Enzymatic and molecular characterization of an acidic and thermostable chitinase 1 from <i>Streptomyces thermodiastaticus</i> HF 3-3

Abstract

Chitinase 1 (Chi1) is an acidic and thermostable hydrolytic enzyme capable of the breakdown of chitin, a resilient biopolymer that is the primary building block of fungi cell walls and marine exoskeletons. In this study, Chi1 was purified from the bacterium Streptomyces thermodiastaticus HF 3-3, and its properties were carefully characterized. The molecular mass of Chi1 was estimated to be approximately 46 kDa and, through sequencing, its N-terminal amino acid sequence was identified as ADSGKVKL. Although the optimal operating temperature and pH for Chi1 were determined to be 65˚C and pH 5.5, respectively, the purified enzyme was stable over wide pH (1.5-9) and temperature ranges. Moreover, Chi1 retained 87% of its activity in the presence of 15% NaCl. While Chi1 activity was inhibited by Ag + and Mn 2+ , other chemicals tested had no significant effect on its enzymatic activity. The K m and V max values of Chi1 for the substrate colloidal chitin were 1.23 ± 0.7 mg/mL and 6.33 ± 1.0 U/mg, respectively. Thin-layer chromatography analysis of the enzymatic reaction end products mainly detected diacetylchitobiose. We also cloned the Chi1 gene and purified the recombinant protein; the properties of the recombinant enzyme were nearly identical to those of the native enzyme. Therefore, Chi1 purified from S. thermodiastaticus HF 3-3 is unique, as it is highly stable under broad range of pH values, temperatures, and chemical exposures. Combined, these properties make this enzyme attractive for use in the industrial bioconversion of chitin.

Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002

“…Organic solvents are used to solubilize hydrophobic substrates in enzymatic assays. The activity of purified CHI was also obviously increased by the presence of Tween 80, similar to chitinase from S. thermodiastaticus HF 3-3 (Take et al, 2018). This indicates that Tween 80 reduces the activation energy of the reaction, promoting the enzymatic reaction.…”

“…Chitinases can be subdivided into three families based on their structural motives: the glycoside hydrolase families 18, 19 and 20 (Take et al, 2018). Chitinases can be further classified as endochitinases, exochitinases, or N-acetylglucosaminases (NAGases) based on the mechanism of chitin hydrolysis.…”

Chemoenzymatic Production and Engineering of Chitooligosaccharides and N-acetyl Glucosamine for Refining Biological Activities

“…A 46kDa Chi1 was purified from Streptomyces thermodiastaticus HF 3-3 with specific activity of 2.4 U mg −1 . The enzyme was found to be remarkably stable over a wide range of pH, temperatures, and chemical exposures (Take et al, 2018 ). Moreover, thin-layer chromatography (TLC) analysis of CC cleavage products revealed that Chi1 exhibited endo- pattern of cleavage with the generation of (GlcNAc) 2 as the major product and minimal amounts of (GlcNAc) 3 and GlcNAc (Take et al, 2018 ).…”

“…The enzyme was found to be remarkably stable over a wide range of pH, temperatures, and chemical exposures (Take et al, 2018 ). Moreover, thin-layer chromatography (TLC) analysis of CC cleavage products revealed that Chi1 exhibited endo- pattern of cleavage with the generation of (GlcNAc) 2 as the major product and minimal amounts of (GlcNAc) 3 and GlcNAc (Take et al, 2018 ). Extracellular chitinases production and its utilization for COS and GlcNAc conversion has also been extensively studied, and some findings have shown high potentiality in terms of catalytic efficiency and/or thermal and pH stability (Halder et al, 2019 ).…”

Peer Review #2 of "Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002 (v0.1)"

“…The activity of purified CHI was also obviously increased by the presence of Tween 80, similar to chitinase from S. thermodiastaticus HF 3-3 (Take et al, 2018). This indicates that Tween 80 reduces the activation energy of the reaction, promoting the enzymatic reaction.…”