Control of product molecular weight in the enzymatic synthesis of a biodegradable polyester, polyhydroxybutyrate

Polyhydroxybutyrate (PHB) is a polymer accumulated by many bacterial species to store carbon and reducing equivalents. PHB is packaged intracellularly in inclusions termed "granules" that contain the polymer in an amorphous state, surrounded by a protein coat. The molecular weight (Mw) of PHB is typically in the range of 1-2 x 106 Da, and it shows the striking characteristic of low polydispersity, (narrow Mw distribution), implying that PHB synthesizing organisms have a mechanism to control PHB Mw. Several approaches have been undertaken in this work to uncover this mechanism of Mw control. An in vitro investigation was conducted, using purified PHA synthase, the native substrate (hydroxybutyryl-coenzyme A), and substrate analogs. The size of PHB synthesized in vitro was found to be dependent on the size of the substrate cofactor and that when hydroxybutyryl-N-acetylcysteamine was used as a substrate, the product was PHB covalently linked to N-acetylcysteamine. Preliminary experiments supported a model whereby substrate cofactor in thiolate form (e.g. -S-CoA) participates in a chain transfer reaction at an internal PHB ester within the enzyme, and the accessibility of the site at which this reaction takes place changes during the course of polymerization.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2006

Author dc:creator

• Lawrence, Adam G. (Adam Gunn)

Advisor dc:contributor.advisor

• Anthony J. Sinskey.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/34574

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/34574