Studies On The Genetic Engineering Of Alginate Lyase And Trehalose Synthase

With the development of genetic engineering, recombinant biocatalyst becomes one of the powerful pathways for obtaining substances. It has been confirmed that a lot of oligosaccharides and digested polysaccharides had some biological functions which are interesting in industrial and agricultural applications. In food industry, some oligosaccharides and polysaccharides have been used as additives to improve nutritional and healthy value. In feed industry, polysaccharide-hydrolytic enzymes and oligosaccharides are widely used as dietary additives. Feed enzymes could improve animal performance and feed conversion efficiency and afford opportunities for new feed resources discovery. Presently, the majority of functional enzymes come from microorganisms, especially bacteria, thus we focus on the genetic engineering study of alginate lyases and trehalose synthase.In this thesis, two sorts of enzymes, alginate lyase with hydrolytic activity with algae polysaccharides as substrate and trehalose synthase with activity to convert maltose to trehalose, were studied. The mature alginate lyase coding sequences from Azotobacter chroococcum and Pseudomonas aeruginosa were cloned, expressed and the enzymes were characterized. In addition, the six trehalose synthase (TreS) producing strains were screened and isolated from a soil specimen obtained from the Tibetan plateau by degenerate PCR. A novel treS gene from Enterobacter hormaechei was amplified and expressed. These main results are formulated as follows:The sequence encoding the mature peptide of alginate lyase of Azotobacter chroococcum, called AcalgL, was cloned. This 1,056bp sequence was inserted into the modified Pichia pastoris vector pPIC9K-6HISEn5'and introduced into the host Pichia pastoris GS115 by PEG method. The purified recombinant AcAlgL had a specific activity of 2,968U/mg and a Mr of 41kDa. The recombinant AcAlgL displayed a maximum activity at 40°C and pH8.5. It was stable over the range of pH5～11. The activity of recombinant AcAlgL decreased by over 50% in presence of 10mM Cu²⁺ and Fe²⁺, furthermore, 10mM Co²⁺, Mn²⁺, Ca²⁺ and 1mM Fe²⁺ decreased it by 20%. The other cations including K+, Na+ and Zn²⁺ did not significantly affect its enzymatic activity.The mature alginate lyase coding sequences of P. aeruginosa, 1,023bp, was amplified and inserted into the modified pPIC9K expression vector. The positive construct was transformed to Pichia pastoris GS115 cells by spheroplasting method. The purified recombinant alginate lyase showed a Mr of 40kDa and a specific activity of 2,440U/mg at an optimal temperature 40°C and pH8.5, respectively. The recombinant PaAlgL was stable below 45°C at a wide range of pH3～12. The recombinant PaAlgL was sensitive to some metal cations negatively including Zn²⁺, Cu²⁺ and Fe²⁺, but Co²⁺ and Ca²⁺ promoted it dramatically. The antimicrobial activity of ampicilin and kanamycin agaist P. aeruginosa was increased by the help of PaAlgL.The gene of Enterobacter hormaechei TreS, containing a 1,626bp ORF encoding 541 amino acids, was cloned. The nucleotide sequence had been deposited in Genbank under No. FJ215664. The recombinant EhTreS had a Mr of 65kDa and a specific activity of 18.5U/mg. The EhTreS displayed a converting rate of 40% at the optimum condition of pH6 and 40°C. Hg²⁺, Zn²⁺, Cu²⁺and SDS inhibited the enzyme activity at different levels whereas Mn²⁺ shows an enhancing effect by 10%.This is the first report for expression of alginante lyases in Pichia pastoris system. It was also provided some detailed characters of Azotobacter chroococcum and Pseudomonas aeruginosa alginate lyases. Their activities are much higher than any other reports. In the research on TreS, the degenerate primers based on the conserved domains of trehalose synthases were proved to be effective for new gene screening. This result provides a prospective biochemical method for the trehalose production.