text: The most recent and most clear cut example of enzyme evolution is the rise of bioremediating enzymes in the past 60 years. Due to the very low number of amino acid changes, these provide an excellent model to investigate enzyme evolution in nature. However, using extant enzymes to determine how the family of enzymes evolved has the drawback that the newly evolved enzyme is compared to paralogues without knowing the true identity of the ancestor before the two genes diverged. This issue can be resolved thanks to ancestral reconstruction.
  First proposed in 1963 by Linus Pauling and Emile Zuckerkandl, ancestral reconstruction is the inference and synthesis of a gene from the ancestral form of a group of genes, which has had a recent revival thanks to improved inference techniques and low-cost artificial gene synthesis, resulting in several ancestral enzymes—dubbed "stemzymes" by some—to be studied.
  Evidence gained from reconstructed enzyme suggests that the order of the events where the novel activity is improved and the gene is duplication is not clear cut, unlike what the theoretical models of gene evolution suggest.
  One study showed that the ancestral gene of the immune defence protease family in mammals had a broader specificity and a higher catalytic efficiency than the contemporary family of paralogues, whereas another study showed that the ancestral steroid receptor of vertebrates was an oestrogen receptor with slight substrate ambiguity for other hormones—indicating that these probably were not synthesised at the time.
  This variability in ancestral specificity has not only been observed between different genes, but also within the same gene family.
  In light of the large number of paralogous fungal α-glucosidase genes with a number of specific maltose-like (maltose, turanose, maltotriose, maltulose and sucrose) and isomaltose-like (isomaltose and palatinose) substrates, a study reconstructed all key ancestors and found that the last common ancestor of the paralogues was mainly active on maltose-like substrates with only trace activity for isomaltose-like sugars, despite leading to a lineage of iso-maltose glucosidases and a lineage that further split into maltose glucosidases and iso-maltose glucosidases. Antithetically, the ancestor before the latter split had a more pronounced isomaltose-like glucosidase activity.