Cytophaga Hutchinsonii ¦Á-isopropyl Malic Acid Synthase Enzymatic Properties

Lignocellulose is widespread and cheap renewable resources, and its main component cellulose and hemicelluloses are a potential raw material of fuel alcohol. A large number of microbes degrading cellulose occur in the nature, such as bacteria, actinomycetes, fungi, etc. Cytophaga hutchinsonii belonging to genus Cytophaga, is of interest because of its high biodegradative abilities, unique digestion mechanism and gliding motility. So far the research focuses on the cellulose degradation mechanism and gliding motility, but few reports are related to the utilization to produce biofuel.Conventional anaerobic fermentation with Clodtridium only produces 1-butanol, but the yield is not ideal. By using metabolic engineering technology including engineering the amino acid biosynthesis in E. coli, various alcohols with three to five carbon atoms in molecular chains could be produced and the yield of isobutanol with good characteristics is higher, which makes it possible to produce all kinds of higher alcohols. During engineering amino acid synthesis to produce isobutanol, a-isopropylmalate synthase (a-IPMS), which is used in pathway of leucine biosynthesis, is especially important. It is encoded by leuA gene and could catalyze intermediate a-ketoisovaleric acid to react with acetyl coenzyme and form a-isopropylmalate; after that it is catalyzed to a-keto acid alien by a-isopropylmalate dehydratase and isopropylmalate dehydrogenase. This enzyme system also catalyzes a-ketobutyrate to form a-ketoisovaleric acid.a-ketoisovaleric acid could produce new fuel isobutanol with promising application by the catalysis of a-ketoacid decarboxylase (KDC) and alcohol dehydrogenase (ADH).The purpose of the study is to research enzymatic properties of a-isopropylmalate synthase for further exploration of metabolic engineering in C. hutchinsonii. Blasting in Gene bank, we found an operon of leu ABCD in C. hutchinsonii genome. We got Leu A fragment by PCR and expressed it in E. coli. At the same time we expressed a-IPMS from E. coli as a control. Comparing enzymatic properties, we found that a-IPMS from E. coli had a lower Km and a higher catalytic efficiency. We created three mutant proteins according to homology modeling results and expressed and purified them. Compared to wild type, enzymatic activity decreased apparently. This indicated these three amino acids played an important role to catalytic activity.