Improvement of Bacillus circulans β-amylase activity attained using the ancestral mutation method

Abstract

Thermostabilization of enzymes is one of the greatest challenges of protein engineering. The ancestral mutation method, which introduces ancestral residues into a target enzyme, has previously been developed and used to improve the thermostabilities of thermophilic enzymes. Herein, we report a study that used the ancestral mutation method to improve the thermostability of Bacillus circulans beta-amylase, a mesophilic enzyme. A bacterial, common-ancestral beta-amylase sequence was inferred using a phylogenetic tree composed of higher plant and bacterial amylase sequences. Eighteen mutants containing ancestral residues were designed, expressed in Escherichia coli and purified. Several of these mutants were more thermostable than that of the wild-type amylase. Notably, one mutant had both greater activity and greater thermostability. The relationship between the extent to which the amino acid residues within 5 A of the mutation site were evolutionarily conserved and the extent to which thermostability was improved was examined. Apparently, it is necessary to conserve the residues surrounding an ancestral residue if thermostability is to be improved by the ancestral mutation method.

Deciphering Modern Glucocorticoid Cross-pharmacology Using Ancestral Corticosteroid Receptors

“…resurrection of an entire protein is not feasible, the insertion of ancestral residues in modern proteins can increase stability and enhance adaptability and tolerance to mutations (30). In addition, we have found that ancestral proteins tend to be more promiscuous to synthetic ligands or drug activation, especially in cases in which the ancestral proteins display a more promiscuous phenotype than the extant proteins for endogenous ligands.…”

Robust enzyme design: Bioinformatic tools for improved protein stability

“…Noteworthy, large alignments of 238-2813 proteins were used in this study to predict the consensus. After screening of 662 colonies the authors were able to identify variants with 8°C increase in T 50 60 . Finally, in a similar study random mutagenesis was complemented with a consensus approach to improve thermostability of Clostridium thermocellum endoglucanase [39].…”

“…Out of eight single and double variants produced, six showed up to 4.7°C increase in half-denaturation temperature. In a similar study thermostability of Bacillus circulans β-amylase was improved [50]. 20 ancestral mutations were selected based on 22 protein sequences.…”

“…In a similar work, a substrate-binding loop of a mesothermophilic polyol dehydrogenase from Deinococcus geothermalis was substituted by a corresponding highly flexible loop of a less thermostable homolog -galactitol dehydrogenase from Rhodobacter sphaeroides [79]. The substrate scope of the resulting chimera represented the average of both wild-type enzymes, and the thermostability was surprisingly improved (increase in T 50 60 by 7°C and 19.7°C, respectively). It was not clear how a highly mobile loop contributed to increased stability which is generally described as a gain of rigidity.…”

Site-Directed Mutagenesis as Applied to Biocatalysts

“…In others, point mutations were used to identify the residues that increased stability. They were then combined to increase the thermostability of the protein (Maxwell & Davidson, 1998;Nikolova et al, 1998;Yamashiro et al, 2010).…”