No 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.

Extracellular α-1,3-glucanase HF90 (AglST2), with a sodium dodecyl sulfate (SDS)-PAGE-estimated molecular mass of approximately 91 kDa, was homogenously purified from the culture filtrate of Streptomyces thermodiastaticus HF3-3. AglST2 showed a high homology with mycodextranase in an amino acid sequence and demonstrated specificity with an α-1,3-glycosidic linkage of homo α-1,3-glucan. It has been suggested that AglST2 may be a new type of α-1,3-glucanase. The optimum pH and temperature of AglST2 were pH 5.5 and 60°C, respectively. AglST2 action was significantly stimulated in the presence of 5-20% (w/v) NaCl, and 1 mM metal ions Mn and Co. On the other hand, it was inhibited by 1 mM of Ag, Cu, Fe and Ni. Regarding the stability properties, AglST2 retained more than 80% of its maximum activity over a pH range of 5.0-7.0 at up to 60°C and in the presence of 0-20% (w/v) NaCl. Based on these results, the properties of AglST2 were comparable with those of AglST1, which had been previously purified and characterized from S. thermodiastaticus HF3-3 previously. The N-terminal amino acid sequence of AglST2 showed a good agreement with that of AglST1, suggesting that AglST1 was generated from AglST2 by proteolysis during cultivation. MALDI-TOF mass analysis suggested that AglST1 might be generated from AglST2 by the proteolytic removal of C-terminus polypeptide (approximately 20 kDa). Our investigation thus revealed the properties of AglST2, such as tolerance against high temperature, salts, and surfactants, which have promising industrial applications.