| Polyhydroxyalkanoates (PHAs) are biological polyesters that are produced by awide variety of bacteria as an intracellular storage material of carbon and energywhen nitrogen is limiting while carbon is sufficient. PHAs have such uniqe propertyas biocompatible, renewable, biodegradable, optically active, piezoelectric and gasseparated, thus they are widely used in medicine, tissue engineering, cosmetic,agriculture, industry fields. However, PHA biosynthesis can not satisfy the demandfor new biomaterials due to low production, high costs and single component. Itseriously restricts the wide applications of PHA.The production of PHA mainly depends on the microbial fermentation. Thesubstrate is an important factor in microbial fermentation to the synthesis of PHA,accounting for28to50%of the total cost. It will largely reduce the cost of PHAsynthese from cheap substrate. Crude glycerol is the mainly by-product in theproduction process of biodiesel. PHA synthesis from glycerol can not only solve theproblems of environmental pollution which are brought by excessive accumulation ofwaste water, exhaust and others, but also reduce the cost of production of the PHA.However, PHA yield is seriously declined when crude glycerol is used to synthesizePHA. It may caused by the high osmotic pressure of glycerol according to thepublished results. How to solve high osmotic pressure of glycerol is the key of PHAsynthesis from crude glycerol. Enolase is a key enzyme in the glycolytic pathway. Itcatalyzes the dehydration of2-phospho-D-glycerate (G2P) to phosphoenolpyruvate(PEP). Enolase is a stress resistance gene which plays an important role in bacterialsurvival.To solve the problem of low PHA yield from crude glycerol, two aspects of workshave been done. On one side, glycerol tolerance of three strains were assessed, a goodstrains with high glycerol pressure tolerance were chosed. On the other side, anenolase gene was cloned from genome of P. putida KT2442, and a recombinantbacterial was constructed through gene engineering techniques to improve the abilityof high osmotic pressure of glycerol. To regulation of monomer composition of PHA,a chimeric enzyme was constructed using the SOE PCR technology, and PHA synthase gene from the R. eutropha (PhaCRe) and A. hydrophila WQ (PhaCAh) werechosed as parental enzymes. Results were as follows:(1) Assess of glycerol tolerance of strains. All bacterial strains (P.oleovorans ZJ03,P.oleovorans ZJ166, P. corrugata CY06) in our lab were cultured in MS mediumcontaining different glycerol concentration, PHA content was detected with GC.According to the result of GC, capacity of PHA production and glycerol tolerancecould be analsised. Result showed that P.oleovorans ZJ03glycerol tolerance wasbetter than the others.(2) Clone of enolase gene. Enolase gene was cloned from the genome of P. putidaKT2442by PCR. After Digesting and ligating, the vector was transformed into E.coliBL21(DE3) and confirmed by restriction enzyme digesting and DNA sequencing.The novel sequence of enolase was deposited in GenBank (JQ864251). Therecombinant was inoculated in LB mediums containing40μg/ml kanamycin untilOD600reached0.6,1mM isopropy-β-D-thiogalactoside (IPTG) was added, cell wascontinuously grown at30oC for4hours to induce expression proteins. Cells werecollected and lysised by ultrasonic, and proteins were purified through Ni2+-NTAagarose column. The molecular weight of protein was approximately46kD. Enolaseactivity was directly detected using spectrophotometer, and the enolase activity was510.9.(3) construction of a novel chimeric enzyme. Single component of PHA greatlyrestrics its wide applications. The construction of chimeric enzymes is a way to createnew enginered enzymes. PHA synthase plays a central role in PHA biosynthesis. Theactivity and substrate specificity of PHA synthase determine the productivity andmonomer composition of PHA. The engineering of PHA synthases can improve thesubstrate specificity and catalytic activity of the PHA synthase, change the monomercomposition of PHAs, obtain novel PHA.To construct a functional chimeric enzyme, PHA synthase gene from the R.eutropha (PhaCRe) and A. hydrophila WQ (PhaCAh) were chosed as parental enzymes.Secondary structure of PhaCReand PhaCAhwere predicted using Predtor、GOR4andSOPMA softwares so as to find the proper junction sites. With this junction sites, achimeric PHA synthase genes PhaCAhRewas obtained. After that, recombinant plasmid pBBR1MCS2-PhaCAhRewas constructed by ligating. Then recombinantplasmid was transformed into R. eutropha PHB-4with conjunction. Recombinant R.eutropha was cultured on mineral salt (MS) medium supplemented with differentcarbon sources in shaking flask at30℃,200rpm,72h. Dry cell was collecting,weighing cells and detecting PHA content with GC. Result showed that chimericenzyme produced3HB only when fructose and gluconate as carbon source. Wihle itproduced a new monomer3HO except3HB and3HHx when octanoate, lauric acid,oleic acid as sole carbon source, gluconate and octanoate as mixed carbon sourcecompared to both parental enzymes. It produced the highest PHA when Octanoate assole carbon source, about57.54%. And it produced the highest3HO when octanoateand gluconate as mix carbon source, about1.932%.In summary, in this study, a good strains which has high glycerol pressuretolerance was choosed, a new enolase was obtained. This has an importantsignificance to improve the tolerance of the strains to high osmotic pressure ofglycerol, and make it possible for the PHA synthesis from cheap biodiesel byproductglycerol. Thus, it can not only solve environmental problems caused by excessiveaccumulation of byproducts of biodiesel, but also reduce the cost of production of thePHA. At the same time, a chiemic enzyme was constructed which produced novelPHA when appropriate carbon sources were added. The study will laid a theoreticalfoundantion for the construction of chiemic enzyme with ability of PHA synthesis bygene engineering techniques and provides a method to improve PHA monomercomposition. It will largely expand the applications of PHA in the future. |
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