Biochemical Characterization Of The Insect Chitin Deacetylases BmCDA1 And OfCDA1

Chitin deacetylase (CDA), a member of carbohydrate esterase family 4, catalyzes the hydrolysis of acetylamino of chitin to generate chitosan. Insect chitin deacetylase 1 (CDAl) is mainly expressed in epidermis during molting, perhaps playing a critical role by participating in chitin assembling in the new cuticle. In this thesis, two insect CDAls including BmCDAl from Bombyx mori and OfCDAl from Ostrinia furnacalis were studied. The following is the results obtained:1. The cDNA sequences of CDA1 from B. mori and O. furnacalis were obtained by using RT-PCR. Both BmCDAl and OfCDAl consisted of an open reading frame of 1620bp and 1617bp respectively. Phylogenic analyses indicated that they both belonged to Group I insect CDA and shared an identity of 96% in similarity. Secquence analyses demonstated that both of them contained five conserved motifs, which were known in fungi and bacteria and were deduced to be responsible to catalysis.2. The tissue-and development-specific expression pattern indicated that BmCDAl was mainly expressed in epidermis and highly expressed at pupa day-1 during development, indicating it might play a vital role during pupation. The expression of BmCDAl was up-regulated at 12h after 20-hydroxyecdysone (20E) treatment, suggesting that the expression of BmCDAl can be regulated by 20E.3. BmCDAl and OfCDAl were cloned and tried to be heterogeneous expressed. In Escherichia coli, BmCDAl was expressed in the form of inclusion body. In Pichia pastoris, BmCDAl and OfCDAl were expressed in a soluble form after induction by methanol. They were then purified through a combination of ammonium sulfate precipitation and metal chelating chromatography.4. The activity of CDAs was determinated by fluorescamine and 3-methyl-2-benzothiazolinone. Unfortunately, the activity of BmCDAl was not detectable. However, BmCDAl possessed the ability of binding colloidal chitin.