineer, whether a first-timer or a seasoned user.
Keywords: enzyme engineering; genetic diversity; library creation methods; sequence space exploration; smart library design.
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