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. Study holds ProTherm entries: 25771, 25772, 25773, 25774, 25775, 25776, 25777, 25778, 25779, 25780, 25781, 25782, 25783, 25784, 25785, 25786 Extra Details: b-amylase; ancestral mutation method; Bacillus circulans mesophile; thermostabilization
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:56 p.m.
|Number of data points||46|
|Assays/Quantities/Protocols||Experimental Assay: activity prot_conc:0.2 mg/mL ; Experimental Assay: Tm prot_conc:0.2 mg/mL ; Experimental Assay: activity prot_conc:0.1 mg/mL ; Experimental Assay: Tm prot_conc:0.1 mg/mL ; Derived Quantity: dTm prot_conc:0.2 mg/mL ; Derived Quantity: dTm prot_conc:0.1 mg/mL|
|Libraries||Mutations for sequence MLHSQKRIWKKIGLCLLSFILGITVFTGSFGSKAEAAVAGDFQVSVMGPLAKVTDWNSFKNQLTTLKNNGVYAITTDVWWGYVESAGDNQFDWSYYKTYADTVKQAGLKWVPIISTHRCGGNVGDDCNIPLPSWLWSKGSADEMQFKDESGYVNNESLSPFWSGVGKQYDELYASFAQNFSAYKDMIPKIYLSGGPSGELRYPSYYPAAGWSYPARGKFQVYTETAKSAFRTAMTTKYGSLDKINAAWGTNLTSMSQISPPTDSDGFYTGGGYNITYGKDFLSWYQSVLENHLGVIGAAAHKNFDPVFGVRIGAKISGIHWQMNNPSMPHSAEHAGGYYDYNRLIQKFKDTDLDLTFTALEMYDSGTAPNYSLPSTLVDTVSSIANSKGVRLNGENALPTGGSGFQKIEEKITRFGYNGFTLLRINNIVNSDGSPTAEMSSFKNYVIKHAKPAGDGGGNPVNSVTIYYKKGFNSPYIHYRPAGGTWTDVPGVKMPDSEISGYAKITLDIGSASQLEAAFNDGNNQWDSNNMRNYFFSPGTSTYIPGTNGTAGSIQAGPPITSSDFQALPAYEMSI|