Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D.
Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.
Laboratory of Molecular Biotechnology, Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, Japan.
Jasmina
J
Damnjanović
Rie
R
Takahashi
Atsuo
A
Suzuki
Hideo
H
Nakano
Yugo
Y
Iwasaki
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D006361:Heating; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism*; D010739:Phospholipase D / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013303:Streptomyces antibioticus / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D001426:Bacterial Proteins; D010716:Phosphatidylinositols; D010739:Phospholipase D