Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution.
We used directed evolution to enhance the thermostability of glycosyl hydrolase family-11 xylanase from Bacillus subtilis. By combining random point mutagenesis, saturation mutagenesis, and DNA shuffling, a thermostable variant, Xyl(st), was identified which contained three amino acid substitutions: Q7H, N8F, and S179C. The half-inactivation temperature (the midpoint of the melting curves) for the Xyl(st) variant compared with the wild-type enzyme after incubation for 10 min was elevated from 58 to 68 degrees C. At 60 degrees C the wild-type enzyme was inactivated within 5 min, but Xyl(st) retained full activity for at least 2 h. The stabilization was accompanied by evidence of thermophilicity; that is, an increase in the optimal reaction temperature from 55 to 65 degrees C and lower activity at low temperatures and higher activity at higher temperatures relative to wild type. To elucidate the mechanism of thermal stabilization, three-dimensional structures were determined for the wild-type and Xyl(st) enzymes. A cavity was identified around Gln-7/Asn-8 in wild type that was filled with bulky, hydrophobic residues in Xyl(st). This site was not identified by previous approaches, but directed evolution identified the region as a weak point. Formation of an intermolecular disulfide bridge via Cys-179 was observed between monomers in Xyl(st). However, the stability was essentially the same in the presence and absence of a reducing agent, indicating that the increased hydrophobicity around the Cys-179 accounted for the stability.
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. miyazaki-kentaro@aist.go.jp
Kentaro
K
Miyazaki
Misa
M
Takenouchi
Hidemasa
H
Kondo
Natsuko
N
Noro
Mamoru
M
Suzuki
Sakae
S
Tsuda
D001412:Bacillus subtilis / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D015723:Gene Library; D006358:Hot Temperature; D006859:Hydrogen / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D013696:Temperature; D013997:Time Factors
D017931:DNA Primers; D004220:Disulfides; D006859:Hydrogen; D004247:DNA; D043364:Endo-1,4-beta Xylanases; D003545:Cysteine