Chitinase from streptomyces griseus

Intracellular trehalose level was determined as previously described [51 (link)]. For accurate chitin determination in the yeast cell wall, an enzyme assay has been used as follows. Lyophilized cell walls (about 10 mg) were suspended in 200 μl of 50 mM potassium acetate buffer, pH 5.0 and boiled at 65°C for 5 min. After mixing and cooling to ambient temperature, the cell wall suspension was treated with 1U of chitinase from Streptomyces griseus (Sigma-Aldrich, France) for 24 h at 37°C. The N-acetyl glucosamine released by the chitinase action was then determined using a colorimetric method as described by Reissig et al. [52 (link)] and adapted for a micro method. Briefly, 125 μl of the enzymatic mixture was heated with 25 μl of 0.8 M potassium tetraborate pH 9.0 at 100°C for 8 minutes. After cooling at room temperature, 750 μl of Reissig reagent (10 g of 4-dimethylaminobenzaydedyde dissolve in 12.5 mL 10 N HCl and 87.5 mL of glacial acetic acid) diluted ten times in deionized water was added, and the tubes were incubated 40 minutes at 37°C. The absorbance was read at 585 nm. The chitin content was obtained from N-acetylglucosamine standard curve (from 0 to 100 μg/mL) made in the same condition.

Eggs were suspended in egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 25 mM HEPES, pH 7.3) before applying shell treatments⁴⁵ . Untreated eggs were taken directly from this suspension. Bleach treatment was performed in a 0.27 M NaOH + 2% NaOCl solution (bleach) for 2 or 5 min, respectively. Egg samples were then washed three times by repeated centrifugation (1680 rcf for 60 s) and resuspended in egg buffer by vortexing. Chitinase-treated shells were obtained by suspending 2-min bleach-treated eggs in egg buffer containing 5 mg mL⁻¹ Chitinase from Streptomyces griseus (Sigma-Aldrich, Buchs, Switzerland) until the start of the experiments (i.e., for ∼15 min).

Samples of R. prolixus first instar nymphs and adult males; adult males and females of L. longipalpis; and adult males and females of A. aegypti were prepared (see item “Preparation of Samples” for details). Aliquots of 70 μL were separated and combined with 70 μL of 1 mg/mL chitinase from Streptomyces griseus (C6137, Sigma) in 50 mM potassium phosphate, pH 6.0, 20 mM sodium azide. Control groups consisted of tubes containing samples and 70 μL of the same buffer. Samples were incubated at 37°C for 18 h, then filled to 350 μL with distilled water and aliquots were taken for chitin content determination following both protocols.

Materials for chromatography have been described elsewhere [10 (link)]. All other reagents and chemicals were of analytical grade (Merck Life Science S.r.l., Milan, Italy). Nuclease-treated rabbit reticulocyte lysate system was purchased from Promega (Madison, WI, USA). Chitinase from Streptomyces griseus (product number Sae0158) was purchased from Sigma-Aldrich solutions (Merck Life Science S.r.l.).