Biochemical characterization of chitinase A from Bacillus licheniformis DSM8785 expressed in Pichia pastoris KM71H
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Chitin is an abundant biopolymer found mainly in the exoskeleton of crustaceans and insects. The degradation of chitin using chitinases is one way to address the accumulation of chitin waste streams in the environment, and research has therefore focused on the identification, improvement and expression of suitable enzymes. Here we describe the production, purification and characterization of Bacillus licheniformis chitinase A in the Pichia pastoris expression system. Optimal enzyme activity occurred at pH 4.0–5.0 and within the temperature range 50–60 °C. With colloidal chitin as the substrate, the Km (2.307 mM) and Vmax (0.024 mM min−1) of the enzyme were determined using a 3,5-dinitrosalicylic acid assay. The degradation products of colloidal chitin and hexa-N-acetylchitohexaose were compared by thin-layer chromatography. The activity of the glycosylated enzyme produced in P. pastoris was compared with the in vitro deglycosylated and aglycosylated version produced in Escherichia coli.... We showed that the glycosylated chitinase was more active than the deglycosylated and aglycosylated variants. © 2018 Elsevier Inc.
Keywords:Deglycosylation / Enzymatic assay / Molecular cloning / TLC
Source:Protein Expression and Purification, 2019, 154, 25-32
- This is peer reviewed version of the following article: Menghiu, G.; Ostafe, V.; Prodanović, R.; Fischer, R.; Ostafe, R. Biochemical Characterization of Chitinase A from Bacillus Licheniformis DSM8785 Expressed in Pichia Pastoris KM71H. Protein Expression and Purification 2019, 154, 25–32. https://doi.org/10.1016/j.pep.2018.09.007