GS115-pPIC9k-ChiA4.0 Microencapsulated Engineered Bacteria Culture Studies

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) is an important chitinase gene, which could hydrolysis the chitin in the shell and the midgut of pest and the cell wall of fungus.The recombinant Pichia pastoris GS115-pPIC9k-ChiA4.0has been constructed successfully by constructing HaSNPV chitinase gene into vector pPIC9k and transferring into host Pichia pastoris GS115. It had all the advantages of Pichia pastoris, which could secrete extracellular chitinase by the induction of methanol. It is important in plant diseases and pest control.To take full advantaged of cell resuspension process in cell metabolism and reduce the process of transition phase by easily separating microcapsules from fermentation liquid.Alginate-chitosan (AC) microcapsules encapsulated recombinant Pichia pastoris GS115-pPIC9k-ChiA4.0were developed. The permeability of AC microcapsule for proteins was studied firstly. Then the growth and metabolism of encapsulated cells in different kinds of AC microcapsules were also stuied. Experimenta results were showed as follow:1. Proteins with a smaller molecular weight were easlier to through the membrane of AC microcapsules. The conditions of the formation of membrane were important for the permeability of AC microcapsules. AC microcapsules with a higher concentration chitosan solution, longer membrane formation time or chitosan with smaller molecular weight had a dense or thicker membrane, which lead the permeability of AC microcapsule for proteins were reduced. Therefore, chitinase could pass smoothly across by changing the membrane formation conditions.2. The AC microcapsules showed a biocompatibility for this kind of recombinant of cells which could grow and metabolize well in AC microcapsules.Due to the block of membranes of microcapsules,The growth and metabolism of cells were both slower than cells without encapsulation.There was an obvious stagnation phase of cell growth in the early train.3. Compared with cells without encapsulation, the growth of the encapsulationed cells had a same growth and metabolism rules. The growth of encapsulated cells was based on the permeability of microcapsule. The better the permeability was, the better the cells growth.4. The orthogonal experimental results showed that the under the condition of that the molecular weight of chitosan50,000, concentration of chitosan3.0mg/mL, membrane formation time10min, the enzyme activitites of chitinase reached to133.8U/mL, which was the highest than other conditions.5. The degraditon of chitin membrane essay showed that the production of encapsulated cells had a good catalytic activity to the chiton and would show good toxicity of tag for insect.Above all, the AC microcapsules can be an ideal reactor for the remmentant cells GS115-pPIC9k-ChiA4.0, which is meaningful for the improvement and formation process of biopesticite------chitinase.