Authors: LiuTzea Tan Tomohiro Hiraishi Kumar Sudesh Mizuo Maeda
Publish Date: 2012/09/01
Volume: 97, Issue: 11, Pages: 4859-4871
Abstract
PolyR3hydroxybutyrate PHB depolymerase from Ralstonia pickettii T1 PhaZRpiT1 consists of three functional domains to effectively degrade solid PHB materials and its catalytic domain catalyzes the ester bond cleavage of the substrate We performed the directed evolution of PhaZRpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved pnitrophenyl butyrate pNPC4 activity Mutated PhaZRpiT1 genes generated by errorprone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface Some cells displaying the mutant enzymes showed a two to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wildtype enzyme These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five to eightfold higher pNPC4 hydrolysis activity than the wild type To further evaluate the mutation effects unfused and undisplayed enzymes were prepared and applied to the hydrolysis of pnitrophenyl esters having different chain lengths pNPCn n = 2–6 and PHB degradation One specific secondgeneration mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis although its PHB degradation efficiency at 1 μg/mL of enzyme concentration was approximately 35fold lower than that of the wild type Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved secondgeneration mutants indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residuesWe are grateful to the NBRP NIG Japan E coli for providing pVUB3 vector used in this work Special thanks go to the RIKEN BSI Research Resource Centre for the DNA sequencing services We are also greatly indebted to Dr Hisano for his invaluable advice on the modeled structure of PhaZRpiT1 This manuscript has been submitted for an English language review This research was supported by grants for Ecomolecular Science Research and Biomass Engineering Program from RIKEN Institute LT Tan is grateful to RIKEN IPA and USM Fellowship for the financial support
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