Production of hydroxyalkanoates for pharmaceutical and medical applications

Abstract

The replacement of traditional plastic by biodegradable plastic, polyhydroxyalkanoates (PHAs) is proposed to enhance the valuable plastic applications and to minimize its side-effects. However, the production cost of PHAs is too high at present, thus hampering the development of its manufacturing processes and widespread applications. The main aim of this research project is to increase the efficiency of PHAs production. Measures to reduce the cost of PHAs production were proposed. The potential application of PHAs in tissue engineering and drug delivery system was also assessed. Two recombinant plasmids pBE2C1 and pBE2C1AB were constructed and then these two plasmids were transformed into Bacillus subtilis DB104 to generate Bacillus subtilis/pBE2C1 and Bacillus subtilis/pBE2C1AB, respectively. The two recombinants strains were subjected to fermentation and showed PHAs accumulation, which was the first report of production of mcl-PHAs in Bacillus subtilis. The result of Gas Chromatograpy (GC) analysis suggested that the product produced by Bacillus subtilis/ pBE2Cl was identified to be a hydroxydecanoate-co-hydroxydodecanoate (HD-co-HDD) polymer while the product produced by Bacillus subtilis/pBE2C1AB was identified to be a hydroxybutyrate-co-hydroxydecanoate-co-hydroxydodecanoate (HB-HD-HDD) polymer. The polyhydroxyalkanoate synthase gene pha, from Bacillus thuringiensis HD-81, was cloned using Polymerase Chain Reaction (PCR) cloning strategy based on the pha loci property. The complete open reading frames (ORFs) were identified from the PCR products. Using the sequence information, the complete PHAs synthase gene was PCR cloned directly from the genomic deoxyribonucleic acid (DNA) and expressed in Escherichia coli confirmed by Fourier Transform Infrared (FTIR) and GC. It was also discovered that the recombinant E. coli yielded higher PHAs content (13.4 %) under the C: N ratios at 50:1. When the concentration of citric acid was 0.5 g/L, 16.72 % PHAs was obtained. With 0.5 g/L of tryptone, 11.03 % PHAs was recovered. A CDW of 8.62 g/L with 10.8 % PHAs was achieved when 2 g/L yeast extract was added. The PHAs content (14.91 %) was recorded when the recombinant E. coli was fed with sodium acetate. Food wastes including tea waste, sugar cane waste, contaminated baked bread and contaminated candies were proposed to act as carbon source for recombinant E. coli nutrient feeding to optimize the PHAs production. The contributions are not only to promote the use of biodegradable plastic but also effectively utilize of the wastes simultaneously. The achievement of this section of study was the optimization of PHAs production by using four proposed food wastes. The results showed that food wastes are the potential carbon source for PHAs production, and the pretreatment methods of waste materials could be further developed for PHAs production and commercial applications. Protein drug delivery has been one of the very intensive studies of PHAs. In this study, PHAs produced by bacteria was proved that it can be used to produce nanoparticle and load with pyrene. It was found that PHAs nanoparticles had high loading capacity, high loading efficiency and low toxicity and they released in a stable manner which had very high potential for protein drug delivery. Valvular heart disease is a significant cause of morbidity and mortality world-wide. Classical replacement surgery involves the implantation of mechanical valves or biological valves. Tissue engineering of heart valves has represented as a new experimental concept to improve current modes of therapy in valvular heart surgery. Preliminary investigation of the PHAs heart valves was also done in this study; it showed that PHAs could be an alternative material of heart valve for tissue engineering.