Thermal Stabilization of Bacillus subtilis Family-11 Xylanase by Directed Evolution

Kentaro Miyazaki,Misa Takenouchi,Hidemasa Kondo,Natsuko Noro,Mamoru Suzuki,Sakae Tsuda

doi:10.1074/jbc.m511948200

Abstract

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.

Highlights

Protein engineering that can complement the weaknesses of the sitedirected approach [8]
Our approach included a global search for weakness using random mutagenesis, an extensive search for the best-fit residue by saturation mutagenesis, and rapid fitness improvement by DNA shuffling
Directed evolution does not require three-dimensional structures but does require a quick and sensitive screening system [10]. From this point of view, the high-throughput screening systems developed for this enzyme are a poor fit except for the clear zone assay [11]

Summary

Introduction

Protein engineering that can complement the weaknesses of the sitedirected approach [8]. We successfully obtained a thermostable variant that retained full activity at 60 °C for more than 2 h, whereas the wild type inactivated in ϳ5 min. Inactivation profiles of wild-type and Xylst enzymes were plotted by incubating the enzymes (0.1 mg/ml) at various temperatures (from 30 to 85 °C) for 10 min followed by determining the residual activity at 37 °C using 10 ␮l of enzymes in 90 ␮l of assay solution containing 0.1 M Mes-NaOH (pH 5.7) and 0.1% (w/v) soluble xylan.

Results

Conclusion