A Preliminary Study On Polyhydroxyalkanoate Synthases

Polyhydroxyalkanoate (PHA) are synthesized by many bacteria under theconditions of nutritional imbalance intracellular energy and carbon storagematerial.PHA has good biodegradable, biocompatible, water-repellent. The different functionalgroups are brought by the unique nature, PHA being used as drug delivery materialsand tissue engineering materials (medical biomaterials can be implanted in the body).PHA is a good potential for development of biological materials, and paid closeattention to the third generation of biological materials, it become the most activebiological materials research focus.However, the biosynthesis of PHA can not meet all areas of demand for newbiomaterials, and the low production and high cost of PHA attracted a greatresearchers concerned about trying toaddress this critical technical issues throughvarious means.PHA synthase is the key enzyme for PHA biosynthesis, use the protein engineeringtechnology to transform the PHA synthase to change the substrate specificity in orderto improve the catalytic activity, change the monomer composition. PHA is acurrentresearch focus. The chimeric enzyme is a fusion of two or more enzymes, andit can show the two pro-enzyme of the characteristics of this enzyme. In recent years,PHA synthase chimeric enzymes become a hot spot. The chimeric PHA synthaseapplication can extend the breadth of its substrate to increase PHA production,reducing production costs, make the PHA close to the chemical synthes of plastic andcan produce a new type of PHA. In the study of PHA chimeric enzyme also found theN-terminal of PHA synthase are related to the nature of the enzyme, such as substratespecificity, enzyme activity, heat-related. For the polymerization reaction, the finalpolymercomposition plays an important role, it can change the substrate specificity ofPHA synthase catalytic activity and other characteristics through transformation ofthe N-terminal.In the present study, we use the Aeromonas hydrophila, Pseudomonas corrugata and Pseudomona putida as the starting strain, delete the N-terminal, cloning andauthentication function PHA synthase. Recombinant bacteria produce the PHA withdifferent monomer composition from the wild bacteria.Results were as follow:1) A. hydrophila WQ of PHA synthase gene cloning type Ⅰ PHA synthaseN-terminal deletion mutant. Primers were designed to clone A.hydrophila WQ PHAsynthase, while the N-terminal2-28amino acid deletion mutant, the recombinantplasmid was transformed into E. coli S17-1, E. coli S17-1-mediated bindingtransformation, the recombinant plasmid pBBR1MCS2-phaC’JWQtransformed into R.eutropha PHB-4. Select different carbon sources in shake flask fermentation to verifythe functional activity of the PHA synthase with wild-type A. hydrophila WQ. Lauricacid as the carbon source when the bacteria the produce the highst PHA, about the65.05%of the dry cell weight, when fructose as a carbon source the recombinantbacteria produced only3HB a monomer, while the wild-type A. hydrophila bacteriaproduce produce about25%PHBHHx of cell dry weight.2) Wrinkles P.corrugata388phaC2reorganization of the functional verification ofplasmid PHA synthase. Our laboratory has been constructed wrinkling thepBBR1MCS2-phaC1,2expression plasmid, but not yet verified the synthasefunctional activity, in accordance with the relevant literature, this study continue touse fructose, glycerol, lauric acid, sodium gluconate, sodium oleate different carbonsources to verifythe the wrinkling pBBR1MCS2-phaC2synthase functional activity.The results showed that the recombinant bacteria can use sodium oleate as the carbonsource, produce1.23%of cell dry weight PHA.3) The stench of a simple cell strains of P. putida KT2442phaC2cloning. Theprimers were designed to clone the P. putida KT2442phaC2PHA synthase, therecombinant plasmid was transformed into E. coli BL21.To constructe the chimeric polyhydroxyalkanoate synthase, we first need to studythe N-terminal of PHA synthase for its function, clone and construct the expressionvector which used to construct the chimeric enzyme. On the basis of this mountain,we ensure that the chimeric enzyme can be constructed successfully. In this study, thedeletion mutant N-terminal of I PHA synthase was researched, demonstrated theN-terminal of PHA synthase, two kinds of type Ⅱ synthase gene were cloned, which in the future to ensure the chimeric synthase is successfully built.